i

MEMCAL SCHOOL

LIBRARY

OUTLINES

OF

PRACTICAL HISTOLOGY.

MORRIS' ANATOMY.

791 Illustrations, Many in Colors.

A Complete Text-Book. Edited by HENRY MORRIS, F. R. c.s. , Surgeon
to, and Lecturer on Anatomy at, Middlesex Hospital, assisted by J. BLAND
BUTTON, F.R.C.S., J. H. DAVIES-COLLEY, F.R.C.S., WM. J. WALSHMAN,
F.R.C.S., H. ST. JOHN BROOKS, M.D., R. MARCUS GUNN, F.R.C.S., ARTHUR
HENSMAN, F.R.C.S., FREDERICK TREVES, F.R.C.S., WILLIAM ANDER-
SON, F.R.C.S., and Prof. W. H. A. JACOBSON. One Handsome Octavo
Volume, with 971 Illustrations, 214 of which are printed in colors.

Cloth, $7.50; Leather, $8.50; Half Russia, $9.50

*** THIS TREATISE is designed for the use of students, and is a sys-
tematic and complete description of all parts of the human body, and
includes a thorough Section by Jacobson on Surgical and Topographical
Anatomy, with 130 illustrations. Whilst each author is alone responsible
for the subject-matter of the article which follows his name, all have
combined, by reading proofs, by consultation, etc. , to give uniformity to
the work.

In no medical text-book of late date is there such a wealth of illus-
tration. Recognizing at the outset that this was a most important part of
the work in hand, no expense was spared in its development. With very
few exceptions, all the illustrations have been drawn and engraved specially
for this purpose from original dissections. Colors have been freely used
in the printing in order to bring out all important details.

"The illustrations are profuse and well executed, numbering in all seven hundred
and ninety-one wood-cuts, two hundred and fourteen of which are in colors from origi-
nal sketches. While it must be admitted that there can be scarcely anything new in
descriptive anatomy, it is refreshing to notice a departure in the arrangement and treat-
ment of this subject, which is PRACTICAL, USEFUL, and INTERESTING. In a word, the
natural method is adopted, and several new features of illustration are introduced. For
instance, the origin and insertion of muscles with exact areas of attachment are noted in
ditferent colored outlines, red for the former and blue for the latter. Thus the reader-
is enabled at a glance to discriminate in the case of a given bone the difference between
the two points, and obtain thereby the direction and extent of action of each muscle. A
similar principle is carried out in other parts of the work, which makes the illustrations
for the most part unique and invaluable, the schematic drawings especially demanding
attention and commendation in this connection. The work as a whole is filled with prac-
tical ideas, and the salient points of the subjects are properly emphasized. The surgeon
will be particularly edified by the section on the topographical anatomy, which is full to
repletion of excellent and useful illustrations."— New York Medical Record.

#&• Descriptive Circular with sample pages
and illustrations free upon application.

P. BLAKISTON, SON & CO., Publishers, Philadelphia.

OUTLINES

OF

PRACTICAL HISTOLOGY:

H fIDanual for Stubents,

BY

WILLIAM STIRLING, M.D., Sc.D.,

BRACKENBURY PROFESSOR OK PHYSIOLOGY AND HISTOLOGY IK THE OWENS COLLEGE,
AND PROFESSOR IN THE VICTORIA UNIVERSITY, MANCHESTER', EXAMINER IN

PHYSIOLOGY IN THE UNIVERSITIES OF OXFORD AND EDINBURGH, AND
FOR THE FELLOWSHIP OF THE ROYAL COLLEGE OF SURGEONS, ENGLAND.

Mill) 368 Illustration*.

SECOND EDITION, 'REVISED ' AND 'ENLARGED

PHILADELPHIA:

P. BLAKISTON, SON & CO.,

1012 WALNUT STREET.
1894.

4C\

K

PRESS OF WM. F. FELL & Co,

1220-24 SANSOW ST.,

PHILADELPHIA.

>

4-

PREFATORY NOTE TO THE SECOND EDITION.

IN the Second Edition of this Handbook I have endeavoured to
introduce a succinct account of the more recent Histological
Methods. Most of these I have tested, a matter of no incon-
siderable labour. The fruitful methods of Golgi, Ram6n y Cayal,
and Ehrlich are fully set forth in the text.

I have availed myself freely of the Journals and Text-books
published at home and abroad, and more especially useful to me
have been the works of Garbini and Ramon y Cayal. For most of
the additional illustrations I am indebted to the Gewebelehre of
SchiefFerdecker and Kossel.

In conclusion, I have to thank my Senior Demonstrator, James
A. Menzies, M.B., C.M., for assistance in reading the proof-sheets.

WILLIAM STIRLING.

PHYSIOLOGICAL LABORATORY, Tiiti OWENS COLLEGE,
MANCIJESTEK,

PREFACE.

THE present work was written primarily to meet the wants of
the students attending the course of instruction in "Practical
Histology " in The Owens College, but it is hoped that it will
be found useful also to students of medicine and science in other
colleges and universities.

The Exercises printed in small type are intended for Senior
Students, and for those attending the course of " Advanced
Histology " given in The Owens College.

Although a drawing accompanies almost every exercise, still
this is not intended to relieve the student from what is a most
important part of the training in Practical Histology, viz., that
the student should make sketches of his preparations. It serves
very little useful purpose to give students sections ready pre-
pared, and ask them merely to mount them. Hence, consider-
able stress has been laid on methods, as a knowledge of these is,
after all, one of the most important parts of a practical training
in Histology. Many methods have been tried and found wanting,
and accordingly those only are introduced which the author, after
experiment, has found to be reliable.

The author is indebted to the various Manuals of Histology
published in this country, and also to those of Friedlander and
Eberth, Stohr, Fol, Kolliker, Martinotti, Francotte, Bolles Lee
and Henneguy, Orth, and Edinger, as well as the various Micro-
scopical Journals, British and Foreign.

Most of the illustrations have been drawn from microscopical
preparations made in the class of Practical Histology, but a few

VI 11 PREFACE.

of them are taken from preparations kindly presented to me by
my friend Professor Swaen of Liege. I am greatly indebted to
my demonstrator, Arthur Clarkson, M.B., C.M., for a considerable
number of the drawings, and also to my pupil, Mr. C. E. M. Lowe,
for similar services. A few of the drawings were made by myself.
To F. W. Stansfield, M.B., Ch.B. (Viet.), my thanks are due for
assistance in reading the proof-sheets.

I have also to express my obligations to several scientific in-
strument makers and others, including Messrs. Zeiss, Hartnack,
Verick, Leitz, Keichert, Jung, Hicks, Eternod, Zimmermann,
Hume, Beck, Swift & Son, Gardner, and A. Eraser.

Finally, I have to thank my publishers for the liberal manner
in which the work is illustrated.

WILLIAM STIRLING.

PHYSIOLOGICAL LABORATORY, THE OWENS COLLEGE,
MANCHESTER,

CONTENTS.

PART I.

CHAP. PAGE

I. APPARATUS KEQU1KED . . . . . . • I

II. THK MICROSCOPE AND ITS ACCESSORIES .....' 5

III. NORMAL OR INDIFFERENT FLUIDS ...... 24

IV. DISSOCIATING FLUIDS 24

V. HOW TO TEASE A TISSUE . . . . . . . .26

VI. FIXING AND HARDENING FLUIDS 2"J

VII. DECALCIFYING FLUIDS 36

VIII. PREPARING TISSUES FOR MICROSCOPICAL EXAMINATION . . 38

IX. EMBEDDING . 40

X. SECTION CUTTING e 48

XI. FIXATIVES . . . , . . . . .60

XII. STAINING REAGENTS . 63

XIII. CLARIFYING REAGENTS ........ 82

XIV. MOUNTING FLUIDS AND METHODS ...... 85

XV. INJECTING BLOOD-VESSELS AND GLAND-TUBES .... 89

XVI. EXAMINATION OF FRESH TISSUES AND FLUIDS .... 92

PART II.

LESSON

I. MILK, GRANULES, FIBRES, AND VEGETABLE ORGANISMS . . 97

II. THE BLOOD I06

III. HUMAN BLOOD, CRYSTALS FROM BLOOD, AND BLOOD PLATELETS 11$
IV. EPITHELIUM (STRATIFIED) AND ENDOTHELIUM . . . .124

V. COLUMNAR, SECRETOHY, AND TRANSITIONAL EPITHELIUM . . 131

VI. CILIATED EPITHELIUM ........ 135

VII. KARYOKINESIS OR MITOSIS .... . 141

VIII. CELLULAR AND HYALINE CARTILAGE. ..... 146

IX. THK FIBRO-CARTILAGES, WHITE AND YELLOW . . . . 15 1

X. CONNECTIVE TISSUE 1 56

XI. TENDON . 163

X CONTENTS.

LESSON PAGE

XII. ADIPOSE, MUCOUS, AND ADENOID TISSUES — PIGMENT-CELLS . l68

XIII. BONE, OSSEOUS TISSUE ........ 174

XIV. BONE AND ITS DEVELOPMENT .,,... 182
XV. MUSCULAR TISSUE ..,,-... . 189

XVI. STRIPED OR STRIATED MUSCLE ,,,... 193

XVII. NERVE-FIBRES ......... 2O2

XVIII. NERVE-CELLS, NERVE-GANGLIA, AND PERIPHERAL TERMINA-
TIONS OF MOTOR NERVES . . . . . . -213

XIX. THE HEART AND BLOOD-VESSELS ...... 223

XX. THE LYMPHATIC SYSTEM, SPLEEN, AND THYMUS GLAND . 234

XXI. TONGUE, TASTE BUDS, SOFT PALATE ..... 246

XXII. TOOTH, (ESOPHAGUS ..... 251

XXIII. SALIVARY GLANDS AND PANCREAS ..... 256

XXIV. THE STOMACH 266

XXV. THE SMALL AND LARGE INTESTINE - .... 272

XXVI. LIVER ........... 285

XXVII. TRACHEA, LUNGS, THYROID GLAND ..... 294

XXVIII. KIDNEY, URETER, BLADDER, SUPRARENAL CAPSULE . . 303
XXIX. SKIN AND EPIDERMAL APPENDAGES . , . . . 315

XXX. SPINAL CORD 328

XXXI. MEDULLA OBLONGATA, CEREBELLUM, CEREBRUM . . . 347

XXXII. THE EYE .......... 358

XXXIII. EAR AND NOSE . . . . . . . . -371

XXXIV. TERMINATION OF NERVES IN SKIN AND MUCOUS MEMBRANES 376

XXXV. THE TESTIS 381

XXXVI. OVARY, FALLOPIAN TUBE, UTERUS ..... 388

XXXVII. MAMMARY GLAND, UMBILICAL CORD, AND PLACENTA . . 393

XXXVIII. TO MAKE PREPARATIONS RAPIDLY FROM FRESH TISSUES . 396

APPENDIX.

A.— SOME GENERAL WORKS OF REFERENCE 406

B. — TABLES OF MAGNIFYING POWER OF OBJECTIVES AND OCULARS . 408
C. —LIST OF MAKERS, BRITISH AND FOREIGN, OF MICROSCOPES,

MICROTOMES, CHEMICALS, AND HISTOLOGICAL REAGENTS . 409
D. — WEIGHTS AND MEASURES, TABLE OF EQUIVALENTS . . .410

INDEX 411

LIST OF ILLUSTRATIONS.

FIG.

1. English form of slide. (Zeiss.) .

2. Square and round cover-glasses. (Zeiss.)

3. Pinewood cabinet. (Swift.)

4. Paper tray for slides. (Jung.) .

5. Scissors. (Beck.) ....

6. Cover-glass lifter ....

7. Cornet's cover-glass forceps

8. Section-lifter. (Beck.)

9. Leitz's microscope, No. 5. (Leitz.)
10. Zeiss's large stand. (Zeiss.)

n. Iris, diaphragm. (Zeiss.)

12. Diaphragms. (Reichert.) .

13. Abbe's condenser. (Swift.)

14. Leitz's IA stand. (Leitz. )

15. Swift's college microscope. (Swift.)

16. Hartnack's No. III.

17. Zeiss's camera lucida. (Zeiss.) .

18. Abbe's camera lucida. (Zeiss.) .

19. Chevalier's camera lucida. (Fertefe.) .

20. Malassez's camera. (Verick.)

21. Eye-piece micrometer scale. (Zeiss.) .

22. Eye-piece micrometer. (Zeiss.) .

23. Microscope lamp. (Swift.)

24. Dissecting microscope. (Verick.)

25. Reichert's dissecting microscope .

26. Cover-glass tester. (Zeiss. )

27. Glass-thimble. (Beck.) .

28. Photophore. (Stirling.) .

29. Mayer's embedding bath. (Jung.)

30. Embedding box. (Stirling.)

31. Celloidin embedding box. (Stirling.) .

32. Valentine's knife. (Beck. )

33. Rutherford's freezing microtome. (Gardner.)

34. Cathcart's freezing microtome. (Fraser. )

35. Planing-iron. (Reichert.)

36. Malassez's microtome. (Fertc&.)

37. Cambridge rocking microtome

38. Minot's microtome. (Zimmermann.) .

39. Silk band for serial sections. (Zimmermann.

40. Thoma's sledge microtome. (Jung.)

41. Malassez' microtome. (Verick. )

42. Williams' microtome. (8h0ift.\ .

43. Swift's ether microtome. (Swift.)

44. Ranvier's hand-microtome. (Reichert.)

45. Vulcanite rings. (Eternod.)

46. Ring for stretching membranes. (Eternod.)

47. Ranvier's support. (Ranvier.) .

48. Capped balsam bottle. (5ecA:.) .

49. Turntable. (.Bedfc.)

50. Turntable with slide.

51. Hand-centrifuge. (Muencke.) .

52. Milk globules. ....

53. Potato starch. (Stirling.)

54. Rice starch. (Blyth.)

55. Air, fat granules in water. (Ranvier.)

PAGE

Xll

FIG.
56.

59-
60.
61.
62.

63-
64.

65-
66.
67.
68.
69.
70.

7i-
72.

73-
74-
75-
76.

77-
78.

79-
80.
81.
82.

83-
84.

85-
86.
87.
88.
89.
90.
91.
92.

93-
94.

95-

96.

97-
98.
99.
100.

IOI.
IO2.
I03.
104.
105.

106.
107.
108.
109.
no.
III.

TI2.

"3-
114.

115-

116.

117.
118.
119.

LIST OF ILLUSTRATIONS.

Silk, wool, cotton, linen. (Stirling.}

Cells from onion -bulb. (Schie/erdecker.)

Mould. (Ainsivorth Davis.) ....

Yeast-cells. (Landois and Stirling.} .

Micrococci. (V. Jaksch.) ....

Blood of frog. (Ranvier. )

Amphibian blood-corpuscles. (Landois and Stirling. )
Action of acetic acid on blood. (Stirling.) .
Action of water on blood-corpuscles. (Stirling.) .
Action of syrup on frog's blood. (Stirling.) .
Tannic acid on blood. (Stirling.)
Boracic acid on blood. (Stirling.)
Blood-corpuscles of lish and bird. (Stirling.)
Aniosboid movements of leucocytes. (Landois.)
Moist chamber. (Ranvier.) ....

Acetic acid on colourless corpuscles. (Stirling.')
Human blood. (Ranvier.) ....

Human blood. (Landois.) ....

Human red corpuscles. (Ranvier.)

Copper hot stage. (Stirling.) . ' .

Reichert's warm stage. (Reichert.)

Crenation of corpuscles. (Stirling. ) .

Fibrils of fibrin. (Landois.) ....

Rat's haemoglobin. (Stirling.) ....

Guinea-pig's haemoglobin. (Landois and Stirling.)

Hsemin crystals. ( V. Jaksch. ) . . . ' .

Leukenric blood. ( V. Jaksch.)

Blood platelets. (Landois and Stirling.)

Isolated epithelial squames ....

Squames of newt's epidermis. (Stirling.)

Hard palate of cat. (Stirling.)

Prickle-cells of epidermis. (Stirling.)

Isolated prickle-cells. (Ranvier.)

Endothelium of central tendon. (Ranvier.)

Omentum of young rabbit, silver nitrate. (Stirling )

Omentum of cat, silvered. (Stirling.)

Columnar and goblet cells. (Stirling.)

Isolated columnar cells. (Stirling.) .

Liver-cells. (Cadiat.) ....

Transitional cells. (Stirling.) .
Gas chamber. (Gscheidlen.)
Gas chamber. (Gscheidlen.)
Ranvier's moist chamber. ( Verick. ) .
Ciliated epithelium. (Stirling.)
Frog's ciliated cell. (Ranvier.)
Ciliated epithelium, ox. (Stirling. ) .
V.S. ciliated epithelium
Goblet-cells. (Stirling.)
V.S. frog's tongue. (Stirling.)
Connective-tissue corpuscle. (Stohr.)
Mitosis ......

V.S. epidermis of salamander. (Stirling.) .
Mitosis. (Stirling.) ....

Cellular cartilage. {Stirling.) .

Hyaline cartilage. (Stohr.)

Hyaline cartilage of thyroid. (Schie/erdecker.)

Costal cartilage. (Stohr.)

V.S. articular cartilage. (Stirling.) .

Branched cartilage-cells. (Stirling. ) .

V.S. intervertebral disc. (Stirling.) .

White fibro-cartilage. (Stohr.)

T.S. epiglottis. (Ranvier.)

Elastic cartilage ear of horse. (Schie/erdecker. )

Transition from hyaline to elastic cartilage. (Schie/erdecker.)

PAGE
102
I03
104
I04
104
I07
107

108
108
108
109
109
no
in
in

112

116
116
116
118
118
119

120
1 2O
120
121
121
I23

127
128
128
129
I29
130
I32
133
133

*34
136

137
137
i37
138
138
139
139
140
142

143
144
144
146

147
148
149
150
iSi
152

155

LIST OF ILLUSTRATIONS. Xlll

FIG. PAGB

120. Elastic fibres. (Stohr.) ........ 157

121. T.S. ligamentiim nuchae. (Stirling.} . . . . . .158

122. Elastic fibres. (Stirling.) ....... 158

123. Fenestrated membrane. (Stohr.) ...... 159

124. Areolar tissue. (Schiefferdecker.) ...... 159

125. Omentura of dog. (Schiefferdecker.) ...... 160

126. Hypodermic syringe. (Hicks.) ...... 161

127. Coarsely granular cells. (Stirling.) ...... 162

128. Cell-spaces in areolar tissue. (Stirling.) ..... 162

129. T.S. tendon. (Stirling.) . . . . . . .163

130. L.S. tendon. (Schiefferdecker.) ...... 164

131. Fibrils of tendon. (Stirling.) ....... 165

132. Tendon of rat's tail. (Stirling.) . . . . . 165

133. Tendon cells. (Ranvier.) ....... 165

134. T.S. tendon of rat's tail. (Stirling.) ...... 166

135. Endothelial sheath of tendon. {Stirling.) ..... 166

136. Cell-spaces of diaphragm. (Stirling.) . . . . 167

137. Fat-cells. (Stirling.) ........ 169

138. Fat-cells. (Schiefferdecker.) ....... 169

139. Empty fat-cells. (Stirling.) .... . 170

140. Fat-cells with margarine crystals. . . . .."•'. . 170

141. Development of fat-cells. \Stirling.). ..... 171

142. Mucous tissue. (Stirling.) ....... 172

143. Adenoid tissue. (Stohr.) ....... 173

144. Pigment and guanin cells. (Stirling.) ..... 173

145. T.S. metacarpal bone. (Ranvier.) ...... 175

146. T.S. femur. (Ranvier.) ........ 176

147. T.S. dense bone. (Ranvier.) ....... 177

148. L.S. dense bone. (Stirling.) ....... 177

149. T.S. metacarpal bone. (Stohr.) ...... 178

150. T.S. Haversi an canal. (Schiefferdecker.) ..... 179

151. Sharpey's fibres. (Stirling.) ....... 179

152. L.S. injected bone. (Stirling.). ...... 180

153. Cancellated bone. (Stirling.) ....... 180

154. Cancellated bone. (Stirling.) ....... 181

155. T.S. embryonic bone. (Ranvier.) ...... 182

156. T.S. foetal bone. (Stohr.) ....... 183

157. L.S. developing bone. (Stohr.) ...... 184

158. V.S. tibia. (Stir/ing.) 185

159. Developing bone. (Jianvier.) ....... 186

160. Membranous bone. (Stohr.) ....... 186

161. Marrow cell. (Stirling.) ....... 187

162. Isolated smooth muscle. (Landois and Stirling.) .... 190

163. Bladder of frog. (Stirling.) ....... 190

164. T.S. smooth muscle. (Stirling.) ...... 191

165. Cement of smooth muscle. (Stirling.) ..... 191

166. T.S. smooth muscle. (Schiefferdecker.) ..... 192

167. Striped muscle of frog. (Stirling.) ...... 194

168. Muscle of fibrils. (Schteffcr decker.) ...... 195

169. Tendon and muscle. (Landois and Stirling.) .... 196

170. Muscular fibre. (Ranvier.) ....... 196

171. Fibril of hydrophilus. (Ranvier.) . . . . " . . 196

172. Crab's muscle. (Stirling.) ....... 197

173. T.S. muscle. (Krause.) . . . . . . . . 197

174. T.S. muscle. (Stirling.) ...... 197

175. Injected muscle. (Landois and Stirling.) ..... 197

176. T.S. and L.S. injected muscle. (Owsjannikow.) .... 198

177. T.S. injected muscle. (Stirling.) ...... 199

178. Heart muscle. ......... 199

179. T.S. frozen muscle. ........ 199

180. Polariser. (Zeiss.) ........ 201

181. Medullated nerve-fibre. (Stirling.) ...... 203

182. Non-medullated nerve-fibre. (Ranvier.) ..... 204

183. Fresh nerve-libre. (Obersteiner.) ..... . 205

xiv LIST OF ILLUSTRATIONS.

FIG. PAGE

184. Nerve-fibre after osmic acid. (Schwalbe.) . ,m . . - . 205

185. Frog's nerve-fibre. (Obersteiner.) . . . . . . 206

1 86. Rauvier's crosses. (Obersteiner.) ...... 207

187. Ranvier's cross and Frommann's lines. (Obersteiner.) . . . 207

188. Peripheral nerve-fibre with axis-cylinder ..... 208

189. Intercostal nerve with Ranvier's crosses . . 208

190. T.S. nerve. (Eichhorst.) ....... 209

191. Non-mednllated nerve. (Obersteiner.} ..... 209

192. Sympathetic nerve. (Schieffer decker.) ..... 210

193. Neuro-keratin network. . . . . . . . .210

194. Frommann's lines. (Obersteiner.) ...... 211

195. Nerve-fibre of frog. (Obersteiner.) . . . . . .211

196. T.S. nerve-fibres, osmic acid. (Stirling.) ..... 212

197. Spinal ganglion. (Cadiat.) . . . . . . .214

198. Cells of spinal ganglion. (Obersteiner.) ..... 214

199. Bipolar nerve-cell of skate. . ..... 215

200. Spinal ganglion herve-cell. (Ranvier.) ..... 216

201. Cervical sympathetic ganglionic cell. (Stirling.) .... 217

202. Multipolar nerve-cell of spinal cord. (Obersteiner.) . . . 218

203. Nerves of frog's sartorius. (Mays.) ...... 220

204. End-plates of lizard. (Kiihne.) . .... 221

205. End-plate of lizard. (Stirling.) ...... 221

206. Sympathetic nerve-cell. (Ranvier.) ...... 222

207. Heart muscle. (Stirling.) ....... 224

208. Purkinje's fibres. (Ranvier.) ....... 225

209. Endocardium. (Ranvier.) ....... 226

210. T.S. tricuspid valve. (Ranvier.) ...... 226

211. L. S. human aorta. (Ranvier.) ...... 227

212. T.S. artery. (Stirling.) ....... 227

213. Eudothelium of artery and vein. (Stirling.) .... 228

214. Capillaries. (Obersteiner.) ....... 229

215. Small artery. (Stirling.) ....... 229

216. Arteriole. (Obersteiner.) ....... 229

217. T.S. small artery and vein. (Stirling.) ..... 230

218. Artery of brain. (Obersteiner.) ...... 231

219. Arteriole, silvered. (Ranvier.) ...... 231

220. Capillary, silvered. (Landois and Stirling.) .... 232

221. Developing blood-vessels. (Stirling.) ..... 233

222. L.S. lymph gland. (Ranvier.) . . . . . . 235

223. Lymph sinuses. (Stirling.) ....... 237

224. Central tendon. (Ranvier.) ....... 238

225. Pleural surface of diaphragm. (Stirling.) ..... 238

226. Stomata. (Stirling.) . . . . . . . 239

227. Tonsil. (Stohr.) ......... 240

228. Thymus. (Stirling.) ........ 241

229. Injected thymus. (Cadiat.) ....... 242

230. Elements of thymus. (Cadiat.) ...... 242

231. Human spleen. (Stohr.) ....... 243

232. Elements of splenic pulp. (Stohr. ) . . . . . . 244

233. Reticulum of spleen. (Cadiat.) ...... 244

234. T.S. tongue of cat. (Stirling.) ...... 246

235. Filiform papilla. (Stohr.) ....... 247

236. Fungiform papilla. (Stohr.) ....... 247

237. Crypt of tongue. (Schenk.) ....... 248

238. Injected tongue of cat. (Stirling.) ...... 248

239. Papillae foliatae. (Stirling.) ....... 249

240. V.S. Papillae foliatae. (Stdhr.) 250

241. V.S. tooth . . . . . . .--".. . . 252

242. 243. Enamel prisms. (Landois and Stirling) .... 253

244. T.S. fang of tooth. (Landois and Stirling. ) . . . . 253

245. Development of tooth. (Landois and Stirling.) .... 254

246. Later stage of 245. (Stohr.) ....... 254

247. Later stage of 246. (Stohr. )....... 254

248. T.S. oesophagus. (Stirling.) ....... 255

LIST OF ILLUSTRATIONS. XV

FIG. PAGE

249. Lobules of submaxillary gland. (Stirling.) ..... 260

250. Acini of submaxillary gland. (Stirling.) ..... 260

251. T. 8. duct of salivary' gland. (Landois and Stirling.) . . . 261

252. Resting serous gland. (Heidenhain.) ..... 261

253. Human submaxillary gland. (Heidenhain.) .... 262

254. T.S. pancreas. (Stirling.) ....... 263

255. T.S. fresh pancreas. (Heidenhain.) ...... 264

256. V.S. stomach. (Stohr.) ........ 268

257. V.S. mucous membrane of stomach. (Stirling.) .... 269

258. T.S. gastric glands. (Landois and Stirling.) .... 270

259. V.S. pyloric glands. (Heidenhain.) ...... 270

260. T. S. small intestine. (Stirling. )...... 274

261. L.S. Peyer's patch. (Landois and Stirling.) .... 275

262. Injected small intestine. (Stohr.) ...... 276

263. Scheme of 262. (Mall.) . ....... 277

264. Injected villi. (Stirling.) ....... 277

265. Auerbach's plexus. (Stirling.) ....... 278

266. Auerbach's plexus. (Cadiat.) ....... 278

267. Section of 266. (Cadiat.) ..... . 279

268. Meissner's plexus. (Stirling.) ....... 279

269. V.S. duodenum. (Stohr.) ....... 280

270. L.S. large intestine. (Schenk.) ....... 281

271. Lieberkiihn's gland. (Heidenhain.) ...... 281

272. T.S. villus. (Heidenhain.) ....... 282

273. Intestinal villus. (Kultschitzky.) ...... 283

274. Villus absorbing fat. (Stirling.) ...... 283

275. T.S. liver of pig. (Stirling.) ....... 286

276. Liver-cells. (Heidenhain.) ....... 287

277. T.S. portal canal. (Stirling.) ....... 287

278. Human liver. (Stohr.) ........ 288

279. Liver of frog. (Stirling.) ....... 289

280. Injected liver of rabbit. (Stirling.) ...... 290

281. In terlobular bile-duct. (Landois and Stirling.) .... 290

282. Bile-ducts iniected. (Cadiat.) ....... 291

283. T.S. trachea of cat. (Stirling.) . .... 295

284. L.S. trachea of cat. (Stirling.) ...... 296

285. T.S. bronchus. (Stirling.) 296

286. T.S. human bronchus. (Hamilton.) ...... 297

287. V.S. lung and pleura. (Hamilton.) ...... 298

288. Silvered lung of kitten. (Hamilton.) ...... 299

289. Injected lung. (Stirling.) ....... 299

290. T.S. dried lung. (Stirling.) ....... 300

291. T.S. foetal lung. (Stirling.) ....... 300

292. T.S. thyroid gland. (Cadiat.) ....... 301

293. L.S. Malpighian pyramid. ....... 303

294. Scheme of renal tubules. (Klein.) ...... 304

295. Glomerulus. (Stirling.) . . . . .' . . 308

296. Rodded epithelium. (Landois and Stirling.) .... 308

297. Irregular renal tubule. (Landois and Stirling.) .... 308

298. T.S. apex of Malpighian pyramid. (Stirling.) .... 309

299. Blood-vessels of kidney. (Landois and Stirling.) .... 310

300. Henle's tubule. (Landois and Stirling.) . . . . .311

301. Cells from renal tubule. (Landois and Stirling.) . . . 311

302. T.S. ureter. (Stohr.) ........ 312

303. V.S. bladder. (Stohr.) ... . 313

304. T.S. penis. (Stirling.) . . . . . . . .313

305. V.S. suprarenal capsule. (Stohr.) . . . . . -314

306. V.S. skin of palm. (Stirling.). ...... 317

307. Human epidermis. (Ranvier.) . ... . . 318

308. T.S. sweat gland. (Stirling.) ....... 319

309. V.S. hair-follicle. (Landois and Stirling.) ..... 322

310. T.S. hair-follicle. (Landois and Stirling.) ..... 324

311. V.S. injected skin. (Taguschi.) ...... 324

312. T.S. nail. (Landois and Stirling.) ... . . . 326

XVI LIST OF ILLUSTRATIONS.

PIG. PAGE

313. Axillary gland. (Landois and Stirling.} ..... 327

314. T.S. spinal cord. (Cadiat.) ..-.-, . . . . . 334
315 T.S. anterior cornu of cord (Obersteiner.) ..... 335
316. T.S. white matter of cord. Obersteiner.) ..... 335
317-20. T.S. spinal cord at various levels. (Obersteiner.) . . 336

321. Neuroglia cell. (Ranvier.) ....... 343

322. L.S. spinal cord to show collateral fibres. (Kolliker.) . . . 347

323. T.S. medulla oblongata. (Ilenle.) ...... 348

324. T.S. medulla oblongata. (Edinger.) ...... 348

325. Leaflet of cerebellum. (Stirling. )...... 350

326. V.S. cerebellum. (Obersteiner.) ...... 350

327. V.S. cerebrum. (Obersteiner.) ...... 352

328. V.S. frontal convolution. (Obersteiner.) ..... 352

329. V.S. injected cerebrum. (Obersteiner.) ..... 354

330. Cell of Purkinje. (Obersteiner.) ...... 356

331. T.S. cerebrum of rat. (Cayal.) ...... 356

332. V.S. cerebrum. (Edinger.) ....... 357

333. V.S. cornea. (Landois and Stirling.) . ... 359

334. Cornea corpuscles. (Ranvier.) ...... 359

335. Nerves of cornea. (Ranvier.) ....... 360

336. V.S. cornea. (Ranvier.) ....... 360

337. Cornea cell-spaces. (Ranvier.) ...... 361

338. V.S. sclerotic and choroid. (Stohr.) ...... 361

339. T.S. sclerotic and cornea. (Landois and Stirling.) . . . 362

340. Lens fibres. (Landois and Stirling.) . ..... 364

341. V.S. retina. (Ranvier.) . . . . . . . 366

342. V.S. cochlea. (Ranvier.) ....... 372

343. V.S. cochlear duct. (Landois and Stirling.) .... 373

344. V.S. olfactory mucous membrane. (Stohr.) ..... 375

345. Olfactory cells. (Landois and Stirling.) ..... 375

346. Tactile cells. (Ranvier.) ....... 377

347. Tactile discs. (Ranvier. )....... 378

348. End-bulb . . . . . . . . . . 378

349. Pacini's corpuscle. (Ranvier.) ...... 379

350. Endothelium of 349. (Stirling.) ...... 379

351. T.S. of 349. (Stirling.) 379

352. Wagner's corpuscle. (Landois and Stirling.) . . . 380

353. Wagner's corpuscle. (Ranvier.) . . . . . .381

354. Organ of Eimer. (Stirling.) . . . . . . .381

355. T.S. testis .......... 382

356. Tubule of testis. (Landois and Stirling.) ..... 382

357. Spermatogenesis of rat. (Stirling.) ...... 386

358. Spermatozoa. (Landois and Stirling.) . . . 387

359. Epididymis. (Schenk.) ........ 387

360. V.S. ovary. (Turner.) ........ 389

361. Ovum .......... 390

362. T.S. Fallopian tube. (Schenk.) ...... 391

363. T.S. fimbriated end of 362. (Stirling.) . . . . .391

364. V.S. uterus. (Stirling.) ....... 392

365. T.S. mamma. (Stirling.) ...... 394

366. T.S. active mamma. (Stirling.) .. . . . . . 394

367. Colostrum. (V. Jaksch.) ....... 394

368. Placental villus. (Cadiat.) . . . . . . -395

[The illustrations indicated by the word " Stohr " are from Stohr's Lehrbuch
der Histologie ; by "Cadiat," from Cadiat's Traite d'Anatomie Generate; by
"Ranvier," from Ranvier's Traite Technique d1 Histologie; by "Schenk" from
Schenk's Grundriss der normalen Histologie ; by " Obersteiner" from Obersteiner's
Anleitung beitn Studium des Baues der nervb'sen Central organe ; by " v. Jaksch,"
from v. Jaksch's Klinische Diagnostik ; by "Edinger," from Edinger's Zwol/
Vorlesungen iiber den Ban, der nervosen Centralorgane ; by " Landuis and
Stirling," from their Text-Book of Physiology ; by " Schie/er decker," from Das
Mikroskop, by " Behrens, Kossel, and Schie/er decker."]

PRACTICAL HISTOLOGY.

PART I.

I.— APPARATUS REQUIRED.

THE student of Practical Histology must be provided with the
following apparatus:—

1. A Compound Achromatic Microscope capable of magnify-
ing from about 50 to 300 or 450 diameters linear.

2. Glass Slides. — The most convenient size is 3 inches by i
inch (or 76 x 26 mm.), with ground edges made of the best
flatted crown-glass. About two gross will be required (fig. i).

76X26 M™

FIG. i.— English form of Slide.

It is convenient to have two dozen or so of a larger size, 3 inches
by j£ inches.

3. Cover-Glasses.— Some are square and others circular (fig. 2).

JflG. 2.— Square and Round Cover-Glasses, showing the most convenient sizes.

The student should be provided with both sorts. Keep the square
ones for balsam preparations, and the circles for those that roquire

• 2

PRACTICAL HISTOLOGY.

FIG 3.— Pinewood Cabinet to hold Sixty Slides.

ringing, i.e., those mounted in glycerine or Farrant's solution.

Only extra-thin covers (or those sold as No. i) should be used.

It is well to measure the thickness of the covers, and to use for

mounting only those that are less than .006 inch in thickness.

Get half-an-ounce
of f-inch circles,
and the same
weight of |-inch
squares. It is con-
venient to have
a few circles and
squares somewhat
larger, viz., i inch
and i^ inch in dia-
meter, for mount-
ing particularly
large sections.
When a large num-
ber of sections are
mounted under
one cover-glass, as

in serial preparations, it is well to have oblong cover-glasses of a

still larger size.

4. A Wooden CaM-
net, fitted with trays
for holding the mounted
specimens. It should be
capable of holding at
least 60 slides, and the
slides should lie on the
flat. A convenient form
is shown in fig. 3. Some
prefer the flat com-
pressed paper trays
shown in fig. 4.

5. Two Mounted
Needles in handles.
The student can easily
make these himself. Fix
a sewing-needle into the
end of a pen-holder,

allowing only about \ inch of the needle to project. The needles
should always be kept bright and polished. A convenient form is
to fix a sewing-needle into a strong wooden " crochet " needle.
6. A Dissecting Case, but failing that, a pair of strong forceps,

FIG. 4.— Paper Tray to hold Slides.

APPARATUS REQUIRED.

and also a liner pair, the latter with long narrow points ; a stout

scalpel. It is well to have a
pair curved on the flat (fig. 5.)

and a fine pair of scissors, and a
straight pair of scissors, and also a
It is convenient to have a pair
of forceps like those shown in
fig. 6 for lifting and applying
a cover glass to a preparation,
or like those in fig. 7 for hold-
ing a cover-glass on which a thin
film with bacteria is spread.

7. Camel-Hair Brushes, at
least two, the smaller crow-size,
and one somewhat larger.

8. A Razor, which is not to
he hollow-ground. It must be
kept very sharp, and stropped
frequently. It is better to have
one ground flat on one side.

9. Watch-Glasses. — Instead
of the ordinary-sized glasses,
the student should provide
himself with at least four 3
inches in diameter.

10. A Section-Lifter. — This may be made by beating out the end
of a piece of copper wire (T^ inch thick) until a thin plate is formed.

FIG. 5.— (a.) Scissors curved on the flat ;
(ft.) fine straight pair.

FIG. 6.— Cover-Glass Lifter.

The plate is then bent at an angle to the stem. It is better, how-
ever, to purchase one made of German silver (fig. 8).

11. Drawing Materials. — As great importance is attached in

FIG. 7.— Cornet's Cover-Glass Forceps.

this laboratory and in this course to making drawings of the
microscopic objects, each student must provide himself with a
drawing-book — a quarto, with unruled paper, and containing i 50
pages or thereby, is sufficient. Suitable dfawing-pendlSj including

4 PRACTICAL HISTOLOGY.

an H.B., and a harder one, e.g., H.H.H. ; both must be of a good
quality of lead. After a sketch has been made in pencil, the
sketches should be coloured. This may be done either with

coloured pencils or water-colours. The latter are greatly to

be preferred.

12. Slips of white bibulous paper, 3 inches by \ inch,
to soak up any superfluous fluid, and to be used for irriga-
tion. For irrigation purposes use small triangular slips.

13. Small glass pipettes, which the student should
make for himself by heating in a gas-flame and drawing
out a piece of narrow glass-tubing at two places, close to
each other, leaving a small part of the tube of the original
width, which acts as the bulb of the pipette. Several may
be made at a time, and their capillary ends sealed in the
flame, and kept until they are required.

14. A pair of narrow glass rods drawn to a point to
tease tissues in such metallic solutions as gold chloride or
silver nitrate, which act on metallic instruments.

15. Labels for the slides. — It is well to have a large
number of pieces of paper cut, 3 inches by i inch, as
temporary labels, on which is written the name of the
preparation. Each label is placed under its appropriate
slide in a tray. These labels arc merely temporary. This
is specially desirable where the slides have to be "ringed,"
as in this process a permanent label is apt to be displaced
or destroyed. In the case of balsam preparations, they
may be labelled at once with the small square permanent
labels. In every case the preparation should be labelled,

sIG't'8'~ an(^ ^ne l^bel should bear not only the name of the tissue
Lifter, or organ, but the direction of the section and the medium
in which it is mounted, and, if desired, the date of mount-
ing. Labels are now printed so cheaply, that for half-a-crown a
student can have a thousand labels printed with his own name.

16. Reagents. — The student should also be provided with the
following reagents, placed in a small wooden framework on the
work-table. Only those reagents that are most frequently used
need be provided for in the framework ; the others can be supplied
as required.

Small bottles — two ounces or thereby — not too tall, and provided
with a glass rod, are necessary. The glass rod has a bulge at the
junction of its upper and middle thirds, and this bulge prevents it
from falling into the bottle, and, at the same time, acts as stopper
for the bottle. Failing this, a piece of glass rod passed through the
cork will answer the purpose.

(1.) Normal Saline, or *6 per cent, salt solution. Dissolve 6

THE MICROSCOPE AND ITS ACCESSORIES. 5

gmis. of pure common salt in 1000 cc. of water. As this fluid is
apt to undergo change, it should not be kept too long.

(2.) Glycerine (either pure or equal parts of glycerine and water).

(3.) Balsam, either Canada balsam or dammar (p. 85).

(4.) Farrant's Solution (p. 85).

(5.) Dilute Acetic Acid (2 per cent.).

(6.) Hsematoxylin Solution (p. 68).

(7.) Picro-Carmine (p. 66).

(8.) Clove-Oil or Xylol.— This should be provided with a small
brush fixed on the end of a wooden rod perforating the cork.

17. Other Apparatus is required, but in a well-equipped
laboratory special articles are supplied as required ; they are referred
to in the context. They include a dissecting microscope, photophore,
mounting block, warm stage, eye-piece micrometer, lamp, turntable,
polarising apparatus, camera lucida, &c., &c.

II.— THE MICROSCOPE AND ITS ACCESSORIES.

1. An account of the optical principles on which the microscope
is constructed is purposely omitted. The compound microscope
consists of a stand fixed to a heavy, usually horse-shoe shaped, foot.
The stand (fig. 9) carries a stage to support the microscopic
preparation, the mirror or arrangement for illuminating the object,
together with the body tube ; the latter consists of a long brass tube,
or one tube telescoped into another. To the lower end of this tube
is fixed a combination of lenses, constituting the lens or objective,
while at its upper end is the eye-piece.

2. The tube is blackened inside, and to its lower end is screwed
the objective, consisting usually of several lenses screwed together.
By means of it a magnified inverted aerial image is produced in the
body of the tube. The lenses on the objective should not be
unscrewed. At least two objectives are required — a low power
and a high power.

At the upper end of the tube is the ocular or eye-piece, com-
posed of two plano-convex lenses, the one next the eye of the
observer being called the eye-glass, the lower one the field-glass.
The two lenses, with their convex surfaces downwards, are fixed in
a brass tube which slips into the upper end of the tube of the
microscope.

3. The tube itself is supported in a vertical position on the stand,
so that it can be moved upwards and downwards vertically, to bring
the objective near to the object, and thus bring the latter clearly
into focus. This arrangement is termed the adjustment. The

PRACTICAL HISTOLOGY.

Body.

Body tube.

mode in which this is accomplished varies. In the cheaper
microscopes the tube of the microscope is moved inside another

tube fixed to the
Eye-piece. stand (fig. 9). This

is done by means of
the hand, while in
the more expensive
microscopes there is a
rack-and-pinion move-
ment for raising or
depressing the tube
(fig. 10).

Usually there are
two adjustments — one
the coarse adjust-
ment, whether it be
by rotating one tube
inside the other or
by a rack-and-pinion
movement ; it is used
to bring the outlines
of the object dimly
into focus. The other,
the fine adjustment,
is a fine screw, usually
placed at the upper
and back part of the
pillar of the stand of
the microscope. By
it the object is brought
accurately into focus.
4. The stand is pro-
vided with a hori-
zontal solid table or
stage, placed at a con-
venient height, and
on which the object
to be examined is
placed. The stage
consists of a flat plate
of brass blackened on
its under surface, or
There are two clips

Stage.

FlG. 9.— Leitz's Microscope, No. V.

of a glass plate fixed on a black ground.

which are used for fixing a preparation in a definite position on the

stafe. It is perforated by a circular aperture in its centre, into

THE MICROSCOPE AND ITS ACCESSORIES. 7

which can be fitted diaphragms of different sizes, or a blackened
circular brass plate with holes of different sizes — from a pinhole to
half an inch — rotates under the stage, so that the desired size of hole
— or diaphragm — can be brought under the central aperture in the
stage. The aperture in the stage must not be too small ; it should
be sufficiently large to enable a section of the spinal cord to be
seen as a whole. A small aperture is used with high powers and a
large aperture with low powers. In the more expensive, and in
some oi the cheaper microscopes also, the stand is provided
with a joint, so that the microscope can be inclined as shown in
fig. 10.

5. Illumination of the Object. — Under the stage is placed a
mirror, movable in all directions, and which is usually provided
with a flat and a concave surface. When it is available, diffuse
light — never direct sunlight — reflected from a white cloud, and a
northerly exposure are to be preferred. For ordinary illumination
the concave side of the mirror is used. The light is reflected from
it, and is transmitted through the hole in the stage, the object on
the stage, and the tube of the microscope, to the eye of the
observer. The flat mirror is used along with a sub-stage condenser
(§ 10).

6. Direct and Oblique Illumination. — When the light is re-
flected from the concave mirror, it strikes the object nearly verti-
cally ; this is called direct or central illumination. But sometimes
it is of importance to detect very fine variations on the surface of
the object; then for this purpose oblique illumination is practised.
This may be done by tilting the mirror slightly, so that the rays
of light fall somewhat obliquely on the object. In this case there
must be no small diaphragm in or under the stage. This may also
be done in the more expensive microscopes by introducing a
diaphragm which permits light to pass only at its sides, its
centre being blocked. This is known as a central stop-dia-
phragm, which shuts off all the axial, and transmits only the;
marginal rays, causing what is called dark-ground illumination

(fig. 12).

7. The Diaphragm, of which there are two forms in common
use. The most common form is a blackened metallic plate — dine
<U«i>li)'aym — perforated with holes of different sizes, placed under
the stage, and so arranged as to rotate on a pivot. The edge of the
plate usually projects a little beyond the stage, so that it can be
readily rotated by the finger, so as to bring the appropriate aperture
under the hole in the stage. The diaphragm is usually provided
with a slightly projecting pin, which gives a click when the hole in
the diaphragm is exactly centred. Another form — rylindrn'nl
diapliraym — consists of a small brass cylinder, into which can be

8

PRACTICAL HISTOLOGY.

fitted small diaphragms perforated by apertures of different sizes
(figs. 10, 12). The cylinder, with its diaphragm, is fitted into a
slot under the stage. These cylinder diaphragms should be so
arranged as to be easily changed.

Perhaps the most convenient form of all is the iris diaphragm,
which can be adapted to any of the larger microscopes (fig. 14),
and in which any size of aperture desired is obtained by turning a
small milled head. The new form (fig. u), as made by English

makers, Zeiss, and others,
and also by Leitz, is
an admirable substitute
for interchangeable dia-
phragms, as by it the
aperture can be readily
increased or diminished.
The smallest aperture is
about 0.5 mm., and the
largest equal to the full
aperture of the con-
densing system.

The management of
the diaphragm is most
important in order to
obtain distinct definition
of an object. The one
general rule — but one
which is very frequently
neglected in practice by
the student — is when
employing a low power
to use a large aperture,
and when employing a
high power, a small or
medium aperture of the
diaphragm.

FIG. io.— Zeiss's Large Jointed Stand, fitted with draw- When O116 wishes to
tube, rack and pinion for the coarse adjustment, a , , . -, , ,

double nose-piece, and cylindrical diaphragm. Observe a brightly col-

oured object with a

homogeneous immersion lens — the object lying in the tissues, such
as stained fungi or mitotic figures — then remove the diaphragm and
allow a flood of light to reach the sub-stage condenser, which is
absolutely necessary in this case.

If, however, it be desired to see certain peculiarities of structure
—as in bacteria — on a black background, then use a central stop-
diaphragm, i.e., one with a central stop (fig. 12), always with an

THE MICROSCOPE AND ITS ACCESSORIES. 9

Abbe's condenser. The object is then seen brilliantly illuminated
on a black background.

8. Objectives. — For ordinary work every microscope requires to
have two objectives of different magnifying powers ; one of these,
when used with an ordinary ocular, should magnify about 60-75

FIG. 12.— Cylindrical Diaphragms
(i, 2, 3), a central stop-dia-
phragm (5).

FIG. ii.— Iris Diaphragm, showing a small
aperture.

diameters linear. This is spoken of as the low power, and in these
pages is indicated by the letter (L). The other should magnify
from 350-400 diameters linear, and is called the high power,
indicated by the letter (H).

If an English make of lens is preferred, let them be an " inch "
and a " quarter inch." The term " one-inch objective " has no direct
relation to the distance between the object and the lens, but indi-
cates that such a lens possesses the same magnifying power as a single
lens of one-inch focus. Swift's new high-angled i-inch lens is a
very good low-power lens. If Continental lenses be preferred, and
if Zeiss's be selected, let them be A and D, equal to §-inch and
J-inch respectively. Zeiss makes lenses AA andDD of slightly better
quality, which cost a few shillings more. If Leitz's lenses be pre-
ferred, use Nos. 3 and 7 ; and if Hartnack's, Nos. 3 and 7 — 3 being
the weaker lens. Crouch's lenses are excellent.

Lcitz numbers his objectives 1-9, and Zeiss A-F. i and A are the
weakest objectives, and the magnifying power increases up to 9 and F.

The microscopes of Reichert of Vienna are also excellent. The
stands arc all provided with a universal thread or screw, so that the
lenses of Zeiss, of English makers, or of others may be adapted to
them.

For certain special purposes much higher powers are required,
but for ordinary work these lenses are sufficient.
3

10 PRACTICAL HISTOLOGY.

The following patterns of microscopes are to be commended : —

Leitz, Stands J^"os. V., III. 17, IV. 19, V. 23, with objectives

3 and 7.
Zeiss, Stands Nos. VI. and VII., with objectives C and E.

9. Eye-Pieces or Oculars. — Two eye-pieces are required, one
medium length and the other shorter, the latter being the more
powerful. The two most useful eye-pieces are Nos II. and IV. of
foreign makers (of Leitz, however, I. and III.), or A and C of
English makers. English makers speak of a deep and a shallow eye-
piece ; the former (IV. or C) is shorter, and is the more powerful ;
the latter (II. or A) is longer, and is a weaker eye-piece.

It is to be remembered that the eye-piece only magnifies the
image of the preparation formed by the objective, and does not
magnify the preparation itself. Hence any fault in the lens, and
consequently in the image in the tube, is magnified by the eye-
piece. Moreover, the field is not so bright as with a weaker eye-
piece. Hence it is expedient to use rather a weak eye-piece. There
is an exception to this rule in the case of apochromatic lenses (p. 13),
which yield a magnification of the object by means of special eye-
pieces, without any loss in the brightness or sharpness of the image.

These comprise the essential parts of the microscope, but if
expense be no objection, the stand may be provided with a hinge-
joint, which enables the microscope to be inclined at any angle (figs.
10, 14). In some microscopes the tube can be elongated by a draw-
tube (fig. 14), which, when it is elongated, increases the magnifying
power of the instrument. It is well to have the draw-tube with a
scale engraved on it, as is done in the more expensive instruments.

10. Sub-Stage Condenser. — When working with high powers, this
is essential, more especially in connection with bacteriological work.
Abbe's condenser is by far the most convenient form. Fig. 14 shows
one of the more expensive stands fitted with a sub-stage condenser.

The essential feature of Abbe's illuminating apparatus is a con-
denser system of very short focus, which collects the light reflected
by the mirror into a cone of rays of very large aperture and projects
it on the object (fig. 1 3).

The cone of light is usually reduced by diaphragms of suitable
size, or by means of an iris diaphragm. By means of the rack-work
the diaphragm can be placed excentrically and oblique illumination
obtained. This apparatus can be used for ordinary work, but it is
specially useful for the investigation of bacteria. The rays of light
from the condenser are brought to a focus in the object, so that an
enormous amount of light is concentrated on the object. The angle
of aperture of Abbe's condenser is 120°. The full aperture of the
illuminating cone of rays is only used when observing deeply-stained

THE MICROSCOPE AND ITS ACCESSORIES.

II

bacteria with objectives of large aperture. In every other case A
diaphragm of suitable size is introduced so as to diminish the
cone of light. When the diaphragm is placed excentrically, oblique
illumination is obtained, while,
with a central-stop diaphragm
all the axial rays are cut off,
and dark-ground illumination is
obtained.

11. Dry and Immersion
Lenses. — By the term dry lens
is meant one in which air is
the medium between the lens
and the object, or at least the
cover-glass on the object. In
immersion lenses some fluid
intervenes between the lowest
lens of the objective and the
cover-glass of the object, and
the liquid chosen is water, or a
medium of higher refractive
index, such as cedar-wood oil,
glycerine, or a mixture of
fennel and ricinus oils. These
oils have nearly the same re-
fractive index as the cover-
glass. In virtue of their greater refractive power, these liquids,
especially the oils, refract more of the rays passing through the
object, and cause these rays of light to enter the lens, so that they
increase the amount of light transmitted to the eye of the observer.
In contrast with dry lenses, they have a larger angle of aperture
and a greater resolving power, and are employed only for the
highest magnifying powers. Powers above \ of an inch should be
oil-immersion lenses. Oil-immersion or homogeneous immersion
lenses are to be preferred to water ones. The oil-immersion are
fast displacing water-immersion lenses.

In using an oil-immersion lens, by means of a glass rod, place a
small drop of thick cedar-oil on the cover-glass and a small drop on
the lowest lens of the objective, and slowly depress the objective
until it touches the cupola of the drop, and focus. Cedar-oil has a
refractive index nearly the same as that of crown-glass, so that
almost all the rays of light passing through the object reach the lens
and pass up the tube of the microscope. Care must be taken that
the drop of oil docs not run on to the cement of the preparation,
else it will dissolve it. It is better to use marine glue to seal up
the preparation, as it is not dissolved by cedar-oil. A very fine

FIG. 13. — Abbe's Condenser, as made by Zeiss
and Swift. (S.) condenser system. Milled
bead for throwing the diaphragm out of
centre ; (Sp.) mirror.

12

PRACTICAL HISTOLOGY.

FIG. 14.— Leitz's Stand la., with a triple nose-piece and Abbe's condenser.

THE MICROSCOPE AND ITS ACCESSORIES. .13

linen cloth moistened with benzene may be used to remove the oil
from the lens. After use, the cover-glass must be wiped dry, as
well as the lens, by means of a clean piece of wash-leather. These
lenses are very good, but very dear, and are not required for ordinary
work. Latterly, instead of oil, a mixture of glycerine and chloral
hydrate has been recommended. The cedar-oil is not so easily
removed from the cover-glass. It may be best removed by a well-
washed linen rag moistened with benzene. The lens itself must be
most carefully cleaned. A lens moistened with glycerine is best
cleaned with alcohol.

12. Angle of Aperture. — Lenses may be of narrow or wide angle
of aperture. The angle of aperture is the angle formed by the
outermost rays coming from a luminous point placed in the focus of
an object, and which enter not only the lowest lens of the objective,
but pass throughout the entire system of the lenses of the objective.
Lenses with a large angle of aperture (130° and upwards), therefore,
will admit more light, i.e., more of the oblique rays will enter the
system of lenses. These lenses are well suited for resolving line
lines on the surface of an object, such as the striae on the scales of
insects' wings and the markings on diatoms, but those parts of the
object superficial to or deeper than the focus are not sharply defined.
Such a lens is said to have greater power of resolution. For ordi-
nary histological work, a high-power (350-400) lens of medium
(8o°-ioo°) angular aperture is to be preferred, for such a lens has
greater penetrating power, i.e., the focal plane is deeper, so that with
it one can see with tolerable distinctness parts of the object lying
immediately above and below the true focus of the lens. It is to
be remembered that the angle of aperture has nothing to do with
the magnifying power of the lens.

13. Abbe's Apochromatic Lenses. — These are dry or immersion,
and are used as high-power lenses when very exact definition is
required. They are very expensive, and are constructed of a
peculiar kind of glass. These objectives secure the union of three
different colours of the spectrum in one point of the axis, i.e., they
remove the so-called "secondary spectrum," and they correct the
spherical aberration for two different colours. The images projected
by them are nearly equally sharp with all the colours of the
spectrum. As there is a very great concentration of light by these
objectives, they permit of the use of very high eye-pieces, thus giving
high magnifying power with relatively long focal length. The natural
colours of objects are reproduced unaltered by these objectives.

Zeiss has constructed a series of compensating oculars to be
used with these lenses. They are classified as i, 2, 4, 8, 12, 18,
and 27, according to their magnifying power. The eye-pieces of
extremely low power are designated —

14 PRACTICAL HISTOLOGY.

(i.) Searchers, which reduce to its lowest limits the available
magnification with each objective, thus facilitating the preliminary
examination of objects, diminishing the labour of searching for
particular parts of the specimen. Thus, No. i enables an objective
to be employed with its own initial magnifying power, i.e., as if it
were used as a simple lens without an ocular (Zeiss).

FlG. 15.— New College Microscope, by Swift & Sons— Tripod Stand.

(ii.) The working eye-pieces begin with a magnifying power of 4.
The most useful are 4 and 6. Other eye-pieces arc —

(iii.) Projection eye-pieces ; but they do not concern us here.

14. In selecting a microscope, there are many points to be
attended to.

THE MICROSCOPE AND ITS ACCESSORIES.

(A.) Mechanical Parts. — It should bo quite stable, so that it
cannot be readily upset. To this end the stand should be solid, and
either of the tripod (fig. 15) or horse-shoe pattern (figs. 9, 10). Very
good stands of the tripod pattern are made by Messrs. James
Swift & Sons (fig. 15) and Messrs. Crouch. Fig. 1 5 has a glass stage,
the body-tube is cloth-lined, which gives a smooth and steady action.
The stage should be at a convenient height, so that when the ulnar
edge of the left hand is resting on the table, the thumb and fore-
finger of the same hand can conveniently grasp find move the slide on
the stage. The stage itself should be a little broader than the length
of the slide. The slide can be fixed on the stage by means of two
brass clips, which are fitted into holes at
the two posterior angles of the stage.
The pillar of the microscope may be fitted
with a joint to enable the instrument to
be inclined, if desired; but of course
this cannot be used when fluids are being
examined ; still in many instances it is
convenient (figs. 10, 14). Fig. 16 shows
a convenient form made by Hartnack of
Potsdam, and one very extensively used
by students.

Itis highly inconvenient tohave to screw
and unscrew a lens every time a change
of lens is required. This is obviated by
using a nose-piece or revolver (figs. 10,
14), which is screwed to the lower end
of the tube of the microscope. The high
and low powers are fitted to this frame-
work, and can be rotated under the tube
as they are required. Xose-pieces are
made for attaching two or three (figs. 10,

/VKir>r>H'-uoe and tVmvnnn Vio
Objectives, and tney can L

iidapted to any microscope.

One must be cautious in using a nose-piece, and take care to raise
the tube high enough to allow the objective to revolve without touch-
ing the cover-glass, which is especially apt to happen on using a high
power after a low power, the lenses not being of the same length.

Test the mechanical parts that they are all solid and work well.
"Raise and lower the tube by means of the fine adjustment, and do
this to the full extent of the threads on the screw, noting parti-
cularly if there is any lateral movement of the tube while this is
being done.

(B.) Optical Parts. — Sec that all the parts arc properly centred by
looking through the tube after removal of the eye-piece. Put on

FlG. 16.— Stand III. of Hartnack,
with joint ami condenser.

1 6 PRACTICAL HISTOLOGY.

the high power ; use a medium eye-piece, and focus a microscopic
preparation ; any thin section of a tissue will do. The field should
be large, well illuminated, and flat. If there are specks in it, clean
all the lenses to see that these specks are not due to dust on the
lenses. If the centre of a flat object in the field does not come into
focus at the same time as the periphery of the object, then the lens
should be rejected on account of its spherical aberration. If coloured
rings appear in an object, then the lens must also be rejected, as it
is not perfectly achromatic. The definition ought to be sharp and
distinct.

The qualities of a good lens are the following : —

The definition should be good, i.e., the correction for spherical
and chromatic aberration should be perfect. The outlines of objects
should be sharply defined and not blurred, and there should be no
coloured halos or fringes round the object. If so, the chromatic
aberration of the lens is not perfectly corrected.

Flatness of field, i.e., all parts of the object in the field at the same
time should be seen with equal distinctness, and, of course, this can
only be so when the field is flat. If the central parts are sharply
focussed, the peripheral parts not, then the field is not flat.

Resolving power, which depends on the angle of aperture. This
is the power to render visible surface markings, superficial lines, or
structural details. -It is better to have a lens of large angle of
aperture for this purpose.

Penetrating power, or the power to see objects in several planes
in the same preparation at the same time. It represents the focal
depth of the lens and its power of focussing images from different
planes in the vertical range. Narrow angled lenses are more suit-
able for this purpose.

Working Distance, i.e., the distance between the lens and the
object. It has no direct relation to " the focal length of a lens.
Wide angle lenses have a shorter working distance than narrow angle
lenses. A good lens should combine all the foregoing qualities, but
of these qualities definition is all important.

15. Drawing of Microscopic Objects. — In this laboratory every
student is required to make sketches of his preparations. This is
of the utmost importance, not only from the point of view of the
student, but also of the teacher.

(A.) Freehand Sketching. — The student must provide himself
with a drawing-booli, (p. 3) and with suitable pencils (H.B. and
H.H.H.), and also with either coloured crayous or water-colours.
It is far more important that an outline sketch in pencil be made
accurately portraying the shape of the object, than that an indifferent
sketch should be covered with a smear of colour. It is astonishing
how some students, after protesting that they cannot " draw,"

THE MICROSCOPE AND ITS ACCESSORIES.

succeed in delineating their preparations when they have given the
matter a fair trial.

(B.) Various Forms of Camera Lucida. — Sometimes, however,
a drawing has to be done to scale, or its outlines and detail*
accurately portrayed. There are many
devices for this purpose. Some ot
these instruments are by no means
easy to work with, but they are
excellent for tracing the outlines of
objects and showing the exact rela-
tion of one part to another, and the
relative size of the parts of an object.

(i.) Zeiss's Camera Lucida (fig.
17). — A collar (c) with a vertical rod
attached (a), carrying a bar (^) which
bears the prism or camera (K). The
collar is slipped over the tube of the
microscope and the eye-piece inserted.
Focus the object, and rotate the prism FIG. 17.— Zeiss's Camera Lucida. (c.)

until it i« -ibnvo tho PVP niopo • t,hr> collar; (a.) and (6.) supporting

eye-pic ^ , l barg . ^K ^ camera.

prism covers one-half of the latter.

The drawing-board is inclined at an angle of 20°, and in looking
down the microscope the paper and the image of the object are
seen simultaneously.

(ii.) Camera Lucida (Abbe). — This apparatus is made by Zeiss
(fig. 1 8). The apparatus is screwed to the tube of the microscope.

FIG. iS.— Abbe's Camera Lucida. (Sp.) mirror ; (IF.) line of reflected light ; (/.) collar for
screwing over eye-piece ; (0.) light to eye of observer ; (£>•) light reflected on paper.

The direction and reflection of the rays from the object and those
from the paper are shown in the figure. In all camera-lucida
drawings it is important to regulate the brightness of the illumination

B

1 8 PRACTICAL HISTOLOGY.

of the paper ; this is effected in this instrument by smoke-tinted
glasses, which fit into the prism mounting. In other cases this is
done by means of a blackened cardboard shade.

(iii.) Chevalier's Camera Lucida (fig. 19). — The microscope
remains erect, the eye-piece is removed, and in its place is inserted

the tube of the camera. Place
a sheet of white paper at the
side of the microscope, and
directly under the horizontal
part of the camera. The in-
strument itself consists of
two tubes at right angles to
each other, and at the angle
is a prism, which reflects at
a right angle in a horizontal
direction the light coming
vcrtically from an object on
the stage. These horizontal

rays have to pass through what is practically an eye-piece of a
microscope, and reach a small prism at the free end of the hori-
zontal part of the instrument. This prism is so arranged that the
rays coming from the object are reflected into the eye of an observer,
who, on looking through the prism, sees an image of the object upon
the sheet of paper on the table. The observer looks through this
prism, but he must so adjust his position as to bring one-half of the
pupil over the prism, and thus with one-half of the pupil view the
object on the paper, while the other half of the pupil receives the
rays of light coming from the pencil and the paper. The distance
between the eye of the observer and the paper is about 10 inches.
It is by no means so easy to sketch the outline of an object with
this camera. The eye should be protected from other rays of light
by means of a blackened shade. The difficulty encountered by
the student is to see the object and the point of the pencil at the
same time.

(iv.} Camera of Malassez (fig. 20). — This consists of two prisms;
one, the ocular prism, is fixed, while the second and larger can be
moved round a horizontal axis. This enables the instrument to be
used in two different positions of the microscope. If one is
examining a fluid, the microscope is erect and not inclined. The
instrument is fixed to the upper part of the tube of the microscope.
Slip the collar over the tube and insert the eye-piece. The image
is projected on the table to the right of the microscope ; but the
paper must be placed at an angle, i.e., on an inclined plane, horizontal
to the direction of the rays from the camera. This is necessary to
avoid one side of the figure being larger than the other. If it be

Till: MICROSCOPE AND ITS ACCESSORIES.

20. — Malasse/'s Camera placed at a
variable angle.

desired to incline the microscope at an angle of 45° (fig. 20), the
camera is so placed that an image is thrown on paper placed behind
the foot of the microscope, the prism being turned to an angle of
45°. This camera is particularly
easy to work with. It is some-
times important not to have too
much light on the paper ; this is
avoided by using the plane side of
the mirror.

16. Magnifying Power of a
Microscope. — This will vary with
the objective and ocular used, and
also with the length of the draw-
tuhe. The magnification is usually
determined when an image is seen
at the range of normal distinct
vision, i.e., 10 inches or 25 centi-
metres. As this is about the
height of a Hartnack's stand or
Zeiss's stand, supposing these
forms of instrument to be used,
place a sheet of white paper on
the table. Let the microscope be upright, with ocular and lens in
place. Supposing we use a No. 7 objective and No. 3 ocular.
Begin with the draw-tube in, place a stage-micrometer on the stage
of the microscope, and focus the scale upon it. The stage-micro
meter is like a glass slide with a fine scale engraved upon it. The
English ones are generally subdivided into thousandths of an inch,
and the Continental ones into hundredths of a millimetre. Look
through the microscope, but keep both eyes open, and part of the
scale will be seen on the white paper. With a pencil mark off sny
ten interspaces, or use a pair of compasses to measure this distance.
Measure off this distance on a millimetre scale, and suppose the
distance thus measured be 28 millimetres. This means that .1
millimetre has been magnified to appear equal to 28 millimetres,
/>., 280 times. The magnifying power for other combinations of
lenses should be determined, and a table made for future reference
and use.

Magnifying Power. — This may be increased—

(a) By using a higher objective.
(f>) By using a higher eye-piece.

(<•) By pulling out the draw-tube and increasing the distance
between the objective and the eye-piece.

Construct a table — according to the following scheme — of the

20

PRACTICAL HISTOLOGY.

magnifying powers of the different combinations of the objective
and ocular supplied with your microscope : —

Ocular.

Shallow, or A or 2.

Deep, or 0 or 4.

Dn.w-tubein (™^orD

One-surt.mc.or

17. Measurement of the Size of a Microscopic Object. — Sup-
pose a red blood-corpuscle is to be measured. Keep both eyes
open ; with one look down the tube of the microscope, and the
other eye will sec an image of the corpuscles on a sheet of white
paper placed beside the microscope. With a pencil mark off the
outlines of a corpuscle, or a sketch may be made of one with a
camera lucida. Remove the preparation, and for it substitute a
stage-micrometer, leaving all the other parts, microscope and paper,
as they were. On looking down the tube of the microscope, the
lines on the micrometer scale are seen. Make a drawing of these
lines. This may be kept for future use, but on the scale should be
noted the combination of ocular and objective, and the extent to
which the draw-tube has been elongated. The distance between
the lines on the micrometer scale being known, say TJo-th of a
millimetre, it is easy to calculate what part of the distance between
any two lines corresponds to the size of the corpuscle.

The most expeditious plan is to use an eye-piece micrometer.
A circular flat piece of glass, with a scale ruled on it (fig. 21), is in-
serted in the ocular between the field-glass (FG)
and the eye-glass (EG) (fig. 22). With this eye-
piece focus the scale on a stage-micrometer — its
markings must be parallel to these in the eye-
piece— and count the number of divisions of the
latter that correspond to one of the former. This
must be determined for each combination of lenses
w^h a known length of tube. Suppose the stagc-
micrometer to be divided into T^yths of a milli-
metre, and that one of these divisions corresponded
to three of those in the ocular, then each of the
spaces in the ocular micrometer is equal to -s J-^th of a millimetre,
or .0033 millimetre.

The histological unit of measurement is loooth part of a milli-

THE MICROSCOPE AND ITS ACCESSORIES.

21

metre or micro-millimetre, expressed shortly as a micron, or by
the Greek letter //,. Thus, each space of the ocular micrometer is
equal to 3-3 //,.

With these data it is easy to estimate the size of any object. The
object is placed on the stage and focussed, always, of course, with
the same combination of lenses and ocular. If other lenses be used,
the value of the ocular divisions must be determined for this particular
combination. Once determined, they can be noted for future use.

18. Artificial Illumination. — White daylight is, of course, to be
preferred, but it is not always available. When artificial light has
to be used, a gas flame, e.g., an Argand burner, or other artificial
light, may be used. An ordinary paraffin lamp with a flat wick and
arranged to burn steadily does very well. An unsteady flame is

FIG. 22.— Micrometer Eye-piece.
(EG.) eye-glass ; (FG.) field-
glass ; (S.) scale.

. 23. — Microscope Lamp.

very injurious to the eyes. It requires some care to find the exact
distance at which the lamp should be placed from the mirror. If
very intense light be required to examine a small part of a pre-
paration, turn the wick edge on to the mirror. Between it and the
mirror place a screen of white paper. If the direct rays from a
lamp are used, to correct the yellow rays place a sheet of pale blue
glass, or a glass globe containing a weak solution of ammonio-
sulphate of copper, between the light and the mirror, or a thin blue
glass may be placed on the stage under the preparation. A paraffin-
oil lamp may be used. Fig. 23 shows a cheap paraffin lamp sold by
Swift & Sons. A remarkably good light is obtained from the

22

PRACTICAL HISTOLOGY.

Welsbach gas-burner, the rays being transmitted through a large
globe containing a dilute blue-coloured solution of ammonio-sulphate

FIG. 24. — Dissecting Microscope by Verick.

of copper. The lamp recently constructed by Kochs-Wolz of Bonn
consists of a petroleum or gas flame, covered by an opaque chimney

FlO. 25. — Dissecting Microscope by Reichert.

provided with a reflector, which directs the rays of light through a
hole in the chimney. Into the hole in the chimney is fitted a glass

THE MICROSCOPE AND ITS ACCESSORIES.

rod, which, bent in a gentle curve, conducts the light from the
flame to the under surface of the stage, the mirror in this case not
being used.

Perhaps the most useful lamp is that known as the albo-carbon
light. It is the one I am in the habit of using when — especially
in winter — good daylight is not available. Ordinary gas is used,
but the gas has to traverse a chamber containing naphthaline before
it reaches the burner. A very white light is thus obtained, and
can be used without the intervention of blue glass or a blue copper
solution.

If expense be no bar, then a small incandescent electric light is
most useful.

19. Dissecting Microscope (figs. 24, 25). — This is very useful.
The lenses usually employed magnify from 5 to 20 times linear,
and are fitted into a framework which can be raised or depressed,
so as to bring the object distinctly into focus.

20. Method of Measuring the Thickness of Cover-Glasses.—
Thick cover-glasses are of no use, and thin ones — extra thin, so
called — are to be pre

ferred. A convenient
thickness is o. 16 mm.
For high powers this1
is essential, and it is
well to measure the
thickness of the cover-
glasses, and to reject
all those above a cer-
tain thickness. This
is conveniently and
rapidly done by means

of the instrument FlG. ^. —Cover-glass Tester.

shown in fig. 26. A

clip projecting from the box fixes the cover-glasses, and the thick-
ness is given by an indicator moving over a divided circle on the lid
of the box. The divisions show hundredths of a millimetre.

21. Camera Obscura Shade. — When one has to continue observing
a microscopic object for a long time, it is convenient to shade the
eyes from all light reaching them, except that transmitted through
the eye-piece. This is best done by a blackened screen cither fixed
to the microscope or arranged in front of the microscope.1 Some
observers place the microscope in a dark chamber, allowing light to
fall upon the mirror through an aperture in its front wall Flbgcl2
has designed a camera obscura for this purpose.

1 Schieffcrdecker, Archivf. iviss. Mikr., p. 180, 1892.

2 Zoolog. Anzeiger, by V. Cams, p. 566, 1883.

24 PRACTICAL HISTOLOGY.

III.— NORMAL OR INDIFFERENT FLUIDS.

Not ^infrequently living tissues or fresh tissue elements have to
be examined in as natural a condition as possible, and obviously, if
a fluid require to be added to it, the fluid must be of such a nature
that it will not injuriously affect the tissue or its elements. These
fluids are spoken of as indifferent or normal fluids. When fresh,
these fluids cause very slight changes in the tissues. Amongst
those used are —

1. Normal Saline. — Dissolve 6 grams of pure sodic chloride in
1000 cc. water.

2. Kronecker's Fluid. — It consists of

Distilled water . . . . . 100 cc.

Sodic chloride . . . . . .6 gram.

Soda .... . .06 „

3. Blood Serum. —The blood is allowed to clot, and the serum,
after a day or so, is poured away from the clot. In a laboratory
provided with a "centrifugal apparatus" any red blood-corpuscles
can be got rid of by " centrif ugalising " the serum. This fluid
does not keep long, and must be fresh. It has been suggested to
add a piece of camphor to it, but this only helps to preserve it
for a short time. Iodine is sometimes added to serum to form
iodised serum, but this is by no means an indifferent fluid

(P- 25).

4. Aqueous Humour. — With a narrow triangular knife puncture
the cornea of a freshly-excised ox's eyeball and collect the aqueous
humour which exudes. If only a small quantity be required, it
may be obtained by puncturing the excised eye of a frog with a fine
capillary glass pipette (p. 4).

5. Fluid of Kipart and Petit :—

Camphorated water
Distilled water
Glacial acetic acid
Acetate of copper
Chloride of copper

It is very useful for examining animal cells.

75 cc.

75 „
i ,,

o. 30 gram.
0.30 »

IV.— DISSOCIATING FLUIDS.

These fluids dissolve or soften the cement substance or inter-
stitial material, e.g., of epithelium, connective tissue, and thus
facilitate the separation of the histological elements from each other.
In some cases, in isolating tissue elements, it is well not to have

DISSOCIATING FLUIDS. 2$

too much fluid in proportion to the morrel of tissue, else hardening
is more apt to take place.

1. Strong Iodised Serum. — A strong solution should be kept,
which may be diluted as required. To the amniotic fluid of a cow
or to blood-serum add crystals of iodine, and keep it in a stoppered
bottle. Shake it frequently. At first very little iodine is dissolved,
but after a time (15-20 days) the solution becomes much stronger,
i.r., it becomes of a deep-brown tint.

For dissociating a tissue, a weak iodised serum is used. A
little of the strong fluid is added to fresh serum until the latter has
a light-brown colour. If an object be placed in the dilute iodised
scrum and the brown colour fades, more of the strong solution
must be added. In using this fluid, take a very small piece of
tissue, the size of half a pea or less, and place it in 5 cc. of the
fluid in a glass-stoppered bottle. After a day or two it may be
dissociated with needles, but the brown tint must be maintained ;
more strong fluid must be added if putrefaction is to be pre-
vented.

2. Dilute Alcohol (Ranvier's Alcool au t'wrs). — This fluid,
devised by Ranvier, and sometimes called "one-third alcohol," is
of the greatest possible service, and is one of the best dissociating
fluids we possess. Mix i part of 96 per cent, alcohol with 2 parts
of distilled water. It dissociates epithelial and other tissues in
24-36 hours. Use a small quantity of the fluid in proportion to
the tissue.

3. Chromic Acid. — One gram in 1000 of water. This requires
two days to a week, according to the tissue placed in it.

4. Potassic Bichromate.— Two parts in 1000 of water, «>., .2
per cent. It is very useful for dissociating epithelium and the
nerve-cells of the spinal cord. It does so in 2-3 days.

5. Ammonium Chromate (5 per cent.). — It is used for disso-
ciating the " rodded " cells of the renal tubules, cells of salivary
glands, Purkinje's fibres of the heart, &c. It acts in 24-36 hours,
and the chromate must be well washed out of the tissues if they
arc to be preserved.

G. Caustic Potash. — Thirty to 35 parts in 100 of water. It acts
in 20-30 minutes, rapidly destroying connective tissue. Water must
not be added to the dissociated tissue, else the tissues are rapidly
dissolved. It is used for isolating the fibres of smooth muscle, or
heart-fibres. Examine the dissociated tissues in the dissociating
fluid. As a rule, tissues so dissociated cannot be preserved, but
there are certain exceptions.

7. Dilute Osmic Acid (.1 per cent.).— It acts in 24-48 hours
according to circumstances, and is well suited for cells containing
fat globules.
4

26 PRACTICAL HISTOLOGY.

8. Landois' Fluid :—

Neutral ammonium ehromate . . .5 grms.

Potassium phosphate

Sodium sulphate . . . . ,,

Distilled water . . . . 100 cc.

This is specially useful for the central nervous system. Small
pieces must lie in it for 1-5 days. (See Lesson on Nervous System.)

9. Methyl Mixture or Schiefferdecker's Fluid :—

Methylic alcohol . . . . 5 cc.

Glycerine . . . . 50 ,,

Distilled water ..... 100 ,,

It is better to prepare it fresh, hut it can he preserved in a
glass-stoppered bottle. The methylic alcohol rapidly
evaporates. The tissues remain in it for several days,
and it is specially useful for the retina and central
nervous system. I find that it isolates epithelial cells
in one to two days. It acts very much like Ranvier's
alcohol.

10. Other fluids are referred to in the text, e.cj.,
baryta water, and 10 per cent, sodic chloride for
tendon. This dissolves the cement substance of epi-
thelial cells and the mucinoid cement of connective
tissue.

11. Digestion Methods, both gastric and tryptic,
FIQ. 27.— Glass arc used (see text).

Thimble for ^ie sPecial llses °^ ^ne above-named fluids are
dissociating referred to in text under the tissues or organs, for
o™\\ss~ueeS which each is specially adapted.

General Directions for Dissociating Tissues.—
Always use a very small piece of the tissue or organ, and place it
in a not too large quantity of the dissociating fluid. Small glass
thimbles (fig. 27) are very useful for this purpose.

V.— HOW TO TEASE A TISSUE.

To separate by means of needles the elementary parts of a tissue
is by no means an easy task. The tissue must be seen distinctly,
and the needles must be so used as not to break up the parts, but
only to separate them. The process may be done by the unaidod
eye or with the aid of a lens or dissecting microscope (figs. 24, 25).
The light must be good, and an appropriate background for the
object should be selected. If the tissue is colourless, use a black

HOW TO TEASE A TISSUE. 27

background, e.<j., black paper; if coloured, a white one. In the
latter case, however, the shadows interfere with exact vision, and it
is better to support the slide upon an object raised slightly above
the white background. This is readily accomplished by placing it
over a white porcelain capsule, or on a photophore (fig. 28), into
which is slipped a piece of white paper.

In a laboratory, one of the most convenient ways is to have the
tables painted of a black or very dark green colour, but the painting
must be flat, with no shining varnish. At the edge of the table is
painted a white strip i J inches broad. Some prefer to burn into
the surface of the table solid paraffin blackened by means of lamp-
black. Others use porcelain slabs one-half black and the other
white.

Photophore (fig. 28). — This is a small wooden box, 5 cm. high,
9 cm. long, and 9 cm. broad. The upper part is formed of glass.
The front wall of the box is Avanting.
Placed obliquely within the box is a
mirror, which reflects the light upwards
through the glass cover and the slide to
the eye of the observer.

In dissociating with needles, we must FlG 28._pi10toi-,ii<>re.

have some knowledge of the arrange-
ment of the parts of the object to be teased, such as the direction
of the fibres, itc. Take a small piece only. Always tease one end
of the tissue, and fix the latter with one needle while the parts are
separated with the other needle.

One of the most convenient combinations is that of Kterhod,
which combines a photophore with a turntable. The wheel of the
latter can be removed, and the block forms not only a photophore,
but also a surface on which tissues of different colours can be teased
and mounted.

VI.— FIXING AND HARDENING REAGENTS.

Most of the tissues and organs must be hardened in suitable
fluids before they can be cut into sections. A large number of
fluids of various kinds are used, each organ or tissue requiring its
own appropriate fluid. Some organs, <?.*/., bone, are too hard to be
cut in their natural condition ; they must be decalcified by appro-
priate fluids. Amongst others, the following fluids are required,
but others are referred to in the text.

A. Alcohol.

Alcohol is one of the most important hardening fluids used

28 PRACTICAL HISTOLOGY.

either by itself, or to complete the hardening processes begun
with other fluids. Moreover, it is almost universally used for
the preservation of tissues already hardened. There are three kinds
of alcohol used for histological purposes, viz., absolute alcohol, recti-
fied spirit, and methylated spirit.

(a.) Absolute Alcohol is alcohol without water, but that sold
usually contains 96 per cent, of pure alcohol, and is sufficiently
strong for microscopical purposes. If an absolutely water-free
alcohol is desired, place well-dried potassic carbonate in the alcohol.
This rapidly absorbs the moisture. Or powdered and heated cnpric
sulphate — a white powder — is added. If water is present, it absorbs
it, and becomes blue again. Lowne uses slips of gelatine.

(b.) Rectified Spirit contains 84 per cent, of spirit and 16 per
cent, of water.

(c.) Methylated Spirit contains a. little wood naphtha, and is
nearly as strong as the ordinary absolute alcohol, and may be made
stronger by placing some well-dried carbonate of potash in it. The
carbonate of potash absorbs any water present in the alcohol. Others
use cupric sulphate well heated in a metallic capsule, until it
becomes a white powder. When it becomes blue in the spirit it
must be replaced by new CuSO4. It can be heated and used again.

Alcohol is used of various strengths for hardening purposes.

Seventy-five per Cent. Alcohol. — To every 75 cc. of absolute
alcohol add 25 cc. of distilled water.

Seventy per Cent. Alcohol. — To every 70 cc. of absolute alcohol
add 30 cc. of distilled water.

Fifty per Cent. Alcohol. — Take equal volumes of absolute
alcohol and distilled water.

To Harden in Alcohol Alone. — The tissues are placed for 12-24
hours in the weaker (50 per cent.) alcohol, and passed through the
stronger alcohols (in each a day or thereby), and finally kept in 95
per cent, alcohol until they are required.

For certain special purposes the tissues, e.g., glands and struc-
tures for the preservation of mitotic figures, are "fixed," and at the
same time hardened by being placed at once, and while as fresh as
possible, in absolute or 96 per cent, alcohol.

B. Chromium and its Compounds.

1. Chromic Acid (Stock Solution). — It is well to prepare a
strong solution — 10 per cent. — and to keep this as a stock to be
diluted when required. Dissolve 10 grams of fresh chromic acid
in 90 cc. of distilled water.

2. Half per Cent. -Solution of Chromic Acid.— To 50 cc. of the
10 per cent, solution add 950 cc. of distilled water, or dissolve i
gram of chromic acid in 200 cc. of water. Similar solutions

FIXING AND HARDENING REAGENTS. 2Q

containing .3 and .2 per cent, chromic acid are frequently used, and
can readily be made from the stock-bottle.

3. Chromic Acid and Spirit, or Klein's Fluid. — Mix 2 parts of
chromic acid (.6 per cent, i.e., 6 grams in 1000 cc.) with i part of
methylated spirit. This should be made fresh, and kept from the
light. If the fluid be changed often it hardens tissues in S-io
days.

4. Chromic Acid and Bichromate Solution. — Dissolve i gram
of chromic acid and 2 grams of potassic bichromate in 1500 cc. of
water.

5. Muller's Fluid. — Dissolve 25 grams of potassium bichromate
and 10 grams of sodium sulphate in 1000 cc. of water. Solution
takes place slowly at the ordinary temperature. Pound the
ingredients in a mortar, add the water, and warm until they arc
dissolved. It takes five to seven weeks to harden a tissue,
according to the size of the tissue placed in it. This fluid is very
extensively used, as it penetrates into the tissues and hardens them
equally throughout. To prevent the formation of fungi, place a
piece of camphor in the solution. Miillcr's fluid preserves blood-
corpuscles in their original form, and they retain their yellow
colour. It also shrivels very slightly the tissue elements.

G. Muller's Fluid and Spirit. — Miillcr's fluid 3 parts, and
methylated spirit i part. Mix, and allow the mixture to cool
before using it. When tissues are hardened in it they should be
kept in the dark.

7. Erlicki's Fluid. — Dissolve 2.5 grams of potassium bichromate
and .5 gram cupric sulphate in 100 cc. water. It should be
prepared fresh. It hardens more quickly than Muller's fluid, and
after the first day or two its action is greatly facilitated by keeping
the tissues in it at 40° C. Ten days or so will suffice for hardening
under these conditions. Experience, however, has shown that the
process of rapidly hardening tissues at a comparatively high
temperature is. not so satisfactory as that conducted at a lower
temperature.

8. Potassium Bichromate. — Make a 2 per cent, solution by
dissolving 10 grams of the salt in 500 cc. of water. It takes
from three to seven weeks to harden tissues, and is one of the best
hardening fluids for the central nervous system.

9. Ammonium Bichromate is used in the same way and of the
same strength. It takes much longer to harden than 8. (See
Central Nervous System.)

10. Ammonium Chromate (5 per cent, solution).— This is used
for hardening the kidney and other secretory glands, the mesentery
of the newt, <fcc. (sec text).

Precautions in Connection with Chromium Compounds. — Solu-

30 PRACTICAL HISTOLOGY.

tions of pure chromic acid do not penetrate well into tissues,
therefore the pieces of tissue must be small. In the case of tissues
placed in Mailer's fluid or potassic bichromate, they are hardened
very slowly. In the case of most organs 3-4 weeks suffice, but in
the case of the brain and spinal cord it takes sever il months to
harden tissues and organs in these fluids. The fluid must be
frequently changed, and it must be large in amount. The forma-
tion of fungi on its surface may be prevented by adding a piece of
camphor, thymol, or naphthaline to the fluid. The hardening
process is accelerated when the fluid and tissue are kept at 3o°-4o°
C., but the result is not so satisfactory as by the slower cooler
process.

Except for special purposes, e.g., Weigert's method for the cen-
tral nervous system, the chromic acid salts must be thoroughly
washed out of the hardened tissue or organ. This is done by
leaving them for many hours or days in a stream of running water.
In all cases it is well to keep the fluids and tissues in a cool place,,
and in some cases in the dark, e.g., 3 and 6.

In connection with the hardening of tissues, especially those of
the central nervous system, it is, with right, insisted upon that the
hardening fluid should be frequently changed. I find, however,
and my observations arc borne out by other observers, that after
the first change of fluid a very satisfactory result is obtained by
placing the organ to be hardened in a very large volume of the
hardening fluid, e.g., the spinal cord of a cat or dog in a litre of the
hardening reagent, and leaving it under proper conditions until the
cord is hardened. Stir the fluid from time to time.

Chromic acid seems to form a compound with the tissues, so
that it is not easily removed from them. Tissues hardened in
chromic acid are not readily stained by carmine, so that for this
purpose it is better to use hsematoxylin or safranin.

C. Acids and Acid Mixtures.

1. Picric Acid. — Make a cold saturated solution of picric acid in
water. There should always be a large excess of crystals on the
bottom of the bottle. Place a small piece of tissue in the solution
for 6-24 hours. The tissues are to be afterwards wTashed in 70 per
cent, alcohol — not water — and transferred to 95 per cent, alcohol.

2. Kleinenberg's Picro-Sulphuric Acid. — To 100 cc. of a cold
saturated watery solution of picric acid add 2 cc. of concentrated
sulphuric acid. This causes a copious precipitate. After twcnt}r
hours filter, and to the filtrate add 300 cc. of distilled water.

Tissues are placed in this fluid for a comparatively short space of
time — -from a few minutes to two or six hours. The time should
never exceed six hours. The liquid must be changed if it becomes

FIXING AND HARDENING REAGENTS. 31

turbid. The hardening process is completed in alcohol. It is well
adapted for embryological work.

3. Picro-Nitric Acid (P. Mayi'r).— Some substitute nitric acid for
the sulphuric acid, but for our purposes there is no advantage in
this. Small pieces of tissue not over J cm. in diameter — prefer-
ably those that contain little connective tissue — are hardened for 1-3
hours. They are washed with alcohol until most of the yellow
colour disappears. Sections can be stained with haematoxylin.

4. Nitric Acid (Altmami). — Use a 3 per cent, watery solution of
pure nitric acid. This has a sp. gr. of 1.02. Use as small pieces
as possible, and leave them in the mixture just until they are fixed,
i.e.t from a quarter to half an hour. Strong solutions, if they act.
too long, dissolve the chrornatin. The tissues are then hardened
successively in 70, 80, and 90 per cent, alcohol. It is particularly
useful for preserving the nuclei of cells, mitotic figures, embryological
tissues, and the retina.

5. Per£nyi's Fluid :—

Nitric acid (10 per cent.) , 40 cc.

Chromic acid (0.5 per cent.) . . . 30 ,,

Alcohol . . . . . 30 „

It is a light greenish-blue liquid, and is specially useful for
hardening ova and embryos. Time, 4—6 hours for a small embryo
and 4-12 hours for the tissues of vertebrates. The tissue is trans-
ferred direct to 75 per cent, alcohol without previous washing in
water. Borax carmine may be added to it, and then this mixture
hardens and stains at the same time (Garbini).

6. Chromo-Formic Acid (flabl's Fluid). — To 200 cc. of J per
cent, chromic acid add four to five drops formic acid. It must be
freshly prepared, and fresh tissues — small pieces — are placed in it
for 12-24 hours. The tissues are thoroughly washed in water, and
hardened in alcohol of gradually increasing strength. Sections can
be stained in hsematoxylin and safranin. It is specially useful for the
study of mitosis and nuclei generally. It has this advantage, that
tissues hardened in it do not afterwards darken.

7. Chromo-Acetic Acid (Fhmming). — As chromic acid by itself
is apt to cause shrinking of some of the more delicate textures, it
has been proposed to mix it with a substance which has an opposite
effect, such as acetic acid. The following combination is specially
recommended by Flemming for fixing the achromatic spindle in
cells : —

Chromic acid .... $-|* grain.

Glacial acetic acid . . . • rs cc-

Water .... 100 ,,

The tissues must be small in size, not above 3-5 mm. in diameter.

32 PRACTICAL HISTOLOGY.

They are hardened for twenty-four hours in this mixture, and after-
wards washed for twenty-four hours in water, and the hardening
completed successively in 70, 80, and 90 per cent, alcohol — each for
12-24 hours. Sections can be stained with hsematoxylin.

8. Osmic Acid. — ^lake a i per cent, solution by dissolving i
gram in 100 cc. of distilled water. This substance is rather ex-
pensive, and is sold in sealed glass tubes. Carefully clean the
surface of the tube, snip off one end of it, and place it with the
requisite quantity of water in a glass-stoppered bottle, which has
been carefully cleaned, so that it does not contain a trace of
organic matter. Organic matter decomposes it very rapidly. It takes
several hours to dissolve. Some prefer to use normal saline solution
instead of water to dissolve it. Its vapour is very irritating to the
eyes and mucous membranes generally. It should be preserved in
yellow-coloured bottles and kept in the dark. If a yellow bottle is
not available, cover a bottle with brown or black paper.

9. Osmic Acid Vapour. — This is most useful for thin membranes
and glands. Tissues stain readily after its use.

10. Chromo-Aceto-Osmic Acid (Flemming's Fluid). — To 45 cc.
of i per cent, chromic acid add 12 cc. of 2 per cent, osmic acid,
and then 3 cc. of glacial acetic acid. This can be kept for a long
time, and need not be kept in the dark. It is specially useful for
fixing the figures in cell-division or mitosis, and for many other
purposes. It " fixes " tissues in from a few hours to twenty-four
hours or longer ; but the pieces must be small, 2-3 mm. thick, as it
does not penetrate deeply. This is Flemming's " strong formula."

A weaker fluid is sometimes used, and is prepared as follows : —

Osmic acid (i per cent.) . 10 c.c.

Glacial acetic acid ( i per cent.) . . .10,,

Chromic acid (i per cent. ) . . . 25 ,,

Water . . . . 55 ,,

They must be thoroughly washed by being kept in running
water for twelve hours, and then hardened in the various strengths
of alcohol, 70, 80, and 90 per cent., each for twenty-four hours.
Sections should be stained as soon as possible after they are made,
as on keeping they do not stain so well. They may be stained witli
safranin, haematoxylin, or methyl-violet, but safranin is the best
(p. 75), it stains the chromatin of the cells a bright red. Tissues
hardened in it are not well adapted for teasing.

11. Fol's Solution. — This is a modification of 10. It contains
less osmic acid, and is used more generally. To 2 cc. of i per cent,
osmic acid add 25 cc. of i per cent, chromic acid, five parts of 2 per
cent, glacial acetic acid, and 68 cc. water.

Precautions with Osmic Acid or Solutions containing it. — If a

FIXING AND HARDENING REAGENTS. 33

tissue is to be hardened in a certain fluid, and to be treated with
osmic acid afterwards, that tissue had better not be put into alcohol
if it contains fat or fatty particles, as the alcohol dissolves the fat.
Thus, a tissue hardened in Miiller's fluid may be put into osmic acid,
and the fat-cells will still be blackened.

It has recently been shown that the prolonged action of turpen-
tine, toluol, xylol, ether, and creasote, but not clove-oil or chloroform,
will decolorise particles of oil (fat) blackened by osmic acid. This
is most important in connection with the osmic method for studying
the absorption of fat in the small intestine (Lesson XXV.).

Tissues containing fat maytherefore be embedded in paraffin after
being passed through chloroform (p. 43).

Osmic acid fixes fresh tissues very rapidly, but it does not pene-
trate deeply ; therefore the tissues must be cut into very small pieces
to get them fixed throughout by the acid. It has the power of
differentiating tissues, the nuclei become yellow, fat, and the nervous
system black. After twenty-four hours or so, the tissues are
thoroughly washed in water and hardened in 90 per cent, alcohol.
To avoid the blackening which is apt to occur in tissues still con-
taining a trace of osmic acid, it has been proposed to treat the
tissues with a weak solution of cyanide of potassium, and then to
harden in alcohol. It enters into the composition of several
hardening fluids (p. 32), and Hermann's fluid (Lesson XXXV.).

D. Other Hardening Fluids.

1. Bichloride of Mercury or Corrosive Sublimate. — A saturated
watery solution contains about 5 per cent, of the salt; but it is
much more soluble in alcohol, especially alcohol of 50 to 60 per
cent. Make a saturated watery solution, and also a saturated
alcoholic solution. A saturated solution in 0.5 NaCl solution is
also used.

A cold saturated solution is best made as follows : — Place 60
grams of it in i ooo cc. of water, and dissolve with the aid of heat.
Filter the warm solution and allow it to cool. On cooling, long
white needle-shaped crystals of the sublimate separate. The
supernatant fluid is used, and the tissues, which must not be more
than J cm. in diameter, remain in the fluid from 1-3 hours, accord-
ing to their size. After fixation they are hardened in 70, 80, and
90 per cent, alcohol. Xo metallic instruments are to be used.
Use glass or wooden instruments.

This is a most excellent hardening reagent, and it hardens tissues
with great rapidity, so that tissues must not be left in it for too
long a time. For small pieces, u quarter of an hour or thereby is
5 C

34 PRACTICAL HISTOLOGY.

sufficient; for large pieces, one to two hours. The pieces when
fixed become whitish throughout.

For glands and glandular structures generally, a half-saturated
alcoholic solution is most useful, i.e., to 50 cc. of a saturated
alcoholic solution add 50 cc. of 70 per cent, alcohol. Vignal
recommends that to 100 cc. of this mixture there be added 5 to
6 drops of nitric acid. The pieces of glands, 4 mm. cubes, are
hardened in one hour or so. The hardening is completed in
alcohol.

N.B. — All the corrosive sublimate must be washed out of the
tissue — by alcohol, not water — otherwise the sections will be
dotted with small black specks or star-like or needle-shaped crystals
of the salt, or the salt may be removed by adding a few drops of an
alcoholic solution of iodine to the alcohol used to complete the
hardening, or before complete hardening in alcohol it may be
steeped for 2-3 days in 70 per cent, alcohol, to which a few drops
of tincture of iodine is added.

The action of the salt may be accelerated by placing the tissues
in the fluid heated to 38° C. Sections stain readily with all the
usual staining reagents.

N.B. — Do not use metallic instruments to transfer the tissues
from one vessel to another. Use glass or wood or horn.

2. Other Fluids for special purposes are mentioned in the
text.

General Directions on Hardening.

The tissues should be taken from the body as soon as possible
after death, and transferred as soon as possible to the hardening
fluid.

Any blood adhering to the parts may be removed by washing
them in normal saline solution.

With a sharp razor cut the tissues in the same plane in which
they are afterwards to be cut for sections. The tissue must be cut
into small pieces, i.e., \ to f inch cubes, except in the case of
tissues to be hardened in Miiller's fluid. They will harden better
if they are small, say i cm. square.

The best way is to suspend the tissues in the upper half of the
fluid, which should always be many times the volume — 15-20 —
of the tissue. If it is inconvenient to suspend them, cover the
bottom of the jar or wide-mouthed bottle in which they are placed
with an old washed rag or blotting-paper to prevent the tissue from
resting directly on the glass.

The liquid must be changed within the first twenty-four hours,
and again on the second day, then on the fourth, eighth, and twelfth

FIXING AND HARDENING REAGENTS.

35

day, and once or twice afterwards, and on each occasion alter the
position of the tissues. If the fluid becomes turbid, change it at
once (p. 30).

Label each bottle carefully, and place it in a cool place. Keep
all chromic acid and solutions containing it or its salts in the
dark.

Tissues hardened in alcohol and picric acid must not be placed
in water, but directly into the various strengths of alcohol,
beginning with 50 per cent, and rising to 95 per cent. Wash
out by means of alcohol as much of the picric acid as
possible.

For other tissues, hardened in chromium salts (p. 30), the excess
of these salts may be removed from them by washing in water (for
certain special purposes this is omitted), and they are then transferred
first to weak spirit, in which they may remain a few days, and then
to the stronger alcohols (p. 28). To avoid the deposits which occur
in chromic acid preparations when they are placed in alcohol, keep
them in the dark. If kept in the dark, as Hans Virchow has
shown, there is no deposit formed when a tissue hardened in
chromic acid or Miiller's fluid is placed in spirit. Kept in this
way, I find that the alcohol remains quite clear and no deposit
forms.

Scheme for the, Hardening of Tissues (Garbini).
Fresh Object.

Picric acid or Chromic acid or

Sublimate.

solution con-
taining it.

Alcohol

its com

>ounds.

Osmic acid.

Running water.

Running water. Distilled water.

Alcohol (50 p.c.) Alcohol (50 p. c.) Cyanide of Potassiu in.
in dark. (iodated).

Alcohol (70 p.c.) Alcohol (70 p.c.) Alcohol (50 p.c.).
in dark. (iodated).

I I

Alcohol (70 p. c. ).

36 PRACTICAL HISTOLOGY.

Garbini divides hardening reagents into the two following classes,
according as the tissues — after hardening — stain readily or do not
stain readily with carmine : —

Tissues stain readily after
hardening in

Vapour of osmic acid.

Corrosive sublimate.

Picric acid and solutions contain-

.in.& it;-.
Nitric acid.

Silver nitrate (weak solution).
Alcohol.

( Chromic acid and its compounds.

Tissues stain with dim- | Osmic acid in solution.

culty after hardening \ Silver nitrate (strong solution).

in | Chloride of gold.

L Perch loride of iron.

Alcohol, picric acid and its compounds, and nitric acid, coagulate
the albumen of the tissues. Chromic acid, or solutions containing
it, or its salts form chemical compounds with substances in the
tissues. Osmic acid, mercuric chloride, and gold are decomposed
when they come in contact with the tissues, and are deposited in
the tissues as an inorganic precipitate.

VII.— DECALCIFYING FLUIDS.

General Directions. — Use small parts of the tissue or organ, and
a large quantity of the decalcifying solution. Renew the latter
often. In all cases harden the tissue thoroughly, e.(/., in alcohol
or Miiller's fluid, before it is decalcified. After decalcification every
trace of the decalcifying fluid must be thoroughly removed by pro-
longed washing (for a day or two) in water or other fluid. The
tissue is then finally hardened in alcohol.

1. Chromic Acid. — .1 to .5 per cent.

2. Chromic and Nitric Acid Fluid.

Chromic acid ..... i gram.
Water ...... 200 cc.

Nitric acid . . . . . 2 ,,

3. Chromic Acid and Hydrochloric Acid Fluid (Bayerl's Fluid}.

Chromic acid ..... I part.
HC1 . . . . i „

Water . . . . 100 parts.

This is specially good for young bones and for ossifying bone,
but it is solely for decalcifying.

4. Saturated Solution of Picric Acid. — The solution must be

DECALCIFYING FLUIDS. 37

saturated and ke^t saturated. This is done by keeping some
crystals of picric acid in the bottom of the bottle. There must be
a large volume of fluid, and the bone should be suspended in the
fluid. It usually requires a fortnight to decalcify a small bone.
Picric acid acts as a fixing, hardening, and staining reagent.

After the bone is decalcified it should be washed and kept in
spirit — not water — until no more yellow stain is given up to the
alcohol. This rule, however, is not rigidly followed.

5. v. Ebner's Fluid. — This fluid prevents the ground substance
of bone from swelling up. Sections should be examined in 10 per
cent, solution of sodic chloride, if it be desired to see the fibrillar
structure of bone, but sections of bone softened in this way may be
mounted in other media, if it be desired to see the other details
in the structure of bone. (See Bone.) It has the following com-
position : —

Alcohol ..... 500 cc.

Water ...... 100 ,,

Sodic chloride . . . . 2.5 grams.

Hydrochloric acid . . . . 2.5 cc.

6. Picro-Sulphuric Acid or Picro-Nitric Acid (p. 3 1 ).

7. Arsenic Acid. — I find that a 4 per cent, solution of this acid
rapidly decalcifies a bone at 30° to 40° C. The tissues, after harden-
ing in alcohol, are well preserved and stain readily.

8. Chromo-Osmic Acid (Haug).

Osmic acid (i per cent.) . . .10 cc.

Chromic acid ( i per cent. ) . . . 25 ,,

Water .... . 65 „

Very useful for delicate tissues, e.g., very young developing teeth.
Wash in water and harden in 70 per cent, alcohol.

9. Phloroglucin Method. — Dissolve with the aid of gentle heat
i gram of phloroglucin in 10 cc. pure non-fuming nitric acid. To
the red fluid add 100 cc. of 10 per cent, watery solution of nitric
acid. The following mixture acts more slowly : —

Phloroglucin ..... I gram.

Nitric acid . . . . 5 cc.

Alcohol . . . . . 70 ,,

Water . . 30 ;,

Phloroglucin itself does not decalcify, it only protects the tissues
from the action of the nitric acid. By this method dccalcification
ran be done very rapidly, even in a few hours. 1 find that a portion
of a human clavicle about an inch in length is softened in 12-16
hours, the outer surface being stained slightly red.

Methods. — The bone should be cut into short pieces and placed

38 PRACTICAL HISTOLOGY.

in a larye volume of the fluid. If chromic acid be used, the bone
must be first hardened in this fluid. Place it in .1 per cent,
chromic acid for twenty-four hours , renew the fluid, but use .2 per
cent., and after a week use .5 per cent. ; shake the vessel from time
to time, to bring new fluid into contact with the tissue.

If a more rapid process is desired, after the bone has been two or
three days in dilute chromic acid (.2 per cent.), use the chromic
and nitric acid fluid. Decalcification requires about fourteen days.

To test for the removal of all the salts, push a needle into the
bone, or make a section with a blunt razor. Obstruction in either
case denotes that the bone has not been sufficiently decalcified. In
most cases, the bony tissues should be hardened before they are
decalcified. This is specially the case in connection with bone
softened in chromic acid. It is better to harden them first in
M tiller's fluid, and then to decalcify them in chromic and nitric
acid fluid (p. 36).

Bone decalcified in chromic acid must be thoroughly washed in
running water for many hours to remove all the chromic salts, and
is then transferred to 70 per cent, spirit and kept in the dark, other-
wise there will be a copious deposit. Renew the spirit, and transfer
the tissue to strong alcohol, still keeping it in the dark.

If a bone is to be softened in picric acid, it may be placed at once
in this fluid, with the precaution indicated at p. 37. It need not
be kept in the dark, but it is better to remove as much as possible
of the yellow stain by means of alcohol. It decalcifies somewhat
more slowly than chromic acid.

VIII.— METHOD OF PREPARING TISSUES AND
ORGANS FOR MICROSCOPICAL EXAMINA-
TION.

As most of the tissues require to be hardened, and it is frequently
impossible to obtain human tissues sufficiently fresh, recourse must
be had to the fresh tissues of animals. As frequently as possible,
however, human tissues should be secured. Most of the tissues
may be obtained from a cat, rabbit, or guinea-pig, and for certain
special purposes the dog, frog, newt, and salamander are used.

The cat, rabbit, or guinea-pig — or, better, all three — are killed
by chloroform. The animals are placed in an air-tight box — a
large saucepan does very well — along with a sponge saturated with
chloroform. Small animals may be chloroformed under a bell-jar.
As soon as the animal is dead, open the thorax by a longitudinal
incision through the costal cartilages — right and left — raise the

METHOD OF PREPARING TISSUES AND ORGANS. 39

sternum, expose the pericardium, open it, and with a pair of scissors
make a snip into the right auricle of the heart, and allow the animal
to bleed freely.

It is best to begin by removing the brain and spinal cord. They
are hardened in Miiller's fluid or potassic bichromate (2 per cent.),
and must be placed in a large volume of fluid. A few spinal
ganglia should also be found and hardened in the same way.

Remove the trachea and lungs, and fill the lungs and trachea of
the rabbit with a \ per cent, solution of chromic acid.

This is readily effected by tying a funnel into the trachea and
pouring in the fluid. By squeezing the lungs gently much air is
forced out, and the fluid gradually runs into and distends the
lungs, which, when distended, are placed in a large volume of the
same fluid. The chromic acid and spirit mixture may be used
instead of pure chromic acid.

Fill the windpipe and lungs of the guinea-pig with J per cent,
silver nitrate. (See Lungs.)

Remove the heart, and harden it in alcohol, after washing away
any blood with normal saline.

The central tendon of the diaphragm may be preserved for
silvering. (Lesson IV.)

The omentum and mesentery, if desired, are silvered. (Lesson
IV.)

( )ppn the abdomen, remove the liver, cut it into small pieces ;
harden some pieces in Miiller's fluid, and others in spirit.

Take out the tongue and oesophagus ; harden them in Miiller's
fluid.

Open the stomach and intestine, wash away any food residues
by means of normal saline. Harden part of the stomach — cardiac
and pyloric — in absolute alcohol, other pieces in Miiller's fluid,
and others in corrosive sublimate, and small pieces in osmic acid.
(See Lesson on Stomach and Intestine.)

The duodenum and small and large intestine are hardened in
the same way, although the bichromate and chromic acid mixture
(p. 29) is particularly good for the small intestine.

The salivary glands and pancreas are removed and hardened by
the methods given under these headings, i.e., small pieces are placed
in each of the following solutions : — Absolute alcohol, osmic acid,
corrosive, sublimate, &c.

Remove the lower jaw, cut it into short pieces, place it in .2 per
cent, chromic acid for a few days, and then decalcify it in chromic
and nitric fluid. This will yield sections of softened tooth.

Remove the kidneys, cut one longitudinally and the other trans-
versely. Using the kidneys of different animals, harden pieces of
each in the following fluids : Miiller's fluid, chromic acid and spirit,

4O PEACTICAL HISTOLOGY.

ammonium chromate, and corrosive sublimate. Other methods of
preparing the kidney are referred to. (Lesson on Kidney.)

The bladder is best hardened in chromic acid and spirit mixture,
or in Miiller's fluid.

Harden the spleen, without cutting into it, in Miiller's fluid.

The suprarenals may be hardened in picro-sulphuric acid.
(Lesson on Suprarenal Capsules.)

Small lymphatic glands from the region of the neck or sul>
maxillary region are hardened in alcohol, while others are injected
with silver nitrate and osmic acid. (Lesson on Lymphatics.)

If desired, the large nerve-trunks may be removed and hardened
as indicated in Lesson on Nerves, or the smaller branches of nerves
may be used for showing the effects of the action of certain reagents
on nerve-fibres.

Remove some of the long bones, leaving in each case the peri-
osteum attached to the bone. Cut the bones into pieces about
J inch long, and place them for a week in J per cent, chromic acid,
and then decalcify them with picric acid, or chromic and nitric
acid fluid, or Ebner's fluid. (Lesson XIII.)

In every case decalcify the ends of the bones, so as to have a
section which will demonstrate the relation between the articular
cartilage and the osseous tissue.

Place small pieces of striped muscle in J per cent, chromic acid,
and other pieces in alcohol.

Nerves, with the precautions given in Lesson on Nerves, are
hardened in osmic acid, potassic bichromate (2 per cent.), alcohol,
or picric acid.

For the methods of hardening the eye, ear, nose, see the Lessons
on these subjects.

The testis — very small pieces — is best hardened in Flemming's
mixture, and larger pieces in Miiller's fluid.

For the methods of hardening the ovary, Fallopian tube, and
uterus, see the Lessons on these subjects.

N.B. — Label every bottle, and write on the bottle the name of
the hardening fluid used, and the dates on which it was changed.

IX.— EMBEDDING.

This is necessary for many tissues ; the piece of tissue may be
either too small to be conveniently held in the hand, or its parts
may tend to fall asunder before or after they are cut.

There are two methods, one simple embedding, where the tissue
is simply iixed or placed in another medium to hold it while it is

KM BEDDING. 4!

being cut, and the other interstitial embedding, where the sub-
stance used for the embedding process is made to penetrate into
the interstices of the tissue.

A. Simple Embedding.— 1. The tissue may be clamped between
two pieces of carrot, scooped out to receive it, or in elder pith, or
(what is very convenient) between two pieces of amyloid or waxy
liver hardened in alcohol.

2. Paraffin. — It is sometimes desirable to surround the tissue
with paraffin or some such medium. The embedding medium
should be about the same degree of hardness as the tissue.

Two paraffins are required, a hard paraffin melting at 60° and a
soft one at 45° C. For use they may be mixed as follows : —

T / Hard paraffin I part

\ Soft paraffin . . . I ,,

•rj ( Hard paraffin . . . i ,,

' \ Soft paraffin .... 2 parts.

Two parts of the hard paraffin and one of the soft yield a mixture
which cuts well when the temperature of the room is 21° C.
(70° F.), but a softer paraffin is easily made by mixing two parts of
the hard paraffin with one part of chrisma or vaseline. The
mixture can be made softer by the addition of a little more
vaseline, and harder by adding more paraffin. The paraffin
mixture is heated on a water-bath or sand-bath until it melts,
but its temperature is raised as little as possible above its
melting-point. It is convenient to melt it in a porcelain
dish with a wooden handle. The tissue is removecT from
alcohol, the surplus alcohol removed by wiping it with blotting-
paper, until the surface is dry. It is then placed in melted paraffin,
and retained in it until the paraffin solidifies. The melted paraffin
can be run into embedding boxes of paper (fig. 30), or the embed-
ding L's may be used (p. 44), but this simple method is now but
rarely used. It has been almost entirely displaced by the following
method.

B. Infiltration Method or Interstitial Embedding.

1. Embedding in Gum. — The tissues after being hardened must
have all their alcohol removed by prolonged soaking in water.
They are then transferred to gum mucilage, or a mixture of gum and
syrup, in which they can be preserved until they are required for
freezing, if freezing is to be the process used for cutting the sections.

Tissues saturated with and embedded in gum mucilage may be
hardened in alcohol and then cut. The sections are placed in
water, which dissolves out the gum.

2. Saturation with, or Infiltration with, and Embedding in
Paraffin— Interstitial Embedding. — In this ease the embedding

42 PRACTICAL HISTOLOGY.

medium is made to penetrate into the tissue, and when it sets, it thus
supports all its component parts. This method is extremely valuable,
especially for brittle and friable tissues, and is largely used. More-
over, the tissues once embedded can be kept in a box, each duly
labelled, for any length of time.

Make a mixture of two parts of hard paraffin and one part of
soft ; place the mixture in a small copper pan or capsule in a hot-
air oven, kept at a constant temperature by means of a gas regu-
lator. The gas supply must be so arranged that the thermometer

FIG. 29.— Mayer's Paraffin Embedding Bath, as made by Jung of Heidelberg.

steadily registers at most i° C. above the melting-point of the
paraffin. Or the paraffin may be melted and kept melted in a
little copper vessel, placed in a hot- water bath, kept at a constant
temperature, as shown in fig. 29. The temperature is kept con-
stant by means of a gas regulator, R • Z is for filling the instru-
ment with water ; a, 7>, c, are embedding vessels and pots.

The tissues to be saturated and embedded should not be large,
and they must be thoroughly dehydrated ; keep them, therefore,
several hours in absolute alcohol. Place them direct into tur-
pentine, creosote, benzol, toluol, or xylol- — some use chloroform, bu'3

EMBEDDING.

43

turpentine, toluol, or xylol is to be preferred. The turpentine or
xylol penetrates into the tissue, displaces the alcohol, and makes
the tissue itself transparent. If the tissue be very small, this will
be done in an hour or so ; if it be larger, of course a longer time
will be required. Thus the time may vary, according to the size
and nature of the tissue, from, one to six hours.

Transfer the specimens from the clarifying medium, and place
them in the melted paraffin, where they may remain 2-10 hours,
according to the size and nature of the tissue. By the end of that
time they will be thoroughly impregnated or saturated with the
paraffin.

For delicate tissues, however, it is better not to transfer them
direct from the clarifying medium to pure paraffin, but to place
them first of all in a mixture of toluol and paraffin, or turpentine
and paraffin, for an hour or two. In the thermostat at 5o°-55° C.
the toluol gradually evaporates, so that nearly pure paraffin remains,
and the saturation with paraffin has been accomplished more
gradually. The object is then finally placed in pure paraffin for
several hours.

Some prefer chloroform as a clarifying agent and as a solvent for
paraffin instead of toluol or turpentine. Objects transferred from
absolute alcohol at first float on the surface of the chloroform, but
as the latter penetrates them and displaces the alcohol they sink.
They are then embedded in a chloroform paraffin mixture and
finally in pure paraffin.

The time (in hours) required for immersion in the several fluids
for pieces of tissue of various sizes is approximately as follows
(Bohm and Oppel) : —

Small Objects
less than
1 mm. diam.

Objects
about 5 mm.
diam.

Larger
Objects about
5 mm. diam.

Very large Objects.

Absolute alcohol
Toluol or turpentine

2
*

6
2-3

24
3-4

( A longer time,
} but at the ex -
\ pense of their
quality.

Objects are now placed in the thermostat or bath.

Toluol or turpen- )
tine-paraffin (
Paraffin .

1
i

4

2

6
3-4

Objects may remain longer in the paraffin without damage,
provided they have been completely dehydrated.

/)/| PRACTICAL HISTOLOGY.

The method of interstitial embedding is particularly useful for
tissues stained "in balk" or en masse. The tissues or organ may
be stained before the process is begun, or sections may be cut and
stained afterwards.

To Stain "in bulk" before embedding. — Pieces of the tissue a
few millimetres square are placed in borax-carmine or Kleinen-
berg's logwood for 10-24 or 48 hours, according to the size and
nature of the tissue. If they be placed in borax-carmine, the pieces
are transferred to acid alcohol for 24 hours, and then transferred to
various strengths of alcohol, and finally to absolute alcohol, by
which they are completely dehydrated. The tissues stained with
Kleinenberg's logwood are well washed in spirit, and transferred
to absolute alcohol to be dehydrated. These dehydrated stained
masses are then placed in turpentine or xylol, and then in melted
paraffin, as described above.

Process of Embedding in Paraffin. —This is the same both for
the simple and the saturation methods. Use embedding |_'s, which
consist of two L-shaped pieces of lead about \ inch high, the long
arm about 2 inches long and the short
one |- inch. Their inner surfaces are
moistened with glycerine, and the L's

themselves are placed on a piece of glass, coated with glycerine, to
enable the paraffin to separate easily, with the long limb of the
one in contact with the short limb of the other, thus making a
rectangular box, the size of which can be increased as required.
Fill the trough with melted paraffin.

Take the tissue — if for simple embedding — direct from alcohol ;
dry its surfaces with bibulous paper, to remove any alcohol which
would prevent the paraffin from adhering to it; pour the melted
paraffin into the trough, transfix the tissue with a fine pin, plunge
it into the paraffin just when the latter begins to set at the edges,
move the tissue in the still fluid paraffin to one end of the trough,
and hold it there until the mass sets around it.

If the tissue has been previously saturated with paraffin, the
trough is filled as before, and the tissue, saturated with paraffin, is
taken, by means of a hot needle, from the fluid warm paraffin, and
fixed in the trough in the same way as described above. It is not
always necessary to transfix the tissue with a pin or needle, but it
is' sometimes convenient to do so. Insert the needle in the
direction of the cutting plane, thus indicating afterwards (when
the mass is set) the direction in which the section is to be cut. A
little paraffin may be poured into the trough, and when it just
begins to set, the tissue is laid on it, and another layer of melted
paraffin is poured over it as soon as its surfaces are set. Place the
whole under the tap, and allow cold water to run over it to

EMBEDDING.

45

/

c d

FIG. 30.— Embedding Box.

accelerate the cooling, as paraffin cooled rapidly is more homogeneous
and cuts better than when it is cooled slowly. In about half-an-
hour the paraffin has set, and can be removed from the mould.

Embedding Boxes (fig. 30) may be used. These are readily
made from a rectangular

piece of writing-paper B' C? • d' D'

folded to the size required.
The paper is first folded
along the lines aa' and bb't
then along cc' and dd't
always folding the paper
towards the same side.
The diagonals AA'-DD'

are indented by means of a! *— r*7 \Q>'

the point of a lead pencil,

or the paper is folded

along these lines. These

corners are then bent up

between the fore-finger and thumb, and then bent round, so as to

be applied to the sides AB and CD of the oblong, and are fixed

there by turning down the flaps/''.

Embedding in Paraffin. — The f olio wingscheme shows the stages : —

(1.) Harden, either from the first or subsequent to other agents

in absolute alcohol.
(2.) Xylol or turpentine (24 hours).
(3.) In warm paraffin fluid at 50° C. (1-12).
(4.) Embed and allow paraffin to cool.
(•">.) Cut sections, and in some cases fix them on slide with

a fixative (p. 60).

(6.) Remove paraffin by turpentine or xylol.
(7.) Alcohol to remove xylol.
(8.) Add water and then stain, &c.

3. Embedding in Celloidin. — This method is specially valuable
where the parts of an organ when cut into sections are apt to fall
asunder. It is specially valuable in such as those of the ovary,
central nervous system, retina, &c.

Celloidin is a form of mtro-cellulose or pyroxylin, or solidified
collodion, and is sold in two forms, one in tablets and the other in
cuttings. E. Schering's is the best, and the form sold in " cuttings "
is to be preferred. Do not let its solution dry, as it is then difficult
to redissolve it.

This method was invented by Duval, who used collodion, and
improved by Schiefi'erdecker. Prepare the solutions of celloidin
by dissolving the latter in equal parts of absolute alcohol and

46

PRACTICAL HISTOLOGY.

--——o

ether. The first, or weaker solution, is made of a thin consistence
like collodion duplex, the other is made stronger, until it has a thick
syrupy consistence.

The hardened tissue is placed for some time in absolute alcohol,
and then for several days, or until it is completely saturated, in a
mixture of equal parts of "absolute alcohol and ether. After this it
is placed in a glass-stoppered bottle in the thin solution of celloidin
until it is completely saturated with it (2—3 days). Transfer the
tissue to syrupy celloidin, and let it remain there for several days
until the tissue is thoroughly infiltrated with celloidin.

Some use three strengths of celloidin solution : —

(1.) A thick syrup consistence.

(2.) One part of (1) diluted with 2 vols. of ether.

(3. ) One part of (2) diluted with 2 vols. of ether.

After thorough infiltration the tissue has to be embedded. For
this purpose make a paper box (fig. 30) ; or use a pill-box, or embed
it on a cork, thus. Take a cork corresponding
to the size of the object, roughen one end of
it, and surround it with a piece of paper
fastened by a pin (fig. 31). Moisten the
roughened surface with absolute alcohol, and
on it place the tissue infiltrated with celloidin,
and surround the latter with the thick solution
of celloidin. Allow it to stand until the cel-

Uloidin begins to harden on the surface. This
takes place in less than an hour. Place the
box or cork, as the case may be, in 80 per cent,
alcohol for 24-48 hours, which hardens the
celloidin to such a consistence that it can be
cut like a stiff cheese, but the sections must be
cut with a knife wetted with 70 per cent.
alcohol. It is immaterial which microtome is
used, as long as the knife is moistened with
not too strong alcohol. The sections may be transferred to alcohol
or water, and stained with any suitable dye. Some dyes stain the
celloidin, especially the aniline dyes, and others do riot.

A section after being stained may be mounted in glycerine or
balsam, but in the latter case absolute alcohol cannot be used to
dehydrate them, as celloidin is soluble in this fluid. Alcohol (95
per cent.) must therefora be used for this purpose. Moreover, the
sections must be clarified by origanum or bergamot oil — not by oil
of cloves, which has a solvent action on the celloidin — and mounted
in balsam. (See also Clarifying Reagents.)

Objects embedded in celloidin may be kept ready for cutting for
an indefinite time in 75-80 per cent, alcohol.

FIG. 31. — Embedding
Paper Box with
Weight for Cel-
loidin with a lead
" Sinker."

EMBEDDING, 47

In cutting sections embedded in celloidin, the knife must be so
placed as to cut with as much of the blade as possible. The
hardened celloidin is fixed to a piece of cork, which is clamped in
the microtome.

If, however, it be desired to cut sections embedded in celloidin
by the freezing method, the following procedure must be adopted.
After the tissue embedded in celloidin has been hardened in alcohol,
whereby it becomes not only " hardened " but somewhat milky in
appearance, the alcohol must be got rid of, which is done by
keeping it in running water for twenty-four hours, when it is
transferred to a freezing mixture of gum and syrup (p. 49). This
freezing fluid gradually penetrates the cheesy-like mass, and dis-
places the water. Such a preparation can be frozen in an ordinary
microtome.

For embedding and cutting in celloidin, the following are the
stages : — •

(1.) Organ hardened, either at first or subsequently, in absolute

alcohol.
(2.) Then in a mixture of equal parts of absolute alcohol and

ether (1-2 days).

(3.) In dilute celloidin mixture (1-5 days).
(4.) In thick celloidin mixture (1-5 days).
(5.) Object placed on cork and exposed to air to dry.
(6.) Then in 80 per cent, spirit (i day).
(7.) Cut sections.
(8.) Stain and \vash them.
"(9.) Dehydrate them in 96 per cent, alcohol (and perhaps 1-2

minutes in absolute alcohol).

(10.) Clarify with origanum oil, xylol, cedar orbergamot oil.
(11.) Mount in balsam.

Suppose a piece of the human spinal cord is to be embedded in
celloidin. After being hardened properly it is transferred from
absolute alcohol to ether, for not more than twenty-four hours. It
is then placed for 6-8 days in solution of celloidin (3), in solution
(2) four or live days and in solution (1) two or three days. It is
then embedded in celloidin as described above, and when it begins
to harden by evaporation of the ether and alcohol it is transferred
to 80 per cent, alcohol for three or four days, which finally hardens
it, but it must be completely covered with spirit. If it is to be
preserved for some time before it is cut, keep it in 70 per cent,
alcohol.

Sections of an organ embedded in celloidin may also be clarified
by means of creosote, or a mixture of i part of creosote and 3 of
xylol, which, however, must be quite water-free.

48

PRACTICAL HISTOLOGY.

Scheme for Infiltration and Embedding (Bohm and Oppel).
90 per cent, alcohol.

Staining in bulk, e.g., borax carmine or hsematoxylin,
and washing out the surplus stain.

Absolute alcohol.

Toluol or turpentine.
Toluol or turpentine-paraffin.

Paraffin,
embedding.

g
I

5
Ether.

Celloidin solution, No. 3.
Celloidin solution, No. 2.
Celloidiu solution, No. I.

Embedding.
80 per cent alcohol.

Object ready to be cut into sections.

X.— SECTION CUTTING.

1. With a Razor. — It is absolutely essential to have a sharp
razor if sections are to be cut by hand, and it is well that the
student should practise this method.

Moisten the blade of the razor with 90 per cent, alcohol, and
place some alcohol in. an oblong glass vessel. Dip the cutting blade or
razor into the alcohol after every third or fourth section. Grasp the
razor tightly with the right hand, so that its blade is horizontal, its
edge directed to the operator, whose fingers are pressed against the

SECTION CUTTING. 49

back of the blade, the back of the hand being directed upwards.
The razor is made to glide thrdugh the tissue, cutting as thin
sections as possible, which are placed in alcohol. The tissue to be
cut may be embedded in any of the ways already described, and it
is held in the left hand while being cut.

A razor ought to be sharp and free from notches in its cutting
edge. A razor with notches in its edge causes striated bands in
the section. Notches can readily be detected by drawing the razor
across one's nail or over a piece of cardboard. In sharpening the
razor use a soft hone moistened with water, then lay the razor flat
on the hone and draw it diagonally from heel to point, with the
edge forwards.

The razor must be frequently stropped to keep it sharp, and it
should not be hollow-ground ; it is better to be flat on one side.

2. Valentin's Knife (fig. 32) consists of two parallel blades,
which can be placed at a greater or less distance from each other by

a

FIG. 32.— Valentin's Knife.

means of the screw (a). The blades are first set apart at the
required distance \ the thickness of the section depends on the
distance between them. The knife was formerly much used by
pathologists for making sections of fresh organs. It is now rarely
used.

3. Microtomes. — For many purposes some form of freezing
microtome will be found most convenient for teaching purposes ;
where a large number of sections is required it is indis-
pensable.

Preparation of Hardened Tissues for Cutting by Freezing.—
In order to secure the full advantages of cutting sections by freez-
ing, the tissues must be previously soaked in and saturated with
proper " freezing fluids." If the tissues be kept in alcohol, first
remove all the alcohol by soaking them for twenty-four hours in
running water. After this the tissue is soaked in gum mucilage,
or, what is preferable, a mixture of gum and syrup. The best
receipt is that of Hamilton.

.Make a syrup of 28.5 grains of pure cane-sugar in 30 cc. of
water ; boil and saturate it with boracic acid. Allow it to cool and
filter. Place 45.6 gnuns of gum acacia in 2400 cc. of cold water,
allow it to "dissolve, saturate, it with boracic acid by boiling, and
filter.

PRACTICAL HISTOLOGY.

Freeziny Fluid.

Take of the syrup
,, ,, mucilage
water

4 parts.

5 ' "

Boil and saturate while hot with boracic acid. Filter through
muslin.

The tissues are soaked for twenty-four hours or longer in this
fluid, i.e., after removal of all alcohol from them. The longer they
are kept in it the better ; in fact, tissues may be kept permanently
ready for freezing in this fluid.

Rutherford's Ice-Freezing Microtome (fig. 33). — By means of a
finely-graduated screw a brass plug can be raised or lowered inside.

FIG. 33. — Rutherford's Freezing Microtome adapted for Freezing with Ether.

a brass cylinder. At the top of this cylinder is a stage or plate (B).
The plug has a small flattened brass knob screwed into it, so as to
catch the frozen mass, and prevent it from being detached (K).
Practically this is the arrangement of the microtome devised by the
late A. B. Stirling, curator of the Anatomical Museum of the
University of Edinburgh. To this arrangement Professor Ruther-
ford added an ice-box (C), which surrounds the upper part of the

SECTION CUTTING. 51

brass cylinder or well. This box is provided with an exit-tube
(H), to allow the water resulting from the melting of the ice to
escape. The size of the cylinder varies from 1-2 inches in
diameter, but for most purposes one with a diameter of i inch will
be found sufficient. The ice-box is covered on the outside with a
thick layer of gutta-percha. Professor Hamilton, of Aberdeen, first
suggested the addition of a glass top to be screwed upon the plate
of the instrument.

By far the most convenient cutting tool for use with this micro-
tome is an ordinary planing-iron fitted with a handle, as recommended
by Delepine (fig. 35).

Above all, the tissue must have been properly hardened, and
previously steeped in a freezing fluid, either gum mucilage or gum
and syrup, after removal of all alcohol from it (p. 50).

In using this instrument, screw the plug down to the necessary
depth, thus making a well of the required depth — at least half the
depth of the cylinder — and into the well drop a few drops of
glycerine, or put a little lard round the line of contact of the plug
and the cylinder. This is to prevent any of the fluid passing down
between the plug and the cylinder.

Fill the ice-box with a mixture of pounded ice and salt, and
pack it well around the central brass cylinder. Keep a cork in
the exit tube H, and only allow the fluid to flow away when it
accumulates in large amount. In a short time the temperature of
the plug is greatly reduced. Pour into the well a little mucilage
(BP), sufficient to form a layer about J- inch thick, and allow this
to freeze. The piece of tissue taken from the freezing mixture is
lifted with a pair of forceps, and put into the well, so that it
touches and adheres to that part of the well farthest away from the
operator.

AVhen the tissue is fixed, fill up the well with mucilage and
cover it with a piece of sheet india-rubber, and keep the latter
in position by a weight. This is to prevent the entrance of the
freezing mixture into the well.

Supposing the tissue to be frozen, the operator seizes the
elevating scre\v P with his left hand, and in his right holds the
planing iron, which is fixed in a wooden handle. With the left
hand the operator turns the screw, i.e., elevates the tissue, while as
rapidly as he chooses with his right hand the planing iron, firmly
pressed on the glass plate at (about) an angle of 45°, is pushed
rapidly forwards and drawn backwards, and in a few seconds
twenty or thirty sections accumulate on the upper surface of the
planing iron. By means of a large camcl's-hair brush they are
transferred to a large quantity of water, whereby the gum contained
within them is dissolved and the sections themselves uncurl. One

$2 PRACTICAL HISTOLOGY.

cannot cut all tissues with equal rapidity. In the case of many
tissues and organs (as elastic tissue, lung, kidney, &c.), after they
are completely frozen, a quarter of an hour will suffice, if a planing
iron be employed, to cut more than a thousand sections. This,
however, cannot be done with sections of the cerebrum, cerebellum,
or spinal cord. With these and with some other organs it is
better to cut each section singly.

I'IG. 34. — CaMicart's Freezing Microtome.

If one has several tissues to cut, the one tissue can be embedded
above the other in the instrument at the same time.

This instrument, however, has other advantages, as it can bo
used also as an ether-freezing microtome. Place the tissue,
previously saturated with the freezing fluid, upon the zinc plate
Z, and cover it with mucilage. By means of an ether-spray
producer N, direct a spray of anhydrous ether from the bottle 0
against the under surface of the zinc plate Z.

In order to economise ether, any non-volatilised is collected
and returned by the tube T to the bottle 0.

SECTION CUTTING.

53

h'\

111 using the instniment in this way, however, the piece of tissue
must he thin, not more than 5-7 mm. in thickness, or thereby.

Gathcart's Freezing Microtome (fig. 34). — The instrument is
screwed to a table by means of a clamp (C). Fix by means of gum
the tissue to be frozen, not more than i cm. thick, upon the zinc
plate. Fill the bottle with ether and put the spray apparatus
under the zinc plate. Work the spray apparatus until the mucilage
and tissue arc frozen.

Sections are cut by means of a flat knife pushed along on the
glass supports (GG),
the tissue being
raised by the large
milled head placed
underneath (M).

The sections are
removed from the
knife by means of
a camel's-hair pen-
cil, and placed in
water.

Fig. 35 shows
Gathcart's micro-
tome clamped to a
table, and the
method of cutting
sections by means
of a planing iron.
The planing iron is
used in exactly the
same way as for Rutherford's microtome.

Roy's Microtome (Freezing), Modified by Malassez (fig. 36).—
This instrument is extremely convenient, the cutting instrument
being an ordinary razor.- Instead of ether, Malassez uses chloride
of methyl, which is preserved in a stout metallic flask. A stream
of the methyl chloride is directed against the under surface of the plate
on which the tissue to be frozen is placed. It freezes much more
rapidly than with ether.

Cutting a Continuous Series of Sections in Paraffin. — For this
purpose the "rocking microtome" (fig. 37) of the Cambridge
Scientific Instrument Company, or Minot's microtome, is most
useful.

Embed the tissue in paraffin in the usual way ; place the razor in
position, and fix the embedded tissue to the end of the brass cap on
the, horizontal bar. Move the brass cap, with its adherent em-
bedded tissue, forwards, until it touches the knife-edge. The

FIG. 35. — Showing how to use a Planing Iroii for
Cutting Sections.

54 PRACTICAL HISTOLOGY.

horizontal bar has an axle which moves in a V-shaped pivot. At

FIG. 36.— Malassez's Modification of Roy's Microtome for Freezing.

the end of the instrument is a knobbed bar, Avhich depresses and

FIG. 37.— Cambridge Rocking Microtome.

raises the horizontal bar, and at the same time moves a toothed

SECTION CUTTING.

55

wheel, which is pushed round by a small eatch, whereby the Lai-
bearing the pivot is raised. At the same time the embedded tissue
in paraffin on the horizontal bar is brought down into contact with
the edge of the razor.

The piece of paraffin should have its sides squared, and the two
faces looking upwards and downwards should be coated with soft
paraffin, i.e., with a low melting-point (48° C.). This is to enable
the one section to adhere to the other. On working the instru-
ment, the sections come off in " chains " or ribbons, and can be
caught upon a plate of glass. If the sections tend to curl up, they
may be " flattened " by being placed in not too warm water.

Minot's Microtome. — In this microtome, which is one of the
best microtomes yet invented for embedded tissues, as shown in fig.
38, the knife is fixed while the embedded tissue — in paraffin — fixed

FlG. 38.— Minot's Microtome.

to a circular disc, is moved vertically upwards and downwards by
means of a wheel. The embedded tissue is fixed to a disc which
can do moved around three axes, and thus the tissue can be cut in
any desired plane. The thickness of the sections is regulated by a
special toothed-wheel mechanism, which is so arranged that sections

PRACTICAL HISTOLOGY.

can be out varying from ^, ^ TJ<y> T5> TH* to TO mm- Instead
of the knife supplied with the apparatus, a razor may be used.

The sections can be received on
a silk ribbon, as shown in fig. 39.
This is clamped to the apparatus.
The sections are received on the
ribbon, which is rotated by means
of a milled head.

Jung's or Thoma's Microtome.
— In this instrument the tissue to
be cut is fixed in a clamp, the
knife is fixed in another heavy
clamp which moves on planed sur-
faces. After each section the
tissue is pushed up an inclined
plane by means of the milled head
on the extreme right of the figure
(fig. 40).

Malassez's Modification of
Roy's Microtome (fig. 41). — Some-
times it is desired to cut sections
of a tissue while it is under fluid,
e.g.) alcohol. This can be done as
shown in fig. 41.
move on its base, and can be placed

FIG. 39.— Silk Band for Catching the Chain
of Serial Sections made by Minot's
Microtome.

The microtome is made to

FIG. 40.— Thoma's Sledge Microtome, as made by Jung.

vertically in such a way that the razor and the piece of tissue to be
cut come to lie in a vessel filled with alcohol.

SECTION CUTTING.

57

FlO. 41.— Malassez' Modification of iloy's Microtome for Cutting Sections under ft fluid

FlG. 42. --Williams' Ice-Freezing Microtome.

58 PllACTICAL HISTOLOGY.

Williams' Freezing Microtome (fig. 42). — This consists of a
wooden, non-conducting tub for holding the freezing mixture.
Vertically in the centre of this rises a brass cylinder, into whose
upper end the roughened brass plate on which the tissue is frozen
can be screwed. The lid of the box is formed by a glass plate fitted

FIG. 43.' — Swift's Ether-Freezing Microtome.

into a framework or kind of cap for the tub. In the centre of the
glass plate is a circular hole into which the freezing-plate projects.
In this instrument the tissue remains fixed, while the knife or blade
is depressed by the movement of a screw. The knife or razor is fixed
in a brass tripod frame-work or knife-carrier.

CUTTING SECTIONS.

59

In using the instrument, fill the tube with the freezing mixture
of ice and salt, and when the central brass pillar becomes sufficiently
cooled, pour on it a little mucilage, and when this is frozen place on
it the tissue, which, must not be more than i cm. in thickness, and
pencil some mucilage on it. When it is solid, the knife in the
tripod is used to cut the sections ; and as the front leg of the tripod
consists of a screw, this is turned, and thus the cutting edge is
brought to touch the tissue.

Swift's Freezing Microtome (fig. 43). — A modification of the
previous instrument is shown in fig. 43, which is adapted for freezing
with ether.

Hand-Microtome (fig. 44). — When only a few sections are required,
this instrument, invented by Ranvier, is extremely convenient. The

FIG. 44.— Ranvier's Hand-Microtome.

tissue is embedded in the well of the instrument in paraffin or elder
pith, and sections made by means of a razor, as shown in the figure.
The tissue is gradually raised by means of the milled head.

Section-Flatteners. — Sometimes the sections saturated with
paraffin when cut exhibit a great tendency to curl up. This can
partly be avoided by pressing the section as it is cut gently ngainst
the knife, by means of a camel's-hair brush. Several section-
flatteners attached to the cutting-knife have been used for this
purpose. Take a wire i mm. in diameter, heat it in a flame, and
bend it twice at right angles, the distance between the angles being
about an inch. The free ends are then bent round in the form of
a hook. These hooks serve to fix the frame on the back of the
razor, forming, as it were, a spring-clip. The part of the wire
between the right angles is so arranged that it lies parallel to, and
about one-hundredth of an inch from, the edge of the knife. In

60 PRACTICAL HISTOLOGY.

cutting, the section has to pass between the spring and the knife>
and is thus largely prevented from curling up.

Curled-up paraffin sections may be made to uncurl by being
placed in water at about 40° C. (Gaskell).

Cutting a Continuous Series of Sections in Celloidin. — The
tissue, embedded in celloidin, is clamped in a microtome, e.g., that
of Jung, and section after section is made. The knife must pass at
an acute angle through the celloidin, and must be moistened with
80 per cent, alcohol. This is easily effected from a wash-bottle
Schanze of Leipzig supplies such a bottle provided with a valve,
which facilitates the outflow of a gentle stream of alcohol upon the
cutting blade. Weigert's method of arranging and fixing the
sections on a slide is the best. Each section in celloidin as it is made
is laid upon a narrow strip of curl-paper by means of a camel's-hair
pencil. The curl-paper is kept moist by being placed on a plate
covered with blotting-paper well moistened with 80 per cent, alcohol.
The sections are laid upon the curl-paper in the order desired.

A slide is coated with a layer of thin collodion, and when it is dry,
the celloidin sections on the curl-paper are transferred to it. This
is done by lifting up the curl-paper, and placing it, sections lower-
most, upon the coating of collodion on the slide. Press on the
whole with a piece of dry blotting-paper. The sections adhere to
the slide, and the curl-paper is removed. Dry the sections with
blotting-paper, and pour over them a layer of thin collodion. They
are now permanently fixed, and can be stained on the slide in any
way that may be desired. This is an extremely convenient method
for serial sections of the central nervous system.

XL— FIXATIVES AND SUBSEQUENT TREATMENT
OF SECTIONS.

Further Treatment of Sections.

This depends on how the sections have been made, and whether
they have or have not been previously stained. Paraffin sections
must be freed from paraffin. If they are unstained, they must bo
stained. In most cases it is found advantageous to fix paraffin
sections to the slide by means of a "fixative." Many sections can
thus be fixed on one slide, and treated simultaneously. The series
of events will then be for unstained paraffin sections :—

(1.) Fixation on a slide.

(2.) Removal of paraffin.

(3.) Staining the section.

(4.) Mounting the specimen.

FIXATIVES AND SUBSEQUENT TREATMENT OF SECTIONS. 6 1

To Fix Paraffin Sections on a Slide. There are several "fixa-
tives" for serial sections, but the following will be found the
most useful.

(1.) Collodion and Clove-Oil. — Mix one part of collodion with
three parts of clove-oil. By means of a brush paint a thin layer on
a slide, and on it place the sections. Heat gently over the flame of
a lamp, to fix them firmly and drive off the clove-oil.

(2.) Albumen and Glycerine (P. Mayer}. — Mix filtered fresh
white of egg with an equal volume of glycerine, add a little car-
bolic acid or morsel of thymol to prevent putrefaction. White
of egg filters very slowly. A very thin layer is painted on the
slide, and made smooth by means of a clean glass rod, which is
thus prepared to receive the sections. The sections are flattened
on the albuminised surface by means of a fine brush, care being
taken that no air-bubbles remain under the sections. Warm the
slide to a temperature just sufficient to coagulate the albumen
(70° C.). This may also be done by holding the slide for a few
seconds over a jet of steam.

Such substances as acids and alkalies which dissolve the
albumen must not be applied to the sections, nor must the sections
be stained with such substances as picrocarmine, which also
dissolve the albumen.

(3.) Method of Gaule. — This method depends on capillary attrac-
tion. The slide is moistened with water or weak spirit, and on
this the paraffin sections are carefully spread out. The surplus
spirit or water is removed by blotting-paper, and the slide placed
in a thermostat at 50° C. for twenty-four hours. Sections so dried
are heated for a moment above the melting-point of the paraffin,
and are then firmly fixed on the slide.

To Remove Paraffin from the Sections. — Sections of tissues
soaked and embedded in paraffin and fixed on a slide are placed in
turpentine, toluol, or xylol. The extraction of the paraffin requires
some time, and takes place more rapidly when the temperature is
raised. The slides may be fitted into a zinc framework and
lowered into a bath of turpentine or toluol. Clove-oil must not be
used if collodion and clove-oil have been used as a fixative. In
that case clarify with creosote and turpentine. The turpentine
dissolves out the paraffin.

After this, if the tissue has been previously stained in bulk,
before it was embedded, drive away the turpentine with xylol or
clove-oil, and mount the section in balsam.

If, however, the sections are from an unstained tissue, after
dissolving out the paraffin with turpentine, the latter must bo
displaced by absolute alcohol, and the slides are passed through
alcohols of various strengths and then into water, i.e., provided the

62

PRACTICAL HISTOLOGY.

sections are to be stained in a watery solution of a dye. The
sections are then coloured in situ on the slide. If the sections are
to be mounted in balsam, they must go through the same process in
the reverse order, viz., increasing strengths of alcohol — a clarifying
agent, clove-oil, or xylol — and finally they are mounted in balsam.

Scheme for the Further Treatment of Paraffin Sections.
Paraffin section.

Fixation on slide.

Toluol or turpentine.

Absolute alcohol.

Staining, if not previously stained.
Wash to remove surplus stain.

Absolute alcohol.

Clove oil.

Toluol or turpentine.

Balsam.

STAINING REAGENTS. 63

XII.— STAINING REAGENTS.

Staining. — This process depends on the fact that different tissues,
or different parts of the same tissue, have an affinity for certain dyes,
and not for others. Thus some dyes stain only the nuclei, others
however may cause a uniform stain, all the tissues being of the same
colour. By using some decolorising reagent, it is possible to remove
the stain from certain parts of the preparation, leaving other parts
stained.

A thin section of a tissue or an organ, as a rule, when examined
shows but little differentiation of its several parts. Only in cases
where pigment is naturally present is this difference very marked.
Some substances when applied to the section stain one part and
leave other parts unaffected, thus enabling one to differentiate more
easily the several parts of a section.

Those substances which stain the nuclei chiefly have been called
nuclear stains. The section is placed in a weak solution of the dye,
e.y., haematoxylin ; and after it seems to be sufficiently stained, the
surplus dye is removed by thoroughly washing the section in water
or alcohol, a part of the dye remaining united with the chromatin of
the nucleus and colouring the latter. Such stains may also colour
to a less degree some other parts of the section. Amongst nuclear
stains are carmine, hsematoxylin, and some of the aniline colours.

"When a section is stained, it is called Section Staining, but the
tissue may be stained in bulk before the sections arc made (p. 44)
— staining in bulk.

A. Carmine and its Compounds.

Carmine. — In order to obtain a strong solution of this dye,
certain solvents require to be employed. It is readily soluble in
ammonia, yielding an ammoniacal solution, which may be made
strong or weak. The ammoniacal solution may be diluted to any
extent required with water, and practically the best results are
obtained by allowing sections to remain for a long time (24-48
hours) in a weak solution.

1. Strong Ammoniacal Carmine Solution. — Eub up in a mortar
2 grams of pure carmine with a few drops of water, add 5 cc. of
strong liquor ammonise, mix thoroughly, and add 100 cc. of water.
Place the whole in a bottle, and after a day or so any undissolvcd
carmine is filtered off and the clear fluid kept as a stock solution.
This solution may he diluted to any required extent. If it smell
Strongly of ammonia, the excess of ammonia must be allowed to
evaporate. When the solution becomes neutral it is very liable
to undergo putrefaction, but this may be avoided by placing a
small piece of thymol in it to preserve it.

64 PRACTICAL HISTOLOGY.

2. Frey's Carmine. — Ordinary carmine has two drawbacks : it
is apt to undergo putrefaction, and as the ammonia escapes the
carmine is precipitated.

Carmine . . . . .0.3 gram.

Distilled water . . . . 30 cc.

Dissolve the carmine in the water, adding ammonia drop by drop
until solution is complete. Then add —

Glycerine . . . . 30 cc.

Alcohol . . . . 4 ,,

and shake the mixture. Keep it in a stoppered bottle. It has
no advantage, as far as coloration is concerned, over ordinary
carmine, but it can be kept for a long time unchanged.

3. Alcoholic Borax Carmine (Grenacher).

Carmine ...... 3 grams.

Borax . . . . . . 4 „

Water . . . . . . . 100 cc.

Dissolve the borax in the water and add the carmine, which is
quickly dissolved, especially with the aid of gentle heat. Add 100
cc. of 75 per cent, alcohol, and filter.

4. Watery Borax-Carmine. — Rub up in a mortar 8 grams borax
with 2 grams carmine, and add 15000. water. After twenty-four
hours decant and filter. Tissues to be stained in bulk — e.g., after
hardening with corrosive sublimate — are placed for twenty-four
hours or longer in this fluid. They are then transferred to acid
alcohol (i per cent. HC1 in 70 per cent, spirit for twenty-four
hours), and then into alcohol.

If it be desired to stain in bulk without bringing the tissue into
contact with water, then use : —

5. Carmine-Solution (P. Mayer). — For staining in bulk, and also
for sections, Mayer recommends the following : — Suspend 4 grams
carmine in 15 cc. water, and then add 30 drops hydrochloric acid,
gently heating the mixture. Add 95 cc. alcohol (85 per cent.) and
boil. Neutralise with ammonia and on cooling filter.

6. Borax Carmine (Grenacher).

Carmine ..... I gram.

Borax ...... 2 grams.

Distilled water . . . . . 200 cc.

The borax dissolves the carmine. The whole is placed in a
porcelain capsule and heated to boiling, when the fluid becomes of
a dark-purplish or bluish-red. Add a few drops of 5 per cent,
acetic acid, until the colour becomes more like that of carmine
dissolved in ammonia. Let it stand for twenty-four hours, and

STAINING REAGENTS. 65

filter. Add a drop or two of carbolic acid to preserve it. This
gives a diffuse stain, so that the sections have to be treated with
acid alcohol (p. 65).1
The original receipt is

Carmine . . . . .5 to .75 gram.

Borax . . . . . 2 ,,

Water ..... 100 cc.

Alcohol (70 per cent.) . . . 100 ,,

Borax-carmine is chiefly used for staining tissues "in bulk."
Small pieces of tissue, to J-inch cubes or larger, may be left in it
for days, and they do not become over-stained. It gives by itself
a diffuse stain ; hence to get its effect concentrated upon the nuclei,
for which it has a special affinity, the pieces of tissue must be placed
for twenty-four hours or thereby in 70 per cent, alcohol containing
i per cent, of hydrochloric acid.

Acid Alcohol. — This is called acid alcohol, and is prepared thus —

Hydrochloric acid . . . I cc.

Alcohol . . . . . . 70 ,,

Water . . . . . 30 ,,

When tissues are placed in the acid alcohol, they change in
colour to a bright scarlet. A certain amount of the surplus
carmine is extracted, but the nuclei become intensely stained.

This method is particularly valuable for a large number of organs,
and especially where nuclear staining is desired.

7. Alum Carmine. — Dissolve 5 grams of potash-alum in 100 cc.
water. Add i gram carmine, and boil for a quarter of an hour.
Make up the bulk with water and filter. Add a drop of carbolic
acid to preserve it, as fungi rapidly form in it. It has the advati
tage of not over-staining tissues left in it for a long time.

8. Lithium Carmine (Orth).

Carmine . . . . .2.5 grams.

Saturated solution of lithium carbonate . 100 cc.

Dissolve the carmine in the cold saturated solution of lithium
carbonate ; solution occurs very quickly. It gives a diffuse stain,
to nearly all tissues very rapidly, and the sections must, therefore,
be transferred, without previous washing in water, to acid alcohol
(p. 65). They can then be mounted in glycerine or balsam as
desired. The nuclei are stained a brilliant red. It cannot be used
for sections fixed on a slide by means of white of egg.

Application.

(1.) Stain (2-3 minutes).

(2.) Washout surplus dye in acid alcohol (\-i minute), i.e., in loo

cc. of 70 per cent, spirit + I cc. HC1.
(3.) Remove all acid by prolonged washing in water.
(4.) Alcohol, oil, balsam.

1 Archivf. Mik. AnaL, vol. xvi. p. 3G3.

66 PRACTICAL HISTOLOGY.

9. Picro-Lithium €armine. — This is even preferable to the fore-
going, because, in addition to staining nuclei red, it stains certain
other parts yellow.

Lithium carmine . . . . . 50 cc.

Saturated solution of picric acid . . . 100 ,,

Mix the two slowly. If, after trying it, one or other colour is
too pronounced, add a little more of the other.

The sections are to be treated with acid alcohol like the fore-
going. The acid alcohol, however, ultimately extracts the picric
acid. This is avoided by not leaving them too long in acid
alcohol. If the picric stain be removed, it may be restored at once
by dipping the section in absolute alcohol to which a little picric
acid has been added.

10. Picro-Carmine. — This most valuable reagent was introduced
by lianvier, and has the great advantage of giving a double stain
without the use of acid or alkali.

(a.) Jtanvier's method of preparing it is as follows : — To a
saturated watery solution of picric acid add a saturated ammoniacal
solution of carmine until precipitation just appears, i.e., until satura-
tion. The fluids must be well mixed. Leave it exposed in shallow
vessels to crystallise, but protect it from the dust. Crystals are
deposited, and also some amorphous carmine. After several weeks,
when its bulk is reduced to one-third, decant the liquid, filter, and
evaporate it to dryness on a water-bath. Redissolve it and the
crystalline deposit in water, filter, and evaporate to dryness. The
brown powder so obtained is dissolved in the proportion of i per
cent, in water. This fluid, prepared in this way, gives very satis-
factory results.

(b.) Stdhr's Method. — A very good solution is obtained by this
method. To 50 cc. water add 5 cc. liquor ammonise and i gram
carmine, which is rapidly dissolved. After complete solution, add
50 cc. of a saturated solution of picric acid. Set the mixture aside
for two or three days in a large open flat evaporating dish, and after
this time filter. To the filtrate add a drop of chloroform to prevent
the formation of fungi.

Precautions. — Preparations stained with picro-carmine are not
liable to be over-stained, and may be mounted in Fax-rant's solution
or glycerine acidulated with formic acid (i per cent.) [formic acid
sp. gr. 1. 1 6].

In staining sections with picro-carmine, cover the section with
picro-carmine, and after a few minutes remove the surplus pigment.
On no account should the section le jtlaced hi water. Water rapidly
extracts the picric acid and leaves the preparation stained with the
carmine only. Add a drop of Farrant's solution or formic glycerine,

STAINING REAGENTS.

67

and cover. Such sections improve with keeping, and the surplus
picro-carmine is really an advantage, for, after a time, the nuclei
become more differentiated — red, and other parts yellow.

If it be desired to mount sections stained with picro-carmine in
balsam, the alcohol through which they are passed, or the clove-oil,
must contain some picric acid to restore the yellow colour.

11. Carmine and Dahlia Fluid ( Westphal}. — Dissolve i gram of
carmine in 2.5 per cent, of alum. Take of this —

Alum carmine .

Glycerine

Sat. sol. of dahlia in absolute alcohol

Acetic acid

100 cc.

100 „

100 „

20 ,,

This fluid is specially useful
(" Mastzellen ") of the liver.
12. Cochineal (Csokor).

Powdered cochineal
Alum .
Water .

for staining the granular cells

50 grama.

5 „
500 cc.

Dissolve the alum in the water, add the cochineal (Coccus cacti),
and boil. Evaporate down to two-thirds of its original volume.
Filter. Add a few drops of carbolic acid to prevent the formation
of fungi. This is an excellent nuclear stain, especially for the
central nervous system.

It stains nuclei of a violet-red and does not overstain, so that
sections may be left in it for many hours. It, however, does not
stain well objects that naturally stain with difficulty.
Application.

(1.) Stain for an hour or more.
(2.) Wash in water.
(3.) Alcohol, oil, balsam.

13. Indigo Carmine (MerJceT).

Solution A. — Carmine .
Borax
Water .

Solution B. — Indigo carmine
Borax
Water

2 grams.
8 „
1 30 cc.

8 grams.
8 „
130 cc.

Keep the solutions separate (B is apt to develop a precipitate).
When required, mix equal volumes of A and B. The mixture
undergoes a change within a week, and hence it is better to make
it fresh when required. Sections must remain in it at least twenty-
four hours — with advantage longer — but the results are certainly
satisfactory. The stained sections are placed for half-an-hour or

68 PRACTICAL HISTOLOGY.

thereby in a saturated solution of oxalic acid, which extracts the
superfluous pigment. Mount sections in Farrant's solution or
balsam. (See Stomach.)

B. Haematoxylin and Logwood.

Hsematoxylin. — This substance was introduced to the notice of
histologists by Bohmer. It is one of the most valuable nuclear
staining reagents we possess, and this is specially the case when its
violet-blue stain is set off by contrast with a ground stain of eosin,
picric acid, or other appropriate dye.

1. Hsematoxylin (Bdhmer). — Make a solution containing —

Hsematoxylin ..... i gram.
Absolute alcohol . . . . 100 cc.

Make a second solution of —

Alum ...... 5 grams.

Distilled water . . . . . 100 cc.

Add drop by drop the first solution to a little of the second until
a deep- violet colour is obtained. The fluid is placed for fourteen
days in an open vessel, protected from dust, and exposed to the
light and air, when it becomes of a bluish tint. This fluid is said
to " ripen." Oxidation processes take place whereby the hsemato-
xylin is converted into hamatein. Filter. Add a fragment of
thymol to preserve it. It stains tissues in 5-15 minutes.

It is sufficient to make a saturated solution of the crystals of
haernatoxylin in a small quantity of absolute alcohol. Add a few
drops to a i per cent, solution of alum, which yields a light violet-
coloured fluid. Expose the fluid to light and air, when it becomes
blue. It is well to prepare this fluid several weeks beforehand to
enable it to " ripen."

The ordinary manipulative procedure for hsematoxylin staining is
as follows : —

(1.) Stain the section (2-10 minutes).

(2.) Wash in distilled water.

(3.) Allow section to remain 12-24 hours in distilled water.

(4.) Remove water with alcohol, — add ethereal oil, balsam.

Bohmer's hsematoxylin is well adapted for staining sections. It
is best adapted for tissues hardened in sublimate, alcohol, picric or
nitric acid, and not so good for those from chromium salts or osmic
acid. Sections to be stained with it before being placed in the dye
are better to be placed first in water or in i per cent, alum solution.
If taken direct from alcohol they may contain a deposit after being
stained. After staining, the sections are thoroughly washed in
ordinary water. Haematoxylin stains the chromatin of the nuclei

STAINING REAGENTS. 69

a (loop blue, and other parts of some tissues light blue, e.g., the
matrix of hyaline cartilage.

2. Strong Nucleus- Staining Hsematoxylin (Hamilton).

Ha-matoxylin
Alum
Glycerine
Distilled water

12 grams.
50 „
65 cc.

130 „

Boil, and while hot add 5 cc. liquid carbolic acid. Allow the
mixture to stand in the sunlight for at least a month.

3. Delafield's Hsematoxylin. — To 100 cc. of a saturated solution
of ammonia alum add drop by drop a solution of i gram hsematoxylin
dissolved in 6 cc. absolute alcohol. Expose to the air and light
for a week. Filter. Add 25 cc. glycerine and 25 cc. of methylic
alcohol. Allow it to stand exposed to the light for a long time.
Filter.

This solution stains extremely rapidly, and may be greatly diluted
when it is used. It keeps for a very long time, and stains well
even tissues which have been hardened in chromic or osmic acid.

4. Kleinenberg's Hsematoxylin. — (i.) Make a saturated solution
of calcium chloride in 70 per cent, alcohol. Shake it well, and
allow it to stand. Decant the saturated solution, add alum to
excess, shake it well, allow it to stand for a day or so, and filter.

(2.) Make a saturated solution of alum in 70 per cent, alcohol.
Filter.

(3.) To one volume of the filtrate from (i) add eight volumes

Of (2).

(4.) To (3) add drop by drop a saturated solution of hsematoxylin
in absolute alcohol, until it becomes of a decided purple colour,
but not too dark, as the solution becomes darker by keeping and
exposure to light. It should be prepared at least a few months
before it is wanted.

It may be diluted to any extent by adding the mixture (i) or
(2). As it contains much spirit, sections placed in it must be
covered, i.e., protected from evaporation, else the spirit will rapidly
evaporate.

This logwood stain is particularly valuable when it is required
to stain a tissue or an organ " in bulk." This a diluted solution
will do in 24-48 hours, provided the pieces be not too large.

5. Acid Hsematoxylin (Ehrlicli).

Haematoxylih ..... i gram.
Absolute alcohol . . . 30 cc.

To the solution add —

Glycerine . . . . . 60 cc. \ Saturated

Distilled water . . . . . 60 ,, /with alum.

Glacial acetic acid . . . • 3 »

70 PRACTICAL HISTOLOGY.

Dissolve the hsematoxylin in the alcohol, add the glycerine and
water, and then the acid. At first the solution is light-red, but
when it has been exposed to the air for 2-3 weeks it gets bluish.
It does not over-stain, and the tissues stained with it, when exposed
to the light, become violet or bluish tinted. It may be used either
for sections or for staining in bulk, and in the latter case it does
not tend to over-stain.

Application. — Sections from alcohol —

(1.) Stain (3-5 minutes).

(2.) Wash in alcohol (90 per cent.).

(3.) Alcohol, oil, balsam.

Garbini finds it better to place the sections after staining in—

(2.) Distilled water.

(3.) Solution of carbonate of lithia (.25 per cent.).

(4.) Alcohol.

6. Glycerine Hsematoxylin (Renaut). — Saturate perfectly neutral
glycerine with potash-alum, and to it add drop by drop a saturated
solution of hsematoxylin in 90 per cent, alcohol, until a deep-violet
tint is obtained. About one-fourth part of the haematoxylin solution
has to be added. Let it stand exposed to the light for weeks, and
filter.

7. Eosin Hsematoxylin (Renaut).- — Add drop by drop a con-
centrated watery solution of eosin to 200 cc. of glycerine saturated
with potash-alum. Filter. Add drop by drop an alcoholic solution
of hsematoxylin. Expose to light for weeks, and filter.

8. Heidenhain's Hsematoxylin.

(1.) This is used for staining in bulk. Prepare J per cent,
watery solution of hsematoxylin, which must not be
kept too long. Boil the hsematoxylin in water and
allow it to cool. Place the hardened tissue in this dye
for 24-48 hours.

(2.) Transfer it for 24 hours to .5 per cent, watery solution
of yellow chromate of potash (24-48 hours). This
causes dark clouds in the fluid, so that the chromate
must be frequently changed.

(3.) "Wash carefully in water. The tissues are then hardened
in alcohol, and may be embedded in paraffin. It is best
adapted for objects hardened in absolute alcohol, e.g.,
salivary glands, pancreas (Lesson XXIII.), or in picric
acid. Besides tinting the nuclei a greyish-blue, the
protoplasm of the cells has a fine steel-gray tint, but
the sections must not be too thick.

STAINING REAGENTS. 71

9. Hamatein. It is well known that a solution of hsematoxylin
(C16H14O6) after being prepared must stand some time to " ripen "
before it is ready for use. The substance ultimately formed chiefly
by the action of the air is hamatein (C16H1206), which occurs in
commerce in the form of a brown powder which is soluble in alcohol
or water. P. Mayer recommends the following solution of this
body in alum or Ham-alum : l —

(«.) Hamatein . . . . i gram.

Alcohol (90 per cent.) . . -5° cc-

Dissolve by heating.

(6.) Alum ..... 50 grams.
Distilled water .... 1000 cc.

Mix the fluids (a) and (b). Allow the mixture to settle, and use
the clear supernatant fluid as a stain. If it be too strong, dilute
with distilled water, or, better still, with alum solution.

I have used this extensively during the last year for staining in
bulk, and find it to be an excellent dye.

Logwood. — Staining solutions were formerly, and sometimes
are, made from logwood chips.

10. Logwood (Mitchell's). — The tannic acid is removed.

Place finely-ground logwood (2 oz.) in a funnel; pack it well,
and allow water to percolate through it until it flows away with
but little colour. Allow the water to drain away ; spread the
logwood on a board to dry.

Dissolve alum (9 oz.) in 8 oz. of water. Moisten the dry logwood,
pack it again tightly into a funnel, and pour on the alum solution.
Close the lower end of the funnel, and allow the alum solution to
extract the dye from the logwood for forty-eight hours.

Allow the coloured fluid to flow off, and percolate 4 oz. of water
through the logwood in the funnel. Add a few drachms of glycerine
and rectified spirit. Dilute largely when using, so that staining
will take place slowly.

11. Other solutions of hsematoxylin, including Weif/ert's, are
referred to in the text. (Lessons on Central Nervous System and
Salivary Glands.)

General Statement regarding Hsematoxylin.— Hsematoxylin is
specially useful for staining tissues hardened in chromic acid.
Ehrlich's haematoxylin is much to be commended. In all cases it
is better to clarify sections stained with hsematoxylin by means of
xylol — not clove-oil — before mounting in balsam.

Haematoxylin and logwood are amongst the best nuclear stains
we possess, and the tissues which stain best are those hardened in
alcohol ; but those also from Miiller's fluid stain well. With those

1 Zeitsch.f. wiss. Mik., viil p. 341.

72 PRACTICAL HISTOLOGY.

hardened in watery solutions of chromic acid it is otherwise. It is
sometimes very difficult to get them to stain. This may sometimes
be effected by soaking the sections previously in a dilute solution of
sodic carbonate.

They are also very valuable in double and treble staining.

In the case of preparations stained by haematoxylin or logwood,
over-staining may be got rid of by placing the sections in dilute
acetic acid. This will rapidly extract the surplus stain, but at the
same time, if allowed to act too long, it will make the remaining
part red. Great care should be taken afterwards by thorough
washing of the sections in water to remove every trace of the acid.

C. Eosin.

1. Eosin. — This substance is readily soluble in alcohol and water.
Make a 5 per cent, watery solution. It gives a beautiful diffuse
rosy hue, and stains very quickly, in a minute or two. The
stronger solution can be diluted as required. It forms one of the
best ground-stains in contrast to logwood or one of the numerous
aniline dyes. When using it as a double stain, e.r/., logwood and
eosin, stain the section first of all in logwood, and if it is to be
mounted in balsam, clarify with clove-oil in which a little eosin has
been dissolved. Sections stained with it can be mounted in balsam,
Farrant's solution, or glycerine. It is a specific stain for the
haemoglobin of red blood-corpuscles, as it stains it, even after
hardening in chromic salts, a copper-red colour, while it also stains
the granules of certain leucocytes of the blood of a reddish tint. It
is used very extensively, and in a very dilute solution is a good
stain for cartilage and striped muscle.

D. The Aniline Dyes.

Watery or alcoholic solutions of the aniline dyes stain sections
with great rapidity. The word "stain" is perhaps not quite the right
word to use. It is rather a process of imbibition than staining
proper. One of the difficulties in using aniline dyes is the rapidity
with which sections have to be transferred from one liquid to another.
They are not used for staining in bulk. After staining and dehydrat-
ing, it is best to clarify the sections (except in special cases) with
cedar or bergamot oil or xylol. The sections are mounted in balsam,
not in glycerine, as the latter dissolves the dyes.

They are amongst the most valuable so-called staining reagents
we possess, and although many of them do riot yield permanent
preparations — the colour fading after a time — still the results
obtained by their use are so important that it behoves the student
to use them frequently. It is most important that they should be
obtained from reliable sources.

STAINING REAGENTS. 73

The aniline colours are divided by Elirlich into ariil, L//xic, and
neutral compounds. Of the three, the basic colours are most used,
us 11 icy are excellent nuclear stains. Some of them have special
affinities for certain tissues, and, as is well known, they are of the
utmost value in bacteriological investigations. They may be kept
in drop bottles. All of them are soluble in alcohol, and most of
them in water ; and so powerful are they, that usually a i per cent.,
or even a much weaker solution, suffices to stain tissues in a few
minutes. Some of them, according to Ehrlich's researches, stain
better when they arc mixed with a mordant.

Aniline-Oil and Aniline- Water (Khrlich's Method). — Shake up
excess of pure aniline-oil with excess of water, and allow it to stand.
The most of the oil sinks to the bottom. This solution should not
be kept too long ; in fact, it is better to make it fresh. Filter a
little of the aniline-water into a watch-glass. To the fluid in the
watch-glass add ten or twelve drops of a concentrated alcoholic
solution of any of the aniline dyes it is desired to use in this way.

Very few of the preparations stained by aniline dyes can be pre-
served in glycerine or Farrant's solution. A 50 per cent, solution
of acetate of potash keeps their colours well, but it is a medium
which it is difficult to keep tightly under the cover-glass. Many of
them can be preserved in balsam, but most of them must be kept
away from the action of acids. In a few cases the colour is partly
extracted and fixed with dilute acids, but in such cases the free acid
must be removed from the section before it is finally mounted.

For convenience these colours may be grouped as follows : —

(A.) Violet Aniline Colours.

Methyl Violet. — Dissolve 2 grams in TOO cc. and filter. This
gives a 2 per cent solution, which may be diluted as required. A
o. i per cent, solution is, in many cases, sufficient. It does best for
porous textures. Sections are left to stain in it for several hours,
washed with water, and then with alcohol, until no more colour
comes away, and mounted in balsam. It stains intercellular sub-
stances but slightly, while cells, and especially their nuclei, are
stained by it.

Gentian Violet always answers very well for staining cell-nuclei,
but it seems to be better for hardened preparations than the
previous dye.

Dahlia is used in the same way as the preceding.

(B. ) Blue (and Purple) Aniline Colours.

Aniline Blue. — Make a i per cent, watery solution, adding a few
drops of absolute alcohol. This is useful for the glands of the
stomach and for a double stain with safranin.

Methylene Blue. — Make a saturated watery solution. Rectified
spirit may be added to make it keep. It is not very largely used
8

74 PRACTICAL HISTOLOGY.

in bacteriology, e.f/., for the tubercle bacillus, but largely for double-
staining of tissues, in contrast to red. It stains axis cylinders of
nerve fibres. (See Nervous System and Epithelial Cement.)

Sections to be mounted in balsam are best clarified by cedar-oil.

Better results are obtained by using a very dilute solution made
as follows (Garbini) : — Add 10 drops of a saturated alcoholic
solution to 100 cc. of a solution of caustic potash (i in 10,000).
Leave the sections to stain (12-24 hours). Leave them in absolute
alcohol (6-8 hours), and then in oil of cloves (2-4 hours) — xylol —
xylol-balsam.

Spiller's Purple. — Use Spiller's purple No. i. Hub up 2 grams
in a glass mortar with 10 cc. of alcohol, and add 100 cc. of distilled
water. It is used as a double stain, and for staining the fibrin in
coagulated blood. The stain requires to be pretty deep, as it is
washed out by alcohol. If a section stained by it is to be mounted
in balsam, use cedar-oil to clarify it.

(C.) Green Aniline Colours. — Amongst these are iodine green,
methyl green (i per cent.), aniline green, and aldehyde green.

Iodine-Green is used as a 5 per cent, filtered watery solution.
It stains nuclei and developing cartilage green, and makes a good
contrast stain. It is not very readily extracted by spirit, and does
not soon fade.

Methyl-Green. — This is a nuclear stain. Make a i per cent,
solution in distilled water, and add 25 cc. of absolute alcohol.
Belgian observers, more particularly Carnoy, use this for fresh tissues
in the following manner : —

Methyl-green ..... i gram.
Glacial acetic acid . . . I cc.

Distilled water . . . . .100,,

Add a few drops of this liquid to a watch-glassful of an indifferent
fluid, e.g.) normal saline.

The others will be referred to in the text.

(D.) Red Aniline Colours. — They are very numerous.

Rosaniline Acetate, Sulphate, and Hydrochlorate (Magenta).—
The term fachsin is sometimes applied to the one, sometimes to
the other, but the acetate is more soluble in water. They give a
rather diffuse stain, but <fre useful for nuclear staining, elastic fibres,
and blood-corpuscles.

Rub up a little (i gram) in a glass mortar with rectified spirit
(20 cc.). After solution add 20 cc. of distilled water.

Magenta for Blood-Corpuscles. — Dissolve .1 gram of magenta
in 5 cc. of rectified spirit and 15 cc. water, and add 20 cc. glycerine.

Acid Fuchsin is a specific colouring-matter for the nervous
system. It was introduced by Weigert, but it has been largely
displaced by Weigert's haematoxylin copper stain.

STAINING REAGENTS. 75

Safranin is specially used as a nuclear stain, and very largely for
the study of mitosis. It is specially useful for tissues hardened in
Flemming's mixture.

Safranin ..... I gram.

Absolute alcohol .... 100 cc.

Water ...... 200 ,,

It may also be used as a much stronger alcoholic solution, 5 per
cent, in 70 per cent, alcohol. There are several varieties of this
dye, and some of them are of little value as dyes. Therefore it is
important to obtain a good sample. That sold as safranin-O can
usually be relied upon, and is to be obtained from Dr. George
( Iriibler, Leipzig.

In using this dye, the sections are, as a rule, left for several
hours in the solution — even twenty-four hours or longer — and ;uv.
then placed in ordinary alcohol or acid alcohol (p. 65) — containing
.5 per cent, hydrochloric acid — to remove the surplus stain. If this
be properly done, the nuclei — the chromatin of the nuclei only — are
stained. Sections may be mounted in balsam.

Application.

(1.) Stain sections in i per cent, watery solution of safranin

(1-24 hours).

(2.) Wash rapidly in water.
(3.) Wash in absolute alcohol or acid alcohol.
(4.) Absolute alcohol, oil, balsam.

Sometimes it is useful to use it in aniline-water, after Ehrlich's
method (p. 73).

As shown by Martinotti, it colours black or dark-purple elastic
fibres hardened in 0.2 per cent, chromic acid (Lesson X.).

(E.) Brown Aniline Colours.

Bismarck Brown or Phenylene Brown.— This is but slightly
soluble in water, but it forms a good ground-stain in contrast to
htematoxylin, and is useful for staining plasma cells and the cells of
the cerebrum. It preserves its colour when mounted in Farrant's
solution or balsam.

((/.) Boil Bismarck brown with 100 of water ( = 3-4 per cent.).
Filter and add one-third its volume of absolute alcohol.

(/>.) Or use a concentrated alcoholic solution in 40 per cent,
alcohol ( = 2-2 i per cent.).

Application."

(1.) Stain (5 minutes).

(2.) Wash out in strong spirit.

(3.) Alcohol, oil, balsam.

It does not tend to over-stain. It is a nuclear stain, and the nuclei

76

PRACTICAL HISTOLOGY.

are brown, and the protoplasm light brown. Such preparations are
well adapted for photographic reproductions. Some use a solution
in 70 per cent, alcohol.

Vesuvin is much more soluble, and is used in the same way as
Bismarck brown.

(F.) Other Aniline Dyes.

Aniline Blue-Black. — This has a remarkable power of staining
nerve-cells, as shown by Sankey. It is best adapted for staining
fresh nerve-tissues, although it is also used for staining in bulk.
It has little power of diffusing through them. (Lessons on Central
Nervous System.)

TABLE showing some of the Aniline, Dyes in most Common Use,
arranged according to their Colour.

Red.

Green.

Blue.

Yellow.

Violet.

Brown and
Black.

Safrauin.

Methyl-

Methylene

Orange.

Methyl-

Bismarck

Eosin.

green.

blue.

violet.

brown.

Fuchsin.

Iodine

Aniline

Violet

Nigrosin.

Ponceau

green.

blue.

BBBBB.

Aniline

BE.

Aldehyde

Quinoline.

Dahlia.

blue-black.

green.

E. Metallic Substances.

1. Nitrate of Silver. — This substance possesses the property of
forming a compound with intercellular substance, which darkens,
and becomes brown or black, on exposure to light. It is unequalled
for the study of the cement substance of epithelium and endo-
thelium, and for the cell-spaces of the cornea, and connective tissue
generally. The tissue, however, must be fresh — the fresher the
better. Use a glass or horn rod to manipulate the tissue in the
fluid, not metallic instruments.

Make a i per cent, solution, i.e., i gram of silver nitrate is
dissolved in 100 cc. of distilled water. This is kept as a stock.
A J or J per cent, solution is the strength usually employed.

If it be desired to " silver " a part of the omentum or mesentery,
this membrane should be pinned out with hedgehog spines, without
being stretched, on a piece of flat cork with a large hole in it, so
that the solution can get to both sides of the membrane. A very
convenient plan is to pass the membrane over a porcelain or ebonite
ring, and fix it with another ring in the manner in which a skin is
fitted on a drum (figs. 45 and 46).

Lave the membrane gently in distilled water to remove any

STAINING REAGENTS.

77

chlorides, and place it in a dilute solution of the reagent (J-J-J per
cent.). The tissue soon — 5-10 minutes — becomes white, and as
soon as it looks grayish remove it and wash it in distilled water.
Place it in ordinary water, and expose it to good daylight, when it
rapidly becomes brown. It can then be preserved in spirit until it
is required.

A membrane may be stained in situ, e.g., the central tendon of
the diaphragm of a rabbit, or the mesentery, e.g., of a frog. Open
the abdomen, irrigate _===_

the membrane by allow- ^^s^ .^••^L B

ing distilled water to
fall on it from a pipette.
This removes all sub-
stances that might com-
bine with the silver and
give rise to illusive ap-
pearances. Drop on the •^j'-i^HJ^ "" Wf W C
silver solution by means
of a pipette. Treat t-he
membrane as in the

FIG. 45.— A. Vulcanite rings used for stretching a mem-
bratie which is to be silvered ; B. Conical rings, the
one inside the other ; C. A single ring.

previous case.

2. Negative Method
(Recldingh ausen). — This

is the usual method by which the intercellular cement substance is
stained black — a weak solution being used — as for the study of
endothelium. The membrane
ought to be kept stretched,
e.g., over the mouth of a
porcelain capsule.

The following modification j,
is recommended by Thanhoffer,
and it works well. After the
membrane is exposed to the w'
action of silver nitrate, it is
washed with a 2 per cent, solution of acetic acid.

3. Positive Method (77^-) is specially designed for showing lacunae
and lymphatic cavities, e.g., the lymphatics of the skin and cornea.
Place the tissue for several hours in i per cent. AgN03 in the dark,
wash in water, transfer in dark to 3-5 solution of sodic chloride.
Wash in water, expose to light. The spaces are found fdled
with black granules. Examine in glycerine.

Sometimes silver nitrate is us"ed in a solid form, e.g., for the
cornea. This will be referred to afterwards in treating of the cell-
spaces of the cornea itself. (Lesson on Eye.)

Silver preparations show the cement substance of epithelium

7 8 PRACTICAL HISTOLOGY.

stained black as " silver lines," and they may be mounted in
glycerine or balsam, either unstained or after staining with logwood,
picro-carmine, or other dye as desired.

In order to stain the lining endothelium of the vascular system,
a solution of silver nitrate is used. The special precautions
required are referred to in the text. (Lesson on Blood-Vessels.)

4. Golgi's Method. — In this method parts of the central nervous
system hardened in potassic bichromate are treated for many days
with silver nitrate or mercuric chloride to demonstrate the processes
of nerve-cells. (Lesson on Nervous System.)

5. Nitrate of Silver and Osmic Acid (Golf/i).— This is specially
useful for the nervous system. Place a fresh nerve of a rabbit just
killed in

Potassic bichromate (2 per cent.) . "* .10 parts

Osmic acid (1 per cent.) . . . 2 ,,

for an hour ; tease the nerve, and let it remain in the mixture for
another hour. Transfer for 8 hours to .5 per cent, silver nitrate and
then to alcohol.

B. Gold Chloride.

Gold Chloride. — This substance has rendered particular service,
especially in connection with the terminations of nerves. It is used
as \-2 per cent, watery solution. Various methods are employed,
according to the end desired.

1. Acetic Acid Method. — Place a small piece of perfectly fresh
tissue, 2 mm. cubes, e.g., hyaline cartilage or a small cornea, in i per
cent, solution in a glass thimble for half-an-hour, keeping it in the
dark all the time. These small glass thimbles are particularly use-
ful, and are better to be somewhat broader relatively than those
shown in fig. 26. The tissue will become yellow ; wash it
thoroughly in distilled water, and expose it to bright daylight in
distilled water slightly acidulated with acetic acid. In a day or
two it will become of a purplish or violet-brown colour. Sections
can then be made and mounted in glycerine.

2. Loewit's Method. — To one part of formic acid (sp. gr. 1.16)
add two parts of distilled water. Place small pieces of the fresh tissue
(1-2 mm. in thickness), e.g., tendon from a rat's tail, in this mixture
for J-i minute, until they become somewhat transparent; transfer
them to a glass thimble or watch-glass containing i per cent, gold
chloride for 15-20 minutes, i.e., until they have become yellow
throughout. During this process, the tissue should be exposed to
light as little as possible. Place the tissue in formic acid (i 13),
and keep it in the dark for twenty-four hours. Afterwards place it
for twenty-four hours in pure formic acid, and keep it also in the

STAINING REAGENTS. 79

dark. Wash it thoroughly with water, and mount in glycerine or
balsam.

3. Ranvier's Lemon-Juice Method. — The fresh tissue is placed
for c^io minutes in the freshly expressed and iiltered juice of a
lenion, until it becomes transparent. Rapidly wash it in distilled
water, transfer it to i per cent, gold chloride solution for from ten
minutes to one hour; the time depends on the tissue under investi-
gation. Wash with water and place the tissues in 50 cc. of water
containing two drops of acetic acid, and expose them to light, when
reduction takes place. Or the tissue may be placed in formic acid
(1:3) after being treated with lemon-juice and gold chloride, and
kept in the dark for twenty-four hours. The latter plan is in many
cases to be preferred, especially where the retention of the super-
ficial epithelium is not desired.

4. Boiled Gold Chloride. — For some purposes, especially for
studying the terminations of the nerves in sensory surfaces, this
method of Ranvicr has yielded me the best results.

Make as required — fresh — a mixture of four parts of gold
chloride (i per cent.) and one part of formic acid. Boil the
mixture and let it cool. Place the fresh tissues (small pieces) in it
for ten minutes to one hour. Wash in water, and place in formic
acid (1:4 water), and keep in darkness, where the reduction
takes place.

5. Rapid Reduction of Gold Chloride. — A tissue may be left in
gold chloride (i per cent.) for half-an-hour or more, and then trans-
ferred to a strong solution of tartaric acid and heated to 45° or 50°
C., when it rapidly becomes of a purplish-brown colour, usually in
the course of a quarter of an hour.

Although it has been stated that for gold chloride preparations
the tissues should be fresh, Drasch, in his researches on the nerves
of the intestine and those of the circumvallate papillae, points out
that he obtained the best results with tissues twenty-four hours
after death ; the tissues, however, must have been kept cool. Any
one who has had the privilege of studying the preparations of
Drasch cannot but have been impressed with the beauty of speci-
mens prepared by his method.

6. Gold Chloride and Chromic Acid (Kolossow). — Place the
tissues for 2-3 hours, according to their size, in i per cent, gold
chloride acidulated with hydrochloric acid (i-ioo). Wash the
tissues with water, and keep them in the dark in chromic acid
(5*0 ^° wo Per cenk) f°r 2~3 days. Wash out the chromic acid
thoroughly.

7. Method of Ciaccio is good for the termination of nerves in
cornea and muscles. Place a small piece of tissue, not more than 2
cubic mm. in size, in fresh juice of lemon (5 minutes) : wash ; place in

SO PRACTICAL HISTOLOGY.

i per cent, solution of chloride of gold and cadmium (30-60 minutes)
in the dark. Wash ; then in i per cent, formic acid (24 hours)
in the dark, and then for 12 hours in sunlight. Finally for 24 hours
in pure formic acid. Wash. Tease and mount in glycerine.

F. Double, Treble, or Multiple Stainings.

It is possible to stain a section so that the several parts of it may
be differently stained. This may be done either by staining succes-
sively with different stains, or by mixing the dyes in one fluid, and
staining the section with the mixture, whereby one part takes up
one of the dyes and another part one of the other dyes. Thus one
gets an elective and differential stain. It is possible thus to combine
a nuclear stain with one which stains only the protoplasm of the
cells, such as eosin or orange.

Amongst double stains are the following : —

1. Picro-carmine (p. 66), one of the most valuable we possess,
and which we owe to Ranvier ; and Picro-litho-carmine. If the
section is to be mounted in glycerine or Farrant's solution, do not
wash it 'in water. If it is to be mounted in balsam, the alcohol in
which it is washed should contain picric acid — the same result is
obtained by using clove-oil with picric acid dissolved in it — other-
wise only a carmine stain is obtained.

2. Carmine and Aniline Blue. — Stain a section in borax-carmine,
and then in very dilute aniline blue, specially useful for the glands
of the fundus of the stomach. In other tissues the nuclei are red
and the perinuclear parts blue.

3. Hsematoxylin and Eosin. — Hsematoxylin is a nuclear stain,—
the sections are first stained in it, and then in eosin, which stains
the general protoplasm of the cell. (See also p. 72.)

4. Aniline-blue and Safranin (Garbini).-The section is trans-
ferred from water.

Manipulation.

Aniline-blue sol. (.5 per cent.), 2-4 minutes.

Wash in water.

Lithium carbonate (.5 per cent.), a few minutes.

Hydrochloric acid (.5 per cent.), a transparent blue colour

is produced.
Wash in water.

Safranin (i per cent.), 10 minutes.
Dehydrate in methylic alcohol.

Clarify in oil of cloves (2 parts) and cedar-oil (i part).
Xylol-balsam.

This is especially useful for some of the salivary glands — thus in
the sub-maxillary one set of cells is red, the other blue ; in the
stomach the parietal cells are red and the inner blue ; the epithelial

STAINING REAGENTS, 8 1

cells of the villi are blue, the goblet cells reddish ; in a hair-follicle
the sheath of Henle is an intense red, and the sheath of Huxley
blue.

5. Ehrlich-Biondi-Heidenhain Stain.

Saturated watery solution of orange . 100 cc.

,, ,, ,, acid fuchsia . 20 ,,

,, ,, „ methyl-green . 50 „

To get complete saturation it is necessary to have an excess of
the crystals for several days. Each fluid is saturated separately.
Before use, the solution is diluted in the proportion of i in 100 with
water, and then on the addition of acetic acid must be bright red.
It is better to obtain the mixture from Dr Griibler.
Application.

(1.) Harden the organ in corrosive sublimate.

(2.) Stain sections in the dilute solution (12-24 hours).

(3.) Wash quickly in 90 per cent, alcohol.

(4.) Dehydrate in absolute alcohol.

(5.) Xylol-balsam.

It is especially useful for sections containing many leucocytes,
and is best used for paraffin sections fixed on a slide. Red blood-
corpuscles are stained red, resting nuclei blue, mitotic figures and
nuclei of leucocytes green-violet.

General Remarks on Staining.— Filter the staining fluid.
When possible, use a weak solution of the dye, and thus let the
sections stain slowly in a fairly large amount of the fluid. Place a
piece of blotting-paper on the inside of a large watch-glass, pour
some of the diluted stain into the watch-glass, and place the sections
in it. The sections should lie as flat as possible, and not overlap
each other. The sections may be moved gently in the fluid by
means of a needle. Cover the watch-glass with another glass of
the same size, or set it aside in a moist chamber, e.g., on a plate
covered by a bell-jar, with a piece of moistened blotting-paper
attached to the inside of the jar (fig. 47). After staining, the
sections are to be carefully washed in distilled water to remove any
trace of surplus dye (except in the case of picro-earrnine). Sections
stained with hsematoxylin should be washed for a long time in
water before they are mounted in glycerine or Farrant's solution.

Be careful not to over-stain the tissue, except in those cases
where the excess can be again removed, e.g., with the aniline
dyes by means of alcohol or acid alcohol.

All acids should be removed from the sections before they are
placed in the dye.

It is convenient on many occasions that the student should
rapidly stain his sections on a slide, but he should also be taught
y F

82

PRACTICAL HISTOLOGY.

to practise the slower method of staining sections in very dilute

solutions of a dye.

There is one method of staining sections which may be profitably

impressed upon the student, viz., that so strongly insisted upon by

Ranvier. Suppose any
delicate object — isolated
epithelial or other cells
— to be mounted in a
watery medium ; a drop
of a solution of picro-
carmine is placed at one
side of the cover-glass.
As the fluid evaporates
at one side of the cover-
glass the picro-carmine
slowly diffuses under the
cover-glass and stains
the preparation. Ex-
posed to the air, the
preparation would soon
become dry. This must
be corrected. This is
best done by placing
the slides to be stained
in this way on a stage
with several shelves

(fig. 47), the whole being placed on a plate moistened with a few

drops of water and covered by a bell-jar. This forms a moist

chamber.

After the cells are stained, glycerine may then be applied at the

side of the cover-glass, with the same protective precautions, so

that the preparation can be finally mounted and preserved in

glycerine.

FIG. 47.— Support of Ranvier for Holding Slides Placed
under a Bell-Jar.

XIII.— CLEARING OR CLARIFYING REAGENTS.

Glycerine, Farrant's Solution, and Glycerine Jelly. — When
any one of these reagents is used for mounting preparations, no
other clarifying substance is used.

Balsam Preparations. — When a preparation is to be mounted
in balsam, be it Canada balsam or dammar, some clarifying reagent
has to be added to the preparation before the balsam is applied.
Before applying the balsam the tissues must have been rendered
transparent.

CLEARING OR CLARIFYING REAGENTS. 83

The following substances are most commonly used : — Oil of
cloves, a mixture of creosote and turpentine, turpentine, creosote,
xylol, cedar-oil, bergamot oil, lavender oil, origanum oil, &c.

Oil of Cloves has this advantage, that it clarifies rapidly and
does not evaporate, so that sections may be left exposed to the air in
it for some time. It renders the sections very hard. It, however,
is not so satisfactory for aniline dye specimens, as it is apt to abstract
their colour. Moreover, it becomes yellow with age.

Creosote is specially useful for preparations which one does not
desire to harden in alcohol ; do not use metallic instruments.

Creosote and Turpentine. — When the fluids are mixed a cloudi-
ness appears, but this disappears on keeping. It is much cheaper
than clove-oil, but it rapidly evaporates (one part creosote to four of
turpentine).

Xylol is perhaps the best, and is specially useful with aniline
dyes. In these cases the balsam — Canada or dammar — should also
be dissolved in xylol.

Cedar- Wood Oil clarifies very slowly. It does not, however,
abstract the aniline dyes, and is used for special purposes, as indi-
cated in the context.

Origanum Oil is used for clarifying sections embedded in
celloidin.

Xylol- Aniline Oil. — Equal parts of xylol and aniline are used for
clarifying sections under certain conditions without the previous use
of alcohol. (Weigert's method, Lesson III.)

Carbolic Acid and Xylol. — A mixture of i part of carbolic acid
and 3 of xylol is used to clarify celloidin sections (p. 47). The
section can be taken from 70 per cent, alcohol, and does not require
to be further dehydrated. To remove the water from the mixture,
keep in the bottom of the bottle containing it a thick layer of
previously-heated copper sulphate.

General Remarks. — Although several essential oils are used for
clarifying purposes, it is not immaterial which one is used. Thus
clove-oil may be used for clarifying sections stained with animal
or vegetable dyes (carmine), while it is inapplicable for aniline
staining, as it dissolves aniline dyes.

In many cases the result may be obtained more gradually by
using a mixture of half alcohol and half essential oil.

Moreover, clove-oil dissolves celloidin, so that it cannot be used
when the section fixed on the slide contains either celloidin or
collodion. Oil of bergamot does not dissolve celloidin.

84

PRACTICAL HISTOLOGY.

Scheme for Staininy (Garbini).
Object.

In alcohol (70 p.c.).

Carmine.

Acid alcohol.

Alcohol (70 p.c.).

Embedding.

Alcohol

90 p.

Alcohol (absolute).

Oil of cloves.

Balsam.

Sections in celloidin.

Sections in paraffin.
Turpentine.

Absolute alcohol.

Alcohol (70 p.c.).

Carmine. Hsematoxylin. Aniline dye,

Acid alcohol. Acid alcohol.

Water. Water.

Carb. of Lithia.

Absolute alcohol.

Oil of cloves or bergamot oil.

Xylol.

Balsam,

MOUNTING FLUIDS, AND METHODS. 85

XIV.— MOUNTING FLUIDS, AND METHODS.

The fluid chosen will depend on the nature of the tissue and
other circumstances.

1. If a section is to be mounted direct from water, glycerine,
Farrant's solution, or glycerine jelly may be used.

2. If a section is to be mounted in balsam, it must have every
trace of water removed by alcohol, and the alcohol must be dis-
placed by one of the clarifying reagents — xylol, clove-oil, &c. —
already mentioned.

Glycerine. — Pure glycerine is only used for such tissues as
have been previously hardened. In the case of tissues — delicate
tissues which have not been previously hardened or fixed — the
direct application of pure glycerine would injure them. In this
case, the best way is to mount the object in normal saline, and at
one edge of the cover-glass to place a drop of a mixture of equal
parts of glycerine and water. Put the preparation in a plate
covered by a bell-jar — an extempore moist chamber. The glycerine
slowly penetrates as the water evaporates.

Some tissues are rendered too transparent by glycerine, and,
moreover, it is very difficult to seal up and keep tight glycerine
preparations.

Glycerine and Formic Acid. — This is sometimes used, especially
for picro-carmine preparations. It is made by adding formic acid
to dilute glycerine (i per cent.).

Farrant's Solution. — This is for many preparations far more
serviceable than glycerine, as it does not render some tissues so
transparent as glycerine, and the preparations can be easily sealed
up or " ringed."

Preparation (Hamilton's receipt}. — Make a saturated solution
of arsenious acid in water by boiling. After standing for twenty-
four hours filter. Take equal quantities of water, glycerine, and
arsenious water, and to the mixture add picked gum-arabic. Let
the latter dissolve until a thick syrupy fluid is obtained, which takes
about a week at an ordinary temperature;, but it must be stirred
frequently. Filter slowly through filter-paper, which must be fre-
quently changed.

Glycerine Jelly. — Melt it in hot water, place a drop on the
soction, apply a cover-glass, and gently press it down. It gelatinises
in a few minute?.

Canada Balsam. — Place some Canada balsam in a capsule or
wide-mouthed bottle near a fire or in a warm chamber (65° C.)
until it becomes hard. Let it cool. This dry balsam is to be dis-
solved in some medium. Some use chloroform, others brn/ol,
others a mixture of both, or turpentine as a solvent. In any case,

86

PRACTICAL HISTOLOGY.

FlG. 48.— Capped Bottle for
Balsam.

the solvent is added until a fairly thin fluid is obtained. Perhaps
the best solvent of all is xylol. It requires nearly twice its volume
of xylol. Filter through paper. The balsam should be kept in a
" capped " bottle (tig. 48) instead of a
stoppered one. If it gets too thick, add a
little xylol.

Dammar Lac (Klein).

Gum dammar . . i£ oz.

Gum mastic . . . ^ ,,

Turpentine . . 2 ,,

Chloroform . . 2 ,,

Dissolve the dammar in the turpentine,
and filter ; the mastic in the chloroform,
and filter. Mix the two solutions and
filter again.

There must be no moisture in the
bottles, and the mixture must be kept in
' capped " bottles, else the chloroform will
evaporate.

Xylol-Balsam. — Dry ordinary Canada
balsam in a sand-bath, to drive off all the
moisture, and until it becomes vitreous.
If it be spread out in a thin layer in a tin

vessel, this is usually accomplished in two hours or so, but the
balsam must not be overheated or change its colour and become
brown. Dissolve the dried balsam in an equal volume of xylol.
Perhaps this is the best form of balsam to use.

Balsam, when prepared, should be kept in a glass bottle with a
ground-glass cap.

To Place a Section on a Slide.— By far the most convenient
method is to place the section in a basin of water. Hold the slide
perpendicularly by the edges in the left hand, plunge the slide into
the water until it is about three-fourths immersed, and with a
mounted needle pull the section on to the slide, and at the same
moment raise the latter out of the water. The section adheres to
the glass, and if it be folded at one end, dip this end in the water,
when it floats out quite flat. Do nob attempt to spread out the
folds on the slide by means of a needle.

Hold the slide vertically to allow the water to drain off, and
remove with a rag or well-washed cloth the remainder of the water
close up to the section.

It may be stained on the slide. After the staining is complete,
remove the surplus dye by means of bibulous paper, taking care,
however, that the section itself does not adhere to the absorbent
paper.

MOUNTING FLUIDS, AND METHODS. 8/

If the section is to be mounted in glycerine or Farrant's solution,
add a drop of either of these reagents and apply a cover-glass.

// the section is to be mounted in balsam, remove as much as
possible of the surplus water or dye, as the case may be, and pour
methylated spirit upon the section. Allow it to remain on the
section for a minute or so, and drain it off at one end of the slide.
Apply fresh methylated spirit again, and finally absolute alcohol.
This is done to secure complete dehydration. The frequent and
prolonged application of strong spirit removes all the water.

Remove as much of the spirit as possible, but do not allow the
section to dry. It is now ready to be cleared up.

With a brush insinuate a drop of the clarifying reagent — clove-
oil or xylol — uwler one corner of the section, and allow the xylol to
How under the whole of the section. It will gradually diffuse into
the tissue ; and if the process be watched under the microscope
with a low power, the section will be seen to become gradually
more transparent, while the spirit will be seen os fine globules
driven out into the essential oil. The success of the process
depends on complete removal of the water by spirit, and the complete
removal of the latter by the essential oil used as the clarifying re-
agent. If any opacity remains, and it looks milky or like an
emulsion, there has been either water or spirit, or both, left in the
section.

More of the essential oil is placed on the section, so that it is
completely bathed in it and rendered quite clear by it. Pour off
the superfluous oil, remove the surplus close up to the edge of the
section, add a drop of balsam, apply a cover-glass, and the process
is complete.

In some cases it is convenient to put the drop of balsam on the
cover-glass, and then to invert this on the clarified preparation.

In all cases where it is directed to mount in balsam, this process
must be gone through, viz. —

(1.) Stain the section.

(2.) Wash it in water.

(3.) Treat the section with strong alcohol (96 per cent.) to remove
water (3-5 minutes).

(4.) Absolute alcohol (3-5 minutes).

(5.) Clarify with an essential oil to remove all the alcohol.

(C>.) If the section be not on a slide already, place it on a slide by
means of a lifter. Remove surplus oil with blotting-paper.

(7.) Add balsam, cover the section with a cover-glass.

(8.) If desired, the hardening of the balsam may be hastened by
gently wanning the preparation on a water-bath.

Sometimes it is not convenient to stain, dehydrate, and clarify a
section on a slide. In this case the sections are stained, dehydrated,

88

PRACTICAL HISTOLOGY.

and clarified in watch-glasses, the sections being transferred from
one fluid to the other, and finally to the slide by means of a " lifter "

(P. 3).

Sometimes the one method is adopted, sometimes the other.

To Clean a Microscopic Preparation. — Any excess of balsam
round the edge of a preparation may be moved with a cloth dipped
in benzol.

In the case of a preparation mounted in glycerine^ any excess of
the latter must be removed with great care, otherwise the cement
will not adhere to the glass.

With preparations mounted in Farrant's solution, leave them in
an airy dry place for ten days or longer ; this gives the medium
time to harden at the edges, and fixes the cover-glass pretty firmly
to the slide. Place the slide in a basin of water, and with a
camel's-hair brush brush away from the edge of the cover-glass
every trace of the medium. There is no fear of disturbing the
cover-glass. Lave the slide in fresh water, and then wipe it
thoroughly dry. It is better to wash a number of slides at a
time.

To cement or " Ring" the Specimens.

Balsam Preparations need not be touched. They keep perfectly
without being covered in by coating the edge of the cover-glass
with an adhesive and resistant cement. If it be desired to cement
them, a thin coating of Hollis's glue must first be applied, and after
it is dry the cement is laid on as directed for preparations mounted
in Farrant's solution.

To Ring a Slide. — The slide should be fixed on a turntable,
the centre of the circular cover-glass corresponding to the centre of
the brass disc of the table. The slide is
fixed in position by means of two brass
clips (figs. 49, a, 1>, 50).

For Farrant's preparations or (jlycerine
preparations, lay on a ring of white zinc
cement with a goafs-hair brush. The disc

FIG. 49.— Turntable for Ringing Slides.

FIG. 50. — Showing how slide
is to be centred on the
Turntable.

is made to revolve with the fore-finger of the left hand, but not too
quickly, and a coating of the cement is laid on evenly. The fore-
finger is applied to the smaller disc (<•}.

INJECTING BLOOD-VESSELS AND GLAND-TUBES. 89

The turntable should be heavy and mounted on a pin-point
centre-piece. The brushes must not be too large, and should be
washed immediately after use in the same fluid as is used to dis-
solve the cement. Thus, for zinc-white the brush is to be washed
in benzol or xylol, and for gold-size in turpentine, and for Farrant's
solution in water.

White Zinc Cement. — Dissolve 3 oz. of dammar in 3 oz. of
benzol, and add 200 grains of finely-ground oxide of zinc. Mix the
whole thoroughly, and strain through several folds of muslin. It is
perhaps more convenient to purchase the cement.

Mounting Block. — It is important that the section be placed
in the centre of the slide. As a guide for this purpose, cut a
piece of paper the size of the slide, and draw diagonal lines from
corner to corner of it ; they will intersect in the centre. Or the
piece of paper may be gummed by means of Hollis's glue between
two slides.

XV.— INJECTING BLOOD-VESSELS AND
GLAND-TUBES.

Transparent Injection Masses. — At the present time, histologists
use transparent injections, consisting of a vehicle — which may be
water, glycerine, or gelatine — and a colouring matter. Most
commonly gelatine is used as a vehicle. The colouring matter of
most red injections is carmine. In this case, the secret is to have
the mass as neutral as possible.

1. Carter's Carmine Injection.

Carmine

Strong solution of ammonia

Glacial acetic acid

Solution of gelatine (i to 6 water)

Distilled water

1 dr.

2 fl. drs.
86 mins.

2 oz.

it,.

Rub up the carmine with a little water in a mortar, add the
remainder of the water, and then add the ammonia, and stir until
the carmine is dissolved. Add the glacial acetic drop by drop,
stirring thoroughly. Add the gelatine solution, and stir briskly.

2. Ranvier's Method.— The following method yields excellent
results. Mix 2-5 grms. of pure carmine with a little distilled
water in a stoppered bottle, and add ammonia solution, drop by
drop, until the carmine, is dissolved, which occurs when the liquid
becomes transparent. Shake up the liquid to get it homogeneous.

Weigh 5 grms. of dry Paris gelatine (Coignet's), and place it in
distilled water for one hour. At the end of this time it is swollen
up and soft. Remove it from the water, wash it in water, and

9O PRACTICAL HISTOLOGY.

place it in a beaker in a water-bath. When the gelatine is dissolved
by the water which it has absorbed, add to it — stirring vigorously —
the solution of carmine, which yields an amrnoniacal solution of
carmine in gelatine.

When the carmine mixture is on the water-bath make a solution
of—

Distilled water ..... 2 parts.
Glacial acetic acid i part.

Pour the acid drop by drop into the mass, stirring thoroughly
all the time with a glass rod. The acid is to neutralise the excess
of ammonia. This requires great attention. It is by the odour
that one recognises when the fluid is neutralised. As the acid is
added the ammoniacal odour diminishes, and there is at last a
faint acid odour. This is the moment to stop adding the acid.
Towards the end of the operation it is best to dilute the acid
somewhat.

Filter the mass through new flannel.

3. Carmine Gelatine Mass (Carter'*) (Fearnley's method).

Carmine

Strong ammonia
Glacial acetic acid
Coignet's French gelatine
Water

3 grams.
6 cc.

6 „

7 grins.
80 cc.

Cut up the gelatine into small pieces and place it in 50 cc. of
the water to swell up, i.e., for four or five hours. Rub up the
carmine in a mortar with a little water and add the ammonia.
Let it stand for two hours and then pour it into a bottle, rinsing
the mortar with the remainder of the water. Place the swollen-
up gelatine, and any remaining water unabsorbed by it, on a
water-bath until it melts. To the dark purple carmine fluid add
the acid (a few drops at a time), mixing the two thoroughly, and
as soon as the fluid changes to a crimson stop adding the acid. To
the melted gelatine add the crimson carmine little by little and
keep stirring all the time.

This mass may be kept in a cool place for a long time if its
surface be covered with methylated spirit. Before using it,
dissolve it on a water-bath, and filter it through fine flannel
wrung out of hot water. The best gelatine to use is French
gelatine — Coignet's.

4. Blue Mass. — The mass is made with gelatine coloured with
soluble Prussian blue or Briicke's blue. It is very difficult to
obtain a pure sample of Briicke's blue, but this can now be had
from Dr. Griibler of Leipzig. Use a saturated watery solution of
Briicke's blue.

Weigh 5 grms. of gelatine, and treat it exactly as described

INJECTING BLOOD-VESSELS AND GLAND-TUBES. gi

for the carmine mass of Ranvier. Take 125 cc. of the blue solution
and heat it on a water-bath, and when the gelatine is fluid and
still on the water-bath, add the warm blue solution in a small
quantity at a time and stir briskly. The glass rod should show no
granules on it when it is withdrawn from the mass. Filter the
mass through new flannel. Even the best gelatine gives a precipitate
at first, but it disappears with heat.

Other injection masses are used, e.g., a watery solution of
Briicke's blue, or gelatine and silver nitrate. These are referred
to in the text.

Brass Syringe. — Many good injections have been made with a
brass syringe. ' The syringe should have a long barrel, and be
warmed by repeatedly sucking up hot water before the injecting
fluid is drawn into it. When the injection mass is forced into
the blood-vessel, the pressure should be applied steadily, and should
not be so great as to rupture the small blood-vessels.

When the blood-vessels of an animal are to be injected, deeply
narcotise an animal with chloroform, e.g., a rabbit or a rat ; make
a vertical incision through the skin from the lower part of the
neck to the ensiform cartilage, cut through the sternal cartilages,
turn up the breast-bone, and pull the sides of the thorax apart
to reveal the contents of the chest. Open the pericardium and
make a snip into the right ventricle. Tie a ligature round the
upper part of the sternum to prevent escape of the injection
through divided vessels. Wash the blood out of the chest. Snip
off the apex of the heart, whereby the cavity of the left ventricle
is opened into.

Insert a cannula into the left ventricle and push its nozzle into
the aorta. Tie it firmly into the aorta with a stout thread.
Place the animal in a bath in warm water at 40° C. If "a syringe
is to be used, by means of a pipette fill the cannula with the
injecting mass, and attach the syringe and force the mass onwards
into the blood-vessels. This is done by slow, steady pressure.
It takes fifteen or twenty minutes to make a good injection.
Any sudden increase of pressure is apt to cause rupture of blood-
vessels and consequent extravasation of the injection mass. We can
judge when a part is well injected by the colour of semi-trans-
parent parts, such as the gums or the skin. They must be deeply
coloured by the injection mass if the injection is successful.

Continuous Air Pressure. — Most frequently injections are now
made by continuous air pressure. The apparatus used should consist
of a tin trough sufficiently large to contain the animal to be injected,
and contain sufficient water to cover it. The water is kept at
40° C. by moans of a gas-burner or spirit-lamp. In the same trough
are placed the injection masses in Wolffs bottles. Each Wolff's

92 PRACTICAL HISTOLOGY.

bottle is connected to a large air-chamber into which water can flow
from the water-tap, and thus compress the air. The pressure within
this cylinder can be registered by means of a manometer. The com-
pressed air acts on the surface of the injection mass in the Wolffs
bottle, and forces it through a tube which is attached to the cannula
fixed in the aorta. The large cylinder for the compressed air may be
made of tin, or one of the large stone jars used by spirit merchants,
or a carboy may be used.

After the tissues are injected, they should be cooled rapidly by
being placed in running water. After the mass is completely set
the injected organs are cut into small pieces and hardened in
alcohol.

The methods of interstitial injection of fluids and the puncture
methods are referred to in the text.

XVI.— EXAMINATION OF FRESH TISSUES AND

FLUIDS.

In examining a fresh tissue or organ snip off a small part with
scissors and tease it in normal saline, or one of the indifferent fluids
mentioned in Chapter III. A convenient plan with some organs,
e.f/.t lymphatic gland or liver, is to make a fresh cut and scrape
the surface with the blade of knife, and then examine the scrapings.

If it be desired to study the tissue elements, it may be placed in
one of the macerating media mentioned in Chapter IV., the parti-
cular fluid selected depending, of course, on what object is sought
to be obtained.

If it be desired to render certain parts of the tissue more trans-
parent, it may be examined in glycerine.

Again acetic acid (1-2 per cent.) may be added to a fresh tissue.
It has the double action of making connective tissue swell up
and become transparent, thus making nuclei more evident, while it
also shrivels the latter somewhat. Albuminous granules are dis-
solved by it, while oil globules are not, so that it may be useful
occasionally in determining the nature of the granules in proto-
plasm. Elastic fibres are not affected by it, and thus can readily
be distinguished from the white fibres of connective tissue. Groups
of micro-cocci are also not affected by it.

The tissue may be stained by acetic fuchsin. This is made as
follows :— To a 2 per cent, solution of acetic acid add sufficient
fuchsin to give a saturated red colour (Kahlden). This reagent not
only makes the nuclei visible, but it stains them as well.

Sometimes a weak watery solution of iodine makes the outlines of

EXAMINATION OF FRESH TISSUES AND FLUIDS.

93

tissue elements more distinct. It is used in the form known as
Lugol's Solution diluted with water.

Iodine .
Potassic iodide .
Water .

part.

2
100

Weak-Alkalies (1-3 per cent.) dissolve most tissues with the
exception of elastic fibres, pigment, fat, and bacteria.

The vapour of osmic acid or the fluid itself (i per cent.) may be
used. It blackens fatty particles.

Finally, the tissue may be stained by means of a watery solution
of methyl-green, methyl-violet 5B, acetic fuchsin, or methylene-blue
in the form of Loffler's Methylene-blue.

Concentrated alcoholic solution of metliyleiie blue . 30 cc.
Caustic potash (o. 10 p. c.). . . . .100,,

Scheme for Liviny or Fresh Objects.

Harden the pieces (§ IV.).

Stain in bulk.

Embed (§ IX.).
Make sections.

Fix on slide (§ XL ).

Stain the sections (§ XII.).

Clarify sections (§ XIII.).

Mount sections.

94

PRACTICAL HISTOLOGY.

Fresh Fluids with Suspended Particles. — In the examination of
a fluid for suspended particles, e.g., bacteria, such as cells, membranes,
&c., especially if these be few in number, it is well to place it in a

Fro. 51.— Hand Centrifuge made by Muencke, Luisen-strasse, 58, Berlin, N.
It costs £3, 10s.

conical glass and allow the deposit to subside. It can then be
removed with a glass pipette.

Centrifugal Apparatus. — To collect the sediment or suspended
particles, a centrifugal apparatus is most useful. By means of it
the deposit can readily be collected at the bottom of a test-tube.

EXAMINATION OF FRESH TISSUES AND FLUIDS.

95

Fig. 5 1 shows a form of hand-centrifuge devised by Litten l and
Muencke of Berlin. The figure is reduced to ~ the natural size.
One revolution of the wheel R — its teeth fit into the thread of the
vertical axis S — causes 50 revolutions of the disc M with the 4 glass
tubes G. The wheel can readily be turned 100 times per minute,
.which gives 5000 revolutions per minute for the disc. Fig. II. shows
the disc in full rotation, and fig. III. the form of glass vessel used.

By means of this instrument, corpuscles of light specific gravity,
such as blood-corpuscles, albumen, micro-cocci, as well as crystals,
e.g. oxalates, can readily be obtained in the form of a sediment, and
it is therefore especially useful for the investigation of the deposits in
urine and exudations. It is also very useful for obtaining tubercle
bacilli from sputum.

Synoptical Statement.

Processes required for Preparing a Specimen for Microscopic Ex-
amination, e.g., the Spinal Cord of a Dog or Cat (Garbkii).

Cut the cord into pieces about 2 centimetres in length, wash them
in normal saline to remove all blood. Place them in

1. Bichromate of potash (2 p.c.)

Wash in running water

3. Alcohol (50 p.c.)

4. „ (70 p.c.)

5. ,, (90 p.c.)

6. ,, (absolute)

7. In xylol or chloroform

8. In paraffin on warm bath

9. Embed in paraffin.

10. Cut sections with microtome, and fix them on a slide

with fixative.

11. Remove paraffin by washing in toluol or turpentine, and

then in absolute alcohol to remove essential oil.

10 days
12 hours
i day
4 days
i day
i day

12 hours
6-8 hours

bb

Harde

| £

12. Place in 70 p.c. alcohol.

13. Stain in strong carmine

or hsematoxylin (5-15
minutes).

14. Wash in water, then in

70 p.c. alcohol, and de-
hydrate in absolute alco-
hol (5-10 minutes).

I i

to

fl

•5

•3

12. Place in distilled water.

13. Pour on a few drops of

the aniline dye (3-5
minutes.)

14. Wash in water and then

in absolute alcohol until
the section has the de-
sired tint.

1 Deutsch. med. Wochensch., No. 23, 1891.

96 PRACTICAL HISTOLOGY.

15. After removing surplus alcohol, pour on a few \ Dehydra-

drops of essential oil (origanum, xylol). V tion and

16. Kemove oil and mount in xylol-balsam. j mounting.

N.B. — In making a balsam preparation, the sections must always
pass through four groups of fluids.

Watery liquid — Alcohol — Essential oil — Balsam.

i «__ _ 2 <-- - 3 «— - - 4

Never take an object from one group to another of the series
without passing it through the intermediate group; it must be passed
from the ist to the 4th, and on the return from the 4th to
the ist.

PART II.

LESSON I.

MILK, GRANULES, FIBRES, AND VEGETABLE
ORGANISMS.

1. Examine the Microscope, the objectives, and the eye-pieces.

(a.) Select the objective and ocular required. For a high power
(H), if a Zeiss' microscope be used, select the objective D and the
ocular 2 ; if Hartnack's, the objective No. 7 and the eye-piece III.
See that the lenses are clean. Place the ocular in the tube, and
screw the H lens to the lower end of the tube, and leave it half an
inch above the level of the stage. For a low power (L) use No. 2
ocular of Zeiss or III. of Hartnack, and objective A or No. 3
respectively. In using a low power, the lens must be i^ inches
above the stage to begin with.

(/;.) With the microscope in front of you, with high-power lens
on it, arrange the concave side of the mirror under the stage so as
to reflect a beam of light up the tube of the microscope into the eye,
looking in at the ocular. Turn the sub-stage diaphragm until a
small aperture in it is under the aperture in the centre of the stage.
If any specks are visible on looking through the microscope, rotate
the ocular ; if they move, of course they are on the ocular itself.
Clean the outer surfaces of the lenses of the ocular with a piece of
clean wash-leather, which should be kept tied to the microscope and
used for no other purpose than cleaning the lenses. Replace the
ocular, and if specks are still present and move when the ocular is
moved, they must be on the inner surface of the eye-glass or field-
glass of the latter. This is easily determined by rotating the eye-
glass of the ocular alone, while looking through the microscope, and
observing if the specks do or do not move with it. Clean the inner
surfaces of these lenses. A general dimness indicates that the objec-
tive itself is dirty. The light used should not be direct sunlight,
but preferably light reflected from a white cloud.

10 G

98 PRACTICAL HISTOLOGY. [l.

The rule with regard to the use of the diaphragm must never be
neglected, viz., to use a small aperture with a high power, and a
large aperture with a low power.

2. Clean a Slide and Cover-Glass.

(a.) The Slide. — Seize the slide by its edges with the thumb and
forefinger of the left hand, dip one half of it into water, withdraw
it, and with a clean old handkerchief rub both wetted surfaces at
once until they are clean and dry. Reverse the slide, still holding-
it by its edges, and dip the other end in water, and clean its surfaces
as before. Lay the slide upon some clean, suitable background,
white or black paper, or on the photophore.

(/;.) The Cover-Glass. — Sometimes the covers have a thin film on
them ; this may be got rid of by placing them in strong sulphuric
acid, and subsequently removing every trace of acid by water. Dip
the cover-glass in water, take it between two folds of a handkerchief
held between the thumb and forefinger of the right hand, and rub
both surfaces at once. After it is cleaned, do not lay it flat, but
tilt it up against some convenient object.

The first lesson is devoted to the examination of a few simple
objects — some of which are occasionally found as foreign bodies in
microscopical preparations — with a view to familiarise the student
with the use of the microscope.

3. Milk. — By means of a glass rod, place on the centre of the
slide a small drop of milk diluted with three or four volumes of
water. To find the centre of the slide, use the mounting block

(P- 89).

Apply a Cover-Glass. — Seize the cover-glass by the edge by means
of a pair of forceps with broad points. The pattern shown in fig.
.6 is convenient. The edge of the cover-glass opposite to the forceps
is allowed to touch the slide close to the drop of fluid, the edge
opposite being gradually and evenly lowered by depressing the
forceps until the fluid touches the under surface of the cover-glass.
By lowering the cover-glass thus gently and obliquely the entrance
of air-bubbles is avoided. Place the object on the stage right under
the lens.

Focut tJte Object (H). — The objective is still half an inch above
the stage. While looking into the eye-piece of the microscope,
seize the tube of the latter between the thumb and adjoining fingers
of the right hand, and with a screwing or twisting movement of
the tube from left to right, gradually depress the tube until the
outlines of the object are indistinctly seen. This is the coarse
adjustment. The focussing process is facilitated by keeping the
slide and object moving slightly. This can readily be done by
moving the slide with the thumb and forefinger of the left hand,
the ulnar margin of the palm conveniently resting on the table.

I]

MILK, FIBRES, VEGETABLE ORGANISMS.

99

Now use the fine adjustment, and bring the outlines of the object
in the field sharply into view.

It is of the greatest importance that the student should be taught
to describe the objects which he sees, and also to make sketches of
them. To facilitate the description of isolated objects, the follow-
ing heads may be adopted : —

Shape.
Border.
Surfaces (upper and lower).

Size.
e. Colour.

/. Transparency and relation to light.
g. Contents.
li. Effects of reagents.

In the object under examination there is a large number of.
minute bodies floating in a fluid. Describe the appearance of the
floating particles under the following heads : —

(a.) Shape. — The milk globules (fig. 52) are spherical, as can be
shown by touching the edge of the cover-glass with a needle, and
then observing them as they
rotate in the field of the micro-
scope. Moreover, if one be
focussed, its outline comes
gradually into focus, and dis-
appears gradually, while optically
with regard to light these bodies
behave as globules, and not as
discs.

(6.) Border. — Smooth and
regular.

(-".) Surftiws. — Elevate the
objective by means of the fine
adjustment until the, upper sur-
face of the globules comes into
view ; depress it again slowly
and then examine the globule

throughout its entire thickness, until its under surface is brought
into view. Both surfaces are smooth.

(d.) tiize. — The globules are not all of the same size. If desired,
measure their actual size (p. 20).

(e.) Colour. — The smaller ones appear colourless, but some of the
larger may havQ tb<i slightest tiuge/rf, a /amkyeJlow.,..

(/) Transparent^ qiidyilaticnl &> Ifyhfttfkey* Jre transparent,
because the outline of a subjacent one cah be eeli Ih'rough a globule

FIG. 52.— Milk Globules, x 400.

IOO PRACTICAL HISTOLOGY. [l.

lying over it. Notice also the highly refractile character of each
globule, characteristic of an oil droplet (iig. 55, 3).

(g.} Contents. — They appear homogeneous and uniform, and no
included body is to be seen. Each globule is, in fact, a globule
of oil.

(h.) Effects of reagents. — To one side of the cover-glass apply a
drop of acetic acid. To the opposite edge of the cover-glass apply
the apex of a triangular piece of blotting-paper. The blotting-
paper sucks up some of the milk, and the acid runs in at the
opposite side to supply its place. This is the process of irrigation.
Move the slide to bring into focus a part of the field which has
been acted on by the acid, and note that the corpuscles, instead of
floating about singly as before, are now aggregated into small groups.
The acid seems to have altered the surfaces of the globules, so that
they adhere to each other. The acid is said to act on the casein
envelopes of the globules, and to soften or dissolve them. This
preparation is not to be preserved.

Make sketches of these objects before and after the action of
reagents.

4. Potato-Starch Granules. — With the blade of a knife gently
scrape the surface of a freshly-cut raw potato ; place the matter
so obtained in a drop of water on a slide. Remove any coarse
fragments, and apply a cover-glass. Focus the object (H).

(«.) Observe that the granules (fig. 53) -are ovoid bodies of
unequal size, not equal at the two ends, clear, and with a sharp
outline. Near the smaller end of each granule
notice a small spot, the " nucleus " or liilum, round
which are concentric layers, giving rise to the
appearance of fine concentric lines arranged with
relation to the nucleus. The lines are more
numerous on one side of the liilum than the other.
Sketch two or three of the granules.

(!>.} Irrigate the corpuscles with a diluted solu-
tion of iodine in iodide of potassium. Each
granu^e becomes blue as the iodine reaches it.
This is due to the formation of iodide of starch.
If the iodine be too strong the granules appear black.

5. Rice-Starch (H). — Examine a little rice-starch in the same
way. Notice the much smaller irregular granules. Each granule
is polygonal, mostly five or six sided. The granules are stained blue
by iodine (fig. 54). Make sketches of the starch corpuscles.

6. Gamboge and Brownian Movement (H). — Rub up a small
piece of solid .gamboge in water, until the Jatter, has a faint yellow
appearance. Place $ $*%> oft a siUfle^ cover, -ah& cxaTiiine.

(a.) Observe 'granules of 'various "sizes 'and shapes floating in the

I.]

MILK, FIBRES, VEGETABLE ORGANISMS.

101

field. The larger ones appear coloured yellow, but the finer ones
cannot be seen distinctly as yellow bodies.

(/>.) Note the finer granules, which exhibit a slow
dancing movement. They are never at rest. This is
called Brownian movement, and appears to be due to
inequalities of temperature in different strata of the
fluid. This movement is exhibited by all finely-
divided particles suspended in a fluid, e.g., China ink,
Berlin blue, provided the particles be small enough,
and the fluid in which they arc suspended be not too
viscid. The fine granules in salivary corpuscles ex-
hibit this movement. (Lesson IV. 1, d).

1. Air-Bubbles in Gum or Water (H).— Make a solution of gum

FIG. 54.— Rico-
Starch.

55-— i- Bubble of air in water (A) when the lower surface is in focus ; (I',) Middle, and
(C) Upper surface of focus. 2. Bubble of air in Canada balsam. 3. Globule of oil in
water

IO2

PRACTICAL HISTOLOGY.

tl.

mucilage and shake it up in a test-tube with air until it forms fine
bubbles. Place a drop of it with its included bubbles on a slide,
cover, and examine.

(a.) Observing larger and smaller bubbles, especially note how
the appearance of the bubble varies with the elevation or depres-
sion of the lens. Sketch these appearances. When the tube is
depressed, the bubble has a small, clear centre, and a wide, black,
sharp, refractile margin (fig. 55, A), because so much of the light
is refracted by the air, and does not pass through the bubbles of
air into the lens. Study the appearance of the bubble when the
centre and then the upper surface are in focus (B, C).

The student will have an opportunity by-and-by of observing
the appearance of a bubble of air in Canada balsam (fig. 55, 2), and
an oil globule in water (fig. 55, 3).

8. Cotton Fibres (H). — Place a few fibres of cotton-wool in
water, cover, and examine.

(a.) Observe the fine translucent flattened threads, which are
really tubes, each looking as if twisted on itself at intervals

(%. 56).

(l>.) Remove the cover-glass and
the water, add a drop of iodine ;
cover and examine ; note that the
fibres are yellowish ; add a drop of
strong sulphuric acid or a drop of the
following mixture : — Glycerine 2,
water i, and sulphuric acid 3 parts.
At the edge of the cover-glass suck
it through \vith blotting-paper, and
note that the fibres become bine.
They are composed of cellulose,
which does not give a blue with
iodine alone, but with iodine and
sulphuric acid.

9. Linen Fibres (H). -Examine

/*..

Linen. Cotter*

n

FIG. 56.— Fibres of Silk, Wool, Cotton,
and Linen.

(a.) Observe the cylindrical or
flattened translucent fibres with no
twist ; but they have a few markings on them here and there, and
at these points the fibre is generally slightly thicker. They are in
reality tubes with" thick walls (fig. 56).

10. Wool (H).-^Examine in water.

(a.) Observe the cylindrical fibres with numerous zigzag trans-
verse lines due to the epithelial covering of the fibre (fig. 56).
It is convenient to examine dyed wools also, as some of these
exhibit the zigzag imbricate scales even better tharwundyed wool.

I.]

MILK, FIBRES, VEGETABLE ORGANISMS.

103

11. Vegetable Cells forming a Membrane. — With a pair of
forceps peel off a thin layer of the covering of a fresh onion bulb.
Examine it either in. water or in water after staining with a solution
of iodine (H). Observe the cells (fig. 57) with well-defined walls,

X

Fro. 57.— Cells from bulb of a fresh onion forming a membrane. M, cell membrane ;
N, nucleus seen from the surface; Nn, nucleolus; V, vacuoles, x 240.

and united to each other by the edges to form a membrane — the
exeentrically placed spherical nucleus and the granular ccll-con-
tents.

12. Cells with sinuous margins, and containing chlorophyll
granules, may be studied in a leaf of the Duckweed or Lemna minor,
also the reaction for cellulose (sulphuric acid and then tincture of
iodine = blue) in the same plant. -

13. The currents in protoplasm are well seen (H) in CJiam ml-
'/a/v'x, which is so common in our streams.

Vegetable Micro- Organisms.— These are frequently found in
microscopic preparations, while others are the cause of various
diseases. They are usually classified as follows: —

1. Mouldy

2. Saccharomi/cetes (yeast-like organisms).

3. Schizomycetes (bacteria-like organisms).

14. Penicillium (H).— This mould is readily found on starch-
paste or on Mailer's fluid preparations which have been left stand-

IO4

PRACTICAL HISTOLOGY.

[I.

ing uncovered. Place a little on a slide, add a drop of water, cover,
and examine.

(a.) Observe the stems, consisting of narrow, clear, oblong cells,
joined end to end, and on the summit of each are several
rows of small spores. The rootlets or mycelium consist
of elongated chains of narrow oblong cells.

The mycelium is composed of much-branched liyphce.
The cells, elongated and narrow, composing these are
separated from each other by numerous partition walls
(fig. 60, e). The walls consist of cellulose and contain
protoplasm with vacuoles.

(b.) Search for one of the hyphas which bear on
their free ends a brush-like group of cells, some of
Gonkiiophore wnicn become constricted to form chains of spherical
bearing stylo- dylogonidia (fig. 58). The ripe stylogonidia have a
onTh^Hyphre green colour, and they give the mould its green
? i m 6 " * C * J appearan ce.

15. Yeast (H). — Place some German yeast in sugar
and water ; keep it for several hours in a warm place. Examine a
drop of the fluid.

(a.) Observe the small oval yeast-cells, each with an envelope, a

large clear vacuole, and granular proto-
plasm.

(6.) Search for one budding, and
notice the small faintly granular bud
adhering to and projecting from the
mother-cell (figs. 59, 60, /).

Each yeast plant is a single cell or
It is composed of a transparent thin delicate
envelope or cell-wall, composed of cellulose. When the cell is

ruptured the empty cell-envelope may
be seen in the field. Within is
granular protoplasm, containing, as a
rule, a clear space or vacuole. Some
W ^f7 '"*$>§& cells contain a nucleus.

^ $*K*&^*& °'°$£ (c.) Stain the preparation with a

watery solution of magenta. Note
that all the cells do not stain equally
well. The buds on the side are stained
of a deep red, a^nd to a less extent the

Yeast-Cells; g. Bacilli and Micro- protoplasm of most of the Cells.

16. Micrococci and Bacteria ( H).—

Set aside a solution of peptone or a watery extract of a piece of flesh
for some time, until a scum forms on the surface due to putrefaction.
Place a little of the scum on a slide, cover, and examine.

FIG. 59.— Yeast-Cells, a. b and c
Budding.

morphological unit.

I.] MILK, FIBRES, VEGETABLE ORGANISMS. 10$

(a.) Observe groups of small round specks, often held together
by a homogeneous medium. These are micrococci (fig. 60, d).

(&.) Small elongated rod-like bodies, each moving across the field
in a zigzag like manner. These are bacteria (fig. 60, g).

N.B. — In all cases sketches must be made of the objects examined.
This holds good for this and all suwading lessons.

17. Determine the magnifying power of the microscope.— Do
this according to the method described at p. 1 9.

ADDITIONAL EXERCISES.

18. Staining of Fission Fungi or Schizomycetes. — They stain readily with
aniline dyes, especially basic aniline colours. Scrape off a little of the coating
which accumulates on the surface of the molar teeth, press it between two cover-
glasses, so as to make cover-glass preparations. Glide the one glass off the
other. Place the cover-glasses, film surface downwards, in a I per cent, solu-
tion of methyl-violet, gentian-violet, or methylene-blue contained in a watch-
glass. Heat the watch glass and its contents over a gas-flame until a faint
cloud of vapour rises ; allow it to cool, and in 5-10 minutes or so the colora-
tion is complete. Remove the cover-glasses, wash them in water, and place
them in absolute alcohol. Remove the cover-glasses and allow them to dry in
the air and mount in xylol balsam. A moist cover-glass preparation from
absolute alcohol may be clarified with xylol.

(«.) Observe many epithelial cells coloured, but so are the organisms.

If it is desired to have only the organisms coloured, the stained cover-glasses
are taken direct from .he staining solution, or laved in absolute alcohol and
placed in a solution of iodine composed of i part iodine, 2 parts potassic iodide,
and 300 water, in which they are kept for a few minutes. Transfer them to
and wash them in absolute alcohol until nearly all colour is gone, and clarify
with xylol. Only the organisms are of a dark-blue tint ; the other tissues are
decolorised or nearly so. This is Gram's method.

Stain bacteria from a putrefying proteid fluid, e.g., peptones 01 meat-
extract, in the- same way.

11

IO6 PRACTICAL HISTOLOGY. [ll.

LESSON II.
THE BLOOD.

UNDER the microscope blood is seen to consist of a clear, transparent
fluid, the plasma or liquor sanguinis, in which are suspended the
blood-corpuscles. The blood-corpuscles are of two kinds, the red
or coloured, and the white, pale, or colourless. Besides these,
i-here fall to be examined the blood-plates, or, as they are also
sailed, blood-tablets or platelets.

( Blood-plasma.
Blood \ ( Red.

I Corpuscles < White.

( Platelets.

BLOOD-CORPUSCLES OF AMPHIBIANS.

(A.) Coloured Corpuscles of Amphibians (Newt or Frog). — After
destroying the brain of a newt or frog, a drop of blood may be
obtained from the cut end of the tail of the former, or from the cut
surface after amputation of the foot of the latter.

1. Bed Corpuscles (H). — Place a small drop of blood in the
centre of a perfectly clean slide and cover it at once with a cover-
glass. Examine it with a high power. To avoid the pressure of
the cover-glass, a short length of a hair may be placed in the blood
droplet before the cover-glass is applied.

(a.) Observe the coloured and colourless corpuscles, the former
much more numerous than the latter (fig. 61).

(b.) Study the red corpuscles. Observe that they are very numer-
ous, elliptical in outline when seen on the flat, slightly yellowish
in colour ; their border or contour is. even and well denned. Select
one seen on edge, and note that it is a thin ellipse, pointed at the
ends, becoming gradually thicker in the centre, so that it is a bi-convex
elliptical disc. Sometimes one corpuscle can be seen overlying a
subjacent one, in which case the outline of the latter can be dis-
tinctly seen through the former, indicating that the corpuscles are
transparent (fig. 61). Notice within each corpuscle a lighter oval,
central area, indicating the existence of an elliptical, colourless,

II.]

THE BLOOD.

107

elongated, granular-looking included body — the nucleus. The long

axis of the colourless nucleus coincides with the long axis of the

corpuscle. At first, the nucleus

may not be very distinct, but

after a time it becomes distinctly

visible, and it can be readily

made so by the action of certain

reagents, especially weak acids.

Small vacuoles frequently appear

in the body of the corpuscle, more

especially in frogs that have been

kept some time. In others there

may be seen a faint radiate

striping of the corpuscles as if

there were fine folds in it due to

partial drying.

* The yellow colour is due to the

presence of haemoglobin, which

is enclosed within, the meshes of

a colourless stroma or delicate

framework.

If the blood be taken from a
frog which has been kept through-
out the winter, vacuoles may be
seen in the hemoglobin of the red corpuscles (fig. 61, v). They are
rapidly produced in frogs after the injection of ammonium chloride.

Sketch two or three corpuscles, both red and white ; the red cor-
puscles both on the flat and on edge, and two
overlapping each other.

2. Acetic Acid. — To the edge of the
cover-glass of the same preparation apply a
droj) of dilute acetic acid (i per cent. — i cc.
glacial acetic acid to 99 cc. of normal saline
solution). The acid runs in under the cover-
glass, but if it does not, apply a small tri-
angular piece of blotting-paper to the opposite
edge of the cover-glass. Let one of the angles
of the blotting-paper touch the fluid under
the cover-glass, and it will suck up some of
the blood, and thus cause the acetic acid to
run in at the opposite side. This is known
as the process of irrigation. Move the slide, and find a part of the
object which lias been acted on by the dilute acid.

(a.) Observe that the red corpuscles, or at least most of them,
become spherical and decolorised, the nucleus becomes very distinct

FIG. 61.— Blood of Frog. a. Red corpuscle
seen on the flat ; b. In Profile ; c. Three-
quarter face ; some of the red corpuscles
show the presence of vacuoles (v) ; n.
Colourless corpuscle at rest; m. One
with amoeboid processes ; p. Fusiform
cell, probably from the vascular wall.

FIG. 62. — Amphibian Coloured
Blood-Corpuscles seen on
the Flat and on Edge,

X 1000.

1 08

PRACTICAL HISTOLOGY.

[n.

and granular, while the outline of the corpuscle may become very
indistinct (fig. 63). The nucleus appears somewhat shrunken or
shrivelled. If the corpuscles are roughly treated, the nucleus may
be seen placed excentrically, or even extruded from the corpuscle.
The acid acts on the haemoglobin, forming a new compound, which
diffuses out of the corpuscles and stains the surrounding plasma a
faint yellow. The nucleus in some of the corpuscles may absorb
some of the yellow pigment, and become stained thereby, especially
if a strong solution of acid has been used. The action on the
colourless corpuscles is referred to at p. 112.

3. Dilute Hydrochloric Acid, i per cent. (H).

(«.) To a fresh drop of blood add, as before, a drop or two of
dilute hydrochloric acid. Watch diligently one or two of the red
corpuscles. They gradually enlarge, become spherical, and may all
of a sudden burst and discharge their contents, the nucleus coming
clearly into view during the process. After the rupture, the residue
of the stroma of the corpuscles may be seen in the field. In other
cases the corpuscles become clear, globular, and transparent, with

FIG. 63.— Frog's Red
Blood - Corpuscle
acted on by Dilute
A.cetic Acid, x 300.

FIG. 64. — Action of
Water on an Am-
phibian Coloured
Blood-Corpuscle.

FIG. 65. —Action of
Syrup on Frog's
Red Blood - Cor-
puscle, x 300.

here and there fine shreds stretching between the nucleus and the
surface of the spherical corpuscle.

4. Water (H). — To a fresh preparation of blood apply a drop of
water to the edge of the cover-glass, and notice its effects upon the
corpuscles.

(a.) The water rapidly diffuses into the corpuscles and renders
them spherical, while at the same time it decolorises them, the
haemoglobin diffusing outwards into the plasma, and staining it
slightly yellow. The nucleus also becomes spherical. Thus the
outline of the corpuscles becomes very faint in the field of the
microscope, the corpuscles themselves now almost consisting of a
nucleated stroma (fig. 64).

5. Strong Syrup (H). — Place a small drop of blood on a slide
and near it a drop of syrup ; mix the two with a needle, and apply
a cover-glass.

(a.) Observe that some of the red corpuscles are rapidly
shrivelled and puckered, especially when seen on edge, owing to
fluid passing out of them by exosmosis (fig. 65). Some of them

II.] THE BLOOD. log

may present here and there a reddish tinge. All tire corpuscles
are not affected equally or at the same time. The same effects ar&
produced by strong saline solutions.

6. Tannic Acid (H). — On a slide mix a drop of blood with a
drop of tannic acid, using a relatively large drop of the acid fluid ;
wait for about a minute, and then apply a cover-glass.

(a.) Observe that some of the corpuscles become globular, while
the haemoglobin passes out of the corpuscles at one or more spots,
and appears on the surface in the form of one or more small
granular buds (fig. 66, c). Sometimes the bud or buds are small.
At others the bud may be as large as the remainder of the corpuscle,
which has become smaller and partly decolorised. In other cases
it may be collected around the nucleus (Robert*). The tannic acid
causes a separation of the haemoglobin from the stroma.

The solution of tannic acid is made by dissolving 2 grains of
tannic acid in i oz. of boiling water, and allowing it to cool.

FlQ. 66.— Actio-i of Tannic Acid

FIG- 67.-Action of Boracic Acid on a Frog's
Red Blood-Corpuscle.

7. Boracic Acid. — Mix a drop of newt's or salamander's blood
with a 2 per cent, solution of boracic acid, which takes a short
time to act on the corpuscles and produce its effects.

(a.) Observe that the haemoglobin is collected around the nucleus,
so that the presence of the latter is obscured, but in many of the
corpuscles fine threads of haemoglobin remain attached to the
circumference of the corpuscle, so that the retracted haemoglobin
may have a stellate form, while the rest of the corpuscle is colour-
less. To the latter Briicke gave the name Oikoid, to the former
Zooid (fig. 67).

If these corpuscles are acted on for twenty-four hours with i per
cent, osmic acid, they are " fixed," and may be mounted permanently
in glycerine-jelly.

8. Magenta. — Mix a drop of blood and a drop of the special
magenta fluid (p. 74) on a slide, cover, and examine.

(a.) Observe that the nuclei of the corpuscles, both red and
white, are stained of a brilliant red, although the surrounding part
of the corpuscle is not so stained, unless the magenta be in great
excess. All the corpuscles are not stained equally brightly. On
the edge of some of the corpuscles at one or more points will be
found small coloured spots or thickenings.

HO PRACTICAL HISTOLOGY. [ll.

9. Osmic- Acid and Picrocarmine (H). — This preparation is
best made by mixing a few drops of blood with an equal volume of
i per cent, osmic acid in a small tightly-corked tube, or by exposing
a thin film of blood to the vapour of a 2 per cent, solution of osmic
acid. After two to four hours pour off the supernatant fluid and
cover the residue of corpuscles with picrocarmine. After twenty-
four hours the picrocarmine can be poured off, and a little of the
deposit placed on a slide and mixed with glycerine-jelly dissolved
by heat, and covered (p. 85).

(a.) Observe that the nuclei of the corpuscles are bright-red, and
the perinuclear part of the corpuscles yellow. Within the granular-
looking nucleus, with a. good lens, may be seen a network of fibrils.

This preparation is permanent, and must be sealed up or " ringed "
after the manner described at p. 88.

10. Blood of Bird (H).— Mount a drop of fresh blood.

(a.) Observe the red corpuscles, which are elliptical, biconvex,
nucleated bodies, but smaller in size and more ^pointed than
amphibian corpuscles (fig. 68).

These corpuscles behave towards reagents as those of amphibians.

11. Blood of Fish (H). — Mount a drop of fresh blood, which is
readily obtained from a gold-fish or salmon, but the blood coagulates
rapidly.

J2.) Observe the red corpuscles, which are elliptical, biconvex,
nucleated, but the ends are not so pointed as in the bird, while,

like the bird's corpuscles,
they are smaller than
those of amphibians (fig.
68, F). The blood must
be quite fresh.

In both cases a few
colourless corpuscles may
be noticed.

FIG. 68.-Red Blood-Corpuscles of Fish (F) and (B.-) Tke White C°r~

Bird (B), x 45o. puscles of the Frog or

Newt.

12. White Corpuscles. — In a fresh preparation of blood (taking
care to place a hair under the cover-glass) search for the colourless
corpuscles, of which there are several varieties. They are much
less numerous than the red.

(a.) Observe that there is: (i.) The finely granular form, con-
sisting of a nucleated mass of protoplasm larger than a red corpuscle
(fig. 6 1, m). (ii.) The coarsely granular variety may be found. In
it the granules are large and refractive, and often lying at one side
of the corpuscles, (iii.) A third variety, much smaller than the
others, may be found (iig. 61, k).

II.]

THE BLOOD.

Ill

(b.) In all the varieties, by careful observation, may be detected
a nucleus, which in some is irregular or subdivided. The surface of
these corpuscles is sticky, as can readily be shown by giving the
cover a push with a needle, when the coloured corpuscles will be
seen to glide over each other, while some of the colourless ones will
be seen adhering to the glass ; and even if a coloured one impinges
on them, they are rarely displaced by the impact, so firmly do they
adhere to the glass.

13. Amoeboid Movements of the White Corpuscles. — Select
one of the large finely granular corpuscles, and at once make a
sketch of its outline ;
in a minute or two
make another sketch,
and do this every few
minutes. Thesketches
will vary, because the
corpuscle has slowly
changed its shape, even at the ordinary temperature. A process of
its protoplasm may bo extruded on one side, while a part of the
corpuscle may be drawn in at another. The corpuscle, therefore,
exhibits spontaneous irregular and indefinite movements, constitut-
ing what is known as amoeboid movements. It may even be seen to
change its place, and thus exhibit locomotion.

In the coarsely (jranular form, if one be found, the processes are
not so pointed, while the granules may be seen to pass suddenly

FIG. 69.— Showing thn Amreboid Movements of
Colourless lllood Corpuscles.

FIG. 70. — Ranvier's Moist Chamber. .<?. Disc on which the fluid to be examined is
placed ; d. Air-Chamber ; c. should be placed on the square part on which the
cover-glass rests.

from one side of the corpuscle to the other. The granules seem to
be passive, and their motion is due to movements of the protoplasm.
These movements may be watched for a long time if the pre-
paration be sealed up either with melted paraffin wax or with oil.
The former is to be preferred. When scaled up in paraffin wax, the
preparation may be kept without evaporation for several days. The
margins of the cover-glass — preferably a square one — are sealed down

112 PRACTICAL HISTOLOGY. [ll.

with paraffin wax by heating a wire bent at right angles to melt
the paraffin and applying it along the edges of the cover-glass.

Ranvier uses a moist air-chamber (fig. 70). In the centre of the
chamber is a small piece of glass, less in height by -^th of a milli-
metre than the height of the walls of the cavity. The fluid con-
taining the corpuscles is placed on this, covered with a cover-glass
which is sealed down by means of paraffin wax. The object is thus
protected from evaporation, while it has in relation to it a layer
of air.

14. Acetic Acid (i per cent.). — On irrigating a fresh preparation
with dilute acetic acid, the protoplasm is made clear and transparent,

and the complex nucleus —
usually consisting of three
parts, and hence called tri-
partite — is distinctly re-
vealed (fig. 71, a). If the
acid act vigorously, it may

FlQ. 7i.-a. Action of Acetic Acid on the Colourless ^e almost impossible to SCO

Blood-Corpuscles of a Frog. Action of Water the Outline of the HOW clear
on the Colourless Blood-Corpuscles of a Frog. i

6. Early, and c. Later Stages. protoplasm.

15. Water.— On irrigat-
ing a fresh preparation with water, the colourless corpuscles are
killed, and they assume a globular form, the protoplasm becoming
at first more granular (fig. 71, ft), and subsequently clear and trans-
parent, thus distinctly revealing the presence of the nucleus. The
granules may exhibit Brownian-movements, while each cell has an
outline round it as if a membrane had been formed round it. So
that by the addition of water these corpuscles may be changed so as
to resemble salivary corpuscles (Lesson IV.).

16. Magenta. — This stains the nucleus deeply and the proto-
plasm to a less degree.

For Fibrin (Lesson III.

Lymph.

17. (a.) Pith a frog, and with a fine pipette withdraw a little
lymph from the dorsal lymph sac, or, better still, curarise a pithed
frog, and next day remove the lymph from its sub-lingual lymph
sac. The lymph accumulates there in large amount.

Lymph (H).

(a.) Observe numerous leucocytes, mixed perhaps with a few red
blood-corpuscles, exactly like the three varieties of leucocytes found
in the blood. A coagulum of fibrin will ultimately be formed.

(ft.) The toad yields a very large quantity of lymph. Destroy
its brain, wipe one leg dry, and cut off the projecting toe. At once
large drops of lymph flow out, which soon coagulates (S. Mayer).

Il] THE BLOOD. U3

ADDITIONAL EXERCISES.

18. Migration of Colourless Corpuscles ( H ). —Heat and draw out a glass
tube so as to form an excessively fine capillary tube. Pith a frog, expose its
heart, make a cut into the latter, and as the blood flows suck up blood into the
capillary tube. Seal the tube at both ends by holding it for a second or two
in a gas- flame.

Place the tube on a slide in a drop of glycerine or clove-oil, rover it and
examine.

(a-) (L) Observe that the blood-clot shrinks, squeezes out serum from the
clot, and by-and-by some of the colourless corpuscles migrate from or are
squee/ed out of the red clot into the serum, where they may be seen exhibiting
amreboid movements. This shows that the vitality of the white corpuscles is
not abolished bv the coagulation of the blood.

19. Feeding White Corpuscles ( H ). — Get some blood from the~heart of a
frog into a capillary pipette, as described in 18. Allow the blood to clot and
exude serum, which it does in about an hour.

liiib up a little Indian -ink in a few drops of normal saline in a watch-glass
till a greyish fluid is obtained. Blow the contents of the capillary tube upon
a clean slide, remove the clot ; the serum contains numerous colourless cor-
puscles. Mix a little of the greyish Indian-ink fluid with the serum, put
a hair under the cover-glass, and seal up the latter with melted paraHin to
prevent evaporation. After a time, observe that the minute particles of
Indian-ink are seen included in the protoplasm of these cells. The cells throw
out processes which surround a particle, meet and coalesce, and thus the
particles come to be included in the corpuscle (Schcifer).

20. Movements and Division of White Corpuscles. — Ranvier1 seals up the
preparation on the slide devised by him, this being done by paraffin wax. A
water immersion lens is placed on the microscope, and then the microscope
with the slide is placed in a glass vessel filled with water at the required
temperature. In this way the movements of the corpuscles, and, as I have
myself seen, even the division of leucocytes, can be readily studied.

21. Glycogen in White Corpuscles (H).— Irrigate a preparation of frog's
or newt's blood with I per cent, solution of iodine containing 2 grams of
potassic iodide. The red corpuscles are stained yellow, the white ones arc
killed ; many of them are also stained yellow, but' in some of them may be
seen mahogany-coloured granules of stained glycogen.

22. Elder Pith Preparation (H). — Into the dorsal sac of a pithed frog
introduce a small piece of elder pith soaked in normal saline, and leave it
there for twenty- four hours. Withdraw it, make a thin section, and examine
it in normal saline solution.

(a. ) Observe the large polygonal cells of the elder pith crowded with lymph
corpuscles — several varieties — which have, in virtue of their amoeboid move-
ments, " wandered" into and permeated the cellular pith.

The elder pith may be hardened in Flemming's fluid (p. 32), and after
thoroughly washing it, sections are made and mounted in Farrant's solution.

23. Serous Fluids of Mammals. — A small mammal, e.g., mouse, rabbit, is
killed by decapitation. Open its peritoneal cavity by means of a sharp red-hot
knife, i.e., by a therm o-cautery. This is to avoid any blood being shed into
the cavity. By means of a fine pipette withdraw some of the "lymph" or
" serosity." It will be found not to be clear like whey, but slightly opalescent.

(a.) Corpuscles (H). — Some are like ordinary lymph corpuscles, many are
ve'-y granular, and there are always red blood-corpuscles also present (llanvier).z

1 Comptcs llendus, vol. 110, p. 686, 1890.

2 Ibid. vol. 110, p. 768, 1890.

H

114 PRACTICAL HISTOLOGY. [ll.

24. Dry Cover-Glass Preparation of Blood.— Place a drop of frog's blood
on a cover-glass, and to it apply another cover-glass. Press the two glasses
together, and then slip them asunder. There will be a thin film of blood on
both glasses. Allow the films to dry.

In a watch-glass place a dilute watery solution of methyl-green, and on this
float the cover-glasses, with the blood -surface next the staining reagent, as in
the method of staining bacteria.

After ten minutes remove the cover-glass ; move it in water ; touch the
edge of it on blotting-paper to remove the surplus green fluid, and allow the
green stain on the glass to dry. After it is dry, add a drop of xylol balsam
and place the cover-glass on a slide. This forms a permanent preparation of
blood-corpuscles, whose nuclei are stained green.

The same process may be practised with other aniline dyes, or eosin may be
combined with methyl-green, as the former stains the haemoglobin, and the
latter the nucleus. A very good stain is eosin-haematoxylin (p. 70).

25. Double-Staining of Blood-Corpuscles (Methyl-green and Eosin}. — Diffuse
a thin layer of frog's blood on a cover-glass, allow it to dry, and pour on
the dry residue a I per cent, watery solution of methyl-green. Leave it on
for ten minutes, and then wash off the surplus stain. Pour on a weak watery
solution of eosin, and after five minutes wash it off also by rinsing the slide
gently in water ; dry, and add xylol balsam ; cover.

Observe that some of the corpuscles have the nucleus green and the sur-
rounding part coppery-red. Eosin is almost a specific reagent for detecting
haemoglobin.

Numerous combinations of this kind may be made, such as magenta and
iodine-green, fuchsin and methylene-blue.

26. A better plan is to place a drop of blood on a cover-glass, and to this
apply another cover-glass, press the glasses together, and then separate them
so that a thin film of blood adheres to each. Allow them to dry. After they
are dry, place them for two hours in a mixture of equal parts of absolute
alcohol and ether, which coagulates the proteids of the corpuscles. Float
the cover-glasses blood-surface downwards upon a saturated solution of methy-
lene-blue. After an hour or more, wash the cover-glasses in water, then in
absolute alcohol, and clear them up with clove-oil in which a little eosin is
dissolved. Remove the clove-oil by immersing the cover-glasses in xylol, and
mount in xylol balsam. A beautiful preparation is obtained. The nuclei are
blue, and the protoplasm of the colourless corpuscles of pale-rose colour.

27. The advanced student should also study the effect of sulpho-cyanide
of potassium (5 per cent.), urea, ammonium chromate, pyrogallic acid, heat,
carbolic acid (i to 1000 of normal saline), and other agents on the coloured
corpuscles. Dilute alcohol reveals a nucleolus within the nucleus. This
preparation may be subsequently stained with magenta.

28. Urea. — A strong watery solution causes the corpuscles to assume
irregular forms and to send out processes, which may separate from the
corpuscle. Changes not unlike these are produced by neutral ammonium
chromate, very bizarre forms being thus produced.

29. Bile, e*g., of mammal or frog, dissolves human red blood-corpuscles.

30. Zinc Sulphate (.25-. 5 per cent.) causes the hremoglobin to separate
from the stroma. One may see it inside the corpuscle, or on the surface as
small buds.

31. Pseudo-Membrane of the Corpuscles. — Irrigate a preparation with
dilute alcohol. This decolorises the corpuscles. Then stain with magenta
or Spiller's purple. The nucleus and so-called membrane of the corpuscles are
thereby stained.

32. Preservation of Blood-Corpuscles by Hayem's Fluid (p. 122). — This is
an excellent method. The corpuscles retain their form, and can be subsequently

III.] HtJMAN BLOOD. * I <

Bi/ii:;ed, e.g., by picro-cannine, and keep well when mounted in glycerine
jelly.

33. Leucocytes of Crayfish Blood or Haemolymph. —The colourless blood of
the crayfish does very well for the study of colourless corpuscles. Make a slit
in the ventral surface of the abdomen between the rinjrs and the blood flows
freely. Receive it in normal saline solution. It clots quickly. Mount some on
a slide, and if it be desired to fix the corpuscles, allow a jet of steam to play on
the cover-glass for a few seconds. If the fluid contain too much blood there is
so much coagulable proteid, that on its coagulation by the steam a white film
obscuring the leucocytes is formed. The corpuscles can be subsequently stained
with picro-carmine and mounted in glycerine. The blood contains two forms
of corpuscles — one with well-marked amoeboid movements and provided with a
large spherical nucleus ; the other filled with highly refractile granules, which
stain red with picro-carmine.

The best method is to allow a drop of blood to fall into a large drop of I
per cent, osmic acid previously placed on a slide. This at once kills and
" fixes " the corpuscles, which can then be stained with picro-carnaine, or I per
cent, watery solution of eosin. The latter stains their protoplasma and its
expansions a rose-pink.

LESSON III.

HUMAN BLOOD— CRYSTALS PROM BLOOD-
BLOOD PLATELETS.

WRAP a twisted handkerchief round the ring-finger of the left hand,
and begin at the base of the finger, gradually constricting the
finger from the base towards the nail. The end phalanx will there-
by become greatly congested. With a sharp clean sewing-needle
prick the skin at the root of the nail ; a drop of blood will exude,
to which rapidly apply a slide ; cover the drop of blood on the slide
with a cover-glass, and examine it as quickly as possible.

Observe various kinds of corpuscles floating in a fluid, the
blood-plasma or liquor sanguinis. Note the red and white cor-
puscles, the former being far more numerous. Blood platelets are
also present ; but it requires special precautions in order to preserve
them.

1. Human Coloured Blood-Corpuscles (H). — (a.) Observe the
field of the microscope crowded with the red or coloured disc-shaped
corpuscles much .smaller than those of the newt ; they are only
•T-Vo^1 °f an m(-h or 7-7 /* (l-2 p-7'% A1) i11 breadth (p. 21), and
, ., ('MMT of an inch or 2 /A in thickness. The observer may notice
that when the corpuscles (-ease to move, after a time corpuscles may
be seen adhering to each other by their flat surfaces, until a chain

PRACTICAL HISTOLOGY.

[ill.

Fro. 72. — Human Red and White Blood-Corpuscles.
a. Ued corpuscle seen on the flat ; b. In profile ;
c. A rouleau; d. Three-quarter face; e,f. Crenated
corpuscles ; g. Spherical ; in. Slightly crenated ;

of these bodies resembling a pile of coins is produced (fig. 72).

This is the so-called formation of rouleaux (fig. 72, c). In order to

obtain rouleaux a fairly
large drop of blood must
betaken. These chains by -
and-by increase in number
and intersect each other, so
as to produce a network -like
appearance in the field. In
the rouleaux all the cor-
puscles are seen edgeways.

(b.) Move the preparation
until a part of it is found
where the red corpuscles
are to be seen not in

L. Large wliite "corpuscle ; I. Small" white cor- rouleaux, but isolated and
puscle; p. Granular leucocyte; n. Free granula- , . ,-, a ,

tions, x looo. lying on the flat.

Study a Single Red Cor-
puscle.— Observe its shape ; when seen on the flat it is circular in
outline, and on bringing its edge sharply into focus, a darker area
is seen in its centre (fig. 74, a). If the fine
adjustment be used, so as to bring the lens
nearer the corpuscle, the dark centre is replaced
by a lighter area, while the rim becomes darker
(fig. 74, b).

Sketch the two appearances.
This is due to the fact that these bodies
are In-concave circular discs. The dark area
in the centre is not due to the presence of a
nucleus, as -they are non-nucleated.

This is confirmed by examining a corpuscle
seen on edge, when it appears somewhat dumb-
bell shaped (figs. 72, b, 73, 2).

In size they are much smaller than those of Amphibia, being only
of an inch (7.7 /x) in their greatest diameter. Their border
is smooth, rounded, and regular, their colour is
pale straw-yellow, their surfaces are smooth, and
they are homogeneous throughout. As to trans-
parency, the outline of one corpuscle can be seen
through one overlapping it. They are soft and
flexible, so that if they impinge on other objects
they may change their form, which they rapidly
regain, so that they are elastic.

(c.) Measure the actual size of several corpusekfj
by the method described at p. 20. It will be found that all tho

FIG. 73.— Red Blood-Cor-
puscles. Human ; i.
Seen on their surface ;
2. Seen edgeways ; 3.
United into a rouleau.

FlG. 74.— Red Cor-
puscle seen on the
flat. a. On rais-
ing, and b. On
depressing the ob-
jective, x 1000.

III.] HUMAN BLOOD. I I/

corpuscles are not of absolutely the same size. This variation in
size becomes more marked in some diseases.

2. Colourless Corpuscles. — With a little care in observing,
especially •when the red corpuscles are in rouleaux, here and there in
the field a few colourless corpuscles will be found. They remain
isolated and do not form groups. They are few in number, only
three to ten being found in one field. The proportion is about 3 to
10 per 1000 of red. They are usually spherical, and although they
exhibit amoeboid movements at 40° C., at the ordinary temperature
they do not do so, and appear as nucleated finely granular masses of
protoplasm. They rapidly alter after they are shed. Some of them
are larger (o-jVo" inch or 10 /x, in diameter), others smaller than the
red corpuscles (fig. 72, L, I).

Move the cover-glass so as to cause a current in the preparation.
Note that the colourless corpuscles adhere to the glass, — they are
sticky and adhesive, — while the coloured ones being smooth, and
polished, glide over each other, and frequently impinge on the
colourless ones without displacing the latter.

3. Blood-Plates (see p. 123).

4. Acetic Acid, Tannic Acid (fig. 66, a, b), Water, Syrup, and
Magenta act in great part in the same manner on the coloured and
colourless corpuscles of man and mammalia as on the corpuscles of
Amphibia. The differences are due to structural differences in the
corpuscles.

Thus in the coloured mammalian corpuscles acetic acid de-
colorises them and renders them spherical (but of course no nucleus
is revealed), leaving a hull or stroma, almost invisible in the field.
Syrup shrivels them, magenta reveals no nucleus, but on the side
of some of the corpuscles a little spot may be observed. Bile makes
them pale, and finally dissolves them. Water decolorises the red
corpuscles, and makes the blood "laky. "

5. Amoeboid Movements of White Blood-Corpuscles. — In order
to study these movements in the white blood-corpuscles of man and
warm-blooded animals generally, the temperature of the blood must
be near the temperature of the body. To keep the preparation
warm, some form of warm or hot stage is necessary.

For the purposes of the student the following simple contrivance
is sufficient. Take an oblong copper plate, 76 mm. long, 25 mm.
broad, and 1.5 mm. thick, with a rod 100 mm. long projecting from
one side of it, as shown in fig. 75. In the centre of the oblong
plate of copper is a hole 15 mm. in diameter. Fix the oblong plate
to an ordinary glass slide by means of sealing-wax.

Make a preparation of blood. Take a cover-glass i inch square,
and on it place a drop of normal saline .solution, and to the latter
add a drop of blood. Mix them. Apply a ^-iiich cover-glass, so as

118

PRACTICAL HISTOLOGY.

[in.

to have a layer of diluted blood in a thin film between the two
cover-glasses, and exactly filling the space between them. Any
surplus fluid at the edge of the cover-
glasses must be removed by blotting-paper.
With a camel's-hair pencil then run a layer
of oil round the edge of the smaller cover-
glass, or the preparation may be sealed up
with melted paraffin wax. This will pre-
vent evaporation. The larger cover-glass
acts the part of a slide.

Fix the warm stage on the stage of the
microscope by means of the clips on the
microscope, and over the aperture in the
warm stage place the cover-glasses. Focus
the corpuscle between the two cover-
glasses, and notice that the colourless
ones do not exhibit amoeboid move-
ments.

Heat the projecting rod by means of a
spirit-lamp. The heat travels along the
copper, and finally warms the cover-glasses
and the layer of blood between them. It
must, however, not be overheated. In
order to prevent overheating, make pre-
viously a mixture of cacao-butter and
white wax, which melts at 38° C., and
place a fragment of this on the copper
stem near the copper plate. Heat should
be applied until the wax just begins to melt.

Observe the colourless corpuscles when heated beginning to

FIG. 75.- Simple Copper Warm
Stage.

FIG. 76.— Warm Stage made by Reichert of Vienna. It costs £i. A, A'. Screws to fix it
to the stage of a microscope ; B. Inflow, and 15'. Outflow of water.

exhibit amoeboid movement. Sketch a corpuscle, and in a few
minutes make another sketch and compare the two.

III.] HUMAN BLOOD. 119

In the more expensive forms of warm stages, as those of M.
Schult/e and others, warm water at a known temperature is passed
through a brass box which rests on the stage of the microscope, and
the exact temperature is determined by means of a delicate thermo-
meter. Fig. 76 shows a convenient form of hot stage, which can be
clamped to the stage of any microscope. It is heated by means of
warm water which passes in at B, and, after traversing a system of
tubes, out at B'. It is provided with a thermometer.

6. Crenation of Coloured Corpuscles (H). — Mix a drop of
human blood with a 2 per cent, solution of common salt. Note
the change of colour. (a.) Observe that

some corpuscles shrink in part, and become
crenate or beset with short spines (fig. 77).
This is due to exosmosis of fluid from the
corpuscles. The colour becomes slightly
deeper than in normal corpuscles. All the
corpuscles are not affected simultaneously
or to the same extent.

In some individuals, merely exposing the
blood to the air for a few minutes before
applying a cover-glass suffices to produce
this condition (fig. 72, e, f) ; but in any
specimen of blood it may be readily produced in the majority of
red corpuscles by acting on them with a saline solution of appro-
priate concentration.

It has been observed that in the blood of a mammal poisoned by
Calabar-bean the blood-corpuscles are crenated.

7. Dilute Alkalies. — Use a 0.2 per cent, solution of caustic
potash (i.e., 2 grams in 1000 cc. of normal saline). It dissolves
both the red and white corpuscles.

8. Fibrin (H). — Make a preparation of human blood, using a
large, drop. Cover it and put it aside for half-an-hour or longer
until the blood coagulates.

(ft.) Observe carefully, and in the meshes between the rouleaux
fine threads forming a delicate network will be seen. They are
fibrils of fibrin.

(b.) A better method, however, is to mix on a slide a drop of
blood with a drop of normal saline solution. Cover and put it
aside for an hour or so to clot, and then irrigate with water or dilute
alcohol (p. 25), which rapidly decolorises and washes away the red
corpuscles, and thus brings into view a fine fibrillar network of fibrin
in the field. Irrigate with a watery solution of Spiller's purple (r
per cent.), which stains the network of fibrin a purplish tinge (fig.
78). Raise the cover-glass, and to it will be found adhering a thin
film of fibrin. Dry the film, apply a drop of balsam, and mount the

120

PRACTICAL HISTOLOGY.

[III.

specimen as a permanent preparation. Numerous purple stained
threads of fibrin are seen stretching from colourless corpuscles, the
latter having their nuclei stained purple.

Crystals from Blood. — The haemoglobin of certain animals
crystallises very readily, e.g., rat, guinea-pig.

9. Haemoglobin Crystals of Rat's Blood (H).— Place a drop of
defibrinated rat's blood on a slide, add two drops of water, and mix,
Apply a cover-glass. After a few minutes, near the edge of the
cover-glass oblique rhombic crystals of haemoglobin will be found.
At first they are small, but they gradually become larger. They
may be single, or arranged in rosettes, or crossing each other

(%• 79)-

10. Crystals from Guinea-Pig's Blood (H). — To a drop of the

FIG. 78.— Fibrils of Fibrin
of Coagulated Blood.

FIG. 80.— Hicmo-
globiu Crystals
from Blood of
Guinea-Pit'.

FIG. 79.— Haemoglobin Crystals from
Rat's Blood, x 300.

defibrinated blood add a drop of Canada balsam or clove-oil, mix,
and apply a cover-glass. It is perhaps better to place the balsam
or clove-oil on the slide first, then to place the drop of blood on
the top of the oil or balsam, and then to mix them ; very soon
(about 10 minutes) large red tetrahedral crystals are formed.
These crystals cannot be preserved for any length of time (fig. So).

Sometimes very good crystals of haemoglobin from human blood
are obtained from leeches which have sucked blood some weeks
previously.

11. Hsemin Crystals (H). — Place a few particles of dried blood
on a slide, add a small crystal of common salt, and two drops of
glacial acetic acid. Cover. Heat over the flame of a spirit-lamp
until bubbles of gas are given off, i.e., until it boils. Allow it to
cool. Or mix fresh blood on a slide with a minute quantity of 2 per

III.]

CRYSTALS FROM BLOOD.

121

cent, salt solution. Heat the mixture until it becomes brownish in
tint, add glacial acetic acid and heat. On cooling, crystals of hsemin
are obtained. Should there be any cubes of common salt present
irrigate with water, which soon removes the latter. Hsemin crystals
are insoluble in water.

(a.) Observe the small brownish or black rhombic crystals, cither
singly or in rosettes, scattered over the field or on the surface of
the larger masses of blood (fig. 81).

o

FIG. 81. — Hncmin or Teichmann's

Crystals.

FIG. 82.— Leuksemic Blood.

(6.) To preserve them, remove the acid, raise the cover-glass,
dry the preparation, and mount them in balsam.

12. Enumeration of the Blood Corpuscles. — See the author's
Outline* of Practical Physiology, Lesson VI., p. 42.

13. Leukaemic Blood (H). — If a small quantity can be obtained
from a patient in the hospital, examine it.

(a.) Observe the great excess of colourless corpuscles. Accord-
ing to the variety of the leukaemia, they may be larger or smaller
in size than most of the coloured corpuscles (fig. 82).

ADDITIONAL EXERCISES.

14. Human Blood and Aniline Dyes (Hj. — Make a cover-glass preparation
of human blood (Lesson II. 24). Ehrlich heated the cover-glass in an air-
oven to a temperature of 120° for several hours to coagulate the proteids. To
avoid this, place the covers for two hours in the alcohol and ether mixture
(Lesson II. 26), and then stain them in methylene-blue as directed for frog's
blood-corpuscles, or stain in I per cent. Spiller's purple or a weak alcoholic
solution of rosein. After washing and drying, mount them in xylol balsam.

In the methylene-blue preparation the coloured corpuscles are unstained,
but some of the colourless ones have their nuclei stained blue, while the
surrounding protoplasm is unaffected ; in other colourless corpuscles the
granules in the protoplasm are stained. These corpuscles correspond to
Ehrljcli's basophile corpuscles. In the other preparations both the coloured
and colourless corpuscles are stained.
12

122 PRACTICAL HISTOLOGY. [ill.

15. Varieties of Leucocytes in Blood. — According to Ehrlich, the following
varieties of colourless corpuscles are present in the blood. (Also Lesson
XXXVIII.)

(a.) Small lymphocytes. They are slightly smaller than the red corpuscles,
and possess a large spherical readily-stained nucleus, which almost tills the
cell, being surrounded only by a small quantity of protoplasm.

(6.) Large lymphocytes are said to represent an advanced stage of (a). They
are twice as large as (a), have a large nucleus surrounded by a well-defined zone
of protoplasm, (a) and (&) together make up 25 per cent, of the leucocytes
in blood.

(c.) Mono nuclear elements, or transition forms, are distinguished from the
large lymphocytes by their nucleus not being quite spherical, and having a
depression in the middle.

(d.) Poly nuclear leucocytes, which are smaller than (0), but larger than
ml blood -corpuscles. They contain a nucleus composed of several lobes, or
several nuclei which stain readily and deeply. They represent 70 per cent, of
all the leucocytes of the blood, and can migrate from the vessels.

(e.) Eosinophilous cells. The nucleus stains less deeply than (d). The
granules which are present in the protoplasm stain deeply with cosin, i.e.,
become intensely red. They occur but sparsely in normal blood.

16. Staining of Leucocytes (Ehrlich).

(1.) Make a cover-glass preparation of blood and dry it for several hours

at 120° C.
(2.) Stain it for several hours in Ehrlich's aoid-hsematoxylin, eosin

solution, or in a strong glycerine solution of eosin.
(3.) Wash in water, — dry and mount in xylol-balsam.

The nuclei of the white blood-corpuscles, as well as the lymphocytes and
polynuclear forms, are deeply coloured ; the nuclei of the mononuclear forms
are "bluish-gray, the red corpuscles copper-red, and the eosinophile granules
red (Kahlden).

17. Eosinophilous Cells. — If it be desired to stain only these cells, stain a
cover-glass preparation with a strong glycerine solution of eosin. The results
of Ehrlich and his pupils, will be found in his pamphlet.1

18. Action of Hayem's Fluid.— This is an extremely useful fluid for pre-
serving and fixing the blood-corpuscles, and can be employed both for the
blood of animals and of man. It consists of

Sodic chloride . . . . . i grm.
Sodic sulphate .... 5 grins.

Corrosive sublimate .... 0.5 grm.
Distilled water .... 200 cc.

The blood is run direct from a blood-vessel into this fluid in the proportion
of i of blood to 100 of the fluid. It takes several hours to harden and fix (he
corpuscles, but twenty-four hours is not too long. By-aml-by the corpuscles
subside, and the supernatant fluid can then be decanted, and the deposit cf
blood-corpuscles well washed with water to get rid of the salts of the mixture.
These corpuscles can then be stained with various reagents, or eosin may be
added to the fluid.

(a.) Make an experiment with frog's blood, and stain the corpuscles fora
day or so with borax -carmine and mount the stained corpuscles in glycerine or
glycerine-jelly.

(b.) Stain another specimen with very dilute eosin-hpematoxylin (p. 70).
The hremoglobin is stained by the eosin, and the nuclei by the hrematoxylin.
It is preserved as (a). Other combinations of dyes will suggest themselves,
(c.) Make similar preparations of mammalian blood.
1 Farbenanaly. Unters. z. Histologie und Klinik d. B lutes, Berlin, 1891.

in.] BLOOD-PLATELETS. 123

19. Blood-Plates or Platelets (H). — Wrap a handkerchief round a finger to
obtain some blood (Lesson III., p. 115). On the skin at the root of the nail
place a drop of normal saline containing methyl-violet (.75 gram in icoocc.).
Through this drop prick the finger, and blood runs into it. Place a little of the
mixed blood and methyl-violet solution on a slide ; cover at once and examine.

It requires a good microscope arid careful observation to see the platelets.
The red corpuscles are unstained while the colourless corpuscles are stained. In
the field are to be seen small oval, refractive, very delicate, non-nucleated
bodies, much smaller than the red corpuscles
— these are the blood-plates (fig. 83, 3). They

are about 2. 5 /tin diameter. They undergo f#jj\ ^ o

cceedingly rapidly in shed blood. % IflpJ ^^

changes exceedingly rapidly in shed blood.

Instead of methyl-violet solution, the skin
may be pricked through a drop of the follow-
ing mixture: — I part of I per cent, osmic
acid, and 2 parts .75 per cent, sodic chloride.

By far the best method of obtaining blood-
platelets in large quantity, and in a condition
in which they do not disappear, is to allow
blood to flow into a solution of oxalate of

. i i-l.l • i i • .1 WltllO. I »1« M M l-l Mill I',-) , l±. I;>II11MI

potash until the mixture contains at least corpuscle surrounded by blood-

I per 1000 of the salt. This prevents coagula- plates.

tion of the blood. On placing such blood in a

centrifuge, when the corpuscles subside a film of grayish material accumulates

on their surface, which consists chiefly of blood-plates. A drop of this spread

on a slide, dried, and stained with a watery solution of methyl-violet 5 B and

mounted in balsam, yields permanent preparations of these bodies (Moser}.

20. Blood-Platelets—Dry Method (H).— Clean a slide thoroughly ; sterilise
it in the flame of a Bunsen-burner, and allow it to cool. Obtain a drop of
blood from the finger in the usual way ; get a drop on the slide, and with the
edge of another slide rapidly spread the drop of blood as a thin film on the
sterilised slide. Move the slide to and fro in the air until the film of blood
dries. The whole process should not occupy more than four seconds. Cover
the dry film with a cover-glass and seal the latter at the corners with paraffin.

(a.) Observe the coloured blood-corpuscles, which for the most part retain
their shape, and between them the blood-platelets or, as Hayem calls them,
the hnematoblasts. They are readily seen thus in human blood. The white
corpuscles are somewhat altered in shape.

21. Weigert's Method of Staining Fibrin. — Embed a thrombus or a piece
of lung affected with acute croupous pneumonia in celloidin, and make a
section. Float the section from water on to a slide. Stain it for ten minutes
or so with a drop of the following fluid : —

Gentian violet (5 per cent.) . . 4400.
Aniline oil ..... i ,,
Alcohol (96 per cent. ) . . 6 , ,

Remove the stain by pressing on the section two or three plies of blotting-
paper, or, better, unsi/ed printing-paper. Add a drop of iodine in iodide of
potassium (iodide of potassium 5 per cent, and saturated Avith iodine). This
quickly decolorises most of the stained parts. Remove the iodine with blot-
ting-paper in the same way as before.

Pour on the section a mixture of equal volumes of xylol and aniline oil,
moving the mixture over the preparation. Remove ihis and apply a fresh
supply of the xylol-aniline oil mixture. This removes all the water. As
long sis there is a trace of whiteness in the section it still contains water.
After all the water is removed, dry the preparation with paper as before, to

124 PRACTICAL HISTOLOGY. [ill.

remove as much aniline oil as possible. Wash the preparation several times
with xylol to remove the last traces of the aniline oil and mount in xylol-
balsam.
The stages are as follows : —

1. Harden in alcohol.

2. Stain 5-15 minutes in concentrated aniline water solution of gentian

violet.

3. Dry with blotting-paper.

4. Apply iodine solution (2-3 mins.).

5. Dry with blotting-paper.

6. Decolorise and wash in aniline-xylol.

7. Remove the latter and mount in xylol-balsarn.

(a.) Observe the threads of fibrin — very fine and numerous — stained a beau-
tiful violet.

22. Solvent Action of Serum. —Place some blood of a rabbit or guinea-pig
in a drop of blood serum of a dog. The red corpuscles are completely dis-
solved in a few minutes. The blood-corpuscles of a pigeon or frog are similarly
but more slowly dissolved, except the nuclei. This property of dog's serum is
set aside by previously heating the serum to 5o°-6o° C. for about half-an-hour.

LESSON IV.
EPITHELIUM (STRATIFIED) AND ENDOTHELIUM.

Epithelium presents the following general characters : —

1. It is always disposed on surfaces.

2. The cells are united by cement.

3. There are no blood-vessels within the cells.
Varieties of Epithelium.

1. Squamous.

2. Columnar.

3. Secretory.

4. Transitional.

5. Ciliated.

Squamous Epithelium may occur either in a single layer, or in
several layers ; in the former case it is sometimes called endothelium.
in the latter it is said to be stratified.

(A.) In a single layer it lines serous and synovial membranes,
heart, blood- and lymph-vessels, air-cells of lung, pigmentary layer
of retina, posterior surface of cornea, anterior surface of iri ;,
membranes of brain and spinal cord, surfaces of tendons and tendon
sheaths, &c.

IV.]

EPITHELIUM AND ENDOTHELIUM.

125

As endothelium, it consists of a single layer of flattened squamous
transparent cells united to each other at their edges by means of a
cement substance.

(B.) As stratified squamous epithelium, it covers the skin (epi-
dermis), and lines the following cavities and surfaces : — mouth,
pharynx (lower half), oesophagus, conjunctiva, over anterior surface
of cornea, vagina, and lower half of cervix uteri, and entrance of
urethra.

I. Isolated Squamous or Scaly Epithelium Cells. — With the
finger-nail or a small section-lifter gently scrape the inner surface of
the lip, place the scrapings on a slide, add a drop of saliva,
skimming off any air-bubbles with a needle, cover, and examine.

1. Squames (H). — (a.) Observe large flat plates or squames
floating in the field, either singly or in groups ; the cells of the latter
may be united by their edges, or by their edges and surfaces.
Select a single squame seen on the flat. ISTote its large size, being
five to ten times broader than a red blood-corpuscle ; its polygonal
shape ; the colourless, transparent, and, it may be, slightly granular
body of the cell, and the small oval excentrically-placed nucleus.

(b.) On some of the cells fine lines may be seen, some of them
due to folds, but most of them to facets, indicating that the cell
has been overlapped and slightly indented
by its neighbour. Select a group of cells
where the cells can be seen adhering to each
other by their edges and surfaces, as the
squames occur in several layers. It is well
also to look for a cell seen edgeways, to
observe that it is really a flat plate (fig. 84).

(c.) Not unfrequently fungi, such as
bacteria, may be seen adhering to the
squames.

(d.) Salivary Corpuscles may be seen.
They are sharply-defined spherical cells, pro-
vided with a membrane, and about the size FlG 84._ceiis of stratified
of a colourless blood-corpuscle. They contain
fine granules, which in the fresh condition
of the corpuscles may be seen to exhibit
Hrownian movement (Lesson I. 6). Each cell may contain one
small, spherical, excentrically-placed nucleus, or sometimes two
nuclei may be present. They seem to be leucocytes distended with
fluid.

2. Magenta. — Irrigate the preparation with a watery solution of
magenta (p. 74), which stains deeply the nuclei of the squames and
salivary corpuscles, while tho po,ri-nuclear parts of these cells are
less deeply stained. As the magenta contains alcohol, the latter

Sqnamous Epithelium de-
tached from the Month.
s. Salivary corpuscles.

126 PRACTICAL HISTOLOGY. [iV.

precipitates the mucin of the saliva either in the form of fine threads
or in membranes, which are stained red by the magenta.

3. Epidermis of Newt — Superficial Layers of Stratified Epithe-
lium (L and H). — Keep a newt or frog for a day or two in a small
quantity of water, and do not change the water. Very soon the super-
ficial layers of squames will be " cast " as thin membranes. Take
these, and harden them in absolute alcohol. Stain a thin piece in
hsematoxylin, and mount it in balsam.

(a.) Try to get a layer sufficiently thin, so that the cells are only
one layer thick. Note the polygonal, large, nucleated cells united to

each other by their edges by means
of a clear cement substance (fig. 85).
(b.) The nucleus is usually ex-
centric, and surrounded by slightly
granular material. In the nucleus
are usually two or three nucleoli,
and sometimes an intra-nuclear
plexus of fibrils is visible, especially
in the cells of the deeper layers.
Sometimes granules of the pigment
melanin are seen in the cells, especi-
ally from a dark-pigmented newt.
4. V.S. of Stratified Epithelium.

FIG. 85.— Superficial Layer of Squames This may be conveniently ex-
cast from the Epidermis of a Newt. , ,.J . -• i A.*

Alcohol and luematoxyiin, x 300. ami ned either in a vertical section

of the skin of the palm of the

hand, or a similar section of the mucous membrane covering the
hard palate of a cat or the conjunctiva on the cornea. The skin
must have been previously prepared by being hardened in absolute
alcohol or chromic acid and spirit mixture (p. 29), while the mucous
membrane may be hardened in the chromic acid and spirit or
Miiller's fluid.

If the mucous membrane of the hard palate be taken, stain it
with picro-carmine and mount in Farrant's solution. Or the mucous
membrane may be stained in bulk in borax carmine, embedded and
cut in paraffin. In the latter case the sections are best mounted in
balsam.

(a.) Examine it first with (L). Observe the epithelium arranged
in many layers, covering a connective-tissue basis — the latter
stained red — and projecting in the form of fine papillae into the
epithelial layer (fig. 86). Neglect the connective-tissue basis
meantime.

(b.) (H) Select a thin part of the deeper layers of the epithelium
near the papillae, and note that the cells there arc somewhat small
and cylindrical, with their nuclei stained red. Study the change

EPITHELIUM AND ENDOTHELIUM.

12'

in the form of the cells towards the free surface of the epidermis
(tig. 86), where they become corneous and less granular.

(c.) Note in the deeper layers of the epithelium prickle-cells.
Adjacent cells are connected by very fine processes or " intercellular
bridges." The fine spaces between the bridges
are called "intercellular channels." When
these are broken across and the cells isolated, the
cells present the appearance of being beset by
very fine processes, and hence they are called
" prickle-cells."

(//.) The cells vary in their shape and characters
from below upwards. It will be easy to detect
the horny layer above, composed of many layers
of flattened, hardened cells. This forms a fairly
well-marked layer, the cells being clearer than
those situated more deeply ; the nuclei are less
conspicuous, and the cells generally stain yellowish
with picro-carmine.

5. Non-Corneous Stratified Epithelium from Fi0""86.-v.s. MUCOUS
the (Esophagus (H). — Macerate the mucous

membrane of the oesophagus of a calf or other
animal for a week in y1^ per cent, bichromate of
potash. This "dissociates" the epithelial cells,
so that when the surface is scraped, one obtains
isolated cells. Make a cover-glass preparation
(p. 114), and stain it in aniline- water-gentian-
violet, i.e., gentian-violet solution dropped into
aniline water (p. 73). Dry and mount in balsam.

(a.) Observe the isolated squamous cells, each with a small ex-
centrically-placed nucleus stained of a violet tint. Numerous facets
are seen in the cells, and their shape both on the flat and on edge
can be carefully studied.

(b.) If desired, stain some in picro-carmine. This is best done by
keeping them in the picro-carmine on a slide placed under a bell-jar,
with water to prevent evaporation, i.e., in a moist chamber.

6. Horny Epidermis. — Macerate a shred of epidermis in 35 per
cent, caustic potash. After a time it is softened and can be broken
u I ) with needles. The cells fall asunder and swell up in the fluid,
and appear as spheroidal cells with a membrane, but no nucleus is
visible. Examine them in the potash solution. No water must be
uddcd, else the cells are dissolved.

7. Prickle Cells (H).— Place for twenty-four hours in i per
cent, osmic acid a small piece of the palmar surface of the skin
— less than one-eighth of an inch cube — from a freshly-amputated
finger. Make vertical sections by freezing, or after embedding in

Membrane of Hard
Palate of Cat. Epi-
dermis with cor-
neous and deeper
layers with prickle
cells. Below, con-
nective tissue of
the mucous mem-
brane with a papilla.
Chromic acid and
spirit, picro - car-
mine, Farrant's
solution.

128

PRACTICAL HISTOLOGY.

[IV.

paraffin, and mount the former in Warrant's solution and the latter
in' balsam.

(ti.) Observe the prickle-cells in situ, i.e., polygonal cells in the
deeper layers of the rete mucosum, with fine processes connecting
adjoining cells, leaving thus a system of fine spaces between the cells
(fig. 87). The fine fibres which pass from cell to cell form "inter-
cellular bridges," and when these bridges arc broken across they
give the appearance as if the cells were beset with fine prickles.

8. Isolated Cells from th9 Different Layers of the Epidermis
(H). — It is usual to macerate very small pieces of any membrane
covered with stratified epithelium in g- or —$ per cent, of potassic
bichromate, which usually takes more than a week to dissociate the
cells.

Fm. 87.— Prickle-Cells from the
Deeper Layers of the Epi-
dermis of the Palm, showing
intercellular bridges and
channels. Osmic acid.

FIG. 88.— Prickle Cells Isolated from
the Human Epidermis by means of
Iodised Serum. n. Prickles ; d.
Space between nucleus and cell-
body, x 800.

A much speedier method is that of Schiefferdecker. Make a
watery extract of " pancreaticum siccum " of Dr. Witte of Rostock.
Filter, and in the filtrate place a small fragment of fresh skin or
the pad from the upper jaw of a sheep. Place the fluid near the
fire or in an oven at 40° C. Within four hours, the epidermis can
be detached, and its cells fall readily apart. Preserve it in a test-
tube in a mixture of equal parts of water, alcohol, and glycerine.
It forms a deposit at the bottom of the tube. A little of the deposit
is mounted in glycerine or Farrant's solution, covered and examined.
It may be stained with picro-carmine or methylene-blue.

(a.) Observe numerous cslis of different shapes, some flattened,
others cubical, and many "prickle-cells" (fig. 88). Many of the
cells exhibit facets where they have been pressed against each
other.

9. Isolated Prickles and other Cells from the Pad of a Sheep's
Mouth (H). — Isolated by means of "pancreaticum siccum" (v.
supra). Many of the somewhat cubical-shaped cells show tho
" prickles " beautifully. These cells may be stained with an aniline
dye or picro-carmine, and some of them show two nuclei.

IV.]

EPITHELIUM AND ENDOTHELIUM.

129

Peritoneal Surface of the
Central Tendon of the
Diaphragm of a Rabbit.
Silvered. /. Lymphatic
slit ; t. Tendon ; c. Ordi-
nary endothelial cells ; n.
Islands of small cells.

10. Endothelium of Central Tendon of Diaphragm. — Mount
in balsam a small piece of the central tendon of the diaphragm of
a rabbit stained in silver nitrate (Method,

P- 77).

(«.) Observe the polygonal areas which
map out the outlines of the single layer of
squames covering the surface of the tendon.
These areas are bounded by brown or black
lines, the so-called " silver lines " (fig. 89).
As a rule, no nucleus is visible in the cells,
but it may be brought into view by staining
with logwood and mounting the preparation
in Canada balsam.

(b.) Amongst these may be seen small
groups or islands of smaller granular cells.
In some specimens stomata surrounded by

granular or germinal cells may be seen. jio._89.— Endothelium of the
These stomata open into a plexus of lym-
phatic capillaries in the substance of the
tendon (Lesson on Lymphatics).

11. Omentum of Young Rabbit (L and
H). — Mount in balsam a small piece of the

silvered omentum of a young rabbit (one week old) (Method, p. 77).

(a.) Observe a thin fibrous membrane mapped into polygonal
areas — some of them
with more or less
sinuous outlines — by
means of black "silver
lines " (fig. 90). ,

The omental mem-
brane composed of
connective tissue is
covered on both sur-
faces with a continu-
ous layer of endo-
thclium.

(/>.) Raise the lens
by means of the fine
adjustment until the
upper layer of squames
conies distinctly into
view ; then depress the
lens and focus through
the thickness of the. membrane to bring into view the. layer of
squames covering the deeper surface, and note that the outlines of
13 I

FIG. QO. — Omentum of a Young linhhit stained with Silver
Nitrate (x 300), showing the epithelium on the upper
and under surfaces ; the outlines of the latter faintly
indicated. Some holes are also seen in it.

130

PRACTICAL HISTOLOGY.

[IV.

the cells do not correspond with those of the upper layer. In
focussing through the membrane, note the plexus of elastic fibres
in it.

(c.) To see the nuclei of the endothelial cells, stain another piece
of the silvered omentum in logwood and mount it iti balsam.

The omentum of a young rabbit is chosen because it is nearly a
complete membrane with few fenestrse or holes in it.

12. Omentum of Cat (L and H).— Mount in balsam a small
piece of the silvered omentum of a cat (Method, p. 77). In cutting
the omentum into small pieces, the easiest way is to spread it out, or
rather float it out, on a sheet of paper, and then cut the paper and
omentum into pieces of the necessary size. The pieces are thus

less liable to fold up, and are
more readily manipulated on
the slide.

(a.) (L) Observe a mesh
work of trabecula3 (T) bound-
ing open polygonal spaces (tig.
91, 111). In the larger tra-
beculse may be seen blood-
vessels (c), an artery or a
vein, or both, surrounded
here and there by groups of
large clear cells — fat-cells (/).
All the trabeculse are com-
pletely covered with endo-
thelial cells, whose outlines,
mapped out by silver lines,

can Just be recognised.

(&.) (H) Select a large

Silver lines, and a. Nuclei of theendothelmm; ', \./ ,, .

m. Meshes; b. Nuclei of the connective-tissue Strand With a blood-VCSSel 111

FIG. Qi.-Omentum of Cat Silvered. T = Trabe-
cula, with c. Blood-vessel; /. Fat-cells; s.

xic silver nitrate and hacmatoxylin' it (T). Focus the silver lines

(s) on its upper surface, and

gradually depress the lens until the fibrous tissue composing the
strand comes into view, and, still lowering the lens, bring the
endothelium on the under surface into view.

(c.) Select a fine trabecula, and note the silver lines on it; also
observe the fibrous tissue of which it is composed.

(tL) If desired, a preparation may be stained with logwood to
reveal the nuclei of the cells of the endothelium (a), as well as
those of the fibrous tissue composing the membrane (l>). The
nuclei of the endothelial cells are superficial, and usually spherical
(a) ; those of the connective-tissue corpuscles are in the substance
of the membrane, and arc usually more flattened and somewhat
oval (b).

EPITHELIUM AND ENDOTHELIUM.

ADDITIONAL EXERCISES.

13. Pogiel's Methylene-Blue Method. — Place any fresh membrane, e.g.,
omentum, mesentery, capsule of kidney, in a 4 per cent, solution of methylene-
blue in normal saline. Allow it to stain for ten minutes or so. Wash it twice
in a saturated watery solution of picrate of ammonia and examine in glycerine.
The outlines of the cells are stained of a purplish colour. This method may
be used instead of the silver method to demonstrate the existence of endo-
thelium on any surface, e.g., on tendons (rat) in blood- or lymph-vessels. It
is also used to demonstrate lymph-spaces.

14. Sections covered by or consisting of epithelial cell structures may
be stained in a weak watery solution of benzo-azurin. This stain is not
removed by alcohol, and the sections can be mounted in balsam. Or benzo-
purpnrin (B. ) may be used. This gives a reddish stain, any excess being
removed by alcohol rendered feebly alkaline, e.g. , by lithium carbonate.
Benzo-azurin stains connective tissue of a bright blue.

LESSON V.

COLUMNAR, SECRETORY, AND TRANSITIONAL
EPITHELIUM.

Columnar Epithelium lines the mucous memhrane of the ali-
mentary canal from the cardiac orifice of the stomach onwards;
the greater part of the ducts of the glands opening into it ; other
gland ducts.

II. Columnar Epithelium. — Slit open the small intestine of a
rabbit or a catt;w*tf killed. Wash the mucous surface with normal
saline to remove any adherent particles.

1. Fresh Condition (H).— With a scalpel gently scrape the
mucous surface and transfer what is on the scalpel to a drop of
normal saline solution on a slide. Diffuse the scrapings in this
fluid, and t<» prevent the pressure of the cover-glass, place in the
iluiil a hair half-an-inch in length. This preparation is not to he
preserved.

(«.) Observe numerous columnar epithelial cells, a few isolated,
but most of them adhering together. Side View of the Cells.
Select an isolated cell, notice its columnar form, usually tapering
to a blunt point at one end, while the body of the cell is faintly
granular, and contains a clear oval nucleus. The free, broad end
is covered wilh a highly retractile disc — seen on edge as a narrow
retractile band— with fine vertical striae in it (fig. 92).

132 PRACTICAL HISTOLOGY fv.

Many more or less complete villi may be seen. Along the edge
of the villus, covered by its layer of columnar cells, the clear disc
is readily seen, and by focussing the surface of a villus the nucleated
mosaic formed by the ends of the cells, with the rounded mouths
of the goblet-cells, come into view.

(ft.) If the animal was killed whilst its food was undergoing
digestion, refractive fatty granules may be seen in the protoplasm
of the cells.

(c.) End View of the Cells. — Move the preparation until a
fragment of detached epithelium is seen showing the free ends of

the cells directed towards the observer
(fig. 92, d).

(d.) Note in such a group of cells the
polygonal outlines of the ends of the
cells, and in each polygonal area a large
rf spherical nucleus, which appears almost

FIG. 92.— a, b. Isolated Columnar to fill the area. A nucleolus can often

Epithelial Cells from the Small ^ TT _ j ^ i

Intestine of Cat ; c. Goblet-cell ; L Ln- Ildy lj

d. Ends of columnar cells and the rounded opening of a goblet-cell.

open mouths of goblet -cells -T-I * n ^ *• j_i

directed towards observer. Focus carefully and notice the appear-
ance of the goblet-cell. When the open

mouth is in focus, it is seen as a circle of small diameter, and on
depressing the tube the broad part of the cell comes into view (fig.
92, d).

(e.) Amongst the cells may be found isolated goblet-cells (fig.
92, c). Each cell has an open mouth, while the lower part of the
cell contains a nucleus embedded in a small quantity of protoplasm.

2. Isolated Columnar Epithelium of Newt (H). — Mount in
glycerine a small quantity of isolated columnar cells which have
been dissociated by maceration in dilute alcohol and subsequently
stained with picro-carmine. Place a short length of hair under the
cover-glass.

The small intestine of a rabbit may be used, but far larger cells
are obtained from the intestine of a newt. Macerate the whole
intestine in dilute alcohol for twenty-four hours and stain in bulk
in picro-carmine for at least another twenty-four hours. On
scraping the mucous surface, the cells are detached and diffused in
formic glycerine.

(a.) Observe the large, tall, columnar cells, often tapering to a
point at their lower end, the red-stained nucleus, and the clear
striated disc. The nucleus usually exhibits a distinct nucleolus
(fig. 93), and sometimes two nuclei are present. Sometimes two
nucleoli are seen in a nucleus, and the cell itself may be branched
at its fixed extremity.

If the intestine of a newt be macerated for twenty-four hours in

COLUMNAR EPITHELIUM.

133

5 per cent, ammonium chromate, then the nuclei and cell-body show
a distinct h'brillar structure.

III. Glandular Epithelium occurs in the secretory glands, e.g.,
liver, pancreas, salivary, gastric, intestinal, and other glands. Neces-
sarily it must vary very greatly in shape and in its functions.

3. Secretory or Glandular Epithelium of Liver. — With a
clean scalpel scrape the cut surface of the liver of an animal just
killed, e.g., a rat. Place the scrapings in dilute alcohol (twenty-
four hours), pour off the alcohol and cover it with picro-carmine
(twenty-four hours).

Liver-Cells (H). — (a.) Examine the isolated cells in glycerine
with the usual precautions. Observe the cubical cells, which may
be isolated or adhering in groups of two or three. The granular

FIG. 93. — Isolated Columnar
Epithelial Cells from the
Newt's Intestine. Dilute
alcohol and picro-car-
mine, x 300.

FIG. 94.— Isolated Hepatic Cells, d. With two
nuclei ; b. Oil-drops ; c. Isolated nucleus of a
cell. Teased fresh.

protoplasm is stained yellowish, and each cell has a spherical bright-
red nucleus. The protoplasm may contain globules of oil, and
occasionally two nuclei may be seen in a cell, especially in the liver-
cells of a young rat (fig. 94).

(/>.) If the cells be much broken up, liberated nuclei and granules
of oil, and sometimes red blood-corpuscles, are seen in the field.

((•-.) Acetic acid clears up the protoplasm, makes the nucleus more
distinct ; it does not affect any fatty particles, but merely makes
them more evident.

Sulpho-cyanide of potassium (5 per cent.) is an excellent medium
for maceration of the liver-cells. The plexus of fibrils in the nuclei
is thus rendered visible.

IV. Transitional Epithelium occurs in the urethra, urinary
bladder, ureters, and pelvis of the kidney. It is confined to the
gcnito-urinary mucous membrane; It consists of several (2-3-4)
layers of cells. The superficial cells are large and flattened
(especially if the bladder has been kept distended), often with two
nuclei, and with depressions on their under surface produced by the
large rounded ends of the cells of the next layer. The cells of the

134 PRACTICAL HISTOLOGY. [V.

next layer are somewhat pyriform (fig." 95, ?>), while the deepest layers

are composed of smaller polyhedral cells.

4. Transitional Epithelium of Bladder (H). — Place the dis-

tended bladder of a frog or cat in dilute alcohol for twenty-four
hours, stain it en masse for the same time in
picro-carmine, scrape off a little of the mucous
surface and diffuse it in glycerine, add a hair
and cover. The cells may also be macerated
by using instead J per cent, bichromate of
potash solution.

(a.) Observe various forms of cells, some
of them more or less flattened with facets on
their surfaces (fig. 95, a, a'), others elongated
with finger-shaped processes (6), some pear-

Fie. 95. — isolated Transi- shaped, and others cubical. All arc

tional Cells from the rmplpqfp/l
Bladder of a Guinea-pig, nucleated. ^

a. A superficial ceil seen 5. To Distend a Frog s Bladder.— By
means of a pin transfix the skin at the

the deeper layers. Dilute margins of the anus, and tie round the pin

alcohol and picro-car- ,-r -, *, , i

mine, x 3oo. a thread so as to completely occlude the

aperture. Open the abdomen, make a slit

into the rectum, and from the latter, after removing its contents,
inject dilute alcohol (p. 25) into the bladder. When the bladder is
distended, ligature it, cut it out, and suspend it in its inflated con-
dition in dilute alcohol for twenty-four hours.

If the bladder of a cat or guinea-pig be used, it is distended from
the urethra with dilute alcohol, and suspended for twenty-four
hours in a large volume of the same liquid.

ADDITIONAL EXERCISES.

6. To Silver the Free Ends of Columnar Epithelium. —A small piece of
the mucous surface of the small intestine of a cat is washed in distilled water,
and then placed for ten minutes in a ^ per cent, silver nitrate solution, and
silvered in the usual way (Method, p. 77). After hardening in alcohol, if the
epithelium be detached and mounted in glycerine, it is easy to obtain a view
of the free ends of the epithelial cells, with the cement substance between them
indicated by "silver lines," and also to see the open mouths of the goblet-cells.
The view obtained is that shown in fig. 92, d.

N.B. — Other preparations of these, forms of epithelium will be obtained in
sections of the organs in which they occur.

VC.] CILIATED EPITHELIUM. 135

LESSON VI.
CILIATED EPITHELIUM.

Ciliated Epithelium occurs in the nasal mucous membrane
(except that of the olfactory region), the cavities accessory to the
nose, the upper half of the pharynx, the Eustachian tube, larynx,
trachea, and bronchi, the uterus (except the lower half of the cervix),
Fallopian tubes, vasa efferentia to lower end of epididymis, the
ventricles of the brain, and the central canal of the spinal cord.

V. Ciliated Epithelium. — A ciliated cell may be any shape, but
usually it is 4more or less columnar. Only the cells of the super-
ficial layer bear cilia. The bunch of cilia are directly continuous
with the protoplasm of the interior of the cell, and are planted on a
clear disc, which is said to be composed of small "knobs " placed side
by side to form a bright refractile disc ; a cilium being attached to
each knob. The deeper cells may be pyriform or polyhedral, accord-
ing to the situation from which the epithelium is obtained. Goblet-
cells may be found between the superficial ciliated cells.

1. Ciliary Motion in the Frog (H). — After pithing a frog, gently
scrape the roof of its mouth with a scalpel, and diffuse the scraping
in a drop of normal saline, add a short piece of hair, and cover.

(a.} Notice groups of cells with cilia on their free surface. The
cilia bend quickly, at the rate of ten to twelve times per second, with
a whip-like motion in one direction, and then rapidly unbend, thus
creating currents in the liquid, and thereby moving the corpuscles,
granules, or other free particles that may be present. They bend
more rapidly in one direction than the other. All the cilia covering
one surface are not in motion at one time, but the movement passes
from cell to cell in a wave-like form. If a portion of cell with cilia
attached is in view, the fragment of the cell may be seen to be
moved in a definite direction by the vibratile motion of its own
cilia. Cilia detached from a cell cease to move.

2. Ciliary Motion in the Mussel (L and H).— Open a salt-water
mussel, collect the salt wrater which escapes, cut out a fragment of
one of the flattened yellow gills, and place it in a drop of salt water.
By means of two needles separate slightly the bars which compose
the gills, cover and examine.

(a.) (L) Observe the bars with their free rounded ends and their
surfaces beset with a fringe of moving cilia, which cause the particles
suspended in the fluid to be carried along in a definite direction.

136 PRACTICAL HISTOLOGY. [vi.

(&.) (H) Select a single, cilium observe that it is a clear homo-
geneous tapering filament, placed on a clear band which covers the
cells on the surface of the bar of the gill. Notice how the cilium
bends more at the top than base, and how it straightens itself again.
The movement may go on for several hours. The backward move-
ment is less rapid than the forward stroke.

3. Heat (H). — By means of a camel-hair brush run a ring of oil

Fia. 96. — Slide with a King of Glass Tube fixed to it, for Studying the Action of
Chloroform on Cilia.

round the preparation (2), and put it aside for an hour or so, until
the ciliary motion becomes slower.

(a.) Place the slide on a hot stage (fig. 75) and gradually apply
heat. As the cilia are warmed they move more quickly; but if the
temperature be too high, of course the proteids are coagulated and
the cells killed. If, while the cilia are moving rapidly, the source
of heat be removed, as the preparation cools the cilia gradually move
more and more slowly.

Use the hot stage described in Lesson III. 5. But in using it,
put the preparation of cilia on a cover-glass moistened with a drop
of sea-water, and invert the cover-glass over the aperture in the hot
stage, so that the drop of fluid and cilia hang in the little circular
cavity.

4. Weak Alkalies. — To a preparation in which the cilia move
languidly, apply a drop of -J per cent, solution of caustic potash.
This immediately revives their action for a short time ; but as the
alkali penetrates into the cells, it ultimately kills them.

5. Chloroform (L and H). — Place a fragment of a mussel's gill in
a drop of salt water on a cover-glass. Put a small drop of chloroform
in a glass cell and place the cover-glass on the cell, with the drop of
fluid hanging into the latter, as shown in fig. 96.

(a.) (H) Observe the movement of the cilia, and, as the chloroform
vapour diffuses into the drop of water and acts on the cilia, how
they move slower and slower. If the action of the chloroform be
pushed too far, their movement will be arrested. If the action of
the chloroform be not too prolonged, and the preparation removed
and freely exposed to the air, the cilia may begin to move again.

6. Action of Gases on Ciliary Motion, e.g., Carbon Dioxide.—
Carbonic acid is generated in the usual way in a flask containing

VI.]

CILIATED EPITHELIUM.

137

marble and dilute hydrochloric acid, and by means of a caoutchouc
tube it is conducted to a glass gas-chamber (fig. 97, C), over which
the preparation of cilia on a cover-glass is inverted.

If it be preferred, the following moist chamber, by Ranvier, for
studying the action of gases may be used. It consists of a brass box

FIG. 97.— Gas-Chamber for Studying the Action of Gases on Cilia. A. Inlet ;
B. Outlet-tube; C. Glass gas-chamber.

about the size of a microscopical slide, and perforated at the centre
by an aperture 2 cm, wide, which is closed below by a plate of glass.

FIG. 98.— Ranvier's Moist Chamber for Applying Gases to a Preparation.

Iii the centre of the aperture is fixed a plate of glass (fig. 98, a), less

in diameter, thus leaving a circular trench all round (b). Moreover,

the height of this circular plate of glass is

less than the height of the brass box by at

least -iV-h inm- ^ne k°x is perforated by

two tubes, through which the gases can

be conducted to the preparation, which is

placed between the top of the circular

glass disc and the cover-glass which covers

in completely the aperture in the brass

box. In this way the gas or vapour can

be applied, to a preparation still in a normal

fluid medium.

(a.) If carbon dioxide be used, observe that it rapidly arrests the
movement of the cilia and renders the cells granular, probably from
the precipitation in them of paraglobulin.

Ciliated Cells from tlie
Mucous Membrane of the
Hani Palate and (Esophagus
of the Frog. Dilute alcohol
and pirro-carniine, x 303.

138

PRACTICAL HISTOLOGY.

[VI.

Fl0:.. Ifx>;7:I11BPlat4.?d

Cihated Cell from the

7. Isolated Ciliated Epithelium and Goblet-Cells (Frog) (H).—
Scrape off a little of the epithelium from the mucous membrane of
the palate of a frog, which has been macerated
for twenty-four hours in dilute alcohol and after-
wards stained by picro-carmine. Before staining,
it is advisable to place the isolated cells for
several hours in | per cent, osmic acid. Diffuse
the cells in glycerine., put in a hair, cover, and
examine.

(a.) Observe an isolated ciliated cell ; it is
short and columnar, perhaps tapering or divided
at one end, while the other end is beset with
cilia, resting on a clear, transparent, refractile
disc ; the protoplasm is granular, and encloses an
oval red-stained nucleus with one or two bright
excentrically-placed nucleoli (fig. 99).

Besides the ciliated cells, there are others
without cilia, oval or elongated, pointed at one
end. These are from the deeper layers of the
ciliated surface (fig. 102).

(&•) Numerous goblet or chalice cells will also

>• ' ..

of a Frog, be seen, ihey are cup-shaped cells, with an open
niouth, and containing mucigen. There is a
small amount of protoplasm at one end of the cell
— the end by which it is fixed — which encloses
a spherical, oval, or compressed nucleus. The
goblet-cells may be seen in two conditions, some clear with their
mucigen discharged, and others full of granules or
" loaded " with mucigen.

The cells may also be isolated or dissociated by
macerating the membrane in iodised serum (p. 25).
A cell isolated in this way, and magnified 1000
diameters, is shown in fig. 100.

8. Isolated Ciliated Cells (Mammal).— Use the
trachea of a cat or other mammal, and macerate
small pieces in dilute alcohol (twenty-four hours).
Stain it in bulk in picro-carmine. The isolated cells
are examined in glycerine or glycerine-jelly. If the
trachea of the ox be used, observe

(a.) The tall narrow ciliated cells (fig. 101), each
with its cilia and clear disc. The ends of the cells
may be pointed or branched. Amongst these may
be seen oval or battledore-shaped cells (a), the
younger cells which exist in the deeper layers of the mucous
membrane.

p. Clear
Nucleus ;
in. Irregular extre-
mity. Iodised serum,

X 1000.

. ioi.— Ciliated
Cells from Tra-
chea of Ox. a.
Cell from the
deeper layers.
Dilute alcohol
and picro-car-
mine, x 300.

VI.] CILIATED EPITHELIUM. 139

9. Ciliated Epithelium (|_ and H). — Mount a vertical section of
the respiratory mucous membrane of the septum of the nose of a cat
or other animal. The tissue has been previously hardened in Miiller's
fluid, or in chromic and spirit fluid, or, what is better, a saturated
watery solution of corrosive sublimate for two or three hours. In the
last case, every trace of the metallic salt must be removed by prolonged
and frequent washing with alcohol. Stain the section with logwood
and mount it in balsam ; or picro-carmine can be used.

(a.) Observe several layers of cells (fig. 102), but only the super-
ficial layer of cells is furnished with cilia, which are placed on a
clear disc on the free end of the cell. Notice the shape of the cells
in the subjacent layers. Those of the lowest layers are nearly
spherical, while in the intermediate layers they are more elongated,

Disc.

Intermediate FIG. 103. —Various Forms of
Forms. Goblet-Cells from the Mucous

Membrane of the Hard Palate
Deepest Layer aiul (Esophagus of the Frog,
of Cells One of the cells shows mucus

exuding from the open mouth
of the cell. Dilute alcohol
FIG. I02.-V.S. of Ciliated Epithelium. and picro-carmine, x 300.

and are described as battledore-cells, arranged in between the others.
They replace the ciliated cells when the latter are shed.

10. Isolated Goblet-Cells (H).— These are readily obtained by
macerating the stomach of a frog in dilute alcohol for twenty-four
hours. Scrape the surface and diffuse the cells in glycerine. They
may be stained with picro-carmine, or they may be diffused in salt
solution and stained with methyl-violet, but the latter preparation
cannot be preserved in glycerine. Numerous goblet-cells will be
found in 7.

(a.) Observe the isolated cells (fig. 103). Each cell is filled for
more than three-fourths of its capacity with mucus, while at the
lower tapering end there is a nucleus embedded in a small quantity
of protoplasm. Sometimes a plug of mucus may be seen exuding
from the, open mouth of a cell.

(l>.) With a high power the interior of the upper part of these
cells may be seen to contain a fine network of fibrils. In the meshes
is a substance, mucigen, and when this is acted on by water it yields
mucin. In a certain sense these bodies are unicellular muciparous
glands.

140

PRACTICAL HISTOLOGY.

[VI-

ADDITIONAL EXERCISES.

11. Cover-Glass Preparation of Goblet-Cells. —Place the oesophagus or
stomach of a frog in dilute alcohol for twenty-four hours. Scrape the mucous
surface and compress the scrapings between two cover-glasses. Separate the
cover-glasses, allow the film adhering to each glass to dry, and then stain
it with eosin or aniline-water-methyl-violet, or safraniii-0. Wash off the
surplus stain with absolute alcohol, allow the film to dry, and mount it in
xylol-balsam.

Perhaps a better plan still is to stain the cover-glass preparations for
twenty-four hours in Ehrlich-Biondi fluid. It is prepared thus :—

Ehrlich-L'iondi Fluid.

Saturated watery solution of orange .

„ ,, acidfuchsin .

,, ,, methyl-green .

100 cc.
20,,

50 „

The solutions used, however, must be saturated. When used as a staining
agent, this strong fluid is diluted with about forty volumes of water.

(a.) Observe the goblet-cells with their characters retained intact. In the
Ehrlich-Biondi preparation the protoplasm is stained red, the nuclei and
nucleoli bluish.

12. Cover-Glass Preparation of Ciliated Epithelium. — The mucous mem-
brane of the oesophagus of a frog is placed for twenty-four hours in dilute
alcohol. A cover-glass preparation is made of the epithelium, and stained as
described under 11, with methylene-blue, safranin-0, gentian-violet, or
Ehrlich-Biondi fluid, and mounted in xylol-balsam.

13. T.S. Tongue of Frog (H). — By means of hedgehog-spines, pin out the
tongue of a frog on a thin layer of cork with a small hole in it. Harden it for
two hours in a saturated watery solution of corrosive sublimate ; remove every
trace of the sublimate by prolonged washing in alcohol — not water. Stain in

bulk in picro-carmine or borax-carmine.
Make transverse sections — best by the
paraffin infiltration embedding method
(p. 41) — and mount the sections in
balsam.

(a.) Observe the fibro-muscular basis of

the tongue' covered on the surface with

ciliated epithelium cells; between the
ciliated cells the goblet-cells, each with an
open mouth and its plexus of fibrils with
muoigen in its meshes (fig. 104).

(b.) Observe, too, how the expanded
ovoid goblet-cells compress the ciliated

FIG. ,04-V.S. Ciliated Epithelium of C,ells «*1 cause the latter to have a peculiar
Frog's Tongue. TO. Muscular fibres, shape, a broad expanded top and a narrow
Corrosive sublimate and picro-car- body.
carmine, x 250. (r. ) The young cells at the base of the

ciliated cells.

If the specimen be stained in picro-carmine and mounted in balsam, the
yellow colour of the picric acid can be retained by putting a little picric acid
into the alcohol used to dehydrate it, or by picric acid placed in the clove-oil
or xylol used to clear up the section.

VII.] MITOSIS OR KARYOK1NESIS.

LESSON VII.

STRUCTURE OF CELLS— MITOSIS OR KARYO-
KINESIS.

Structure of the Animal Cell. — To see all the structures in an
animal cell is by no means easy. Speaking generally, the tissues
of the articulata, amphibians, and reptiles yield the largest tissue
elements for examination. The cells may be examined in the fresh
condition or after "fixing," hardening, and staining.

In Fresh Condition. — Examine a teased preparation in an
indifferent fluid, e.g., normal saline, the liquid of Ripart and Petit
(p. 24), or in solution of methyl-green (p. 74). The preparation may
be sealed up with paraffin wax (p. in) and examined after some
time.

After Hardening. — The best osmic acid "fixing" fluids for this
purpose are the fluids of Flemming, Rabl, and Fol, and the best
stains safranin and gentian-violet. Mount in xylol-balsam.

To see the finer details an oil-immersion lens and Abbe's con-
denser must be used.

Mitosis or Karyokinesis. — By these terms is meant the remark-
able series of phenomena which take place in cells — animal and
vegetable — when they undergo a process of indirect division. In
this connection it is important to remember the constitution ' of a
cell and some of the terms which have been applied by different
authors to its several parts. A cell may or may not possess a dis-
tinct cell- wall, but the cell-body appears to be made up of two
substances, which Flemming names as follows : — One composed of
threads, seldom forming a network, and called by him cyto-mitoma
or mitoma (/xtVos, thread), also called spongioplasm, and the
other, homogeneous and lying in the meshes of the latter, is the
paramitoma or hyaloplasm. The cell-contents are generally
described as consisting of a finely-granular soft substance, the so-
called pr<>tt>i>1<i#m (fig. 105). This protoplasm consists of a network,
sometimes called a "filar mass" or spongioplasm, which lies em-
bedded in a homogeneous ground-substance or "interfilar maw " or
hyaloplasm. The filar oaass corresponds to the mitomaot Flemming
and t<> the Bponfjioplasm of some other authors, while the interfilar
mass corresponds to the p<traniito)na, or the hyoiofAasn^ or para-
of some authors.

142

PRACTICAL HISTOLOGY.

[VII.

Protoplasm

Nucle

Xuclc ar Membrane.
Xuclear Network.

The nucleus (fig. 105), bounded by a nuclear membrane, com-
posed of two layers, an outer one, which does not stain (achromatic),
and an inner one, which does (chromatic). "Within the membrane

is an intranuclear network or
karyomiton (Kapvov, a kernel)
or karyomitoma, consisting of
a reticulum of threads or fine
fibres, arranged sometimes in
the form of a regular network.
As these threads, or at least
particles in them, stain readily
with certain dyes, e.g., safranin,
FIG. 105.— Connective-Tissue Corpuscle from they have been called chromatiii

or composed of chromoplasm.

In the meshes of this more or less perfect network lies the nuclear
fluid, which, however, does not stain with certain pigments : it has
been called achromatin.

In the meshes of the reticulum lies one — usually more than
one — -nucleolus. It is more refractile than the rest of the nucleus.
Generally, however, as stated, two or more nucleoli are present,
and they seem to differ in their chemical constitution, so that
Flemming speaks of principal and accessory nucleoli. Many of
the bodies described as nucleoli are really parts of the intranuclear
fibrillar network seen in optical section. Other observers have
applied different terms to these structures, but here it is not
necessary to multiply terms. The "attraction sphere" existing in
the protoplasm of some cells seems to exercise some influence on
the dividing nucleus.

The great majority of cells reproduce themselves by indirect
cell-division or mitosis, and in this process the network within
the nucleus plays a most remarkable part. The division of a cell
is always preceded by the division of the nucleus. Starting from
the resting nucleus, where the threads are not well developed, soon
two poles appear in the nucleus, and then the threads grow
thicker, more numerous and tortuous, forming the convolution
stage. The various stages are indicated in the following
scheme : —

Mitosis.

1. Resting nucleus.

2. Skein or spirem.

the

The mother nucleus showing the fibrils
reticulum or network stage.

Close skeiu of fine convoluted fibrils, then thicker
loops running from polar to antipolar regions, the
nucleoli disappear.

3. Cleavage of fibrils. Each loop (usually v-snal)(1(l) splits longitudinally
into two and the achromatic spindle appears.

VII.]

MITOSIS OR KARYOKINESIS.

143

4. Monaster, Star, or
equatorial stage.

5. Metakinesis or

Divergence

6. Dy aster or

Double Star.

7. Dispirem or

Double Skein.

8. Network or

Reticulum.

The achromatic spindle distinct with two poles,
terminating in two polar corpuscles (with
cytasters), nuclear membrane lost. The V-shaped
chromatin fibres arrange themselves in the equator
of the spindle. Cytoplasm separates into a clear
and a granular zone.

Sister threads separate and move towards poles along
fibres of the spindle.

Complete separation of the two sets of V-shaped
sister thread", towards the poles of the spindle.

Open skein iu daughter nuclei passing into a close
skein. Nuclear membrane forming. Cell itself
divides.

Kesting condition of daughter nuclei. Cytoplasm
divided, remains of spindle disappear. Chromatic
fibres more twisted.

In the preparations one readily finds examples of these and the
other stages of mitosis.

Mitosis. — ])y far the best animals to use for studying the process
of mitosis are the larvae of the water-salamander. The young

FlG. 106.— Mitosis. A. Nuclear reticulum, resting-stage ; B. Preparing for division ; C.
Wreath stage ; I). Mouaster with achromatic spindle ; E. Barrel or pithode stage or
metakinesis, i.e., chromatin fibrils travelling along the achromatic spindle towards the
jioles ; F. Diaster ; G. Daughter wreath stage ; II. Daughter cells passing to resting-

animals must be carefully'fed, else the mitotic figures are not well
seen. They are killed at various stages, when they vary in length
from | to i inch in length. They may be hardened in J per cent.
chromic acid.

A very good hardening reagent is one-sixth p.c. chromic acid.
From a week to ten days is sufficient. If Flemming's mixture (p. 32)
lie used, the tissues must remain in it twenty-four hours or less.
Some prefer KabFs mixture (12-24 hours) (p. 31), others absolute.
alcohol or picric acid. After being " fixed " the tissues are placed in

144

PRACTICAL HISTOLOGY.

[VII.

30 to 50 per cent, alcohol, the alcohol being renewed frequently
until no colouring matter is given off. They can then be preserved
in strong alcohol, but on prolonged keeping the nuclei change
somewhat.

1. T.S. Tail of Larva of Salamander (H). — Stain a section for
12-24 hours, or even longer, in a solution of safranin (p. 75) — a
saturated alcoholic solution diluted with half its volume of water.

In dealing with such delicate sections,
it is well to " fix " the section on a
slide beforehand, especially if it be
cut in paraffin. The section may be
fixed by albumin and glycerine, the
paraffin removed by turpentine or

FIG. 107. — V.S. Epidermis of a Young T-,nn1iflin A ffpv efaininrr \viQliin cjnirif
Salamander with Kesting Nuclei, naplltlia. Attei Staining, wasll 111 *pll it

Monaster and Blaster Stages of and place ill acid alcohol (lOOCC.

amiilSfa^in,Cxi3ooChr d absolute alcohol to 3-5 drops of

hydrochloric acid). This rapidly de-
colorises it (J-i min.). Instead of acid alcohol the absolute alcohol
may be used. It removes the surplus stain more slowly. The
difficulty is just to hit the moment when the dye is washed out of
the nuclear matrix, the fibrils being still stained. The section is
then transferred to absolute alcohol and clarified, and mounted in
xylol-balsam. The best agent to clarify the section is cedar-wood

oil, as it does not dissolve
the safranin, which clove-oil
does.

(a.) Observe the layers of
epithelium of the epidermis
(fig. 107). In several of
the nuclei the characteristic
mitotic figures are to be
seen, and in one or two
sections it is not difficult to
pick out examples of nearly
FIG. 108. -Mitotic Figures from the Epidermis .^ t|ie stages of nuclear
of a Young Salamander. Chromic acid and . . ^

safranin, x 300. division. While the nuclear

fibres are well seen in

safranin-stained specimens, the nuclear spindle is not usually well
seen.

2. Surface-Scraping of tha Epidermis, Cornea, or External
Gills. — Instead of making a section, scrape the surface of the skin
of the tail, or break up the external gills in water, or stain (safranin)
and mount the cornea in xylol-balsam. Stain either with safranin
or logwood, mount in balsam, and numerous mitotic figures will be
found (fig. 1 08).

VII.]

MITOSIS OR KARYOKINESIS. 145

Some prefer to stain the sections,-— e.</., after hardening in picric
acid — -with Kleinenberg's logwood diluted with the alcoholic
solution of alum and calcium chloride (p. 68), allowing the sections
to stain for 12 hours or longer. They are mounted in balsam.

N.B. — It is important to note that tissues must be treated
differently according as one wishes to see the chromatic fibres or the
achromatic spindle. To see the ordinary mitotic figures, osmic acid,
or any fluid containing it, is good (12-24 hours) ; but in order to see
the achromatic spindle, it is better to use a chromo-acetic mixture
(p. 31) for 12 hours.

ADDITIONAL EXERCISES.

Mitosis, however, can also be studied in mammalian tissues.

3. Mitosis in Omentum of New-Born Rabbit.— Orth recommends the
omentum of a new-born rabbit. Harden it for twenty-four hours in Flem-
ming's fluid (p. 25) ; wash it thoroughly, and stain it in safranin-0 ; wash in
water, and remove the surplus dye, if necessary, by means of alcohol acidu-
lated with hydrochloric acid (p. 144). In the cells of the milk-spots and in the
walls of the blood-vessels it is easy to detect mitotic figures, but they are much
smaller than in the salamander.

4. Mitosis in the Amnion. — One of the readiest sources is the amnion of a
pregnant rat, as recommended by Solger. After the rat is killed, the uterus
is excised ;md placed in a saturated watery solution of picric acid. The uterus
and the membranes round each fcetus are opened under the picric fluid.
Harden for twenty-four hours ; wash well in water, and harden in the various
strengths of alcohol, beginning with 70 per cent. Better results are, I think,
obtained by removing tlie picric acid by washing in alcohol instead of water.
Select the amniotic membrane and tinge a small part of it in Ehrlich's acid
hsematoxylin (p. 69) diluted one-half. The membrane may ako be hardened
in Flemming's fluid and stained with safranin.

5. Method of Martimotti and Resegotti. — Small pieces of the tissue, e.g., a
rapidly growing tumour, are hardened in absolute alcohol. Sections are made
and coloured in a watery solution of safranin-0. The decolorisation, how-
ever, is obtained by a hydro-alcoholic solution of chromic acid. Take one to
two parts of a I per cent, solution of chromic acid to eight or nine of alcohol.
After it is sufficiently decolorised — i.e., the colour is removed from every part
except the fibrils of the nuclei — wash the section in absolute alcohol, clarify
in oil of bergamot, and mount in balsam. This method yields excellent
results.

6. Mitosis in Plants. — Various plants have been recommended, but my own
experience is limited to the following : —

(a. ) Take the fruit of Fritillaria iinperialis when they are 30-40 mm. in
length, and place them in absolute alcohol for a week. Then in equal parts
of glycerine find absolute alcohol for 24 hours. Then cut the fruit in two ;
with a dissecting microscope search for the embryo-sac. It shows various
stages of mitosis after staining for 12-24 hours in safranin. Mount in xylol-
balsaro.

(b.) A transverse section of the fruit of Liliumcandulum also does very well.
14 K

146 PRACTICAL HISTOLOGY. fvlll.

Harden the buds in absolute alcohol when they are about i-i| cm. long.
Stain in safranin and mount in xylol-balsam.

(c. ) I have also tried the young growing shoots of an onion bulb placed in
water. The tips of the growing rootlets were hardened in Fol's or Rabl's fluid
and stained with safranin. Fairly good specimens were thus obtained.

LESSON VIII.
CELLULAR AND HYALINE CARTILAGE.

Cartilage. — The varieties of cartilage are classified as follows : —

1 . Cellular or Parenchymatous occurs in the chorda dorsal is, ear

of mouse and rat.

2. Hyaline encrusts the articular ends of bones, occurs in costal,

trachcal, bronchial, laryngeal (except epiglottis and cornicula
laryngis), nasal cartilages, external auditory meatus, and in
the " temporary " cartilages of the foetus.

Fihrmm / ("') White nbro-cartilage.
3' ' '• 1 (&.) Yellow fibro-cartilage.

(a.) White fibro-cartilage occurs in the intervertebral discs,
interarticular nbro-cartilages, as marginal cartilages on
the edge of joints (hip, shoulder), lining tendon
grooves, in sesamoid bones, the sacro-iliac synchon-
drosis and symphysis pubis.

(b.) Yellow fibro-cartllaf/e occurs in the epiglottis, cartilages
of Wrisberg and Santorini, external ear, and Eustachian
tube.

I. Cellular Cartilage (H). — Kill a rat or mouge ; snip off the ear.
With a stout pair of forceps remove the skin and the other tissues
from the ear until the thin lamella of cartilage
which forms the basis of the ear is exposed.
Harden the cartilage in absolute alcohol. Mount a
thin part of the cartilage in Farrant's solution.
1. Cellular Cartilage (H).

(a.) Observe the clear cdh (fig. 109, c) ; some of
them may be spherical, but most are polygonal in
FlGCarUiageelfrom snaP°> closely pressed together, and united by a very
the Ear of a small amount of matrix or intercellular substance (m).
5co'ho£x 250. By focussing, rows of them in several planes may be

seen. Usually no nucleus is visible in the cells.
If desired, a section can be stained with hsematoxylin and
mounted in balsam.

VIII.] CELLULAR AND HYALINE CARTILAGE. 14?

II. Hyaline Cartilage. — This consists of cells or corpuscles em-
bedded in a hyaline matrix.

2. Cartilage of Newt (L and H).— Snip off a small piece of the
thin cartilage of the sternum of a freshly-killed newt, and with a
scalpel scrape away any librous tissue or

muscle adhering to it. Mount it in normal >«

saline solution or J per cent, solution of alum. \

(a.)Observe a homogeneous matrix (fig. no,
m\ like ground glass, in which are embedded m-~-^
here and there cartilage-cells or corpuscles
('•). The matrix is comparatively small in
amount, and hyaline. v'Cy

(?>.) Each corpuscle consists of a spherical
mass of transparent, finely-granular cell-sub- , k-
stance (c). Sometimes the protoplasm con- °*tftS. «5 iSSix?*
tains refractile granules of oil, and in it is Body of cartilage-cell ;

, , , . °, , , , , , n. Nucleus, x 250.

placed a spherical, clear, granular nucleus (n),

Near the margin of the preparation may be seen cavities or capsules
from which the cell-contents have fallen out ; others where the
cell-contents have shrunk from their capsule ; while at other places
the cells completely fill the spaces in which they lie. On focussing
through the thickness of the tissue, the cells are seen to be two or
more layers deep, i.e., in a section of moderate thickness they lie in
several planes. The cells may lie singly or in groups.

3. Effect of Acetic Acid (H).— Irrigate with a 2 percent, solution
of acetic acid.

(a. ) Observe that the nucleus becomes more distinct and granular,
the cell-contents clearer, and the cell shrinks from its capsule, so that
a space is left between the capsule and the irregular shrunken cell-
contents.

4. Action of Gold Chloride (H). — Mount in Farrant's solution
a section of articular cartilage from the head of the femur of a
fivshly-killed frog which has been stained by the gold chloride
method (p. 78). If the gold be reduced by formic acid the bone is
thereby softened, so that both bone and cartilage can be cut together
in a freezing microtome.

(a.) Observe the matrix faintly stained, and the corpuscles or
cell-contents, but not nucleus, stained of a purple hue. The cell-
contents have shrunk very little. Here and there an empty
cartilage-capsule may be seen. The gold chloride has a special
affinity for the protoplasm of the cartilage-cells, so that they stand
out distinctly in contrast to the less-stained matrix in which they
lie embedded. This preparation represents, as it were, the " positive
picture," in contrast to that obtained by the use of silver nitrate,
\vhii-h yields the "negative picture" (p. 77).

148

PRACTICAL HISTOLOGY.

[VIII.

As a general rule, it may be stated that gold chloride stains the
cellular elements, whilst silver nitrate stains the cement substance,
e.g., connective tissue, cornea, &c.

5. Costal or Tracheal Cartilage (L and H). — With a razor make
a thin transverse section of a fresh rib cartilage or tracheal cartilage,
e.g., of a dog, cat, or rabbit, and examine the section in normal
saline.

(a.) (L) Observe the circular or oval outline of the section
surrounded by the perichondrium firmly adherent to the cartilage,
which consists of cells embedded in a hyaline matrix (fig. 1 1 1, Pch).

Chb

FIG. in. — Hyaline Cartilage. T.S. Human thyroid hardened in alcohol, x. Fibrous
matrix ; Chb. Cartilage-capsules ; Pch. Perichondrium.

(ft.) (H) Observe that the cells are smaller and flattened near the
periphery, fusiform farther in, oval or spherical nearer the centre of
the section. They may lie singly, or in groups, or in rows.

(c.) The matrix is generally hyaline, but in some places it may
be fibrous (figs, in, x, and ii2,/).

(d.) Around each cell, or, it may be, each group of cells (fig. 112,
3 or 2), look for a cartilatje-capsule (fig. in). It is firmly united
to and continuous with the matrix ; but by tilting the mirror
slightly, so as to modify the light, it may be seen distinctly as a

VIII.]

CELLULAR AND HYALINE CARTILAGE.

149

well-defined membrane bounding the cell-cavity. Not unfrequently
several so-called " daughter-cells" may be seen within one capsule.

6. Hardened Costal Cartilage. — Mount a transverse section of
costal cartilage which has been hardened in a saturated solution
of picric acid. The picric

acid is removed by washing
in alcohol.

(a.) Stain a section with
picro-carmine and mount it
in Farrant's solution.

(/>.) Stain a section in
haonatoxylin and mount it
in balsam. The matrix is
stained of a light blue, and
the corpuscles of a deeper
tone.

(c.) A very good stain for
hyaline cartilage is MerkePs
indigo-carmine stain (p. 67).

The preparation can be X J

mounted in balsam, and is
not too transparent.

(d.) Carmine. — Place a
similar section for twenty-
four hours in a strong solu-
tion of ammoniacal carmine
(p. 63). Wash away the
surplus carmine, and allow a
drop of strong glacial acetic
acid to fall on the section. After a minute or so, wash the section
thoroughly in water to remove all the acid, and mount it in Farrant's
solution.

Observe the same arrangement of the cells as before, but the
cells are stained red while the matrix is colourless. The connec-
tive-tissue corpuscles of the perichondrium are also red. Round
each cartilage-cell is a thin outline deeper stained than the rest,
indicating the presence of a cartilage-capsule.

7. Fat in Cartilage-Cells (H).— Place a section of costal cartilage
(preferably made from a piece of costal cartilage taken from a
person over fifty years of uge) in i per cent, osrnic acid for an hour,
wash it in water, and mount in Farrant's solution.

(a.) Observe in some of the cells small and somewhat larger
aek spots, which are globules of oil blackened by the osmic

FIG. ii2.— T.S. Human Costal Cartilage, z. Cell
shrunk from the wall of its cavity, h ; i. Two
cells ia one cartilage-capsule, k; at a; is the
commencement of a separation wall ; 2. Five
cartilage-cells within one capsule, but the
lowest cell has fallen out of its cavity ; 3. Car-
tilage-capsule cut obliquely, so that it appears
thicker at one side ; 4. Cartilage-capsule not
opened into ; g. Hyaline matrix ; /. Fibrous
matrix, x 300.

black
acid.

8. Eosin (H).

-Stain with a watcrv solution of eosin a section

150

PRACTICAL HISTOLOGY.

[vni.

of human costal cartilage from an adult. The section becomes
uniformly red. Wash it in dilute acetic acid and mount it in
Farrant's solution.

(a.) The cells are more deeply stained than the matrix, and
numerous cells will be seen in groups or in rows due to the pro-
liferation of cartilage-cells. The cartilage-capsules are usually
more deeply stained than the surrounding matrix. Look for a
part of the matrix which has become fil>rous. It is deeply stained.
If the mirror be slightly tilted, or the light shaded from the pre-
paration by the hand, the cartilage-
capsules are usually distinctly seen.

9. Articular Cartilage. —Decalcify
the head of a long bone (e.fj., the femur)
of a cat or other animal in picric acid
Hyaline or chromic and nitric acid, with the
Cartilage. procautions indicated at p. 37. When
it is thoroughly decalcified, make — by
freezing— vertical sections, so as to in-
clude the encrusting cartilage and the
subjacent cancellous bone. Place some
sections in i per cent, osmic acid for
twenty-four hours, and stain others in
picro-carmine. Mount examples of
both in glycerine-jelly, as glycerine or
Farrant's solution makes the tissues
rather too transparent.

(a.) (L) Observe the layer of en-
crusting cartilage fixed upon the can-
cellated bone beneath (fig. 113), a
bold, irregular, wavy line separating
the cartilage from the bone, but the
one dovetails into the other. In the
FIG. n3.— v.s. Articular Cartilage, cartilage notice two areas, an upper and

Chromic and nitric fluid. Picro- } ° •,, V.val; ' mftfwC . «

carmine. ne, \\itn a nyai nx, an<

a lower, narrower one, with a more

granular matrix. The latter is the zone of calcified cartilage. A
fine wavy delicate line indicates where the hyaline matrix ends and
the calcined matrix begins. In the matrix note the cartilage-cells,
flattened at the circumference — i.e., next the joint cavity — in small
groups deeper down, and in vertical rows in the substance of the
cartilage.

(/>.) (H) Study the shape of the cells from the free or joint surface
downwards. At the circumference they are flattened or fusiform, and
deeper down they are more or less polyhedral and arranged in vertical
rows. Some of the cells may be somewhat shrunk within their

Calcified
Cartilage.

Bone.

VIII.

CELLULAR AND HYALINE CARTILAGE-

capsules. A row of cells may be seen partly in the hyaline and
partly in the calcified matrix. Note the finely
granular character of the calcified matrix.

(''.) Observe the bone with its lamellse and
bone-corpuscles, and its open meshes containing
bone-marrow.

10. Cartilage of Cuttlefish (H).— Mount in
Farrant's solution a section of the cephalic car-
tilage of a cuttlefish. The cartilage must have
been hardened previously in picric acid, alcohol,
or osmic acid.

(a.) Stain a section in picro-carmine. Observe
that the cells lie in groups of three or four, and
from the periphery of the group processes are
given off which anastomose with processes from
adjoining groups of cells (fig. 114).

Eosin and Hsematoxylin. — Stain a section
slightly with a dilute solution of eosin and after-
wards with dilute hsematoxylin. Mount in
Farrant's solution. The matrix is reddish, and the cells and their
processes purplish in hue.

FIG. 114. —Branched
Cartilage-Cells of the
Cartilage of Loligo.
Picric acid, eosin, aud
logwood.

ADDITIONAL EXERCISE.

11. Silver Nitrate and Cartilage Matrix (H).— Rub a piece of solid silver
nitrate upon the cartilaginous end of the freshly-excised femur of a frog.
Kxpose the cartilage in water to sunlight. It rapidly becomes brown. Make
a thin surface section with a razor, and mount it in Farrant's solution.

(a.} Observe the matrix stained brown, and a large number of unstained
spaces apparently empty. The latter are the cavities in which the cells lie,
but the cells themselves are too transparent to be readily seen. This picture
is the reverse of that obtained with gold chloride (p. 147).

LESSON IX.
THE FIBRO-CARTILAGES (WHITE AND YELLOW).

III. Fibro-Cartilages.
A. White Fibro-Cartilage.

1. Intervertebral Disc. — Decalcify in chromic and nitric acid
fluid (p. 37) or picro-sulphuric arid (24 hours) an intervertehral disc

152

PRACTICAL HISTOLOGY.

[IX.

and its adjacent pieces of bone (rabbit or cat). By freezing make
vertical sections to include the disc and its adjacent bones ; place
the sections for twenty-four hours in i per cent, osmic acid, wash them
thoroughly in water, and mount in Farrant's solution or glycerine-

jelly. '

(a.) (L) Observe the decalcified bones, and between them the
disc (fig. 115). The bone consists, for the most part, of cancellated
bone, and the disc stretches between the two plates of denser bone
which cover the ends of the vertebrae. A thin layer of hyaline
cartilage exists on the surface of the bodies of the vertebrae.

(b.) The disc itself is of considerable thickness, and consists of
many parallel bundles, between which, and at right angles to them,

are other bundles cut
transversely, the fibres
of adjoining bundles
being arranged some-
times in a zigzag
fashion (fig. 115). In
these bundles are carti-
lage-cells, more numer-
ous in the central
bundles and fewer in
the outer ones. In
the centre of the disc
may be seen a more
pulpy tissue, the re-
mains of the chorda
dorsal is.

(c.) Externally on
both sides is a ligament
of connective tissue

which passes from one vertebra to the other. It gradually shades
into the fibres of the disc.

(d.) (H) Observe the fibres, with a greater or less number of large
oval cells lying between them. The fibres can perhaps be traced
into the matrix of the bone.

(e.) The cells — oval and with a hyaline capsule — are most numer-
ous in the central part of the disc, and usually there is no difficulty
in seeing groups of cells in the hyaline cartilage forming a thin
coating on the bone. The boundary-line between the disc and the
bone is never straight, but wavy. This can readily be made out by
tilting the mirror slightly.

2. "White Fibro-Cartilage (H). — Snip off a small piece of the
intervertebral disc of an ox or sheep or man, after hardening a small
portion for a day in a saturated solution of picric acid or spirit, or

FIG. 115 — V.S. Intervertebral Disc of Cat. B. Bone ; D.
Disc. Chroino-nitric acid fluid and osmic acid, x 15.

IX.] THE FIBRO-CARTILAGES. 153

picro-sulphuric acid (twenty-four hours). The hardening is com-
pleted in the various strengths of alcohol. Tease it with needles
in Farrant's solution.

(a) Observe the fibrous matrix, consisting of very fine, wavy
unbranohod fibrils (fig. 116), and between them oval
nucleated cells, each one with a
distinct thick hyaline capsule.
In some of the latter, concentric
rings indicating the deposition of
successive capsules, may be seen.

(b.) The bundles of fibres run
in various directions, and each
fibril is unbranched. The cells
— with thick capsules — are not
very numerous, and lie either
singly or in groups of two or
three between the fibres.

3V Another Method. — The
cells' of this cartilage do not liu-<; u>

stain very readily, but the follow- FIG. 116.— From Human Intervertebral Disc.

., , . , •,, m. Matrix or fibrous ground-substance;

ing method gives good results : c. Cartilage-cell ; k. Capsule surrounded

-Harden the cartilage-small j[J* °™£S^!? x £"»"»*•
pieces— for a day or so in

Kleinenberg's fluid (p. 30), and after washing the pieces free from
the picro-sulphuric acid, place them for several days in borax-
carmine, and stain them en masse. Concentrate the pigment in
the cells by placing the pieces in acid alcohol for twenty-four hours
(\\ 65). Sections can be "made either by freezing or embedding in
paraffin. The cells are stained bright red.

B. Yellow Fibro-Cartilage. — (i.) Harden the epiglottis of a sheep,
dog, or cat for forty-eight hours in absolute alcohol.

(ii.) Harden a part of .the ear of a pig in a saturated solution of
picric acid for twenty-four hours. Wash away the picric acid with
alcohol, and in the various strengths of alcohol complete the
hardening.

By freezing make sections of the epiglottis and ear.

4. Epiglottis. — Stain a section in picro-carmine and mount it in
Farrant's solution.

(«.) (L) Neglecting the stratified epithelium and glands which
are present, observe the perichondrium (fig. 1 1 7, c,/), embracing the
mass of cartilage, and firmly adherent to the latter, which has a
fibrous, yellow-stained matrix, studded with cells — stained red —
embedded in it. The mass of cartilage may appear to be interrupted.
or it may even be perforated.

(b.) (H) Observe the perichondrium, composed of connective
15

154

PRACTICAL HISTOLOGY.

[IX.

tissue, with numerous elastic fibres ; the latter can be traced into,
and become continuous with, the elastic fibres of the matrix (iig.
117, e, /). The matrix consists of fine branched and anastomosing
fibres of elastic tissue, stained yellow with picric acid. Where the
fibres are cut transversely, they appear as yellow dots or granules.

In addition to these, however, there
are numerous granules of elastin
scattered in the matrix. In this
meshwork notice the nucleated
cells stained red. Each cell has a
capsule, but near the perichondrium
they are smaller and flattened (/),
while in the substance of the carti-
lage they are larger, oval, or
spherical (?•).

5. Acid Fuchsin Method. — Stain
a section with a watery solution of
acid fuchsin. Wash the section for
a long time in absolute alcohol and
mount in balsam. The network is
intensely red, and the other parts
uncoloured.

6. Double Staining of the Epi-
glottis.— (i.) Stain a section Avith
picro-carmine, and then faintly with
logwood. Mount in balsam. To
preserve the yellow colour of the
fibrils, the clove-oil, with which the
section is cleared up, must be made
yellowish by dissolving in it a little
picric acid. The fibres are yellow,
the cells red, and the nuclei
purplish.

(ii.) Stain another section with

,.™- ,~ >™«, .- , ,, ~ dilute eosin-hsematoxylin, and mount

Elastic fibres ; i. Superficial layers of it in Farrant's solution or balsam.

The cells take the logwood tint, and
Alcohol and picrb- the fibres the colour of eosin.

7. Ear of Pig or Horse. — Stain
a section in picro-carmine, and mount it in Farrant's solution.

(L and H) Observe the skin, its glands and muscles. Neglect
these, and note the perichondrium enclosing the cartilage with a
characteristic arrangement of the cells. The cells near the surface
are small, flattened, and parallel to it, while those in the centre
are larger and arranged across the long axis of the section.

n

FIG. 117 — T S. Epiglottis

Fat-cells in perichoudrimn

f a Dog.
c

smaller cells ; r. Layer of larger
cells with elastic grannies, l.\ f.
Pericliondrium.
carnune.

IX.]

THE FIBRO-CARTILAGES.

155

Between the cells is a matrix, which may be partly hyaline and
partly yellow fibrous.

8. Transition of Hyaline to Elastic Cartilage (H). — Dissect
out the arytenoid cartilage of an ox or sheep. Harden and pre-
serve it in alcohol. Cut sections through the part where the
hyaline cartilage merges into the elastic variety. This is quite
visible to the naked eye, the elastic part being more opaque and
yellowish white in tint. Stain a section with picro-carmine, and

. 1 18.— Elastic Cartilage Ear of Horse,
hardened in alcohol. El. Elastic fibres cut
in various directions ; K. Nucleus of
cartilage-cell ; Knh. Contour of cartilage-cell
cavity ; Knh'. Empty cartilage capsule ; Knz.
Cartilage-cell.

FIG. ng. — Elastic Cartilage developing in
Hyaline Cartilage in Arytenoid Carti-
lage of a Calf, x ioo. The clear spaces
indicate the position of the cells, the
shadow part the hyaliue matrix.

mount it in Farraiit's solution. On making a section of such a
cartilage in the fresh condition, one part has the pale-bluish colour
of hyaline cartilage, and the other part is very faintly yellow.

(a.) At one part observe hyaline cartilage, whose matrix
gradually becomes fibrous. At first only a few scattered granules
of elastin are seen, then the hyaline matrix is traversed by elastic
fibres, and gradually the matrix loses its hyaline character, and
becomes distinctly fibrous. Around each cell there is a clear area
— hyaline — devoid of fibres (fig. 119).

156 PRACTICAL HISTOLOGY. [X.

LESSON X.
CONNECTIVE TISSUE.

THE group of Connective Tissues includes cartilage, ordinary
connective tissue (with adipose tissue), adenoid or retiform tissue,
mucous tissue, bone and dentine, (i) These all subserve more or
less mechanical functions in the organism; (2) they all have much
in common in structure, i.e., they are composed of cells, and an
intercellular matrix, but usually the development of the matrix
exceeds that of the cells ; and (3) they are all developed from the
mesoblast of the embryo.

ORDINARY CONNECTIVE TISSUE.

It consists of the following structural elements : —

A. Structural Elements.

-r,.-, ( White or gelatinous.
Fibres. | Ye]lQw or&elastic>

f fl. Flattened or lamellar cells, called also fixed oon-

j§ j nective-tissue corpuscles.

5 j Fixed . . •{ 2. Granular cells (eosinopliilous cells ?).
1,1 j 3. Vacuolated or plasma cells of Waldeyer.

^o I I .Clasmatocytes of Ranvier (?).

I Migratory. Wandering cells or leucocytes.

B. Arrangement qf these Elements.

(a.) Areolar, e.g. , subcutaneous and submucous tissues.

(6.) Bundles in parallel groups, e.g. , tendon (with parallel fibres) and

fasciae (fibres crossing at right angles),
(o.) Fenestrated fibrous membranes, e.g., omentum.
(d.) Compact bundles crossing in all directions, e.g., skin.

The lamellar cells are flattened or winged plates which lie on
the bundles of fibrils. They have a large oval nucleus lying in a
clear plate.

The granular cells, or " Mastzellen " of Ehrlich, are often found
near blood-vessels, and in the -fat present in areolar tissue, in
the submucous tissue of the intestine, and in Glisson's capsule.
The cells are often spherical, and the granules are numerous and
proteid in nature, and stain with aniline dyes, e.g., cosin, hence
the term sometimes applied to them "eosinopliilous cells."

The plasma cells were formerly confused with the foregoing,

X.]

CONNECTIVE TISSUE.

157

but in the plasma cells the protoplasm is vacuolated, and the
vacuoles contain fluid. They sometimes have short processes.
What the relation of the clasmatocytes to these other cells may be
is so far not determined ; nor, indeed, do we know the relation
between the granular and the plasma cells.

The migratory cells are identical with the white blood-cor-
puscles or lymph-corpuscles, and may therefore be regarded as an
adventitious element.

Yellow or Elastic Fibres occur in the ligamentum nuchse of
animals (large fibres) ; lig. subflava ; stylo-hyoid ligament ; con-
nective tissue generally ; in the walls of the air-tubes and lungs ;
the larger blood-vessels, especially arteries ; the vocal cords and
some ligaments of the larynx ; many organs, e.g., spleen.

1. Yellow or Elastic Fibres— Thick Fibres (H).— Tease out
in water a fragment of the ligamentum nuchse of an ox ; cover
and examine it. It can be mounted in Farrant's solution.

(a.) Observe the broad
fibres with a definite
outline, yellow in colour,
refracting the light
strongly, branching and
anastomosing, and some-
times curling at their
ends where they are
broken across (fig. 120,
/). A small quantity
of white fibrous tissue
will be found between
and supporting the
ill .res (/,).

(/>.) Measure the size
of one of the larger
fibres. They are about
7_8 /A (;nWth mch) in
diameter.

((-.) Irrigate with acetic
acid. The fibres are not affected, and no nuclei are revealed in
them. They consist of the substance elastin, which is unaffected
by acetic acid.

Make longitudinal and transverse sections of the ligamentum
nuchse (hardened in alcohol). Stain both in picro-carmine and
mount in Farrant's solution. The connective tissue is thereby
stained red, and the elastic fibres yellow.

2. L. S. Ligamentum Nuchae (H). — Observe the fibres (yellowish),
with a small amount of connective tissue (red) between them. The

FIG. 120.— f. Elastic fibres from the
undue ; f>. Fine white fibrous tissue, x 300.

158

PRACTICAL HISTOLOGY.

[X.

lil)res ; c. Connective-tissue
between them; n Nuclei
of connective-tissue cor-
puscles. Alcohol and borax-
carmine, x 300.

fibres are broad with well-defined margins, have a feeble yellow

tint, and are transparent. They branch and anastomose, and where

ruptured curl up at their ends.

3. T.S. Ligamentum Nuchse of Ox (H). — Observe the polygonal

ends of the broad fibres— yellow — and nearly as broad as, or broader
than, a coloured blood-corpuscle, sometimes
single, mostly in groups of three or more
(fig. 1 2 1, a) — homogeneous throughout. A
small amount of connective tissue (c) (red)
between the groups.

4. Another section may be stained with
a watery solution of magenta and mounted
in Farrant's solution. The fibres are stained
red, but the pigment is ap6 to diffuse into
the Farrant's solution.

5. A good plan is after hardening the
ligamentum nuchse in alcohol to stain it in
borax-carmine for several days, with the

precautions stated at p. 65. Transverse sections show the white
fibrous tissue between the elastic fibres, with its nuclei stained red
(fig. 121, n).

6. Fine Yellow Elastic Fibres (H). — Harden the mesocolon or
mesentery of a young rabbit in Flemming's fluid, and stain it in

methyl-violet as directed under Lesson X. 14,

^^^^^^^^^ or stain it with magenta, when the elastic
fibres are stained red ; or with safranin after
hardening in chromic acid.

(a.) Observe the network of fine elastic
fibres. Many of the fibres have a diameter
equal to one-sixth, or less, of that of a
coloured blood-corpuscle (i p. or TT^.^Q- inch
Mesocolon of Rabbit, in diameter). The fibres branch and anasto-

J lemming s fluid and j i r 1 1 j?

safranin. mose, and by carefully focussing, one can

observe that the fibres do not all lie in
the same plane (fig. 122).

7. Fenestrated Membranes (H). — Sometimes the elastin is so
arranged as to form sheets or plates of elastic tissue, e.g., in the
large arteries ; at other times these are perforated with holes, and
are called fenestrated elastic membranes.

With a pair of forceps tear off a little of the endocardium from a
sheep's heart, spread it on a slide, and treat it with caustic potash.
Or use the basilar artery, slit it up and scrape away the outer coats,
and use caustic potash as before.

(a.) Observe near the margin of the preparation the elastic
membrane with holes in it (lig. 123).

X.]

CONNECTIVE TISSUE.

159

8. White Fibres of Areolar Tissue (H). — Dissect off a thin
lamella from an intcrmuscular septum, or remove a little of the
subcutaneous tissue of a rabbit
or rat. Place it on a dry slide,
and rapidly spread it out into a
tli in film, but do not let it
become dry, which can easily be
avoided by breathing on the
] (reparation. This is known as
the "half-drying" or "semi-
desiccation method," and is a
very useful one, especially for
sections containing much con-
nective-tissue. Place a drop of

iim-iivil Qilinp enliitmn rm fhr* FIG. 123.— Network of Thick Elastic Fibres.

n. Pa*siiiff into a fenestrnted membrano.
COVer-glaSS illld apply it to the »«•• Human endocardium. Fresh and

preparation.

(a.) Observe the unbranched white fibres, wavy in their course,

EL¥-

Bilgf

Bilgf

Bdgf Lc Lc

FIG. 124.— Areolar Tissue from Intermiiscnlar Tissue of a Oalf, x 200. I'.dirf. ( 'ouuective-
tissiie fibres, i.e., bundles of fibres ; Bdgz. Connective-tissue cells ; EIF. Elastic fibres ;
Lc. Leucocytes.

with a faint, ill-defined outline, crossing each other in various
directions. They are colourless, of feeble refractive power, and

i6o

PRACTICAL HISTOLOGY.

[X.

transparent. The fibres are striated longitudinally, and are seen to
be made up of excessively delicate fine unbranched fibrils. The
fibres vary from 6 /u to 8 /* in diameter (-^^-^^ inch). They
may be round or flattened, and are of indefinite length. Amongst
the white fibres may be seen a few fine elastic fibres (E1F), recog-
nised by their sharper contour, and by the fact that they branch
and anastomose. . They run between, but never in the white fibres.
It is not often that the corpuscles are visible without the action of
special reagents. The corpuscles are best seen in young animals
(fig. 124, Bdgz).

(b.) Irrigate with a 2 per cent, solution of glacial acetic acid ;
observe that the white fibres swell up, become clear, gelatinous,

and homogeneous ; the elastic fibres
being unaffected, come clearly into
view. The latter have a sharply-
defined outline, branch and anasto-
mose, and sometimes curl at the ends.
The corpuscles, or at least their
nuclei, come into view. Observe
the oval or fusiform nuclei of the
fixed connective - tissue corpuscles
(Bdgz)— they may be surrounded
with some soft protoplasm — and
the much smaller compound nuclei
of the wandering cells or leucocyte*
(Lc). In the rat especially one is
very likely to find the very granular
nucleated cells known as granular
cells. They frequently lie along
the course of the small blood-
vessels. Do not preserve this
specimen.

If the areolar tissue be taken from the sub-arachnoid space of
the brain and treated with dilute acetic acid, the fibres lose their
librillated structure, nuclei appear, and the fibres themselves may
be seen to swell up here and there, while they are constricted at
irregular intervals by a thin fibre. This is due to these fibres
being partly embraced by connective-tissue cells, which have long
branches which partly encircle the fibre.

9. Fenestrated Fibrous Tissue (L and H). — Harden the
omentum of a dog or cat in Miiller's fluid. Stain a piece in logwood,
and mount in balsam.

Note the meshes bounded by areolar tissue. The fibrils which
compose the fibres are readily seen, and sometimes an cnclotliolial
cell may be seen partially detached (fig. 125, E'), for the omentum

Flo. 125.— Omentum of Dog. E', E". Par-
tially detached endothelial cells. E.
Nuclei of endothelial cells, x 130.

X.]

CONNECTIVE TISSUE.

161

lying in a serous cavity is covered with endothelium. The nuclei on
the surface are the nuclei of endothclial cells, and those in the sub-
stances of the trabeculffi belong to connective-tissue cells.

ADDITIONAL EXERCISES.

10. Martinotti's Reaction for Elastic Fibres ( H ). — Harden elastic tissue,
e.g., ligamentum nuchse, or an organ containing elastic fibres, e.g., skin,
artery, lung, trachea, in .2 per cent, chromic acid for three weeks. Cut
sections and place them for twenty-four to forty-eight hours in a saturated
alcoholic solution of safranin-0. Wash them in acid alcohol (p. 65), and then
in absolute alcohol, to remove the surplus dye ;

clear in xylol, and mount in xylol-balsam.

(a.) All the elastic fibres, and they alone, are now
either purplish, or, if the fibres be fine, black.
This is a most excellent method for differentiating
elastic fibres. The one thing of importance is to
secure a good sample of safranin ; some samples are
quite inactive.

11. Elastic Fibres (ffcrxheimer's Method).— Place
the sections containing elastic fibres in an alcoholic
solution of hjematoxylin, to which is added a few
drops of a saturated solution of lithium carbonate.
Stain them for a few minutes. Place them for five
to twenty seconds in tincture of perchloride of iron,
which rapidly decolorises all except the elastic
fibres, which remain bluish or blackish. Wash in
water and mount in balsam. This method is admir-
ably adapted for demonstrating the longitudinal
layer of elastic fibres in the trachea and bronchi.

12. Violet-B Method. — Cut out the hyaloid mem-
brane of a frog's eye, or a piece of the omentum of a
young rabbit, or the suspensory ligament of the liver
of a rabbit. In normal saline pencil away the epi-
thelium covering the membrane. Stain the section
with violet-B (i gram violet- B in 300 cc. of normal
saline). This stains the cells and the elastic fibres.
The preparation cannot be mounted in glycerine
or Farrant's solution, as these dissolve out the dye,
but a strong solution of common salt may be used
(,V. Mayer).

13. Areolar Tissue— Permanent Preparations.
(i.) By means of a hypodermic syringe (fig. 126)

make an interstitial injection of silver nitrate

(i : 1000) into the subcutaneous tissue of a dog or

rabbit. In this way an artificial cedema is produced

and the tissues are " fixed." With a pair of scissors

curved on the flat, snip out a little of the now cedematous connective tissue

and stain it with picro-carmine. It requires some time to stain, and the

preparation should be left for ten to twelve hours in a moist chamber

.^fig. 70), and then the picro-carmine is slowly displaced by acid glycerine,

i.c., glycerine slightly acidulated with formic acid. In this way the

FIG. 126.— Hypodermic Sy-
ringe for making a Sub-
cutaneous or Interstitial
Injection.

1 62

PRACTICAL HISTOLOGY.

[X.

various elements — fibrous and cellular — are usually brought distinctly into
view (Ranvier}.

(ii.) An excellent plan is to inject picro-carmine interstitially, and to leave
the bulla several hours before snipping out a small part of it and mounting
it in formic glycerine, hi this preparation connective-tissue fibres with con-
strictions at intervals are frequently seen.

(iii.) A fibre may be stained with acid haernatoxylin and mounted in gly-
crrine (p. 69).

14. Coarsely Granular Cells (" Mastzcllen " of Ehrlicti). —Place part of the
omentum of a young rabbit — or the fat from around the kidney of a rat or
rabbit — in a watery solution of gentian-violet, to which a filtered watery solu-
tion of aniline-oil has been added. Heat the
whole in a capsule until the vapour begins to
rise, and allow it to cool.

After staining for twenty-four hours, remove the
tissue and wash it in acid alcohol until most of the
blue is gone. Dehydrate it in absolute alcohol,
dear with xylol, and mount it in xylol-balsam.

(H) Search for a blood-vessel, and along its
course will be found large oval cells crowded
with numerous granules stained blue (fig. „ 127).
These cells are found also apart from the blood-
vessels.

15. Gentian-Violet and Carmine Preparation.
— The preparation may be double stained, thus :
After washing in acid alcohol, stain the prepara-
tion in lithium-carmine for a few minutes, and
again extract with acid alcohol. Mount as before
in balsam. Observe the granules of the cells,
blue as before, the nucleus red. All the other
nuclei in the field are now red.

16. Clasmatocytes. — Ranvier1 has given the name clasmatocyte (tc\d(ru,a,
fragment, KVTOS, cell) to cells which can be seen in thin connective-tissue mem-
branes of vertebrates. In Triton cristatus these cells may be I mm. in length.
Stretch a membrane, e.g., the omenturn of a mammal or mesentery of a small
reptile on a slide. Fix its elements by dropping on it a drop of I per cent,
osmic acid, and then stain it with methyl-violet-BBBBB (i part) dissolved
in distilled water (10 parts). Examine the preparation in the fluid or in
water. Large branched cells are seen stained of
a bluish tint. At the extremities of the branches
are small islands or granulations similarly tinted,
and it is for this reason Ranvier has given them
this name.

17. Cell-Spaces in Areolar Tissue (H). — From
a freshly-killed rabbit snip out a small piece of
the subcutaneous tissue as free from fat as pos-
sible. Spread it upon a dry slide, and drop on it
FIG. 128.— Cell-Spaces in Areo- from a pipette a half per cent, solution of silver
lar Tissue. Silver nitrate, nitrate. Allow the silver to act for ten to twelve
minutes, remove it, cover the film with glycerine

and expose it to light. It rapidly becomes broun. It is better to use con-
nrctive-tissue from a calf, but the layer used must not be too thin.

(a.) If successful, note a brownish ground — the cement substance — and in it
clear branched spaces corresponding in shape to the fixed connective-tissue cor-
puscles. These are the cell-spaces (fig. 128).

1 Comptcs Rcndus, vol. 110, p. 165, 1890.

FIG. 127.— Coarsely Granular
Cells, the " Maslzellen " of
GcTinnn authors, from Rat.
/. Fat cells ; v. Vein ; in.
" ^lastzelleii.'

XI.]

TENDON.

18. Perioesophageal Membrane of Frog. —To Rnnvier1 we owe our know-
ledge of the value of this membrane. The oesophagus is surrounded by a lymph-
sac, which is separated from the pleuro-peritoneal cavity by an excessively thin
membrane. The sac is readily distended by insufflation, after pulling out the
oesophagus through the mouth by means of a hook. This membrane is covered
011 both surfaces by epithelium which can be stained with silver nitrate ; its
texture consists of fine connective-tissue with elastic fibres, but containing
clasmatocytes, and in it are also to be found blood-vessels and non-medullated
nerves. Treat it as described in 16, to see the clasmatocytes. By the same pro-
cess the non-mt'dullated. nerve fibres and elastic fibres will also be stained.

LESSON XI.
TENDON.

Tendon is composed of white fibres arranged longitudinally and
parallel to each other. The fibres are arranged in bundles, the
tendon-bundles, which are held together by a slieath and sepia of
connective-tissue. The fibres are united to
each other by a cement substance, and on
the- primary bundles of the fibres are placed
the tendon-cells, which vary in their shape
and arrangement in different tendons. It
is supplied by few blood-vessels, and con-
tains only a few elastic fibres.

Tendon. — Harden a small tendon of a
man, calf, dog, or cat, in Miiller's fluid,
alcohol, bichromate of potash, and com-
plete the hardening in alcohol. By freez-
ing, make transverse and longitudinal sec-
tions, or use the celloidin method. Tendons
cannot be cut after being embedded in
paraffin, they become too hard.

1. T.'S. Tendon (L).— (a.) Stain a sec-
tion in picro-carmine, and mount it in
Warrant's solution. Observe the sheath
(fig. 129, #), composed of connective tissue
arranged circularly, sending septa (/) into
the substance, of the tendon, thus breaking it up into polygonal
areas of different sizes, which are filled by the cut ends of the
longitudinally-arranged iibn-s.

1 CoMjiffs lli-mtiis, vol. Ill, p. 863, 1890.

FIG.

129.— T.S. Tendon. s.
Slieath, with b. Blood-ves-
sel ; t. Trabeculsc or septa ;
c. l.ranchfd spaces in the
tendon for tendon-cells;
I. Matrix or cut ends of
the fibres, x 50.

164

PRACTICAL HISTOLOGY.

[XI.

(b.) The branching stellate spaces, interfascicular spaces (fig.
129, c), between the fasciculi or bundles of fibres. If these spaces
contain air, they appear somewhat dark. These spaces can readily
be seen as branched dark spaces if a transverse section is made —
by means of a knife, not a razor — of a small tendon dried at the
ordinary temperature.

(c.) (H) Observe the cut ends of the fibres (I), which appear
almost homogeneous, but amongst them here and there may be
seen a few dots, which are the transverse sections of elastic fibres.

and the branched tendon-
spaces (c), some of them
with a nucleated branched
cell.

2. L.S. Tendon.— Stain
a section with logwood, and
mount it in balsam. (H)
Observe the longitudinal
arrangement of the fibres,
and between them rows of
fusiform tendon - cells, or
rather the long fusiform
nuclei of the tendon cells
arranged between the
fibres. The other parts of
the cell become too trans-
parent to be seen (fig. 130).
Sometimes a L.S. of one of
the septa may be seen.

3. Fibrils in Tendon
(H). — Macerate a tendon
from the tail of a rat for
twenty-four hours in a satu-
rated solution of picric acid,
or for 3—4 hours in baryta
water. Tease a small part,
and examine it first in

water. Mount it in Farrant's solution. This is apt, however, to
render the fibrils too transparent. Perhaps a better method is to
place the fine tendons for twenty-four hours in equal parts of
i per cent, osmic acid and i per cent, silver nitrate. Mount a
teased preparation in Farrant's solution.

(«.) Observe the isolated fibrils (-^-Q^-Q-Q inch in diameter), exces-
sively fine; wavy, and unbranched (fig. 131).

4. Tendon of Eat (Gold Chloride Method).— Kill a rat, cut off
its tail, forcibly rupture the tail, when a long leash of fine white

FIG

u

Rows of nuclei of tendon-cells.

XL]

TENDON.

165

glistening threads or tendons will be obtained. Prepare the
tendons by one of the gold chloride methods. One of the best
methods is the lemon-juice method of Ranvier (p. 79), but the
boiled formic acid and gold method also
yields excellent results. It is to be re-
membered that it is not necessary to use
gold chloride to demonstrate the tendon-
cells ; this can be done by hsematoxylin.

With regard to the action of gold
chloride, my experience leads me to
believe, that in order to see the rows of
tendon-cells with their lateral protoplasm
expansions, the lemon-juice method is
very good; while the old acetic acid
method makes the fibres less swollen up,
and on teasing they are readily isolated,
thus enabling one to see cells either
singly or in rows clasping them. . Not un-
frequently isolated tendon-cells are to be
seen in the field of the microscope.

Tease a small part of the gold tendon in Farrant's solution.

(a.) (H) The fibres are swollen up and transparent, and lying 011
them are rows of tendon-cells (fig. 132, b, b) stained of a violet
tint. Each cell is somewhat oblong with a distinct nucleus, and

. i3i.— Fibrils of Tendon of
Rat Isolated by Picric Acid,
x 300.

li.. i 52.— Gold Chloride,
Tendon, Tail of Rat. a.
Tendon - cells seen on
edge and embracing a
fibre; bb. On the fiat,
the cells with a ridge.

FIG. 133.— Tendon-Cells, Tail of Rat. c. Cells ;
p. Lateral prolongation or expansion uf the
cell protoplasm ; «. Nucleus ; a. Stripe or
ridge.

bears a flattened wing-shaped expansion (fig. 133). Along the colls
is usually to be seen a stripe or ridge (fig. 133), produced by the
cells being compressed between several adjoining fibres. This ridge
may be seen to be interrupted in some of the cells. The nuclei of
the adjacent cells may be seen to be close together.

1 66

PRACTICAL HISTOLOGY.

[XL

(b.) If a side view of the cells is obtained (fig. 132, a), they
partially clasp the fibre, but never envelope it completely ; in this
respect these cells differ from endothelium.

5. T.S. of Gold Tendon (H). — Kemove the skin from the tail of
a young rat, cut out a piece of the tail a quarter of an inch in
length with its tendons, and subject it to the gold chloride process
(p. 79). When, after reduction, it has become purple or brownish,
decalcify the bone, harden it in alcohol, and make transverse
sections. Mount one in balsam.

(a.) Many tissues will be seen, including muscle, nerve, fat, and
bone. Neglecting these, observe the small rounded areas at the
circumference, the transverse sections of the small tendons, each
surrounded by its own sheath of connective tissue (fig. 134, t). In
each observe the branched stellate spaces (fig. 134, c), frequently
anastomosing with each other. These interfascicular spaces are

..I

FIG. 134.— T.S. of a small Ten-
don, Tail of Kat. t. Sheath ;
c. Interfascicular spaces
with tendon -celU. Gold
chloride.

thellal Cells on the Sur-
face of a Tendon, Tail of
Rat. Silver nitrate.

purplish in colour ; they contain the tendon-cells, and also a
purplish deposit due to the gold chloride acting on the lymph which
they contain in the fresh condition.

6. Endothelial Sheath of Tendon (L and H) —Silver a leash of
the fine tendons from the tail of a rat. Mount a short length of
one of them in balsam.

(a.). Observe the tendon made up of parallel fibres, and note on
their surface a single layer of endothelium. The squamesare large,
polygonal, and mapped out by "silver lines," but no nuclei are
visible.

7. Fresh Tendons and Acid Logwood (H).- — Place three or four
tendons (rat's) i| inch long, on a dry slide, and fix their ends with
paraffin, so as to keep them extended. Make a solution of acid
logwood by adding one part of i per cent, glacial acetic acid to
three parts of. logwood solution. This solution is red. Place a
drop of it on a cover-glass, and lay it on the tendons. The acid
brings into view rows of narrow, granular, nucleated cells between

XI.] TENDON. 167

the fibres of the tendon, while at the same time the logwood stains
them. Instead of acid logwood use picro-carmine or acid hsema-
toxylin (p. 69). Displace the dye with water and mount in
glycerine. The tendons are purposely taken longer than the
breadth of the cover-glass, so that they may remain stretched.

8. Fresh Tendon. — On a black surface, tease in normal saline a
small piece of any tendon of a calf. Observe the fibres and fibrils,
but no cells are visible. Irrigate the preparation with 2 per cent,
acetic acid. The mass becomes clear and transparent to the naked
eye, and now under the microscope one sees the fusiform nucleated
cells singly or in line in order between the swollen-up fibres. The
preparation may be stained with magenta, which brings into clearer
view the cells, and any elastic fibres present,

ADDITIONAL EXERCISES.

9. Tendon of Eat (Dogicl's Method). — Very good preparations are obtained
by placing the fresh tendons for several days — the longer the better — in
Grenadier's alum-carmine (p. 65). This fluid stains but slowly. The cells,
however, are stained, and if a tendon be teased, isolated cells, and cells on the
fibres are easily seen. It is a good method for showing the relations of the
cells to the fibres.

10. T.S. Tail of Rat (Corrosive Sublimate and £ or ax- Carmine).— Harden
short lengths of the tail of a rat, the skin being first removed, in corrosive
sublimate for three hours or so. Remove every trace of the mercuric salt
by prolonged washing in alcohol. Stain the tissue in bulk in borax-car-
mine, and then decalcify it in dilute hydrochloric acid. Make transverse
sections, after embedding it by the interstitial method in paraflin. Sections
may also be made by freezing, but they are apt

to fall asunder. This method also yields
beautiful preparations, comparable to those by
the gold chloride methods. The transverse
.sections of the tendons are very characteristic.

11. Dried Rat's Tendons. — A very convenient
method is to dry the tendons of a rat's tail,
keeping them extended during the process.
After drying, they can then be used at any
time. By acting on them with dilute acetic
acid they swell up slowly, and the rows of cells
are thereby revealed. The cells — after washing
away the acetic acid— can be stained with picro-
rnrmine, and the preparation mounted in dilute

glycerine.

'12 Cell-Spaces (Saft-CaiMcten) in Central FlQ. I36._Cell-Spaces in the Cen-
Tendon (Silver Method). — Place the central tral Tendon of the Diaphragm
tendon or the whole diaphragm for five minutes of Rabbit. I Lymphatic ; s.
in a £ per cent, silver nitrate. Remove it, and Cell-spaces. Silver nitrate,
with a camel's-hair pencil brush both surfaces

of the tendon to remove the endothelium. Replace it in the silver solution
for fifteen minutes. Remove it ; wash it in water, and expose it to light to

1 68 PRACTICAL HISTOLOGY. [XII.

reduce the silver. One piece may be mounted in balsam ; another piece should
be stained with acid logwood or picro-lithium carmine (in this case use dilute
hydrochloric acid), and mounted in balsam.

(a.) Observe a large number of clear, branched anastomosing spaces, sur-
rounded by brown areas of ground-substance. The former are the cell-spaces
and Saft-Cancilchcn, or juice-canals, and some of the latter may be seen to
communicate with the lymphatics (fig. 136).

(b.) In the stained specimen, stained nuclei are seen in the spaces, i.e. , the
nuclei of the cells which occupy these spaces.

13. Cell-Spaces (Iron Sulphate Method}. — Using the fresh central tendon of
the diaphragm of a mouse or guinea-pig or rat, place it for a few minutes in I per
cent, sulphate of iron. Pencil away the surface endothelium, and leave it in
the iron solution for five to seven minutes. Remove it, wash it, and place it
in i per cent, ferricyanide of potash, in which it becomes blue. Mount it in
Farrant's solution or balsam. In this preparation the cell-spaces and juice-
canals are again clear, but the ground-substance is blue.

14. Cell-Spaces in Kat's Tendon. — The fine tendons are placed in silver
nitrate (| per cent.) for two minutes, and then the epithelium is brushed off
by a camel's-hair pencil. Five or six sweeps of the brush usually suffice. The
tendons are stained for other ten minutes in silver, washed, and exposed to
light in alcohol. Rows of clear, somewhat quadrangular spaces in a brownish
matrix are obtained.

LESSON XII.

ADIPOSE, MUCOUS, AND ADENOID TISSUES-
PIGMENT CELLS.

ADIPOSE TISSUE (FATTY TISSUE).

Adipose Tissue. — A fat-cell consists of a membrane enclosing a
globule of oil, which pushes the oval flattened nucleus (surrounded
by a small amount of protoplasm) to one side, so that it lies close
under the cell-wall. Size, 40 //, to 80 p (^-Q-^-Q inch).

Fat-cells are arranged in groups, which form lobules, and these
again form lobes. Each lobule has an afferent artery, one or two
efferent veins, and a dense network of capillaries between the fat-
cells, each capillary surrounding one or more fat-cells.

It is to be remembered that cells in connective tissue containing
fat may have a two-fold origin. Fat may be formed in ordinary
connective-tissue cells, but there are other cells of a connective-
tissue nature, which seem to be more specifically fat-cells. During
development this tissue is formed at certain parts, e.g., in the groin,
axilla, and neck, and presents a grayish-yellow appearance in the
form of lobules, surrounded by connective-tissue — readily seen
in a young animal. The cells at first contain granules. The

XII.]

ADIPOSE TISSUE.

169

fat-cells derived from this tissue form lobules, and are supplied
with blood-vessels very much as a gland is
supplied with blood; so that each lobule is
provided with an artery, vein, and capillaries.

1. Fat-Cells. — Cut out a small piece of the
omentum of a cat, selecting a piece that con-
tains a little fat, and mount it in normal saline.

(ti.) (L) Observe the large, highly refractive
fat-cells arranged singly or in groups (fig. 137).

(b.) (H) The large fat-cells of variable size FIG. 137.— Fat-CeiK some
— some of them polygonal — highly refractive
contents, but no nucleus visible; connective-
tissue passing between some of the cells.

showing a Nucleus.
The central one shows
crystals of margarine,

X ioo.

Place

mil part of the omen-

2. Action of Osmic Acid (H)

tum of a cat or embryo
ox in ] per cent, osmic
acid for an hour. Wash
it thoroughly, and mount
in Farrant's solution.

(fi.) (L) Observe the
fat-cells, which first be-
come brown and ulti-
mately quite black. The
fat-cells are in groups,
and their relations to the
blood-vessels can also be
seen in fig. 138.

3. Membrane and
Nucleus of Fat-Cells
(H). — Place a piece of
the omentum or subcu-
taneous adipose tissue
in absolute alcohol for
several days, and after-
wards in ether for a day
or two ; or the tissue
may be boiled for a few
minutes first in alcohol
and then in ether.
Transfer a small piece
to hsematoxylin and
allow it to stain for
several hours. Wash it
in water and place it in

absolute alcohol. Extract it with turpentine, clear it up with clove-
oil, and mount it in balsam.
16

B

FIG. 138.— Fat-Cells stained with Osmic Acid from lh:
Omentum of an Embryo Ox. A. The fat-cells in
groups or lobules, blackened by the osmic acid, and
showing their relation to the blood-vessels; 11. a-c
fat-cells in different stages of development.

PRACTICAL HISTOLOGY.

[XII.

(a.) Observe the collapsed membrane of the fat-cells, with a small
oval blue-stained nucleus immediately under the cell-wall. A small
quantity of protoplasm surrounding the nucleus may be visible.

4. Or harden a small piece of the omentum of a rat or other
animal in absolute alcohol. Select a piece which contains some fat.
Stain it for twenty minutes in lithium-carmine, then place it in acid
alcohol (i per cent, hydrochloric acid in 70 per cent, alcohol) ;
place it in absolute alcohol ; clear it up with clove-oil or xylol, and
mount in Canada balsam.

(a.) Observe the envelopes of the fat-cells and the nuclei of the
fat-cells, the latter stained bright red (fig. 139). Other red-stained
nuclei are visible, but they are the nuclei of blood-capillaries between
the fat-cells.

5. Margarine Crystals (H). — Place a small piece of fat for forty-
eight hours in glycerine. Tease a piece in Farrant's solution.

(a.^ Notice the large cells some of them with granular contents,

FlO. 139. — Empty envoi opes of
Fat-Cells. Alcohol and
ether.

PlG. 140. — Fat-Cells containing Crystals of
Margarine.

others with a stellate arrangement of needle-shaped crystals of
margarine (figs. 137 and 1 40). If the star of crystals — really palmitic
and stearic acids — be broken up, then the needle-shaped crystals are
distributed throughout the cell.

6. Blood- Vessels of Adipose Tissue (L and H).— Make a rather
thick section of a mass of adipose tissue in which the blood-vessels
have been injected. This can be done by injecting the blood-vessels
of a rabbit with a carmine-gelatine mass. As the fat is very soft, the
best method of obtaining such sections is to saturate the tissue with
paraffin and cut it in paraffin. Mount sections — not too thin — in
balsam. A section which has been saturated with paraffin and cut
in this substance must have the paraffin removed by soaking in

XII.

ADIPOSE TISSUE.

171

turpentine or xylol, which dissolves out the paraffin ; the section is
afterwards mounted in balsam.

(L) Observe the very vascular small lobules, composed of fat-cells.
To each lobule there passes one artery, and from it emerge one or
two veins.

(H) Observe the loop of capillaries round each fat-cell or around
several fat-cells.

7. Development of Fat-Cells. — These may be studied in the
subcutaneous tisyie of a newly-born rat, or in the omentum of a
newly-born rabbit. Stain a small

piece of any of these tissues in osmic
acid.

(a.) (L) Observe the fat-cells in
groups surrounded by connective
tissue.

(b.) (H) Observe the shape of the
cells, with small globules of oil —
black — scattered throughout the
protoplasm, the nucleus pushed to
one side in the more developed fatty
cells by a globule of oil, which is
stained black (figs. 138, 141).

8. Atrophic Fat-Cells.— These are
readily obtained from the yellow

bone-marrow of an old person who has died from some wasting
disease, or from the sub-pericardial fat of a person who has died
from phthisis.

(H) Observe the envelope of the fat-cell, now no longer completely
filled with fat, but containing a little protoplasm and some serous
fluid.

9. Injection Method. — By means of a hypodermic syringe, inject
under the skin of a dog or cat silver nitrate (i in 1000), which
c.-iuscs a local oedema and isolates the elements of the areolar tissue,
including the fat-cells (JRanvur).

FIG. 141.— Developing Fat-Cells from
the Subcutaneous Tissue of a
Foetus. Osmic acid.

MUCOUS TISSUE.

Mucous Tissue. — In the embryo it exists under the skin ; it
forms Wharton's jelly of the umbilical cord, and in the, adult it forms
the. vitreous humour. It is essentially an embryonic tissue.

10. Mucous Tissue (H). — Harden the umbilical cord of a three-
inonths foetus in Mailer's fluid and then in alcohol. J\ lake trans-
verse sections by freezing, and stain them in picro-cannine or
h;eni;itoxvlin or gentian-violet. Mount in Warrant's solution.

(a.) Observe the large, branched, granular, nucleated cells, which

172

PRACTICAL HISTOLOGY.

[XII.

FIG. 142.— Mucous Tissue of Umbilical Cord
of Fretus. MUller's lluid and logwood, x
300.

anastomose with each other. Between the cells is a fluid which
contains mucin, and according to the stage of development of the cord,
there, is a grejitoror less number of fibres. The older the cord, the

more the fibres increase in num-
ber, and its characters approach
those of ordinary connective-
tissue. A better view of the

finer processes is obtained by
examining the. tissue in normal
saline (fig. 142).

ADENOID TISSUE.

Adenoid, Ketiform, or Re-
ticular Tissue consists of a re-
ticulum or network of fine fibrils,
which run in all directions,
forming a meshwork in several
planes. Some regard it as made
up of branched corpuscles, the processes of which anastomose.
In the meshes are leucocytes or lymph-cells, which usually occur
in such numbers as to obscure the presence of the fine meshwork
in which they lie.

It is very widely distributed, e.g., in lymphatic glands, simple
and compound, tonsils, solitary glands, and Peyer's glands ; in the
bronchial, pharyngeal, nasal, intestinal mucous membrane, spleen,
thymus, and a few other situations.

11. Adenoid Tissue of Lymphatic Glands. — This may be pre-
pared in several ways.

(i.) Harden an abdominal lymphatic gland of a calf or kitten for
two weeks in MUller's fluid. Make sections, and shake up one in a
test-tube with some water ; this dislodges the lymph corpuscles, and
in places leaves the fine reticulum visible.

(ii.) A better plan is to inject into a fresh lymph gland a J per
cent, solution of osmic acid, or J per cent, solution of silver nitrate.
In either case an oedema is produced which separates the parts and
reveals the network. The injection is made by means of a hypo-
dermic syringe (fig. 1 26). The syringe is filled with the solution, and
the sharp nozzle is thrust into the gland, and the contents of the
syringe rapidly injected haphazard into the organ. It passes in, and
forms, as it were, an oedema, and separates, and at the same time
hardens, the constituent parts of the organ. This is the method of
interstitial injection, one which is frequently employed.

(iii.) The gland may be hardened for twenty-four hours in picric

XII.]

ADENOID TISSUE.

173

acid, and the sections stained with eosiii-hsematoxylin and mounted
in balsam.

(H) Observe some parts crowded with lymph corpuscles, but where
these are wanting, note the very fine network of fibres, with nuclei
here and there at the points of intersection (fig. 143).

The lymph corpuscles may be got rid of by applying to a fresh
preparation a dilute solution of caustic potash which dissolves them.

12. Pigment-Cells and Guanin-Cells. — These may be studied
by pinning out on a frog-board one of the webs between the toes of
a frog (Lesson XIX. 11, e).

(r/.) (Land H) Observe large, branched, corpuscles loaded with

Leucocyte

FIG. 143. — Adcm^d Tissue. Human lymphatic
glunil. Picric acid and eosiu-heematoxylin.

FlG. 144.— Pigment and Gnanin-Cells of
Frog. A Contracted: B,C. Partially
relaxed pigment-cells. G Guairin
cells.

black granules of melanin (fig. 144, B), also smaller black spots,
which are cells with their processes retracted. Every intermediate
stage between these two states may be seen.

A permanent preparation is readily made by stripping off the skin
from the web of the toe of a dead frog, hardening it in alcohol, and
mounting in balsam. The web should be fixed in an extended
position before it is placed in the alcohol.

In such a preparation, not only will pigment-cells be found, but
guanin-cells also, i.e.., small oval cells filled with white refractive
granules of guanin (fig. 144, G). To see the guanin-cells turn off
the light reflection from the mirror, when the granules in the guanin-
cells will appear bright and refractile on a black ground.

For pigmcnted connective-tissue corpuscles from the choroid, see
Lesson on Eye.

1^4 PRACTICAL HISTOLOGY. [XIII.

ADDITIONAL EXERCISE.

13. Mucous Tissue.— Many branched cells are seen in a T.S. of the tail of a
tadpole, young triton, or salamander, hardened in I per cent, osmic acid and
cut in paraffin.

It is also to be found under the skin of the flanks in frogs at the breeding
season. It gives good preparations when stained with methyl-violet-5B
(S. Mayer).

LESSON XIII.
BONE, OSSEOUS TISSUE, &c.

THE essential elements of osseous tissue, of which bone consists,
are a calcined fibrous matrix or ground-substance, with cells or
bone-corpuscles embedded in it; the latter are lodged in spaces
called lacunw. A bone, however, is a complex organ. The follow-
ing scheme may facilitate the comprehension of its minute
structure : —

BONE.

In a longitudinal section of a long bone, observe with the naked
eye —

Periosteum covering the bone.

Compact or dense bone, the substantia dura (with Haversian

canals).
Cancellated or spongy bone, the substantia spongiosa (with

Haversian spaces and cancelli).
Medullary cavity with marrow.

Histologically dry compact bone shows —
[Peripherie or circumferential.
2| | Haversian or concentric.
ce'i Intermediary, interstitial or ground.
[Perimedullary,

^ ( white.
Sharpey s perforating fibres j yellow>

HAVERSIAN SYSTEM.
Dry bone. Recent bone.

( Blood-vessels, connective tissue,
Haversian canal . . . . lymphatics, osteoblasts.

i Between the lamellae, branched
Iaraell8e ' ' ' bone-corpuscles.

Lacunae
Canaliculi

j Processes of bone-corpuscles
| and lymph.
Lymph.

XIIL]

BONE, OSSEOUS TISSUE, ETC.

175

f i. External layer, fibrous, with the larger blood-
vessels.

Periosteum J\ 2. Internal or osteogenic layer, with finer blood-
vessels, numerous elastic fibres, and
blasts, and sometimes osteodasts.
Osteodasts or myeloplaxes of Robin.
Blood-vessels, nerves, and lymphatics.
, . ( i. Endochondral in cartilage.

* \ 2. Intra-membranous or periosteal.

1. T.S. of Dense or Compact
Bone. — It is better to buy a pre-
pared transverse section of the
shaft of a small, dry, long bone.
In it most of the smaller spaces,
being filled with air, appear
black.

(a.) (L) Observe the medullary
cavity, bounded by a ring of bone,
less dense internally.

(b.) Sections of tubes which
appear round, or oval — Haver-
sian canals — surrounded by con-
centric lamellae (fig. 145, c), and
between these lamellae are lacunae
(e), with fine channels passing
from them — the canaliculi. Some
lamellae are arranged parallel to
the circumference of the bone —
the peripheric (a)— while others
of larger radius are incomplete,
and jnmmed in between the
Hayersian system : they are inter-
mediary (jl). Around the medul-
lary canal are the perimedullary
lamellae (b).

(c.) (H) Observe the shape of
the lacunae — flattened branched •

spaces — with their numerous ' * -~" " - 1^

wavy branching canaliculi, and ^^^^^^^ ^-^*
how adjoining canaliculi anasto- *^

IM< iso by traversing the lameike FlG- MS-— T.S. Human Metacarpal Bone.
/(> \ A , ,, o. Peripherie lamelle ; A. Perimedullary

(fig8- r45> I47> e)' Afc tne 0«ter lamelhu; ft Ilaversian canals snr-

part of each Hayersian system ro«»ded ^r their Hftrerriwi tanelto ;

,. , •"„ ' ''• Intermediary lamella?; e. Lacuiiai,

some 01 the canaliculi of the x 20.

I76

PRACTICAL HISTOLOGY.

[xnt.

outermost row of lacunae will be found to form loops and open into
the lacunae from which they arose : these are recurrent canaliculi
(fig, 147, a). The canalicular system is for the distribution of
lymph to all the parts of the calcined fibrous matrix. Notice also
that the intermediary lamellae are parts of circles with a much

larger radius than those
of the Haversian system.
Each Haversian canal,
with its system of lamel-
lae, lacunae, and canali-
culi, forms a Haversian
System. The greatest
diameter of the lacuna) is
about 14 p. (T¥Vo iw'h)-

(d.) The lamellae in a
Haversian system on
transverse section appear
as thin concentric bands,
a clear transparent one
alternating with one which
looks more granular.
These are not due to
different kinds of lamellae,
but in the clear ones one

FIG. 146.— T.S. The Shaft of a Human Femur.
Haversian canals; s. Haversiau lamellae; si. In

looks upon the long axis

of the fibrils composing the lamella, and in the others upon the
ends of the fibrils.

2. L.S. of Dense Bone, prepared in the same way.

(a.) (L) Observe Haversian canals running chiefly in the long
axis of the section, with here and there oblique, short, junction
canals. Near the surfaces some open externally, and others com-
municate with the medullary cavity. The lamellae of any Haver-
sian system run parallel to its own Haversian canal. In the
system of canals — each 20-100 ^ wide — the canals frequently
divide dichotomously, and ultimately form a network in the com-
pact bony substance.

(b.) If the section be near the surface of the bone, so as to
include the peripheric lamellae, canals for blood-vessels, perforating
the lamellae and not surrounded by lamellae as in the Haversian
systems, may be seen. They are called Volkmann's canals, and
contain the perforating vessels. They are connected with the
Ilaversian canals proper. They are well seen in sections of the
femur of a guinea-pig, but, unlike Haversian canals, they are not
surrounded by Ilaversian or concentric lamellae.

(c.) (H) Observe the flattened oval lacunae with their canaliculi;

XIII.]

BONE, OSSEOUS TISSUE, ETC.

177

their arrangement, as well as that of the lamellae (fig. 148). If a
canal be viewed carefully many fine dots will be seen in it. These
are the openings of the canal iculi.

3. T.S. Decalcified Shaft of a Bone.— The bone, cut into short
lengths, must be decalcified in picric acid or chromic and nitric
fluid, with the precautions laid down at p. 38. AVhen sufficiently

FIG. 147.— T.S. Shaft of Human Femur. 77. Haversian

canals ; c. Lacunae with bone-corpuscles ; a. Lacuna) j,-IG> I48.— L.S. Dense
with recurrent canaliculi ; s. Intermediary lamella? with (]ry bone, x 40.

Sharpey's fibres, h; p. Large fibres of Sharpey in inter-
mediary lamella; ; I. Confluent lacunre. These Ranvier
supposes are bone-corpuscles and lacuna1 undergoing
atrophy, x 300.

soft — ascertained by pricking it with a pin — it is hardened in
alcohol in the usual way. Sections are best made by freezing.

(i.) Stain a section in picro-carmine and mount it in glycerine-
jelly. Glycerine or Farrant's solution tends to make the prepara-
tion rather too transparent.

(ii.) Place some sections in i per cent, osmic acid for twenty -four
hours and mount them in glycerine-jelly.

(a.) (L) Observe the periosteum (fig. 149), embracing and
adherent to the bone. In the bone itself the lacunae, and especially
the canaliculi, are no longer black, and are not so visible as in the
non-decalcified bone. Each lacuna contains a highly refractive,
branched, nucleated, and stained corpuscle.

Bone-Corpuscles. — Observe their arrangement following that of
17 M

t;8

PRACTICAL HISTOLOGY.

[XIII.

the lamellae, but the latter are not so distinct as in dry bone.
Several lamellse lie between two consecutive rows of bone-corpuscles.
The Haversian canab contain blood-vessels, connective tissue, and
other cells, or osteoblasts.

(6.) (H) The periosteum consists of an external layer stained
red, and composed chiefly of white fibrous tissue. Attached to it
may be found small fragments of striped muscle. The internal
layer contains many elastic fibres, and, especially in young bones,
there may be seen one layer or more of flattened or cubical cells,

Periosteum.
Peripherie lamellse.
Huversiau
canals.

Haversian
lamella).

Interstitial lamellae.

Perimedullary
lamellae.

Marrow.

FIG. 149.— T.S. Part of a Human Metacarpal Bone, x 50.
marrow. Dilute nitric acid.

. Haversian space with

called osteoblasts (fig. 156, c). The latter may be seen not only
under the periosteum, but also passing along with blood-vessels into
the Haversian canals.

Fibres may be seen passing from the deep surface of the perios-
teum into the bone — the perforating fibres of Sharpey.

(c.) Observe the lamellae, but their outline is not very distinct,
while the canaliculi will not be distinctly visible, being indicated
by fine lines traversing the lamellse. The bone corpuscles are
nucleated refractive cells, each lying in a lacuna. In such a
preparation, one cannot make out that they send processes into the
canaliculi.

(d.) Select a large Haversian canal and study its contents. Note
the presence of an artery and vein with very thin walls, and the
cavity lined by osteoblasts (fig. 150, Obi.), and the remainder filled
ii}) with medullary tissue.

4. Perforating Fibres. — (i.) From a membrane bone of the
skull (e.g., the parietal or frontal bone, which has been softened
in 2 per cent, hydrochloric acid or in v. Ebner's fluid (p. 37)

XHL] BONE, OSSEOUS TISSUE, ETC. 179

t

and from which the acid has been removed by steeping in water,

and subsequently in spirit) with forceps remove the periosteum, and

tear off a thin lamella of

osseous tissue. Place its

under-surface uppermost on

a slide in water or v.

Ebner's fluid.

(H) Observe fine tapering
fibres like nails — perforat-
ing fibres — projecting from ^z-
the surface. Some aper-
tures may be found from

which corresponding fibres v x,-^ _^-- / ;

have been withdrawn. . . - '

(ii) These are far better FlG. I50._T.S. of alarge Hav.rsia^anal with its

developed 111 the bones of Soft Parts. Femur of a Doff, x 130. A. Aitcry;

i v • 1 n f -U- j Q (i. V. Vein ; Kz. Bone-cells ; M. Medullary tissue ;

the skllll Of birds. Soften Obl. Osteoblast ; pkl. Osteoclast.

the vault of the skull of a

fowl in v. Ebner's fluid and harden in alcohol. Make not too thin
sections, and with needles tear the lamellae asunder. Examine it
in v. Ebner's fluid. Or make sections of a human frontal bone
softened in dilute hydrochloric acid, and examine it in water.

(H) Numerous perforating fibres passing between the separated
lamellae, and, it may be, the sockets from which they have been
withdrawn, will be seen (fig.
151). In some of the sections
branched perforating fibres are
visible. The important point
is to make the sections as S \ I I 1

nearly as possible parallel 1 v / V^

the course of the fibres. The •<
preparation is apt to be made '£§> ij^ / "r^'^-^&r-
too transparent by Farrant's
solution, so that the fibres are

not SO distinctly seen in this *'W. 151.— Sharpey's Perforating Fibrus.

medium as in water or v.

Ebner's fluid. Observe that there are no Sharpey's fibres in the

Haversian systems.

5. Blood- Vessels of Bone (L). — These are not easily injected.
Inject with a fluid carmine mass (p. 89) the posterior half of
the body of a rabbit. Do this from the alxlominal aorta. Or use
the injection fluid mentioned at p. 181. It requires considerable
pressure to cause the injection to traverse the blood-vessels of bone.
Therefore clamp the inferior vena cava to prevent the exit of the
injection mass. Decalcify the injected bone, and afterwards make

i8o

PRACTICAL HISTOLOGY.

[XIII.

V B

T.S. and L.S. The sections, however, must not be too thin. L.S.

tire the most instructive, and are mounted in glycerine-jelly.

(a.) Observe that the blood-
vessels lie in the Haversian
canals, and follow the arrange-
ment of the latter (fig. 152).
If the marrow of the bone be
preserved, fine blood-vessels
may be seen in it. Perhaps
"perforating vessels'7 lying in
Volkmann's canals may be
noticed, especially in transverse
sections.

6. Cancellated Bone (L and
H). — In the vertical section of
the head of a long bone showing
articular cartilage a view of the
open lattice-work will be ob-
tained (Lesson XIV. 3). Or a
T.S. may be made across the

head of a long bone' e'ff'> a
femur, preferably of a young

animal. Stain a section in
haematoxylin, picro-carmine, or

COsin-logWOOd, and mount it ill
<"rlycerine-ielly.

(a.) (L) Note the network of

osseous trabeculse (fig. 153) bounding the spaces or cancelli. In
the latter lies red marrow, and on their walls are osteoblasts. In

the centre of some of the
trabeculse may be seen a
deeper stained irregular bar,
the remains of calcined carti-
lage (fig. 153, CC). On the
calcified cartilage is deposited
osseous tissue.

(6.) (H) In each trabecula
note the fibrous matrix and
the bone-corpuscles. In the
interior of some of the tra-
beculse the remains of un-
absorbed calcified cartilage.
Note also how the osseous
tissue with spherical bulgings advances upon the calcified cartilage.
By carefully shading the light, it will be seen that a more or less

FIG. I52.-L.S. Injected Bone. P. Perios-
teum ; B. Bone; V. Blood-vessels.

FlG. 153.— Cancellated Bone. C. Canceling;
T. Trabecula; CC. Calcified cartilage; B.
Bone ; 0. Osteoblast.

XIII.]

BONE, OSSEOUS TISSUE, ETC.

181

spherical mass of osseous tissue surrounds, and in fact is formed V.y,
each bone-corpuscle (fig. 154). There are thus spherical masses —
cell-territories, as it were
— and in the centre of
each a bone-corpuscle.

7. Fibrillar Structure
of LamellsB (H).— De-
calcify a bone in v. Ebner's
fluid (10-15 percent, sodic
chloride and 1-3 per cent,
hydrochloric acid). Either
this fluid or that given at
p. 3 7 may be used. Scrape i'i«-
off a thin lamella and
examine it in water.

(H) Observe the fibres of which it consists. They are com-
posed of fibrils arranged in bundles. They are best seen near
the edge. Fibres in different planes cross each other at a right
or obtuse angle.

[54;

-Simi'.lPartof Fi

CC. Calcified

LS- !53>

cartilage; B. Bone; O. Osteoblast; BC. Bone-
corpuscles.

ADDITIONAL EXERCISES.

8. Blood-Vessels of Bone. — These may be injected from the descending
aorta of a rabbit with a saturated and filtered watery solution of Briickc's
IVrliii blue. One must remember that the injection often fails. Inject from
the abdominal aorta, after a time using pretty high pressure. It is well
to ligature the inferior vena cava after some injection has flowed from it.
Decalcify the bone in chromic acid. The sections may be stained with very
dilute fuchsin.

9. Decalcified Bone. — Make a T.S. of dense bone — rather a thick section —
steep it in alcohol, transfer it to a slide, and mount it in glycerine-jelly, or in
a mor.-el of dry Canada balsam. In the latter rase the slide must be heated
to melt the balsam. Examine it at once, when the lacunae and canaliculi will
be seen black on a clear ground. They still contain air, hence their black
appearance. Gradually, however, the jelly penetrates into the canalicular
system, and ibis characteristic appearance vanishes, but it is an instructive
exercise for students to perform this experiment. Afterwards the lamella1
remain quite distinct in the preparation, there are also faint indications of the
canaliculi.

10. Bone in Polarised Light. — Examine a non-decalcified transverse section
of the shaft of a long bone in polarised light (Lesson XVI. 16). When the
Nicols arc crossed, each llaversian system has a bright cross on a dark ground,
while ihe lamellse are alternately bright and dark. The crosses arc also seen
in decalcified bone, so that they arc produced by the organic basis or ossein
of the bone.

1 82

PRACTICAL HISTOLOGY.

[XIV.

LESSON XIV.
BONE AND ITS DEVELOPMENT.

BONE is developed either in connection with cartilage or membrane.
The former is called endochondral and the latter intramem-
branous ossification. With the exception of a part of the skull —
its sides and vault — and nearly all the facial bones, all the bones
are laid down in hyaline cartilage.

1. Development of Bone— T.S. of Fostal Bone. — Decalcify the
shaft of. the femur or other long bone, e.g., the radius and ulna,

FIG. 155.— T.S. Radius and Ulna of an Embryo Dog. I- Interosseous ligament ; p. Peri-
osteum ; me. Medullary cavity ; insp. Subperiosteal tissue; lo. Osseous trahecuhu ;
n. Point where the ligament enters the bone ; n'. Union of ligament with the perios-
teum. Observe that at first the interosseous membrane is inserted into a depression
in the bone ; when the membrane becomes ossified a ridge is formed.

of a newly-born kitten in picric acid (p. 37). Make transverse
sections, and stain them with picro-carmine. Mount one in
Far rant's solution.

(a.) (L) Observe the periosteum (fig. 156, a, &), composed ex-
ternally of connective tissue, with fusiform corpuscles stained red.
Under this, one or more layers of cubical or somewhat flattened
nucleated cells, osteoblasts (<"). They pass into and line the Haver-
sian spaces, thus reaching the cancelli and medullary cavity, which

XIV."

BONE AND ITS DEVELOPMENT.

they also line, so that every spicule or surface of young bone is
covered by them. From them the bone-corpuscles are formed.

(b.) The osseous tissue is stained red, and forms an anastomosing
series of trabeculae bounding large spaces — Haversian spaces-
containing blood-vessels and marrow, and lined by osteoblasts.
Processes from the trabeculse project into the deeper layer of the
periosteum. At this stage a concentric arrangement of the lamellae
leading to the formation of Haversian systems has not yet taken
place. In the bone matrix, the bone-corpuscles (/), irregular, re-
fractive, nucleated cells, each lying in a lacuna.

(c.) (H) Under the periosteum and in the spaces may be seen
larger multinucleated cells.

Osteoclasts or Myeloplaxes (fig. 1 56, If). — The cells are much
larger than the osteoblasts, con-
tain many nuclei, and lie in little
depressions of the bone eroded
by themselves. These depres-
sions are called Howship's
lacunas. These cells are con
cerned in the removal of bone.

2. Intra-Cartilaginous For-
mation of Bone (L and H).—
Decalcify in picric acid the pha-
langeal bones of a four-months
footus. Make longitudinal ver-
tical sections, stain in picro-
carmine, and mount in Farrant's
solution.

(a.) (L) At the head of the
bone observe a mass of hyaline
cartilage (fig. 157), and lower
down bone, and where the two are continuous an irregular festooned
margin, the line of ossification.

(b.) The cartilage at the upper part is hyaline, with the cells
small, and arranged singly or in groups, while deeper down the
cartilage-capsules are beginning to be arranged in rows, and below
this are larger cartilage-capsules with clearer contents, and a some-
what refractive matrix between them.

(f.) Under this, the line of ossification, with its festooned
margin, are spicules of calcified cartilage passing downwards
towards the medullary cavity. In the bony part, the primary
medullary spaces, with their osteoblasts, blood-vessels, osteodasts,
and tho. newly-formed hone deposited on the calcified cartilage.

(d.) Tho first none is formed under the, periosteum (fig. 157), by
means of the subperiosteal osteoblasts. As these osteoblasts

FIG. 156.— T.S. Fcctal Bone of Kitten, a, b.
Superficial and deep layers of perios-
teum; c. Layers of osteoblasts, with
Ic. Osteoclasts ; m. Matrix of bone ; I.
Lacunae, with bone-corpuscles.

1 84

PRACTICAL HISTOLOGY.

[XIV.

become embedded in the bone matrix they become bone-corpuscles.
This piece of bone is perforated by blood-vessels, which pass into
the primary medullary spaces in the hollowed-out cartilage.

(e.) (H) Examine the several parts. Search for an osteoclast
lying in a little cavity — Howship's lacuna — and notice that while
the margins of the trabeculae, covered by recently-formed bone,
are stained of a deep red colour by the carmine, no such red stain

Osteogenic tissue
Emloclionclral bone

Blood-vessel
Perichondral bone

Enlarged cartilage-capsules

Enlarged cartilage-capsules.
^7 Calcified cartilage in the form of

Pointed processes projecting into
the

Primary medullary spaces.
Perichondral bone.

Fro. i57.— Dorso-palmar Longitudinal Section of the Second Phalanx of the Finger of
a Four-Months Foetus.

is seen where the osteoclast is embedded. It, in fact, is eroding
or eating away bone.

3. Epiphysis and Epiphys'al Cartilage (H and L).— Make

longitudinal vertical sections of a young rabbit's femur or tibia
to show the cpiphysial cartilage. Stain it in picro-carmine and
mount in glycerine-jelly ; or, better still, double stnin it in hsema-
toxylin and picro-carmine or haematoxylin and eosin. In the last
case the cartilage will be blue, the rest red or copper-red. The
method of double staining is particularly valuable for the study
of bone development.

(a.) (L) Observe the thin layer of encrusting cartilage on the
head of the bone (fig. 158, (7), and under it the cancellated bone
of the head of the tibia. This cartilage is continuous below
with —

(/>.) A broad layer of cartilage between what is to be the head

XIV.

BONE AND ITS DEVELOPMENT.

I85

of the bone — the epiphysis (E) — and the future shaft or diaphysis
of the bone (Z>).

(<•.) In the epiphysial cartilage (EG) the vertical rows of carti-
lage-cells, smaller above, and larger and more quadrilateral below
(fig. 158 and iig. 159, C).

(d.) The shaft with longitudinal spaces — the primary medul-
lary spaces (fig. 158, MS)— bounded by trabeculse of calcined
cartilage partly covered by bone (fig. 159, b). The spaces con-
tain young marrow (c).

(e.) (H) Study specially the cells of the epiphysial cartilage
(fig. 159, 0), and notice that the cells and capsules are smaller
above and larger below — zone of enlarged cartilage-capsules. Some
of the enlarged cartilage-
capsules of the lowest row
may be seen opening into
the primary medullary
spaces. The line of ter-
mination of these spaces is
called the line of ossifica-
tion. The bone grows in
length by the proliferation
of the epiphysial cartilage-
cells.

(/'.) Bounding the pri-
mary medullary spaces,
directive trabeculse of cal-
cified cartilage (//) with a
deposit of bony matter on
them. Note specially the
osteoblasts (p) partially em-
bedded in the osseous matter which they themselves secrete or
form. When they become embedded in the osseous products
of their own activity, they are then called bone-corpuscles. Note
also that the bone on the cartilage is bounded by convex surfaces
which fit into corresponding depressions in the cartilage. On
the larger trabecnlse may be found osteoclasts
cavities— Howship's lacunae — which they have
spares themselves are filled with red marrow
vessels (v).

4. Intra-Membranous Formation of Bone (H and L). — Take the
parietal bone of a foetus when the parietal bone is only partially
ossified; scrape off the periosteum from a part near the periphery
of the ossified part; stain in picro-carmine and mount in Far-
rant's solution.

(a.) Observe at one part the fibrous matrix, and shooting from

FIG. 158.— V.S. Head of Tibia of a Young Rabbit,
greatly reduced to show details. C. Encrust-
ing cartilage ; E. Epiphysis ; EC. Epiphysial
cartilage ; D. Diaphysis ; p. Periosteum ; MS.
Primary medullary spaces.

lying in little
eroded. The
(c) and blood-

1 86

PRACTICAL HISTOLOGY.

[xiv.

it calcified fibres of connective tissue, these covered with osteo-

blasts (fig. 1 60).

Marrow of Bone.— It fills
the medullary cavity of long
bones, the cancelli of spongy
bone, and it occurs in some of
the larger Haversian canals.
There are two varieties, yellow
and red, the difference in
colour being due to the former
containing a large amount of
fat-cells.

Yellow marrow occurs in
the medullary cavity or canal
and the larger cancelli of long
bones. Besides a small amount
of connective tissue and blood-
vessels, it consists principally of
fat-cells.

Bed marrow (H) occurs in
the spongy tissue at the ends of
long bones, in the short bones
of the hands and feet, flat
c. Epiphy- bones Of the skull, epiphyses of

sial-cartilage ; 0. Bone; a. Row of carti- i • i -i

lage-cells; b. Cartilaginous trabeculre; m. long bones, clavicle, ribs, and

Primary medullary space; n. Osseous • ii moflnllarv rinal of tho

deposit; V. Directive cartilaginous trabe- m l Jliary canal <

culne ; p. Bone-corpuscle being embedded long bones of SO111C animals, e.<l..

iksviia TvioftMv • /» "\Tnr-pn\xr_r>^lla • 11 . . 11- T

guinea-pig, rat, rabbit. It con-
sists of delicate connective
tissue with blood-vessels and numerous cells of several varieties,
(i.) Medullary or Marrow Cells (fig. 161, a, b, c).— They arc

the most numerous, and are
nucleated cells, not unlike
large leucocytes. They are
spherical, with finely granular
protoplasm and a spherical
pale nucleus. Sometimes
they contain two nuclei.
Examined quite fresh in
serum, no nucleus is visible,
but it is revealed by the
action of acetic acid or
dilute alcohol. The proto-
plasm of some of them contains numerous highly refract! le granules,
and that of others some brownish granules. There is also a smaller

FIG. 159. — L.S. Head of a Metacarpal Bone of
Rabbit, aged three months.

in the osseous matrix
Injected blood-vessel,

c. Marrow-cells ; v.
240.

Connective tissue bundle?

Osteoblasts. Calcined.

Non-calcified.

FIG, 160. — Surface Section of a Parietal Bone
Human Embryo x 240.

XIV.]

BONE AND ITS DEVELOPMENT.

187

variety of corpuscle. They exhibit amoeboid movements under
proper conditions.

(2.) Cells with Budding Nuclei (fig. 161). — Far less numerous,
but easily distinguished, are large finely granular cells, each with
a single large, often twisted, nucleus, which in some looks as if
it were composite, in others it would seem to consist of several
parts united by some
substance like the nuc-
leus itself. There is
very great variety in the
shape of those nuclei,
which are visible in the $•
fresh condition of the *
cell. These cells are fi '
not amoeboid (fig. 161.
//, i).

(3.) Myeloplaxes. -
These are larger than

the, foregoing, consisting FIG. 161.— Cells from the Red Marrow of the Tibia of a

^f o ^,,Ql-,r mlo-r. Young Rabbit, a, b, c. Marrow-cells examined in

iciy gra normal saline ; rf, e. Marrow-cells after dilute alco-

protoplasm with liumer- ho1? /. 9- After dilute alcohol; h, i Large cells

i . / n .. \ with budding nuclei ; m. Myeloplaxes. x 300.

ous nuclei (fig. 161, m).

What relation there is between (2) and (3), or if there is any relation

at all, is entirely unknown.

(4.) There are to be found cells smaller than but not unlike (i),
with a homogeneous protoplasm which has a reddish tint and a
spherical nucleus. Some of them have a small bud at the side
(fig. 1 6 1, /, g). They are regarded as cells from which coloured
blood-corpuscles are formed. They are regarded by Bizzozero as
similar to the nucleated red blood-corpuscles of the embryo.

(5.) Always a few fat cells.

(6.) Numerous red blood-corpuscles from the blood-vessels in the
red marrow.

(7.) Sometimes osteoblasts may be detached along with the other
constituents of the marrow.

Lay open a rib or a long bone of a guinea-pig or rabbit ; remove
a little of the red marrow and diffuse it in blood-serum or normal
saline. With a high power search for examples of each of the

foregoing kinds of cells,
from it the red marrow

The vertebra of a calf may be used, and
is readily expressed by squeezing it in a

vice.

The, nuclei in some of the cells are best revealed by the action of
dilute alcohol.

5. Red Marrow. — (i.) Squeeze out some of the, red marrow from
a rib of a guinea-pig or rat; shake it in a test-tube containing

1 88 PRACTICAL HISTOLOGY. [XIV.

normal saline tinged with methyl-green, aldehyd-green, or aniline-
green. The marrow will fall as a precipitate. Examine a little of
this in normal saline. Stain some in picro-carmine, and mount in
dihite glycerine.

(H) Observe the various forms of marrow and other cells met
with, the nuclei tinged red. The large myeloplaxes with numerous
nuclei have their protoplasm green and the nuclei red. The fat-cells
are quite green.

(ii.) Place some of the red marrow for 12-24 hours in dilute
alcohol, mount and stain the cells with picro-carmine. In this way
the nuclei of all the cells are stained red, their protoplasm yellowish,
while nucleoli are revealed.

(iii.) Harden some of the red marrow in Hayem's fluid for
twenty-four hours. Wash the deposit, stain (picro-carmine) and
mount a little in glycerine. This is a good hardening medium for
this purpose ; the red blood-corpuscles can be readily distinguished,
while the nuclei in all the cells are distinct.

6. T.S. Red Marrow. — Small pieces of the ribs of a young
rabbit are placed in the following fluid for one day : —

Sodic sulphate . . . 2.5 grams.

Mercuric chloride . . 0.25 ,,

Water . . . 500 cc.

Rinse in water, and transfer to picric acid to decalcify the bone.
The tissue can then be soaked in gum, then hardened in alcohol
and sections cut. Sections may be stained with eosin and logwood.
The eosin stains any cells containing haemoglobin of a reddish-orange
tint.

ADDITIONAL EXERCISES.

7. Cover-Glass Preparation of Red Marrow (H). — As described for blood,
get a thin layer of red marrow on a cover-glass and stain it for twenty-four
hours in Biondi's fluid (p. 140). Dry cover-glass preparations may also be
stained with methylene-blue, eosin-harnatoxylin. or methyl-green. Mount
the cover in xylol-balsam. If desired, clear it up with oil of cloves in which
a little eosin is dissolved, and remove the clove-oil by xylol. This method
yields excellent preparations.

8. Squeeze on a slide a little of the red marrow from the rib of a young
rabbit. Squeeze a little between two cover-glasses to get in a thin film, find
expose the latter for a minute or two to the vapour of osmic acid. Stain it
with picro-caruiine and mount it in glycerine.

XV.] MUSCULAR TISSUE. 189

LESSON XV.
MUSCULAR TISSUE.

Muscle histologically occurs in two varieties — (i.) Non-striped;
(2.) Striped. Non-striped muscles are involuntary, \vhile striped
muscles, as a rule, are voluntary ; but the heart-muscle is an
exception, for though striped it is involuntary.

Non-Striped (smooth, involuntary). Occurs in the outer coats
of the lower half of oesophagus ; muscular coat and muscularis
mucosse of stomach and intestines ; villi ; ureter, bladder, and
urethra ; pelvis and capsule of kidney ; trachea (trachealis muscle) ;
bronchi ; oviduct ; uterus ; iris, ciliary muscle ; erector pili muscles
of skin, sweat glands ; coats of blood- and lymph- vessels ; capsule
and trabeculse (lymphatics, spleen), and ducts of some glands, sali-
vary, bile-ducts, &c.

It consists of nucleated elongated fusiform contractile cells, held
together by a clear cement. The cells taper towards their extremities,
and although they appear homogeneous in reality they seem to consist
of longitudinally arranged fibrils held together by a sarcoplasm, or at
least a transparent material. The nucleus is oval or rod-shaped.
Some state that the cells have an elastic sheath. Each cell is from
4 to jo /* GroVrr- 25*00 inch) in breadth and 40-200 //, (^-^
inch) in length.

1. Non-Striped Muscle (H). — Place thin strips of the muscular
coat of the intestine for forty-eight hours in a 25 per cent, solution
of nitric acid, which softens the connective tissue and renders the
tissue yellow. Wash it thoroughly in water. Tease a small part
in glycerine or Fun-ant's solution. Or macerate a small piece of
intestine in | per cent, bichromate of potash for 48 hours. Cells
can then be readily isolated.

(a.) Select an isolated fibre. It is spindle-shaped, elongated, or
fusiform, tapering to both ends, and in its centre there is an oval
nucleus, distinguished by its being rather more refractive than the
rest of the cell (fig. 162). At the poles of the nucleus there may
b<- a few granules.

It is very difficult to stain these cells after the action of nitric
acid, but this may be done with magenta, provided the nitric acid
be entirely washed out of the tissue beforehand. Each cell is said
to have a sheath, but that cannot be seen in fibres prepared in this
way.

190

PRACTICAL HISTOLOGY.

[XV.

FIG. 162. — Isolated Smooth
Muscular Fibres,
acid, x 300.

2. Muscle-Cells from the Frog's Bladder (|_ and H).— Distend
the frog's bladder with dilute alcohol thus. Transfix the skin on
each side of the anus with two pins, and tie round them a thread so
as to occlude the anus ; open the abdomen,
make a slit into the large intestine, clear out
any residues it may contain, and inject dilute
alcohol through the intestine into the blad-
der. When the latter is full, ligature it at
the neck, and suspend it for twenty-four
hours in a large quantity of dilute alcohol.
Then open the bladder, and with a camel's-
hair brush pencil away all the lining epi-
thelium. Stain a portion of the bladder in
logwood and mount it in balsam, or stain
in picro-carmine and mount it in Farrant's
solution.

(a.) Observe the thin membrane, traversed
in every direction by thicker or thinner tra-
beculaB of smooth muscle (fig. 163, a). The
trabeculae consist of numerous long fusiform
Nitric nucleated cells (c) — the nuclei, long, narrow,
and oval. Some of the cells are triradiate
(/;). These are what S. Mayer has called atypical cells. Oval nuclei
with blunter ends are seen in the fibrous covering of the bladder (d).
They are the nuclei of connective-tissue ceils.

3. T.S. Non-Striped Muscle
(H). — This is obtained by mak-
ing transverse sections of the
circular muscular coat of the
small intestine (cat), previously
hardened in chromic acid and
spirit, or Miiller's fluid. Stain
a section in haBmatoxylin and
mount in balsam.

(a.) Observe polygonal areas
of unequal size, mapped out
from each other by a refractive
(fig. 164) cement substance.
Each area corresponds to the
transverse section of a tibre.
Some of the areas contain a
nucleus (n), others not. Sur-
rounding groups of these areas are fine septa of connective tissue
(s), which map out the fasciculi or bundles of cells. The fibres are
arranged in bundles or fasciculi, each surrounded by an envelope of

FlG. 163. — Bladder of Frog. a. Large strands
of smooth muscle ; b. Triradiate, and c.
Fusiform muscle-cells ; d. Nuclei of con-
nective-tissue corpuscles.

XV.]

MUSCULAR TISSUE.

lyi

connective tissue. The fasciculi are large or small according to the
number of fibres entering into their composition.

(b.) In some preparations one can see intercellular bridges between
adjacent cells, like those that occur in squamous epithelium.

4. L.S. of Non-Striped Muscle (H). — This may be obtained by
making a longitudinal section of
the longitudinal coat of the in-
testine, but it is better to strip
off a thin lamella of this coat from
the intestine of a rabbit hardem-d
in spirit or Muller's fluid. Stain
in hsematoxylin and mount in
balsam.

(«.) Observe the oval fusiform
nuclei (fig. 164, n) lying in narrow
fusiform areas — the cells. The
boundary-lines between the cells

FIG.

164.—
estim

—A. T.S. non-striped muscle, in-
testine of cat ; B. Longitudinal strip of
intestine of rabbit; c. Cell; n. Nucleus;
s. Septum of connective tissue. Chro-
mic and b chromate fluid, hsematoxylin.

are usually not well defined.

5. Cement Substance of Non-
Striped Muscle (H).— With dis-
tilled water wash out the contents
of the small intestine of a freshly-killed rabbit, or the large intes-
tine of a frog. Tie one end of the gut, and fill it with .5 per cent,
solution of silver nitrate, and tic the other end of the gut. Suspend
the whole in J per cent, solution of silver nitrate for ten minutes
or so. Slit up the gut along the line of
attachment of the mesentery. Wash it
in water and expose it to light. It soon
becomes brown. Lay it on a glass plate,
mucous surface uppermost, and with a
scalpel* scrape away all the mucous and
submucous coat, which is very easily
done, especially if the intestine has been
macerated for about twenty-four hours in
water. There remain only the muscular
and thin serous coats. Harden in alco-
hol. Snip out a piece, dehydrate com-
pletely in absolute alcohol, and mount in FIG. 165.— Cement Substance of

L i Smooth Muscle, Intestine of

Rabbit. Silver nitrate.

(a.) Observe the narrow elongated

fusiform areas bounded by silver lines (fig. 165) ; they indicate the
outline of the fusiform cells. On focussing upwards and down-
wards, notice the longitudinal and circular direction of the fibres
crossing each other. In this preparation also many lymphatic
paths lined by sinuous epithelium may be seen. They are recog-

PRACTICAL HISTOLOGY.

[XV.

nised by the dilatations in their course, and by the character of the
endothelial cells lining them. By focussing deeply, the silver lines
of the endothelium of the serous membrane may be seen.

6. Methylene-Blue Method (S. Mayer).— Into the blood-vessels
of a cat — e.g., from the aorta — inject the following solution : —

Methylene-blue (S. Mayer's)1 i gram.

Normal saline ....... 300 cc.

After an hour or so, open the abdomen ; the intestines appear
blue ; cut out a portion of the muscular coat and place it in the
following mixture : —

Picro-Grlycerine Mixture.

IOO CC.
100 ,,

Saturated watery solution ammonium picrate
Glycerine ......

On teasing a small piece of the muscular coat in the same mixture,
isolated muscle cells are readily found, the nucleus being stained of
a faint rosy-pink.

ADDITIONAL EXERCISES.

7. FibriUar Plexus in Muscle-Cells (H).— Kill a newt, open its abdomen,
and pin out its intestine and mesentery on a thin piece of cork with a hole in
it corresponding to the mesentery. Place it for
twenty-four hours in a 5 per cent, solution of am-
monium chromate. After this wash away all the
chromate, stain a piece of the mesentery in logwood,
and mount it in balsam.

(a.) In the membrane observe narrow strands of
non-striped muscle composed of very large fusiform
cells. In the large nuclei a plexus of fibrils, while a
leash of line fibrils will be seen in the perinucloar part
stretching from the poles of the nucleus to the ends of
the fibre.

8. Fibrils in Smooth Muscle. —These may be seen
for a short time by macerating small pieces of the.
stomach of a frog for twenty-four hours in dilute
alcohol, or in 8-10 per cent, of sodic chloride (Enyel-
mann).'2

9. Isolated Smooth Muscle-Cells. — To get these
cells quickly macerate a small piece of the muscular
coat of the stomach, intestine, or bladder in 33 per
cent, caustic potash for 15-20 mins. Tease and
observe in the same solution. Water must not be

added, else the effect of weak caustic alkalies is produced, viz., solution of
the cells.

1 To be obtained from Dr. Grubler, Leipzig, or Bindscheidler & Busch,
Basel. '2 Pfliiger's Arch., xxv.

FlQ. 1 66. — T.S. Smooth
Muscle of a Cat's Intes-
tine. Chromo - acetp -
osmic acid. Shows in-
tercellular bridges, and
at a an artificial slit,
X 1000.

XVT.] STRIPED OR STRIATED MUSCLE. 193

10. Grooving on Smooth Muscular Fibres.— Harden the muscular coat of
the small intestine of a cat in chromic and bichromate fluid or in Flemming's
fluid. Stain and cut sections in paraffin. (H) Observe the polygonal areas,
but note that fine intercellular bridges connect adjacent cells (fig. 166).
The surface of each cell seems to be grooved with canals, but what these canals
contain is not known. In each cell the cut ends of fine fibrils are seen.

LESSON XVI.
STRIPED OR STRIATED MUSCLE.

Striped Muscle — sometimes called voluntary or skeletal muscle
— occurs in the muscles of the skeleton, pharynx, upper half of
the oesophagus, diaphragm, the sphincter of the bladder, external
anal sphincter, and the muscles of the outer and middle ear.
The muscular fibres of the heart are also striped, but they are
involuntary.

A Muscular Fibre is cylindrical in form, and tapers at its
extremities. They vary in breadth from 10 to 50 fi. (^Vo" TSTT
inch), but they are broadest in the muscles of the extremities.
The fibres are i to i^- inches in length.

A muscular fibre consists of the following parts : —

(i.) Sarcolemma, or sheath.

(2.) Sarcous substance, which is transversely striated.

( 3 . ) Muscle-corpuscles.

1. The Sarcolemma (H). — (i.) To avoid the effect of the con-
tractility of the muscle, kill a frog several hours before it is
required. Dissect out the sartorius muscle, because it is com-
posed of parallel fibres. Tear off a thin strip, and with needles
tcaso it in iU*ti1lnd water.

(a.) Observe the cylindrical shape of the fibres, marked trans-
versely by alternate light and dim stripes. Run the eye along
the edge of a fibre, and perhaps a clear, transparent bulla or bleb
will be seen. If so, it is the Sarcolemma raised from the subjacent
sarcous substance by water diffusing into the fibre (Hg. 167, A).

(/A) The surcolenima is a clear, transparent, colourless, homo-
geneous clastic membrane, forming a tubular sheath for the sarcous
substance. It is allied to, but not identical with, elastic tissue.
It is much tougher and less easily ruptured than the sarcous sub-
.stancc which it contains.

(ii.) A much better plan is the following: — Tease out a few
18 N

194

PRACTICAL HISTOLOGY.

[xvr.

fresh muscular fibres on a slide in normal saline, and across the
direction of the fibres place a hair. Cover, and press the cover-glass
down firmly on the fibres. The hair ruptures the sarcous sub-
stance, which retracts and leaves the tougher unbroken sarcolcrnma
between the ends of the ruptured fibre. Remove the hair, and
the now empty sarcolemma will be seen (fig. 167, C).

(iii. ) Another excellent method is to leave the muscle in a
saturated solution of ammonium carbonate. The sarcolemma will

FlQ. 167.— A. Striped muscle of frog, sarcolemma raised' in the form of a bleb; B.
Ruptured fibre with sarcolemma ; C. Fibre ruptured by a hair ; D. Effect of acetic
acid on a muscle-fibre ; E. Muscle-discs. Ammonium carbonate.

be seen raised for long distances from some of the fibres, with a
little of the sarcoglia sometimes adhering here and there to its
under-surface.

2. Muscle-Corpuscles, or Nuclei (H). — Tease a piece of a fresh
frog's muscle, and irrigate it with 2 per cent, acetic acid. I have
often found the nuclei beautifully stained in a piece of muscle that
has been in ammonium carbonate and then in picro-carmine.

(a.) Observe the sarcous substance becoming swollen up and
more homogeneous, while a number of fusiform, somewhat shrivelled
or shrunken nuclei, with their long axis in the long axis of the
fibre, come distinctly into view. They are now slightly more
refractive than the altered sarcous substance, hence they are seen
with greater distinctness. Focus carefully, and note that these
nuclei lie not only under the sarcolemma, but also in the substance
of the fibres. Had a mammalian muscle been used instead of one
from an amphibian — in the case of most muscles — the nuclei would
have been found directly under the sarcolemma only. The presence
of the nuclei is merely revealed by the action of the acid, which
alters the refractive index of the sarcous substance, and thus brings
the nuclei into view (fig. 167, D). Sometimes faint longitudinal
striation is exhibited by such a fibre.

3. Isolated Muscular Fibres. — (i.) Pith a frog, and plunge it in
a beaker of water at 55° C. Leave it in the water, and allow the

xvr.]

STRIPED OR STRIATED MUSCLE.

195

water to cool gradually (Ranrier\ It will now be found that the
fibres of any muscle can be dissociated with great ease ; the muscles
to be preserved in 70 per cent, alcohol until they are required.
By careful manipulation very long fibres may be isolated from the
sartorius. These muscular fibres exhibit the ordinary characters
of striped muscle. This is by far the easiest method of obtaining
isolated fibres.

(ii.) Place small pieces of a fresh muscle in the following mixture.
Nitric acid saturated with potassic chlorate. There must be crystals
of the latter in the fluid. The tube or „,
vessel is speedily filled with yellow 'i^3Sl
nitrous fumes. It is usually advised ^3^-':
to leave the muscle several hours in
this fluid. I find, however, if this be
done, that the muscle is dissolved.
Half an hour is usually sufficient.
With glass rods remove the now
softened orange-coloured muscle, and
place it in water. It becomes whitish.
Shake it in a tube with water. The
fibres fall asunder quite readily, and
after having all the acid removed by
prolonged washing, they can be stained
and mounted.

4. Fibrillse of a Muscular Fibre
(H). —Place a frog's or mammal's
muscle in water (two hours), and after-
wards in dilute alcohol for twenty-four
hours. Tease a small fragment of the
now softened muscle in glycerine, or
tease a fresh muscle of a calf in white
of egg. If a fibre be split up, bundles
of fibrils may be seen as in fig. 168.

Select a fibre, and note that at its
free end it splits up longitudinally into
a large number of very fine fibrils or fibrillae, each of which is
transversely striated like the original muscular fibre. Much larger
fibrils are obtained from the muscles of insects, e.g., Hydrophilus or
Dytiscus (fig. 171).

5. Muscle-Discs (H).— The usual directions for obtaining these
are to place dead muscle for several days in dilute (.2 percent.)
hydrochloric acid. The, muscular fibre then cleaves transversely.
I have not found this to be a very satisfactory method. A much
better plan is to place small pieces of the muscle in a saturated
solution of ammonium carbonate for several hours.

FIG.

Lped

lar Fibre of a Calf, teased in
white of egg, and showing iso-
lated bundles of fibrils, x 200.

196

PRACTICAL HISTOLOGY.

[xvi.

(a.) Not only will the sarcolemma be seen, but inside it, here
and there, the sarcous substance will be seen cleft
transversely into discs (fig. 167, E).

6. Ending of Muscle in Tendon. — This is readily
seen by taking the lower end of the sartorius with
its tendon from a frog treated as in Lesson XVI. 3,
(i.), and which has been afterwards placed in 70 per
cent, alcohol. Tease out the muscular fibres, when
their conical ends will be seen ending abruptly, and
the small tendons beginning as abruptly (fig. 169).

7. Crab's Muscle (Sarcous Substance) (H).—
Plunge the living muscles of a crab — or better still,
use a stag-beetle — into absolute alcohol for twenty-
four hours. Stain a fragment in dilute eosin-liaema-
toxylin, and mount it in Farrant's solution or balsam.
In the latter case the clarifying reagent must contain
a little eosin to restore the eosin colour to the pre-
paration.

(a.) Observe the fibre striped as in x

fig. 170, with alternate light and
dim discs, the dim discs (a) stained
of a logwood tint, while the light
discs are of a faint eosin tint.
(b.) In the light disc (b) observe a fine line or
series of dots running transversely, sometimes called
Dobie's line or intermediate
•6 line (figs. 170, I, 171, c),
dividing the light disc into
two equal parts, these ad-
hering to the ends of the
dim disc. They are then
called lateral discs.

(c.) The dim disc may
exhibit slight vertical stria-
tion, indicating a tendency
72 to cleave longitudinally. If
transverse cleavage be as-
sociated with simultaneous FlQf

FIG. 170.— Muscular Fibre of longitudinal cleavage, small Hydrophilus. a.
Great Adductor of Rabbit 4. » \ 4. • i Dim, c. Light
Living and Extended, a. elements are Obtained, disc; b Inter-
Dim disc; b. Light disc; which were called "sarcous "^ picroc-i?-

mineand formic
glycerine.

FIG. 169. — Rela-
tion of a Tendon
T. to its Muscu-
lar Fibre, the lat-
ter with a Coni-
cal Termination.

.#

171. — Fibril
Muscle of

elements " by Bowman,
profile. Examined in its /,/ \ Nuclei may be seen.

own juice, x 300. v / ., * .. „

A similar preparation of
an insect's muscle may be stained in picro-carmine

Tease it so

XVI.] STRIPED OR STRIATED MUSCLE. 197

as, if possible, to isolate a fibril. Mount in formic glycerine. The

FIG. 172. — Crab's Muscles,
partly schematic. A.
Non-stretched ; B. Ex-
tended ; A'. Fibrils ; D.
Dim ; L. Light stripes.

. ,73._T.S. Muscle, with its Sheaths. P.
Perimysium ; E. Endomysium.

dim disc is stained red (fig. 170, a), and if the fibre be stretched

Or"'

XT^'/VN,
•"-' /Q^r-N VN

FIG. 174.—^. T.S. muscle fibres of newt ; B.
Of mammal; C. T.S. muscular fibres
(mammal), with endomysium: b. Blood-
vessel: /''. Fasciculus: P. Perimysiurn.

FlQ. 175.— Capillary Plexus in Muscles.
a. Artery; v Vein; e. Capillaries,
x 250.

tli P. details of its structure can be better seen, especially if a very
high objective be used (fig. 171).

1S8

PRACTICAL HISTOLOGY.

[XVI.

8. T.S. of Muscle.— Make transverse sections of a small mamma-
lian muscle which lias been kept stretched and hardened in 0.5 per
cent, chromic acid and afterwards in alcohol. Stain one in logwood
and mount it in Canada balsam.

(a.) (L) Observe the sheath of connective tissue or perimysium
surrounding the whole muscle, and that from it septa pass between
groups or fasciculi of the muscle-fibres, and also a small amount
between the muscle-fibres, forming the endomysium (figs. 173,
174).

The ends of the muscular fibres somewhat polygonal or rounded,
with stained nuclei (one, two, or three) immediately under the
sarcolemma. In amphibian muscles (fig. 174, A) and in a few

mammalian muscles, e.#.,
the semi-tendinosus of
the rabbit, nuclei also
occur within the sarcous
substance.

(6.) (H) The ends of
the fibres appear finely
dotted with clear inter-
spaces, the dots corre-
sponding with the ends
of the bundles of fibrils
or muscle - prisms or
sarcostyles, while the
clear areas are due to
what is called sarcoglia.
9. L. S. Injected
Muscle (L). — Make a
longitudinal section of
an injected muscle, i.e.,
parallel to the direction
of its fibres. It is better
to inject the whole of
the posterior half of the
body, e.g.) of a rabbit,
from the aorta. The
injected bone will yield injected muscle.

FIG. 176.— T.S. and L.S. Injected Striped Muscle.
and 2. T.S. Muscular fibres ; 3. L.S.; a. Arteries.

preparation made for
Mount it in balsam.

(a.) Observe the elongated quadrilateral meshes of capillaries
between the muscular fibres, but outside the sarcolemma, and that
capillaries run between the fibres with short transverse connecting
branches. Trace their origin from an artery and their termination
in a vein (fig. i 75).

10. T.S. Injected Muscle. — Mount, either strained or unstrained,

XVI.]

STRIPED OF STRIATED MUSCLE.

igg

ill balsam. In this the cut ends of the capillaries between the
fibres will be seen (figs. 176, 177).

It is to be noted that there is a difference in the T.S. of con-
tracted and uncontracted muscles (Spatlahnlz). Beautiful figures
are given in.1

11. L.S. Bed Muscle of Rabbit Injected (L).— Use the semi-
tendinosus or soleus of a rabbit. This shows the same general
arrangement of the blood-vessels, but some of the transverse
branches and some of the veins have small dilatations or ampullae
upon them, while the capillaries are usually more tortuous than
those of the pale muscles.

CARDIAC MUSCLE.

12. — Harden small pieces of the heart in 20 per cent, nitric
acid (forty-eight hours), or 2 per cent, potassic bichromate or
ammonium chromatc for thirty-six to forty-eight hours. Tease a
small piece in Farrant's solution. Small pieces of muscle cardiac
placed fresh in picro-carmine for several days show the structure
well when mounted in glycerine.

Kid. 177. -T.S. Muscle In-
jected. M. Muscle,
with n. Nuclei, ft. l.lood-
vessel (capillaries).

FIG. 178.— Muscular Fi-
bres of the Human
Heart.

Firs. 179. -T.S. of a
Fresh Fro/en M oscu-
lar Fibre, showing
Cohnheim's areas.

(a.) Observe the faintly transversely -striated fibres made up of
short quadrilateral pieces with short oblique processes, which join
other muscular fibres. The muscle-cells branch and anastomose.
A rather indistinct line of clear cement joins the ends of adjacent

cells.

(/;.) There is no sarcolemma, but a well-defined nucleus lies in
the substance of the fibre, while the transverse striation is much less

1 " Die Vertheilung d. Blutgcfiisse iin Muskcl," Abhand. d. math. phys.
$la$scd. k. Sciehsig. Gesdl. d. Wissensch., 1888.

2OO PRACTICAL HISTOLOGY. fxvi.

distinct than in skeletal muscles (fig. 178). In a transverse section
of a cardiac muscle the nucleus lies in the centre of the fibre, and
radiating from it are fine lines (fig. 207) ; an appearance somewhat
similar to this is shown in T.S. of insects muscles.

ADDITIONAL EXERCISES.

13. T.S. Frozen Muscle (H). — With a freezing microtome make a transverse
section of a muscle taken from a recently-killed animal.

(a.) The ends of the fibres are mapped out into a large number of small
polygonal areas — Cohnheim's areas — separated from each other by a clear net-
work of lines. The darker areas correspond to the ends of a bundle of fibrils —
the so-called muscle-prisms, while the clear material between them is the
sarcoglia (fig. 179).

14. Living Muscle (H). — Remove, with as little injury as possible, some of
the muscular fibre from the leg of a water-beetle (Dytiscus or Hydrophilns),
place the muscle on a slide without the addition of any other fluid, and cover it.
Examine it as quickly as possible, and with the highest available objective.
In insects' muscles there is far more protoplasmic matter or sarcoplasm between
the muscle prisms or sarcostyles than there is in vertebrate muscle.

(a.) Observe the alternate cross stripes, some of which will be distinctly seen,
while at other parts they may be very close together, or the fibre may exhibit
contraction waves.

The dim disc or band may exhibit slight longitudinal striation, while a
dotted line — Dobie's line — will be seen running across the bright or light disc,
dividing it into two so-called lateral discs.

The nuclei, surrounded by a small quantity of protoplasm, may be visible.

15. Crab's Muscle (Methyl-Violet). — Stain a fragment of a crab's muscular
fibre (hardened in alcohol or M filler's fluid and spirit, p. 29) with methyl-violet
as described for fibrin (Lesson III. 18). Decolorise it with Lugol's solution of
iodine in iodide of potassium, clarify it in aniline-oil and xylol, and mount it
in balsam. Use all the precautions detailed under Weigert's method for fibrin.
In successful portions, the dim disc, and it alone, will be obtained of a deep
violet.

Do this with a contracted fibre, one extended, and one relaxed. In the
extended fibre observe that it is chiefly the light disc which has been elongated
by the extension (fig. 172, B). In the contracted muscle, the discs are closer
together and narrower, while the fibre is broader at the contracted part.

16. Polariscope. — An ordinary microscope can be fitted with a polariscope,
which consists of two Nicol's prisms ; one is placed below the object, and is
called the polariser (fig. 180), while the other, the analyser, is placed above
the ocular.

The light reflected from the mirror as it passes through the polariser is
polarised. With the analyser in position, look into the ocular, and slowly
turn the analyser. The best forms are provided with a graduated circle to
indicate the extent of the rotation. There are two positions of the analyser in
which the field is quite dark, caused by the polarised rays being cut of}'. This
occurs when the planes of polarisation of the two prisms are at right angles to
each other, i.e., when the Nicols are crossed. Between these two positions of
the analyser a greater or less amount of polarised light is transmitted. Certain
transparent histological preparations when placed on the stage of the micro-

XVI.]

STRIPED OK STRIATED MUSCLE.

201

scope are dark when the Nicols are crossed, others under the same conditions
cause the light to reappear, and appear bright on a dark field. They are said
to be doubly refractive. The dim disc is doubly refractive or anisotropous,
while the light disc is singly refracted, and is isotropous.

Either a preparation of fresh muscle or a balsam preparation may be used.
It is well to take a muscle which has broad and distinct stripes to see the
phenomena, one set of bands bright and refractive, and the others dark on a
dark ground, i.e., with crossed Nicols.

Instead of a glass-cover slip, cover the preparation with a thin slip of mica,
or place a thin plate of gypsum under a preparation of striped muscle. The
field shows various colours, red, pink, green, &c., according to the thickness of
the mica plate, the position of the Nicols, and the relation of the axis of the
mica to that of the Nicols. Suppose the general tint of the field to be pink,
then any doubly refractive substance assumes a tint complementary to the pink,
i.e., of a greenish hue. On turning the analyser, the tint of the field varies,

FIG. 180.— A. Polariser to fit into Zeiss's large stand in a frame under Abbe's condenser ;
B. Section of A showing arrangement of the prisms.

and with it the colour of the anisotropous substance, while the isotropous sub-
stance, being singly refractive, and having no effect on the direction of the
polarised ray, has the same colour as the field.

The doubly refractive property is possessed by bone (p. 181), smooth muscle,
and the white fibres of connective tissue.

17. Obliquely striated Muscle of Anodon.— Place a wedge between the
partially opened valves of Anodon, the fresh-water mussel, so as to put the
fibres of the posterior adductor on the stretch. The whole animal may then
be placed in dilute alcohol, or I per cent, potassium bichromate, for two days.
On teasing a portion, isolated fibres showing oblique striation are obtained.
Or the muscle may be examined fresh in sea-water or in the blood of the
animal.

Retro-lingual Membrane of Frog (Ranvier).1 — Under the tongue of the frog
is a lymph-sac which is separated from the buccal cavity by a thin membrane.
It contains striped muscular fibres which anastomose with each other, thus
forming a plexus— in this respect presenting a peculiarity. Cut off the head,
pull out the tongue, and remove the membrane. It can be floated in salt
solution to see it. Place the membrane for twenty-four to forty-eight hours
in dilute alcohol, then pencil away the epithelium, and place it for twenty -

1 Comptes Retidus, 1890.

19

202 PRACTICAL HISTOLOGY. [XVII.

four hours in dilute methyl-violet-sB. Wash and mount in glycerine. The
elastic fibres are stained of a bright blue, and they seem to spring from the
ends and sides of the muscular plexus — also stained blue. The elastic fibres
arc fixed to the sarcolemma — which, however, is not stained by the methyl-
violet — and the union is a very firm one. Thus the muscular fibres divide,
and arc in elastic sheaths connected with elastic fibres.

Ranvier has used the same membrane for studying the changes which take
place in a striped muscular fibre during contraction.

LESSON XVII.
NERVE-FIBRES,

NERVE-FIBRES are of two kinds, medullated and non-medullated ;
the former are found chiefly in the white matter of the nerve-
centres and the cerebro-spinal nerves, while the latter occur in
large numbers in the sympathetic system.

I. Medullated Nerve-Fibre.— The essential part is the axis-
cylinder, a soft, transparent rod or thread running from end to end
of the fibre, and composed of primitive fibrils. It is covered by
the myelin, or white substance of Schwann, or medullary sheath,
which envelops the axis-cylinder everywhere except at the termina-
tion of the fibre and at the nodes of Ranvier (fig. 181). The
myelin gives the nerve-fibre its highly refractive appearance and
its double contour, and it can be shown to consist of a gtroma or
network of fibrils of a peculiar chemical substance called neuro-
keratin, enclosing a semifluid fatty-like substance, containing,
amongst other chemical substances, protagon, a complex phos-
phorised fat. Histologically it consists of cylinder-cones or medul-
lary segments, whose ends are bevelled and fit one into the other,
but separated from each other by oblique clefts or incisures.

Outside the medullary sheath is a thin, transparent, tough elastic
sheath, the primitive sheath, sheath of Schwann, or neurilemma.
It is not present in all nerve-fibres, being absent from the fibres of
the central nervous system.

Between the axis-cylinder and the myelin is a thin layer of
matter, called by Kiihne axilemma. By others it is regarded as
an albuminous cement.

At fairly regular intervals — about i mm. — along the course of a
fibre are constrictions, the nodes of Ranvier, where the myelin is
absent, so that the neurilemma appears to produce a constriction
at these points. The part between any two successive nodes of

XVJL]

NERVE-FIBRES.

203

Ranvier is an interannular segment, or

internode, and about the centre of this,
under the neurilemma, is a flattened oval
nucleus — nerve-corpuscle — surrounded by
a small quantity of protoplasm, and lying
in a slight depression of the myelin.
Nodes of Ranvier are absent from the
nerve-fibres of the brain and spinal cord.
Osmic acid blackens the myelin ; silver
nitrate produces the so-called Ranvier's
crosses. The manner of their production
is given in the text. Nerve-fibres do not,
as a rule, branch except towards their
terminations.

The fibres vary greatly in diameter,
some being only half as broad as a red
blood-corpuscle (4 p, ^-^tr inch), others as
broad (8 ^ -g-^Vo" inch)? and others broader
still; so that they vary in diameter from -g
2 /A to 20 /JL. 1

II. Non-Medullated Nerve - Fibres. — f
They occur specially in the sympathetic S
system, but are also present in the cerebro- |
spinal nerves. Each fibre consists of a |
bundle of fibrils enclosed in a transparent c
structureless sheath. Some observers doubt
the existence of this sheath. The fibres
are somewhat flattened ; they branch and
anastomose, and in their course are oval
nuclei (fig. 182). As they have no myelin,
they are not blackened by osmic acid, so
that this reagent serves to distinguish the
two kinds of fibres.

Nerve-trunks consist of bundles or funi-
culi of nerve-fibres, each bundle containing
a greater or less number of fibres. Several
bundles are held together by a common
connective-tissue sheath — the epineurium.
The sheath around each funiculus is com-
posed of lamellated connective tissue,
covered on both surfaces by endothelial
cells, and is called the perineurium or
lamellated sheath. Lymph spaces exist
between the lamellae. Delicate fibrils of
connective tissue lie between the nerve- *IG'

.Node of
Uanvier.

Primitive
sheath.

.Nerve
corpuscle.

. Axis-cylinder.

White substance
of Schwann.

Node of
"llunvier.

PRACTICAL HISTOLOGY.

[XVII.

fibres, and constitute the endoneurium. The larger blood- and
lymph-vessels lie in the epineurium and
A perineurium, while the endoneurium sup-

ports the few capillaries which are distri-
buted to the nerve-fibres (fig. 190).

The term Sheath of Henle is applied to
the prolongation of the perineurial sheath —
usually a single lamella — around a small
branch, or even one or two nerve-fibres.

The following statement may facilitate the
study of the parts to be investigated : —

NERVE-FIBRES.

I. Medullated (chiefly in cerebro-spinal
system).

II. Non-Medullated (sympathetic or fibres
of Remak).

A. Medullated Nerve Fibre consists of—

( i . ) Primitive sh eatk, sheath of Schwann
(or neurilemma).

(2.) Nerve-corpuscles occur under the
primitive sheath in each inter-
annular segment.

(3 . ) W h ite substance of Schwann, myelin,
or medullary sheath (with cylin-
der cones and incisures). It
contains a net-work of neuro-
keratin.

(4.) Axis-cylinder, composed of primi-
tive fibrils (surrounded by a
sheath called the axilemma).

(5.) Nodes of Ranvier and internodal or
interannular segments between
two successive nodes.

Ranvier 's crosses and Frommanris lines,
tained by using nitrate of silver.
Sheaths of a Nerve-Trunk. — Epineurium,
perineurium, and endoneurium.
B. Non-Medullated Nerve-Fibres consist of —

FIG. 182. — Non-Meduiiated
Nerve-Fibre, vagus of Dog.

b. Fibrils ; n. Nucleus ; p.

Protoplasm surrounding it.

(i.) A bundle of fibrils, usually enclosed in

!2.) A transparent sheath or neurilemma (?).
3.) On the fibres are oval nuclei.

XVII.]

NERVE-FIBRES.

205

1. Medullated Nerve-Fibres (H).— Select a small nerve, e.g.,
the sciatic or one of its branches, of a frog. Cut out half an inch
of it, and place it on a dry slide, but add no fluid. Fix one end of
the thread by pressing on it with any thin blunt object, e.g., the
flat surface of a mounted needle, and fray out the opposite end in
a fan-shaped manner with a mounted needle, so as to isolate some
nerve-fibres. To prevent it from
drying, breathe on the specimen
from time to time. Add a drop
of normal saline, cover, and ex-
amine (fig. 183).

(a.) Observe highly refractive
medullated nerve-fibres of vari-
able size, some as broad as a red
blood-corpuscle and others nar-
rower. Each fibre has a dmille
contour, i.e., two thin lines on
each side of the centre. The
double contour may be inter-
rupted here and there. The
double contour is due to the
white substance of Schwann,
medullary sheath, or myelin.

(!>.} In the centre a clear
bright rod, the axis-cylinder.

(c.) Outside the myelin is a
thin transparent sheath, primi-
tive sheath or neurilemma,
scarcely to be detected as such
unless the fibre is ruptured or
the sheath raised from the
myelin, or stretching as a funnel-shaped prolongation from the end
of a torn fibre.

(d.) Selecting a fibre isolated for a considerable distance, trace
its outline, and observe at intervals slight constrictions — nodes
of Ranvier — where the myelin is absent.

(e.) A small quantity of delicate connective tissue — endoneurium
— with, perhaps, a capillary and a few blood-corpuscles between the
fibres.

(/.) The myelin tends to exude from the ruptured ends of the
fibres, and appears as highly refractive spherical droplets — myelin
drops — often with concentric markings, but there is no nucleus
in them (fig. 183, I). The myelin drops exude more rapidly in
a preparation irrigated with i per cent, acetic acid, or 2 per cent.
caustic soda, or even with distilled water alone.

to. 183. — Fresh
Nerve -Fibre Ex-
amined in Nor-
mal Saline. a.
A short piece of
the axis-cylinder
projecting ; I.
Myelin drops.

FIG. 184. — Nerve-
Fibre of Frog. a.
Ilanvier's node ; b.
Nucleus ; ?'. Inci-
sures ; m. Myelin
blackened. Osmic
acid, x 400.

2O6 PRACTICAL HISTOLOGY. [XVII.

Stain the preparation with picro-carmine, and note that this
reagent diffuses into the fibres at their cut ends and
at the nodes of Ranvier. It stains the axis-cylinder
red, and also the nerve-nuclei or corpuscles which
iie Just under the neurilemma.

2. Nerve-Fibres in Osmic Acid (H). — A nerve-
fibre is rapidly blackened by osmic acid, as can be
shown by applying a drop of i per cent, solution to
a fresh nerve, but for good permanent preparations
it is well to stain the nerve after the action of the
osmic acid. Place a small piece of nerve in a small
glass thimble along with 2 cc. of .5 per cent, osmic
acid. Cork the thimble, and after twenty-four hours
thoroughly wash the preparation ; tease it a little,
and place it for twenty-four hours in a solution of
picro-carmine. In fact, if it be left for days in this
dye it is better, as the fibres can then be more readily
dissociated. Tease a small fragment, and mount it
in glycerine acidulated with formic acid.

(a.) Observe in each fibre the myelin stained black.
Search for a node of Ranvier, a narrow constriction,
and note .that the myelin is absent at the constriction,
although the axis-cylinder and neurilemma are pre-
sent (figs. 184, a, 185). Find the next node, and,
between the two adjoining nodes, the stretch of
nerve — the internodal or interannular segment
(fig. 181).

(6.) In the interannular segment, just under the
neurilemma, and lying in a slight depression of the
myelin (lig. 184, 6), a red-stained oval nucleus sur-
rounded by a small quantity of protoplasm, and
about midway between the two nodes (fig. 185, n).
The axis-cylinder stained red, and continuous
throughout the fibre.

(c.) In the myelin what look like oblique slits —
incisures — running obliquely outwards from the
axis-cylinder to the neurilemma (fig. 185, i). They
correspond on each side, and break up the myelin
FlFibre85'~(Osmfc into a number of short lengths — cylinder cones-
Acid), a. Axis- the bevelled end of one cylinder-cone fitting into the

cylinder; r. Node -,11 n i i c Ji i v i

of iianvier ; ». in- oppositely bevelled end oi the next cylinder-cone.

cleS^'w "prokt Many cones lie in an internode.

piasn'; s. Neuri- (cZ.) Some of the fibres are broad, and others

narrow, about half the breadth of the others.
(e.) Some fibres are not blackened by osmic acid at all. They

XVIL]

NERVE- FIBRES.

2O7

FIG

appear as flattened bands with oval nuclei at intervals in their
course. They are non-medullated nerve-fibres, which, as they
have no myelin, are not blackened by the osmic acid.

(/.) A small quantity of connective tissue and capillaries. To
get a good view of the incisures and nodes, the best plan is to
stretch the sciatic or other nerve of a frog on a match before
placing it in osmic acid. In this way the fibres are kept straight,
and the cylinder-cones pulled asunder as far as possible, thus
making the incisures wide and distinct.

3. Ranvier's Crosses (H). — Rapidly tease out on a dry slide one
of the branches of the sciatic nerve (frog), and stain it for five
minutes with .3 per cent, solution

of silver nitrate. Wash off the
silver, apply a drop of glycerine,
and expose it to daylight. It
rapidly becomes brown.

(a.) Observe the fibres, but at
each node will be seen a brownish-
hlack cross (fig. 186); the silver
nitrate diffuses into the nerve-fibre
only at the nodes, stains tho
cement joining one internode with
another, thus making the trans-
verse bar of the cross, and as it
diffuses along the axis-cylinder it
stains some cement substance on
the latter, and thus makes the

vertical bar of the cross. Occasionally a number of transverse
(fig. 187) lines — Frorcmann's lines — are seen on the axis-cylinder,
i.e., on the vertical bar of the cross (p. 210). Place a sciatic nerve
of a frog for twelve hours in i per cent. AgN03, and keep it in
the dark. Wash and harden in absolute alcohol, and mount
in balsam. The crosses and lines are then seen with great dis-
tinctness.

4. Endothelial Cells of the Perineurium. Intercostal, or other
Small Nerve. — (i.) It is well also to stain with silver nitrate one
of the small intercostal nerves of a rat or some other small nerve.
Stain the whole nerve in silver nitrate.

(ii.) Open the abdomen of a frog, remove the abdominal viscera,
so as to expose the nerves coining from the vertebral canal. Pour
on the nerves .3 per cent, silver nitrate. After three minutes, cut
out the nerves and place them for half an hour in fresh .3 per
cent. AgNOr Wash in distilled water, tease a piece in glycerine,
and expose it to light.

(a.) The crosses are seen as before, but above them is the

lO. 186.— Nerve
with Ranvier's
Crosses. Silver
nitrate, x 30.

187. — Nerve-
Fibre. «. Node
and cross of Ban-
vier with Fro-
mann's lines, x
200. Silver nit-
rate.

208

PRACTICAL HISTOLOGY.

[XVII.

endothelial sheath of the nerve-fibre, composed of polygonal
squamcs (fig. 189).

5. Axis-Cylinder. — Harden for two or three days a nerve in
chromate of potash, tease a piece, and stain it in carmine. Observe
the axis-cylinder stained red (fig. 188). Or treat a fine nerve for
four days to a week in -^ per cent, chromic acid or -^ per cent,
bichromate of potash. On teasing long stretches of
isolated nerve axis-cylinders are readily found.

6. T.S. of a Nerve. — Select a rather large nerve,
e.g., the human sciatic, and harden about an inch of
it in picric acid for forty-eight hours, or in 2 per
cent, ammonium bichromate for two weeks. Wash
out the bichromate.
Complete the harden-
ing in alcohol. Sec-
tions may be made, and
stained with logwood
or carmine, but they
are very apt to fall to
pieces. It is prefer-
able, therefore, to stain
the hardened tissue
"in bulk." Place it
in borax carmine for
three days, then trans-
fer it to acid alcohol,
and pass it through
absolute alcohol and
turpentine, and embed
it in paraffin. Cut
sections, when the
paraffin keeps all the
parts in their places.

Fix a section on a slide with white of egg, remove
cylinder; 6. Ran- a}} t]ie paraffin by placing the slide in turpentine,
Nucleus, c'hro- clear it up in clove-oil, and mount in balsam.

(<i.) (L) Observe the connective-tissue sheath —
epineurium— or sheath surrounding the whole
nerve, sending processes into the nerve, — numerous
bundles — some large, others smaller — orfuniculi of nerve-fibres, each
surrounded by a lamellated sheath — perineurium — which sends fine
septa — endoneurium — into each funiculus. In each bundle the cut
ends of the fibres are directed towards the observer (fig. 190).

The large blood-vessels are in the epineurium, and a few in the
endoneurium.

FIG.

Fro. 188. — Peri-
pheral Nerve-
Fibre, a. Axis-

mate of potash
and carmine, x

200.

Mouse, Ranvier's Crosses and
Endothelial Covering. AgN03,

x 300.

XVII.]

NERVE-FIBRES.

209

(/>.) (H) Select a bundle. Observe the perineurium, made up
of several concentric lamellae with nuclei between them. The cut
ends of the fibres varying
in diameter.

(r.) Xote in each fibre
the section of the stained
axis-cylinder. Surround-
ing this a clear trans-
parent ring, indicating
the position of the myelin.
which has been dissolved
out in the process of pre-
paration.

(d.) Outside this a thin
circle — the primitive
alieath. Between the
nerve-fibres a small
quantity of connective
tissue or endonemium.

Non-Medullated Nerve-
Fibres.

FIG. 190.— T.S. of Several Funiculi of the Median
Nerve, p. Perineurium ; ep. Epineurium ; ed. En-
doneurium.

7. Non-Medullated or
Sympathetic Nerve -

Fibres. — (i.) These are readily found in the large splenic nerve of
the ox, or in a portion of the sympathetic nerve of the thoracic or
abdominal chain. The nerve has a pretty thick sheath. Cut this
open, and cut off a small piece. Tease it in
normal saline. The process of teasing is
greatly facilitated by placing the nerve for
twenty-four hours in dilute acetic acid (5 drops
in 100 cc. water).

(H) Observe that there are very few medul-
lated fibres, the great majority being non-
medullatod (iig. 192). Their outlines are not
very distinct ; they arc faintly striated longi-
tudinally, and have oval nuclei at intervals.
They may be stained with picro-carmine.

(ii.) Place the cervical sympathetic nerve of
a rabbit in .25 per cent, osmic acid for

twenty-four hours. Wash it in water, and stain it for several
hours in picro-carmine. Tease a fragment in glycerine.

(iii.) Or tease the vagus of a rabbit upon a dry slide, taking care
that the nerve does not become dry. Cover it for 5-10 minutes

O

FIG. 191. — Non-Medullater
Nerve - Fibres, Sympa-
thetic Nerve of Rabbit,
x 200.

2IO

PRACTICAL HISTOLOGY.

[xvn

with i per cent, osmic acid, wash away the osmic acid, add picro-
carmine, and place the whole for 24-48 hours in a ruoist chamber

a,

FIG. 193. — Neuro-

192. — Part of the Human Sympathetic Nerve in keratin Network

Osmic Acid. There are two medullated fihres in a Medulluted

amongst Remak's flhres, x 350. Externally the Nerve-Fibre,

epineurium, Epn; mF. Medullated fibres with
Ranvier's nodes; K. Nuclei of Remak's fibres;
inlF. Remnk's fibres ; K'. Nucleus of medullated
fibre ; Bz. Connective-tissue cells.

(fig. 47), and afterwards displace the picro-carmine by glycerine,
as recommended at p. 82.

(H) Observe a few small medullated fibres (blackened by Os(>4),
and numerous non-medullated fibres, some of which may bo seen to
branch. Note the oval nuclei on the fibres (fig. 191). If the part
be taken from near a ganglion, often nerve-cells may be seen.

ADDITIONAL EXERCISES.

8. Neurokeratin Network and Axis-Cylinder. —Place the fresh sciatic
nerve of a frog in a dilute solution of ferric chloride consisting of —

Liquor ferri perchloridi . . . . I part
Distilled water or spirit .... 3-4 parts.

XVII.]

NERVE-FIBRES.

211

Leave it in this fluid for three or four days. Wash every trace of the iron salt
out of the preparation, and preserve it in alcohol. Place small pieces of the nerve
several days in a saturated solution of dinitrosoresorcin in 75 per cent, alcohol.
Tease a fragment, dehydrate it with alcohol, clarify with xylol,
and mount in balsam (Plainer). This is an excellent method.

(a.) Ohserve the axis-cylinder stained green. It can be
seen with the utmost distinctness passing from one internode
to the next one, and across the nodes of Ranvier, which are
particularly sharply denned. In the myelin, a network of
fibres — the neurokeratin network — stained green. The axis-
cylinder appears as distinct as in fig. 193.

(ft.) Klihne and Ewald's Method.1 — Harden a nerve for
twenty-four hours in absolute alcohol. Boil it in absolute
alcohol, and extract it with ether. To remove everything
except the network, a teased preparation is digested in pan-
creatic juice.

(c.) Heidenhain's hoematoxylin (p. 70) may be used to
stain nerve-fibres. It stains the axis-cylinder and the neuro-
keratin network.

9. Frommann's Lines and Ranvier's Crosses (H). — Place

a fresh nerve in .c-i per cent, silver nitrate for forty-eight Vlf. ,,. „

11 • • i i t t-ir i A i -T IG. IQ4- — rioni-

hours, and keep it in the dark. Wash it in water, and mann s Lines
expose it to light for 2-3 days in equal parts of formic acid, on an Axial
water, and glycerine, and preserve it in glycerine. Tease Cylinder,
a piece in glycerine.

(a.) Observe the crosses of Ranvier sharply denned, and on the axis-cylinder
well-defined transverse markings, extending for a long
distance along the axis-cylinder. Frommann's lines. — If
an axis-cylinder be dislodged from its fibre, a biconical
swelling may be seen (a). It corresponds to that part of the
axis-cylinder opposite a node of Ranvier (fig. 194).

10. Axis-Cylinder— (a.) Action of Collodion (H).— Tease
a fresh sciatic nerve of a frog without adding any fluid.
Add a large drop of collodion and apply a cover-glass, or
tease a fresh nerve in chloroform. Examine quickly, as the
preparation soon spoils. The neurilemma is distinct, the
myelin is transparent and finely granular, while the axis-
cylinder appears as a dark cylindrical rod — often with a
curved course — in the centre of the fibre, and it may even
project beyond the end of the fibre.

(ft.) Isolated axis-cylinders are readily obtained from the
central nervous system after maceration of the white matter
of the cord in methyl-mixture (p. 26), or Miiller's fluid or
ammonium chromate.

11. Schwann's Sheath.— Macerate a peripheral nerve for
several days in ammonium chromate (i : 3000). The myelin
is dissolved while the neurilemma and axis-cylinder remain
(Schiefferdccl-cr}.

12.' T.S. Nerve, Osmic Acid (H).— Stretch a nerve on a
piece of wood, and place it — wood and all — for two days in
.5 per cent, osmic acid, or, better still, in Flemming's
mixture for one day. On the second day, add a little more
osmic arid to Flemming's mixture, and harden the nerve
for another day. It is better to embed the nerve in paraffin
and make transverse sections. The section.? are fixed on a slide by a fixative,
1 K Mint's Untersuch. , Heidelberg, 1878.

FIG. i9v— Periphe-
ral Nerve-Fibre
of Frog. a. Lon-
gitudinal fibrillne
in axis-cylinder ;
b. T.S. of nerve-
fibre. Osmic acid
and Bismarck
brown, x 1000.

212

PRACTICAL HISTOLOGY.

[XVII.

the paraffin is extracted by turpentine, and the sections mounted in balsam.
They may be stained with a watery solution of Bismarck brown. Instead of
this, after fixing the nerve in osmic acid, and hardening in 90 per cent, alcohol,
place it for 24-48 hours in a strong saturated watery solution of acid fuchsin
(S.N. 30), wash in alcohol, embed and cut in paraffin. The ends of* the fibrils
on the axis-cylinder are stained red (fig. 195).

(a.} Note the axis-cylinder in the centre surrounded by a dark ring (figs.
195, 196, b), the myelin blackened by the Os04. If the section of a nerve-
fibre is through incisures, the double contour of the myelin may be seen
sometimes with a narrow black edge, at others with a broad black edge
externally.

(ft.) If a piece of the nerve be placed for forty-eight hours in a solution of
Bismarck brown, and then teased, the appearance shown in fig. 195, a, is
obtained, when the axis-cylinder presents a longitudinally striated appearance.

13. Size of Nerve-Fibres. — The osmic acid method has yielded the best
results. Some of the fibres are broad, and others are narrow or fine. Thus the
anterior roots of the upper cervical nerves, and the third cranial nerve, contain
only broad nerve-fibres, while the second and succeeding thoracic nerves contain
broad and fine fibres (fig. 196). In nerves going to muscles there are many
large and few small medullated fibres, while in nerves going to viscera the
fine medullated fibres are far more abundant than the broad fibres.

FlG. 196.— A. T.S. of the anterior root of a spinal nerve below the first dorsal nerve.
B. T.S. of a part of a cervical nerve. Osmic acid.

14. Living Nerve-Fibres are readily studied in the inflated lungs of a newt
or frog. The frog's lung is best kept inflated by Holmgren's apparatus.1
Nerve-fibres are also readily seen in the tongue of a frog arranged as for
studying the circulation of the blood (Lesson XIX.).

15. Marchi's Method for Degenerated Fibres. — Harden a nerve in Miiller's
fluid for eight days and then in the following fluid : —

Marchi's Fluid.

Miiller's fluid ... 2 parts.

Osmic acid (i per cent) . . I part.

Embed in celloidin and mount in warmed balsam. It is well not to employ
balsam dissolved in chloroform, as then the darkened parts lose their dark
colour. The degenerated parts of nerve-fibres are black. This method is
particularly useful for degenerations in the nerve-centres before sclerosis has

1 Beitrage z. Awit. u. Fhys., C. Ludwig, gewidmet, Leipzig, 1874, p. cxvi.

XVII.] NERVE-FIBRES. 2 1 3

set in, but it is also applicable to nerve-fibres that have undergone degenera-
tion, e.g., after section of a nerve, constituting Wallerian degeneration. It
will also detect any degenerated fibres in an ordinary nerve.

16. Isolated Schwann's Sheath. — Place a stretched nerve of a frog in the
following lluid for twenty-four hours in the dark : —

Boveri's Fluid.

Silver nitrate ( i per cent.) . . . 10 cc.
Osmic acid (i per cent.) . . . 10 ,,

Wash it in water, and place for twenty-four hours in very dilute caustic
potash (2-3 drops of a concentrated solution in 15 cc. water). Tease in
glycerine. The axis-cylinder shrinks and Schwann's sheath may be traced as
a continuous sheath without any interruptions at the nodes.

17. Nerve-Fibres of the Spinal Cord. — These are devoid of Schwann's
sheath, but they possess both Ranvier's nodes and incisures of Lantermann.
Boveri's fluid may stain both. It is evident then that these two structures
have no relation to Schwann's sheath, but are related entirely to the myelin.
The cylinder-cones are readily isolated by Schiefferdecker's methyl-mixture
(p. 26).

18. Degeneration of Nerve-Fibres. — This is readily studied in the rabbit.
The skin is first disinfected with solution of corrosive sublimate, and then the
median and ulnar nerves are exposed on the inner aspect of the upper arm,
the nerves divided, and the wound sealed with collodion (C. Huber).1 In
different animals the nerves are excised 2, 3, 4, . . . 8 or 10 days after the
operation. The excised nerves are kept extended on wood and fixed for
twenty-four hours either in Hermann's fluid (Lesson XXXV.) or the picro-
osniium mixture of Benda, prepared by saturating a I per cent, solution of
osniic acid with picric acid and filtering. They are then washed in water and
hardened in alcohol. The sections are stained with safranin and light-green.
Besides showing the usual degeneration phenomena, they show mitotic division
of the nuclei of Schwann's sheath, showing that these proliferate.

LESSON XVIII.

NERVE -GANGLIA, NERVE -CELLS, AND PERI-
PHERAL TERMINATIONS OF MOTOR NERVES.

Spinal Ganglia. — Harden a spinal ganglion of a cat or dog in
2 per cent, ammonium bichromate for three weeks, and subse-
quently in alcohol. Make transverse and longitudinal sections
of the ganglion, stain with logwood or carmine, and mount in
balsam.

1. L.S. Mammalian Spinal Ganglion (L). — (<.<.) Note the cap-

1 Archivf. mik. Anat., xl. p. 409, 1892.

2I4

PRACTICAL HISTOLOGY.

[XVIII.

sule (fig. 197, c) surrounding the ganglion; nerve-fibres (a) enter
the ganglion at one end and leave it at the other. They run in
groups, chiefly through the central part of the ganglion, so
that they are cut in different planes.

(6.) Numerous spherical cells (tig. 197, b) lying singly or in
groups between the nerve-fibres, but chiefly towards the surface.

(c.) (H) Select a single ganglion-cell; note its spherical shape,

FIG. 197.— L.S. Spinal Ganglion, a. Nerve-fibres; b. Nerve-cells ;
c. Capsule of the ganglion.

its granular contents, and single, large, distinct, excentrically-placed

nucleus, often with one or more distinct nucleoli (fig. 198). The

nucleus has a well-defined nuclear membrane.

(d.) Around each cell is a capsule, which is lined by a single

layer of flattened cells, but only the nuclei of these cells are seen.
The cell-substance is frequently somewhat re-
tracted from the capsule, so that a space may
intervene between the two.

In a T.S. of such a ganglion, notice the capsule
of the ganglion sending in coarse septa, the nerve-
cells near the circumference, and the nerve-fibres
chiefly in the centre.

2. Isolated Cells of a Spinal Ganglion
(Mammal) (H). — Into a dorsal ganglion of a
young rabbit make an interstitial injection of
osmic acid (2 per cent.). Tease a small piece in
picro-carmine and mount the preparation in

, . 0 ,. n • j i • i i

glycerine. bometimes a cell with its single
process may be found. The cells are unipolar. It is more difficult
to find the connection of the issuing axis-cylinder with a nerve-
fibre, forming what Ranvier has described as T-shaped nerve-fibres,
but with care such processes can be found.

plasm shrunk from

the Capsule, x 200.

XVIII.] NERVE-GANGLIA, NERVE-CELLS, ETC. 215

3. Spinal Ganglion of Frog. — These ganglia lie under cover of
the small white calcareous sacs situated on each side of the verte-
bral column, which are seen at once when the abdominal cavity
is opened and the abdominal viscera removed. Remove the white
chalky mass, and the greyish semi-transparent small ganglion will
be seen. With sharp-pointed forceps it is not difficult to tear away
the capsule of the white calcareous mass. These sacs contain arra-
gonite ; some of the crystals are large, but the smaller arragonite
particles when examined in water exhibit Brownian movement.
Treat it in the same way as directed for the frog's Gasserian
ganglion.

(H) In a carefully-teased specimen (use a dissecting microscope,
p. 22), it is by no means difficult to find large unipolar cells, each
cell with a distinct hyaline capsule, and the cell itself with a
relatively large nucleus and well-defined nucleolus. Moreover, the
continuation of the body of the cell with a nerve-fibre is not
difficult to establish. The methylene-blue method may be used
(p. 222).

4. Spinal Ganglion of a Skate. — Make an interstitial injection
of osmic acid (2 per cent.) into such a ganglion. Stain a piece in
picro-carmine and tease it in glycerine. Bipolar cells are readily
found. Each cell shows a distinct capsule enclosing a nucleated
cell with a pole at either end

continuous with a nerve-fibre
(fig. 199).

5. GaSSerian Ganglion. — FIG< I99._Bipolar Ganglion Cell of the Spinal

(a.) The Gasserian ganglion Ganglion of a skate,

of a sheep does very well ;

harden it in the same way as for spinal ganglia or in Miiller's fluid.
The same general arrangement of fibres and cells is seen, only the
cells are larger, and their protoplasm frequently contains granules
of a yellow pigment. Very instructive results are obtained by
double-staining it with eosin and haematoxylin, first with hsemato-
xylin and then with eosin, or use eosin-haematoxylin, and mount
in balsam. The nuclei are blue, the other parts reddish. If a cell
be isolated after interstitial injection of osmic acid, as recom-
mended for spinal ganglia, the cells have the form shown in
fig. 200.

(6.) A fresh ganglion may be teased in salt solution. The large
spherical cells are readily isolated, but they usually shell out of their
capsule. Stain them with magenta solution.

6. Gasserian Ganglion of Frog. — Destroy the brain and spinal
cord of a frog, remove the lower jaw, divide the skull into two longi-
tudinally by a vertical incision. Tear off the mucous membrane
covering the roof of the mouth. From a foramen just behind the

216

PRACTICAL HISTOLOGY.

[XVIIT.

eyeball there issue a few fine threads, branches of the fifth nerve.

Scoop out the brain. These threads are readily recognised by being

usually somewhat pigmented. With a pair of scissors make a snip
in the base of the skull at right angles to the cut
already made. Turn up the bone, and on the fifth
nerve, which runs towards the foramen behind
the eyeball, will be found a small oval swelling
surrounded by a tough capsule. Divide the latter
and remove the ganglion. I have usually found
that the little ganglionic swelling is somewhat
pigmented. At any rate, it is easily found by
tracing the fifth nerve backwards. The nerve is
accompanied by an artery.

Tease the ganglion in .25 per cent, osmic acid,
and let it stain in this fluid for two hours. Stain
it for several hours in picro-carmine (under a
moist - chamber, p. 82), tease a fragment in
glycerine. Try to find a cell with its single pro-
cess prolonged into a nerve-fibre. In most of the
cells, however, the process is apt to be detached.

7. Sympathetic Ganglia (Frog). — Lying in
contact with the spinal column of the frog is a

FIG. 200.— Nerve-Ceil row of sma]i semi-transparent ganglia, the sym-

from Rabbit's

Spinal Ganglion, pathetic chain, and between them and the roots
ceii^ca^uie^n6 °^ ^he spinal nerves pass fine nerve filaments.
Nucleus of nerve- Open the abdomen of a freshly-killed frog, re-

flbre ; a. Nerve- ,-, . , ,. •, , , i i* . j_-i i

fibre; Fibre di- move the intestinal tract and liver, cut through
the peritoneum above the kidneys, raise the
kidneys and excise them. There will be seen
the white nerves issuing from the cord. Between
these and the sympathetic ganglia fine nerves run transversely.
Cut out the sympathetic ganglia and treat them with chloride of
gold by the method (p. 79, 3) ; or cut out the aorta and the
adjacent tissues, and subject them to the gold chloride method.
After the piece of tissue has acquired a purplish colour, examine
it with a low power to find nerve-cells. The nerve-cells may be
isolated or arranged in groups. It requires great care to get a satis-
factory preparation. Note the pyriform shape of the cell, each with
a large nucleus, the cell-substance continued into a straight process,
which may be seen to be encircled by a spiral process. The body
of the cell is surrounded by a well-marked capsule, which is con-
tinued over the cell-processes, and has nuclei on its inner surface.

8. Sympathetic Ganglion (Mammal) (H). — Harden the first
thoracic sympathetic ganglion or the superior cervical ganglion of a
man or rabbit in 2 per cent, ammonium bichromate (2-3 weeks),

viding at e at. a
node of Ranvier,
T-shaped fibre.

XVIII.]

NERVE-GANGLIA, NERVE-CELLS, ETC.

Capillary; V. Vein; K. Capsule;

N' Nerve-cell> x 3°°-

and subsequently in alcohol. Make transverse sections. Stain in
picro-carmine or a watery solution of nigrosin (several hours), and
mount the former in Farrant's solution, and the latter in balsam.

(a.) Observe the fibrous capsule of the ganglion ending in
septa, and numerous bundles of non-medullated nerve-fibres cut
obliquely or transversely. A few blood-vessels.

(/>.) The nerve-cells, each with a capsule, showing nuclei. The
nucleated cell substance is frequently somewhat shrunk from its
capsule, and at one side it usually
contains some yellowish-brown pig-
ment granules, especially if the
human cervical ganglion be used.
It is difficult to see the process,
which becomes continuous with
a nerve-fibre, but with care it may
be seen passing out of one or more
of the cells (fig. 201).

(c. ) The veins have long fusiform
dilatations upon them ; this is not
unfrequently seen in teased prepara-
tions of a human ganglion, but can
only be fully demonstrated in an
injected specimen of the ganglion.

9. Isolated Multipolar Nerve-Cells of the Spinal Cord.—
There are several ways of preparing these.

(i.) Cut out a small part of the anterior cornu of the spinal
<4.ml of an ox, calf, sheep, or other animal, and place it in very
dilute chromic acid (.01 per cent.) or .2 per cent, potassium
bichromate for a few days, and do not change the fluid. Wash,
and place it for twenty-four hours in strong carmine solution
(p. 63). Place a little of the red pulp on a slide, and, with the
aid of a dissecting microscope, try to isolate one or more multi-
polar nerve-cells.

(ii.) Or, what is a better method, take small fragments of the
anterior cornu of the spinal cord of an ox or calf, and place them
in dilute alcohol for forty-eight hours or longer. After this time
we can see better the distinction between the grey and the white
matter. Shake the fragments in the dilute alcohol, and allow the
debris to subside. Pour off the alcohol, and "fix" the cells with
.25 per cent, osmic acid (one hour); pour this off, and stain the
cells for forty-eight hours with picro-carminc. Pour off the picro-
carmine and replace it by glycerine-jelly. When the glycerine-
jelly is warmed, a drop of the fluid placed on a slide is almost
certain to contain one or more isolated multipolar nerve-cells.

With a low power find a cell.

PRACTICAL HISTOLOGY.

[xvul.

(a.) (H) Observe the large size of the cell (100 ^, ^Jo" inch, and
therefore visible to the naked eye), with numerous processes —
branched processes — which run in all directions (fig. 202). The

processes branch ; and then
branch again and again to
form a fine protoplasmic
system of processes — the
protoplasmic processes.
One process is always un-
branched, it is by no
means difficult to see — the
axis-cylinder process —
which becomes continuous
with, or in fact is, or be-
comes, the axis-cylinder of
a nerve-fibre (fig. 202, a).

(b.) The multipolar cell
itself has no cell- wall, and
it contains a large, con-
spicuous, spherical, nucleo-
lated nucleus, the latter
with a distinct envelope.
The protoplasm is fibril-
lated, and the fibrils may
be seen to stretch into the
branched processes. Some-
times the cells contain pigment (fig. 202, I).

10. Cover-Glass Preparation of multipolar Nerve-Cells.—
(i.) From a perfectly fresh cord of a sheep or ox snip off a small piece
of the anterior cornu; press it between two cover-glasses, so as to form
a thin film. Separate the cover-glasses and allow the film adhering
to each to dry. Float the cover-glass — film surface downwards —
on a concentrated watery solution of methylene-blue for several
hours. Wash the cover-glass in water mixed with alcohol, drain it,
allow it to dry, and mount it in xylol balsam ; the cover-glass can
be passed through absolute alcohol, cleared with xylol, and mounted
in xylol balsam. The multipolar nerve-cells are all deeply stained
blue (T/tanko/er).

(ii.) If the use of aniline colours be objected to, the following
method gives good results : — For three or four days macerate a small
part of the grey matter of the anterior cornu in 20 cc. of water,
containing i gram of each of the following : Neutral ammonium
chroma te, potassic phosphate and sodic sulphate (Landois' fluid,
p. 26), and then stain it in bulk for 24-48 hours in equal parts of
the above solution and strong ammoniacal carmine.

FIG. 202. — A Multipolar Nerve-Cell from the An-
terior Coruu of the Grey Matter of the Human
Spinal Cord. a. Axis-cylinder process ; b. Pig-
ment, x 150.

XVIII.] NERVE-GANGLIA, NERVE-CELLS, ETC. 2IQ

Squeeze a little of the red pulp between two cover-glasses, and
treat it as recommended for the methylene-blue preparation. It
would bo difficult to get preparations that surpass in beauty those
prepared by the methylene-blue method of Thanhoffer.

(iii.) Make a cover-glass preparation from a fresh spinal cord.
Heat one of them by passing it two or three times through the flame
of a Bunsen-burner. Thereby the proteids are coagulated and
partially charred. In some of these preparations good views of the
blood-vessels may also be obtained.

(a.) Observe — especially in the methylene-blue preparation — the
large cells with numerous branched processes ; some of them are
very long, and each shows distinct fibrillation.

(/>. ) The unbranched axis-cylinder process.

(e.) The body of the cell, nucleated and nucleolated, with its cell-
contents, traversed by blue-stained fibrils, running in certain definite
directions through the cell.

Other forms of nerve-cells are referred to under Cerebrum and
Cerebellum.

ADDITIONAL EXERCISES.

11. Motor Nerves to Muscles. — (i.) If the skin over the sternum of a small
frog be divided longitudinally, on raising the skin a small thin muscle —
musculus cutaneus pectoris — will be seen running from the skin to the sternum.
Keep the muscle stretched and " fix " it by pouring on it a little osmic acid.
Cut out the muscle, and after dehydrating, mount it in balsam. It is apt to
darken on exposure to light.

( L) Observe the nerve is black — sending branches over the muscular fibres ;
trace these onwards over the muscular fibres until a single nerve-fibre is found.

(H) Note that when a nerve-fibre divides, it does so always at a node of
Ranvier. The nerve-fibre can be traced to a muscular fibre, but it apparently
stops abruptly, because the myelin stops where the nerve pierces the sarco-
lemma. Other methods are required to see the termination within the sarco-
lemma.

(ii.) May's Method. — Select a thin muscle, e.g., the cutaneus pectoris,
sartorius, mylo-hyoid, &c., and place it in water containing 2 per cent, glacial
acetic acid for twelve hours. Make— fresh — the following mixture : —

| per cent, potassio-gold chloride . . . I cc.
2 ,, osmic acid . . . . . I ,,

2 ,, glacial acetic acid . . . 50 ,,

and place the muscles in it for 2-3 hours. Then transfer them to the following

mixture : —

Glycerine . . . . . . . 40 cc.

Water 20 ,,

Hydrochloric acid (25 per cent.) . . . . I ,,

for several hours. They become very transparent, and can be investigated in
glycerine or Farrant's solution.

220

PRACTICAL HISTOLOGY.

[XVIII.

12. Nerves of Frog's Sartorius. — Suppose the sartorius to be selected. A
beautiful view of the distribution of the motor nerves — black — is obtained
(fig. 203).

The single nerve-trunk enters the muscle on the median
aspect and on its imder surface about the level between
the middle and lowest thirds, Several large brandies —
usually two — run nearly parallel towards both ends of the
muscle — two longer, towards the upper end of the muscle
A — and two or three shorter, towards its lower end, the
latter following a slightly more oblique course. Numerous
branches form elongated quadrilateral meshes. At two
points in the muscle, towards its ends, there are more fine
branches than elsewhere. The fibres form plexuses and
divide. Note specially that the knee and pelvic ends are
devoid of nerve-fibres.

13. Motor Nerve-Endings.— (i.) Take a thin muscle,
e.g., the eye-muscles or intercostal muscles of a small
mammal, the thin leg muscles of a lizard, or the thin
cutaneous muscles which pass between the skin and the
wall of the chest in snakes, and stain them with gold
chloride by the formic acid gold chloride method (p. 79).
They must remain in the gold solution about one hour.
The gold may be reduced, either in water acidulated with
acetic acid, by exposure to the light, or in the dark, in
25 per cent, formic acid.

(H) Tease a piece of the purplish- violet muscle in
glycerine, and search for a purple nerve-fibre termination
in an arborescent branched end-plate lying on the sarcous
substance of the muscle (fig. 204). Nuclei are present in
the protoplasm of the end-plate.

(ii.) Golgi's Method.— Place the muscles of a newly-
killed lizard for a minute or two in a .5 per cent, solution
of arsenic acid, and directly afterwards in a solution of
.5 per cent, solution of chloride of gold and potassium for
1 5-20 minutes, and reduce the tissue in sunlight in a I per
cent, solution of arsenic acid. Instead of the above gold
solution, use the following mixture, devised by Kiihne : —

Arsenic acid (.5 per cent.) . . . . 60 cc.

Osmic acid (2 per cent.) . . . 3 ,,

Chloride of gold and potassium (i per cent.). 12 ,,

The tissue is then placed in I per cent, arsenic acid, and reduced by exposure
to sunlight. The process may be greatly hastened by doing the reduction
process at a temperature of 50° C., but it must be done in the direct rays of the
sun. The pieces of tissue can be preserved in the following fluid, devised by
Mays : —

Glycerine . . . . . . . 60 cc.

Arsenic acid ( I per cent.) . . . .10,,

Methylic alcohol . . . . .10,,

Water . . . . . . . 20 ,,

In working with solutions of gold, do not use steel instruments. They must
be either glass, platinum-iridiurn, or the substance known as " nickeline."

In birds, reptiles, and mammals the nerve-fibres terminate in " end-plates,"
which are disc-shaped bodies lying under the sarcolernma, i.e., they are hypo-
lemmal in position, 40-60 /u, long and 40 ^ broad. They consist of a finely
granular protoplasm with nuclei. As the gold chloride stains only the axis-

FlG. 203. — Distribu-
tion of Nerve-
Fibres in the
Frog's Sartorius.
A. Upper, B.
Lower end ; aa
and bb. Numer-
ous fine branches ;
P. Pelvic end, and
A'. Knee end, with
no nerve-fibres.

XVIII.]

NERVE-GANGLIA, NERVE-CELLS, ETC.

221

cylinder, one sees its branched arborescent terminations in the protoplasm
(tig. 205). A good plan to see the unaltered end-plates is to examine the

Nerve-fibre.

End-plate.

Muscle nucleus.

FIG. 204. — Termination of a Xerve-Fibre in End-Plate of a Lizard's Muscle.

muscle in a freshly-prepared I per cent, solution of sulphate of iron or ammonio-
sulphate of iron (Kiihnc, Mays}.

14. Frog's Motor Nerve-Endings. — There is no " end-plate," as in mamma-
lian muscles, but the axis-cylinder splits up into bayonet-shaped branches
under the sarcolemma. The myelin is continued up

nearly to the sarcolemma, but as the nerve-fibre
perforates the sarcolemma and becomes hypolemmal,
the myelin stops, so that within the sarcolemma the
branches are pale, and consist of branches of the axial-
cylinder only. The gold method (p. 79) may be used.
A simple method is to stain a small piece of a fresh
muscle (e.g., sartorius, near its middle, not at the ends)
with Delafield's logwood. It stains the hypo-sarco-
lemmal nerve-terminations. The mode of terminal ion
of nerves in sensory surfaces will be found under Skin,
Eye, and the sense-organs generally.

15. Terminations of Nerves in Tendon. — This is
best shown by one of the gold- chloride methods.
Perhaps the following by Manfredi is the best : —

Place the tendinous ends of the gemelli muscles of a
rabbit (or the enucleated eyeball with its muscles
attached) in

Potass bichromate (2 per cent.) . for 3 days.

Acetic or arsenic acid (i per cent.) . ,, 30 mins.

Gold chloride ( i per cent.) ... ,, 30 ,,
Wash and leave exposed in sunlight in I per cent, arsenic acid until it assumes
a violet-blue colour.

16. Pyriform Nerve-Cells (Frog).— These are most readily found in the
ganglion of the vagus as it issues from the skull. Pith a frog, distend the
(esophagus by pushing a small test-tube into it, place the frog on its belly,
relleet the skin over the shoulder-blade, divide the trapezius and remove the
fore-limb. The vagus will be seen coming out, along with the glosso-pharyn-
geal, through a large foramen immediately in front of the occipital condyle.
Clear away the muscles from the region of this foramen, snip and excise a

Fro. 205.— End-Plate of
a Lizard's Muscle.
One end -plate seen
in profile, the other
from above, with a
nerve - fibre axis -
cylinder terminating
in it. Gold chloride,
Golgi's method.

222

PRACTICAL HISTOLOGY.

[XVIII.

small part of the bone, so as to trace the nerve as far Lack as possible. Its
greyish gelatinous semi-transparent ganglion is seen. Remove the nerve with
the ganglion and place it for twenty minutes in I per cent, osmic acid and then
stain it in picro-carmine. Tease a small piece in glycerine, and it is by no
means difficult to find pyriform cells, each with a large nucleus, usually near
the broad end of the cell. The protoplasm frequently contains large refractile
granules. The straight process from the cells is readily seen, and with care
the spiral process also can be seen.

The methylene-blue method (p. 192) shows very well the straight and the
spiral fibre. Place the fresh tissue in the methylene fluid.

17. Isolated Cells of Sympathetic Ganglion (Mammal). — (a.) Tease a sym-
pathetic ganglion — best done after maceration for 24 hours in weak acetic acid
(2 drops to 100 cc. water). To isolate a cell showing its connection with a

nerve-fibre requires much patience.

(b.) Place a small piece of the superior
cervical ganglion of a rabbit in £ per cent,
osmic acid for 2-3 hours. Leave it in water
for a day or two to macerate, and then
tease it in diluted glycerine. A better plan
is to make an interstitial injection of the
osmic acid. It requires to be carefully
teased to get isolated cells showing several
nerve-fibres passing off from them.

(H) Note the spherical cell, with a
nucleated capsule. It gives off many pro-
cesses, each of which becomes continuous
with a nerve-fibre, i.e., it is multipolar.
These cells in the rabbit usually contain
two nuclei (fig. 206).

(c.) Double Impregnation Method of
Ramon y Cayal. — It is better to use the
sympathetic ganglia, e.g., G. stcllatum of
embryos (dog, rabbit), or those of new-born
animals. The fresh ganglion is placed in
Golgi's bichromate-osmic acid fluid (3 days),
then wash it with distilled water and after-
wards with .75 per cent, silver nitrate.
Leave it for 1-2 days in fresh silver nitrate
(•75 Per cent.). AVash it again, and place
it again in osmico-bichromate mixture (4-
4! days), and then again in silver nitrate.
Sometimes even a ''treble impregnation " is
useful. This process — "intensivo" or
" impregnacion doble " — we owe to R. y
Cayal, who has obtained good results with
it, and so has L. Sala,1 whose paper1
contains figures of his results and a resume

the literature. He finds that the cells are multipolar with a single
unbranched process and numerous branched protoplasmic processes.

18. Methylene-Blue Method. —This is an admirable method, and depends
on the fact that this substance stains blue the axis-cylinders or fibrils of
nerves in vivo. It is applicable for studying the terminations of nerve-fibres
in any tissue where they terminate, and also for the connection between nerve-
fibres and nerve-cells. When injected into the blood-vessels of an animal, it

FIG. 206. — Isolated Nerve-Cell from
Superior Sympathetic Ganglion of
a Rabbit. /. Remak's fibres;
n'n'. Nuclei of these libres ; nn.
Nuclei of cell.

6f

1 Archiv ital. de Biologic, 1893, p. 439.

XIX.]

THE HEART AND BLOOD-VESSELS. 223

acts on the nerves, and on the latter being exposed to the air they become
blue. It may also be applied to fresh tissues.

Inject some of the following solution into the blood-vessels : —

Methylene-blue I gram.

Normal saline .... 300 cc.

Or introduce a 3 per cent, solution, or even the solid substance, into the
'lymph-sac of a frog. After an hour or two, expose a muscle to the air,
or use the cornea, or any other tissue with nerves in it, and on examining
it under the microscope the nerves will be found stained blue. To preserve
such specimens, mount them in a solution of picrate of ammonia and glycerine
(p. 192). Fresh tissues, e.g., cornea or a thin muscle, may be immersed in a
weak solution of methylene-bliie with the sjime result.1

The methylene-blue method may boused for the study of nerve terminations
in any organ, e.g., the sense organs, and in arteries one can see most beauti-
fully the plexus of non-medullated fibres in the muscular coat.

19. Nerve-Cells of Crayfish. — Select a small individual and inject into its
abdominal cavity I to 2 cc. of a 0.2 per cent, solution of methylene-blue.
After 8 or 10 hours, remove the chitinous covering over the ganglia ted nerve-
cord and expose the latter in a vessel, which admits air and yet prevents
evaporation. In 24 hours or so excise a ganglion and observe it in a drop of
glycerine tinged with picrate of ammonia (Ketzius).

LESSON XIX.
THE HEART AND BLOOD-VESSELS.

Heart. — The wall of the heart consists of — (i.) Pericardium;
(2.) Myocardium ; (3.) Endocardium.

The pericardium covering the heart is a serous membrane
composed of fibrous tissue, with numerous elastic fibres, and covered
on its free surface by serous endothelium. It is sometimes called
epicardium. The fibrous tissue is continuous with that which
invests the bundles of muscles of the myocardium itself. Under-
neath the epicardium are the blood-vessels, nerves (ganglia), and
the lymphatics.

The myocardium is composed of striated muscular fibres, whose
characters have been described already (Lesson XVI. 12). The
iibres are arranged in bundles separated from each other by a
greater or less amount of connective tissue, in which run the blood-
vessels and nerves.

The endocardium in structure resembles the pericardium, but
it is thinner. It consists of a fibrous basis, with elastic fibres
covered by a single layer of endothelium. It contains a few smooth
muscular iibres.

An artery consists of three coats : —

(i.) Tunica intima, or inner coat, composed of a single layer

1 S. Mayer, Zcitsch. f. wiss. Mikrox., vi. 422, 1889, gives numerous references.

224 PRACTICAL HISTOLOGY. [XIX.

of endothelium resting on an elastic lamina composed of elastic
networks, or an elastic membrane. In many arteries, however,
there is a layer of connective tissue between the epithelium and the
elastic lamina — the sub-epithelial layer.

(2.) Tunica media, or middle coat, consists of a varying number
of layers of circularly-disposed, short, smooth, muscular fibres ;
but in most arteries, and chiefly in the large ones, it is intermixed
with elastic fibres or laminae.

(3.) Tunica adventitia, composed of fibrous tissue with elastic
fibres, the latter especially numerous near the middle coat.

There are, however, great variations in structure in arteries,
according to their size and other conditions.

Veins. — Speaking broadly, the veins have the same general
structure as the arteries. They are, however, much thinner, and
some of them have valves. They consist of three coats ; the inner
coat is thinner than in arteries, and the elastic lamina thinner

FIG. 207.— A. Fibres of the heart cut longitudinally ; B. Transverse sections of the
heart-fibres ; c. Cell ; n. Nucleus ; a. Connective tissue ; v Vein.

and often incomplete. The shape of the endothelial cells is different
(fig. 213, V). The middle coat is also thinner, and has less
muscular and elastic tissue, and relatively more connective tissue.
The outer coat is relatively very strong, and is composed of fibrous
tissue, which sends processes into the middle coat. There are,
however, great variations in the structure of veins.

Capillaries. — They form networks of fine tubes of uniform
diameter, sufficient to allow blood-corpuscles to pass along them
freely in single file. The arrangement of the network varies in
different tissues. The walls, when examined fresh, appear to be
homogeneous, but they are composed of flattened epithelial cells
or endothelial cells united to each other by their edges by cement
substance, which is blackened by silver nitrate.

1. Heart. — Harden small pieces of the heart (human) in alcohol.
Stain a small piece in bulk in borax-carmine, and then place it for
twenty-four hours in acid alcohol. It is best to cut sections by the
paraffin method and mount them in balsam. A very instructive

XIX.] THE HEART AND BLOOD-VESSELS. 225

preparation is to make a transverse section across both ventricles of
the heart of a small mammal, e.g., guinea-pig. This is best done
in a heart stained in bulk.

Sections may be cut by means of a freezing microtome, and then
stained with picro-carmine, and mounted either in Farrant's solution
or balsam. In both cases it is well to include a section of the peri-
cardium and also of the endocardium. Transverse sections of the
papillary muscles are very instructive.

(a.) (L) Observe the branched and anastomosing fibres, but in
addition some of them will be cut obliquely, and others trans-
versely, with the nuclei stained (comp. p. 199).

(b.) (H) The faint transverse striation, short branches, absence
of sarcolemma, the nucleus in the substance of the fibre, and the
indistinct cement substance (fig. 207).

(c.) The fibrous character of the pericardium, which sends fine
septa between the bundles of muscular fibres. If the pericardium
be not included in the section, still connective tissue will be seen
between the bundles of fibres, especially in transverse sections of
these (fig. 207, B).

2. Purkinje's Fibres (H) occur under the endocardium in the
heart of the sheep and some other animals. Open a ventricle of a
sheep's heart, observe the network

of fine glistening lines ; strip off the
endocardium, snip out a little piece
of the heart-muscle, and place it for
thirty-six hours in dilute alcohol or
5 per cent, ammonium chromate for
two days. Tease a very small piece
in picro-carmine, and mount in
glycerine.

(a.) Search for isolated polygonal
cells, each with usually two nuclei,
and the edges only of the cells
striated. These are heart-cells ap- FlG. 2oS._^injVs Fibres. Dilute
parently arrested in the process of alcohol, x 300.

striation (fig. 208).

3. Endocardium (H). — Harden a part of the ventricle of the
human heart in alcohol or potassic bichromate. Make sections tc
include the endocardium.

(a.) Observe oval nuclei on the surface, the nuclei of the endo-
thelial cells. Under this a superficial layer of fibrous tissue (fig.
209, a), with a few smooth muscle-cells (?«/), and underneath this
fibrous tissue the basis of the membrane (tc).

(b.) Outside this is the myocardium (me).

4. T.S. Heart-Valve (H). — Harden a cusp of a human tricuspid

21 P

226

PRACTICAL HISTOLOGY.

[XIX.

valve in chromic and spirit fluid (two weeks), make transverse
sections, stain in logwood, and mount in balsam. Alcohol does

well as a hardening re-
agent, and the sections
can then be readily
stained in picro-carmine.

(a.) On the surface,
toward the auricle (A),
note a superficial layer
of lamellated connective

FIG. 209.— Endocardium of Left Ventricle (Human), fjccmp whir-h iq rovprprl
a. Superficial layer ; ml. Smooth muscular fibres ; l U6» W»
tc. Fasciculated fibrous tissue; inc. Muscle of With eildofclielium (ll£.

heart, x 150.

2IOj

anj_ underneath

this a fibrous basis with elastic fibres (re).

(b.) If the section passes through the insertion of one of the
chordae tendinese, it presents the appearance shown in fig. 210, ct.

5. Aorta. — (i.) Make transverse (and longitudinal) sections of
the human aorta, or of that of an ox, which has been hardened in
alcohol, or, preferably, in 2 per cent, potassic bichromate (ten days).
Stain a section in picro-carmine, and mount it in glycerine.

(ii.) Another good method is to slit up any large artery, pin

FIG. 210. — T.So of the Cusp of the Human Tricuspid Valve, vertical to the axis of the
cusp. A. Auricular, B. Ventricular surface; a. Superficial lamellated layer of the
auricular, and a', of the ventricular surface; ct. T.S. of one of the chorda; tendineee,
where it is inserted into the valve ; re. Fibrous tissue, the basis of the valve, x 100.

it, inner surface upwards, upon wood. The pins must be close
together to prevent too great shrinking of the tissue. Place it in
a dry, well-aired place, say near a fire, so that it dries within a few
hours. Make transverse sections with a sharp razor. This is best
done by making a slit in a cork and clamping the dried membrane
in the slit. Place the sections in water; they swell up greatly.

Remove them,
glycerine.

stain with picro-carmine, and mount in formic

XIX.]

THE HEART AND BLOOD-VESSELS.

227

Fio. 211. — L.S. Human
Thoracic Aorta. A. In-
ternal, E. Middle, and
.C. External coat, x 20.
Drying, picro-carmiue,
and acid glycerine.

(a.) (L) Observe the subdivision into three coats (fig. 211), the
tunica intima (inner), media (middle), and adventitia (outer).

(b.) (H) The inner coat is lined by a layer of squames, whose
nuclei may be detected as slight oval swellings (not seen in the
dried specimen). Under this several layers
of yellow elastic membrane, with a small
amount of pink-stained connective tissue
between them. The outermost layer of
elastic membrane is generally thicker than
the others, and marks the outer limit of this c
coat.

(c.) The middle coat, composed also of
numerous elastic laminae, stained in this case
bright yellow, and between them patches of
smooth muscle with a somewhat brownish
tint, and some connective tissue stained
pink. The colour of these two tissues is quite distinct.

(d.) The outer coat, composed of white fibrous tissue (pink),
some elastic laminae, and a few smooth muscular fibres.

It is obvious, therefore, that elastic tissue enters largely into the
structure of the larger arteries.

6. Fenestrated Membrane of Henle. — Tear off a thin lamella
from the inner surface of a large artery, e.g., the aorta of a sheep.
Irrigate it with acetic acid, or place it

in 35 per cent, caustic potash, and
mount it in Farrant's solution.

(H) Observe the elastic laminae,
some of them with holes in them
(fig. 123). These laminae tend to
curl up at their edges. (See also
Lesson X. 7.)

7. T.S. Medium Sized Artery, e.g.,
the femoral artery of a child, prepared
and stained as the aorta.

(a.) (L) No te the three coats.

(b.) (H) The inner coat, with its
endothelium and internal elastic
lamina. In many arteries a layer of
sub-epithelial connective tissue lies between the endothelium and the
elastic lamina. The elastic lamina is thrown into folds in an empty
artery owing to the contraction of the middle coat (fig. 212).

(c.) The middle coat is composed of several layers of smooth
muscle arranged circularly. In a Canada balsam preparation the
nuclei stand out distinctly. Amongst the muscle-fibres are twisted
elastic fibres (fig. 212).

M.

FIG. 212.— T.S. Artery. 7. Tunica
intima ; M. Media ; E. Externa.

228

PRACTICAL HISTOLOGY.

[XIX.

(d.) The outer coat, chiefly composed of white fibrous tissue
intermingled with elastic fibres, some of which are cut trans-
versely. The elastic fibres are more numerous towards the inner
part of this coat. Here and there a section of a blood-vessel may
be seen.

8. Endothelial Lining of Veins and Arteries. — Cut open the
external jugular vein of a rabbit just killed. Pin it to a piece of
cork — inner surface uppermost — by means of hedgehog-spines.
Wash the internal surface with distilled water, and then apply
to it for five minutes J per cent, solution of silver nitrate. "Wash
off the silver, place the vein in water or alcohol and water, and
expose it to light. Do the same with any large artery.

(A.) Snip out a small portion of the vein, and mount it in
balsam, inner surface uppermost.

(H) Observe the "silver lines," indicating the existence of a
layer of polygonal squames composing part of the inner coat. The

FIG. 213. — A. Epithelial lining of an artery of a calf, and V. of the jugular vein of a
rabbit. The arrows show the direction of the blood-stream. Silver nitrate.

long axis of the squames lies across the long axis of the vein itself
(%. 213, V).

By focussing through the thickness of the wall, narrow fusiform
areas, bounded by black lines, may be seen, indicating the existence
of the smooth muscular fibres in the middle coat.

(B.) With a razor shave off a thin layer from the brown inner
coat of the artery. Mount it in balsam.

(H) Observe the elongated lancet-shaped endothelial cells (fig.
213, A); the long axis of each cell in the long axis of the tube.
The variation in the shape of the epithelial lining of vessels seems
to have relation to the velocity of the blood-stream in these
vessels.

9. Pia Mater, Capillaries, Small Arteries, and Veins (H).—
Carefully remove a small piece of the pia mater from the brain of
a sheep recently killed. Lay it on a glass plate, outer surface

XIX.

THE HEART AND BLOOD-VESSELS.

229

lowest. With a camel's-hair pencil dipped in normal saline solution
brush away all the brain matter, leaving a somewhat villous-looking
surface. Cut out a small part of the membrane, and mount it in
normal saline.

(a. ) Find a capillary, note its diameter, uniform calibre, and oval
nuclei bulging slightly into the lumen of the tube.
The rest of the wall is homogeneous (fig. 214).
Trace it backwards until the small artery or arteriole
with which it is continuous is found.

(b.) A small artery or arteriole with a thin
outer coat, and a middle coat composed of a single
layer of smooth muscular fibres arranged circularly FIG. 214. — Capii-

,, laries of Brain,

(fig, 2l6;. fresh, x JOG.

(c.) Select a small vein which is somewhat
thinner than the corresponding artery ; the muscular coat is very
i m perfect. Irrigate with 2 per cent, acetic acido Nuclei wherever
present come distinctly into view.

(</.) The oval nuclei of the lining endothelium, the long axis of

FIG. 215.— A A small artery with the
lumen in focus. B. Small arteriole
just before it passes into a capil-
lary, x 300.

FlG. 216. — Small Artery from
Human Brain, Pigment
Granules in the Adven-
titia, x 150.

the nuclei in the long axis of the vessel. Outside this the elastic
lamina appears as a somewhat refractive membrane with longi-
tudinal folds.

(e.) The vessel crossed transversely by the long, oval nuclei of
the muscular fibres of the middle coat (fig. 216). Note in some
cases they are not distributed at equal distances, but in groups
(fig. 215). One point must he particularly studied, viz., to focus
tli rough the thickness of a small vessel, and observe carefully that
the- appearance of the vessel varies with the position of the lens,
i.e., whether the upper surface, lumen, or deeper part of the artery
is in focus.

PRACTICAL HISTOLOGY.

[XIX.

(/.) In the outer coat the elongated fusiform nuclei of the con-
nective tissue cells are arranged longitudinally.

For a permanent preparation, the pia mater after being removed
is hardened in 2 per cent, potassic bichromate and preserved in
alcohol. A thin piece is selected and stained with logwood or with
eosin-logwood, and mounted in balsam.

10. T.S. Small Artery and Vein.— Select a small artery and
vein, harden in M tiller's fluid, stain in bulk with haematein, and
cut in paraffin.

(H) Observe the three coats in each, but they are much thinner
in the vein than the artery. In the former the intima is very thin
and the outer coat relatively thicker (fig. 2 1 7).

FIG. 217.— T.S. Small Artery and Vein. A. Artery ; V. Vein ; N. Nerve.

11. Injection of Silver Nitrate into Blood- Vessels. — (i.) From
the aorta inject the blood-vessels of a rabbit with .25 per cent,
silver nitrate. Before doing so wash out the blood-vessels with
normal saline to remove all the blood, and then with distilled
water. Slit up the intestine, wash out its contents, and expose it
to light in alcohol and water. Scrape away the mucous membrane,
leaving only the muscular coats. Dehydrate a small piece and
mount it in balsam. It is easy to find large and small vessels as
well as capillaries.

(a.) (H) Select a small artery, and note in it the endothelial
lining (fig. 219, E) and the circular muscular fibres mapped out
from each other by silver lines (m).

XIX.]

THE HEART AND BLOOD-VESSELS.

Arterioles and Small Arteries. — (a.) (H) Select a small artery
or arteriole. Note the layers already described. If the circular
muscular fibres be arranged in one layer, note that the fibres are
arranged in alternate groups on opposite sides of the vessel (fig.
215, A).

(b.) Trace an arteriole into a capillary, and note the change in
structure, especially how the muscle disappears (fig. 215, B).

(c.) Select a larger vessel, and in it observe the structure already
described and shown in fig. 218.

(d.) Find capillaries, and note the endothelium of which they

FlQ. 218.— Middle-sized Ar-
tery of Brain, a. Endo-
thelium ; b. Fenestrated
membrane; c. Middle
or muscular coat ; d.
Adventitia; e. Pigment,
x 300.

FlQ. 219.— Arteriole of the Rabbit's Small
Intestine. E. Endothelial cells of the
intima; in, Circular muscular fibres ;
indicated by silver lines, x 200.
Nitrate of silver.

are composed (fig. 220)0 In order to see the nuclei of these cells,
UK; preparation should be stained with logwood.

(ii.) Instead of a rabbit, the blood-vessels of a frog may be
injected in the same way from the aorta or ventricle. Use a glass
syringe. Wash out the vessels first with normal saline, then with
distilled water, and, finally, inject the silver nitrate solution. .The
bladder, intestine, and mesentery are particularly serviceable for
obtaining small vessels and capillaries.

12. Circulation of Blood. — A day before the. frog is required,

PRACTICAL HISTOLOGY.

[XIX.

FIG. 220.— Capillaries Injec
ted with Silver Nitrate.

let the brain of the animal be destroyed. An hour before the web
is to be examined, place two or three drops of a . 5 per cent, watery
solution of curare in the lymph-sac under the skin of the animal's
back. The drug should not act too rapidly. After a hour or so it
paralyses the extremities of the motor nerves,
and thus makes the frog motionless. A small
dose of the drug is given in order not to
affect the calibre of the blood-vessels.

Make a frog-plate by taking a piece of
stout cardboard or thin slip of wood 1 5 cm.
long (6 inches) and 5. cm. (2 inches) broad.
At one end of it cut a triangular slit whose
base is 2 cm. or less in width. Tie a thread
round the tip of, e.g., the third and fourth
toes of the hind-limb, place the frog on
its belly on the board, and by means of
the two threads gently stretch the web
across the triangular slit. It must not be
drawn too tightly. The threads can be fixed
in slits made in the horns bounding the
triangular aperture. To prevent evaporation from the frog, it had
better be placed in a moist cotton rag or surrounded with moist
blotting-paper. Moisten the web with a drop of water, and cover
it with a narrow fragment of a cover-glass. In selecting a frog,
choose a light-coloured one.

(a.) (L and H) Find an artery, and note that the blood flows
from larger into smaller vessels with what appears to be consider-
able velocity. Contrast it with a vein, in which the blood flows
in an opposite direction, i.e., from smaller vessels — capillaries — to
larger ones, but the current is slower in the veins than in th«
arteries. The walls of the vein are slightly thinner than those of
the artery.

(b.) The capillaries, small and of uniform diameter, with the
corpuscles moving in single file. The flow is uniform.

(c.) In the arteries and veins note the rapid red central stream,
or axial zone of coloured corpuscles, and next the wall, on either
side, the peripheral zone or space of Poiseuille, narrow and free
from red corpuscles, but containing a few white ones rolling lazily
along the vascular wall (H).

(<i.) If a red corpuscle happen to be arrested at the bifurcation
of a capillary, other corpuscles impinge on it and bend it. As
soon, however, as it is dislodged it regains its shape, so that the
red corpuscles are highly elastic.

(6.) Numerous pigment-cells, some of them contracted, others
expanded, are visible (fig. 144).

XIX.] THE HEART AND BLOOD-VESSELS. 233

If desired, the phenomena of inflammation can readily be
studied by applying some irritant to the web, e.y.t mustard or
creosote (one minute).

ADDITIONAL EXERCISES.

13. Elastic Fibres in Arteries (Martinotti). — Harden the blood-vessel in
chromic acid, make sections, and stain them with safranin as directed in
Lesson X. 10. All the elastic fibres are purplish or black. Herxheimer's
Method (p. 161) also yields good results.

14. Development of Blood- Vessels. — (i.) Harden the omentum of a newly-
born rabbit in Flemming's fluid for twenty-four hours. Wash it thoroughly to
remove all the hardening solution, Stain a piece for 24-36 hours in safranin.
Remove the surplus stain in the usual way with acid alcohol. Mount in
balsam.

(ii. ) Kill a rabbit five days old with chloroform ; do not bleed it. Open
the abdomen, remove the stomach and spleen, and attached to them the
omentum. Place all in a saturated
watery solution of picric acid for one
hour ; wash away all the picric acid, cut
out a small piece, stain it in logwood and
then with eosin, or double stain it at
once in eosin-hsematoxylin. Mount in
Farrant's solution.

(a.) (H) Search for a network of capil-
laries, which is easily found. Try to
find one of them which gives off a long,
narrow, blunt process. The process may
be found partially channelled (hg. 22l). a New-Born Rabbit. Flemmiug's

By the union of two such processes, which fluid and safranin.

ultimately become hollow, new capillary

arches are formed. The blood-corpuscles in a preparation of (ii.) are stained
with eosin.

15. Nerves and Nerve-Cells in a Frog's Heart.— Pith a frog, expose its
heart, cut away the pericardium, divide the frjenuin. which connects the pos-
terior surface of the ventricle to the pericardium ; raise the heart, find the
sinus venosus, ligature the inferior and two superior vense cavae which open
into the latter, make an incision into otie of the aortae, and into it tie a fine
glass cannula. Inject normal saline so as to wash out the cavities of the
heart. Distend the heart-cavities with the following mixture : — Four parts
of gold chloride (2 per cent.) and one of formic acid boiled together and
allowed to cool. Ligature the other aorta, so as to get the heart-cavities fully
distended. Place a ligature below the cannula, cut out the heart and place it
for ^-i hour in 5 cc. of the gold mixture. Open the auricles, wash out the
heart in water, and expose it to light in distilled water — 50 cc. — containing
three drops of acetic acid. Reduction of the gold takes place slowly in 3-4
days. Cut out the auricular septum and examine it in glycerine.

Pyriform nerve-cells, each with a straight and a spiral process, will be
found along the course of the nerves in and near the auricular septum.

The nerve-fibres in the auricular septum are readily found by using instead
of the gold a. 2 per cent, solution of osmic acid.

The Methylene-blue method yields excellent results, if the fresh auricular

234 PRACTICAL HISTOLOGY. [xX.

septum be placed in a weak solution of the blue, and then mounted in
ammonium picrate glycerine (p. 192).

16. Circulation in the Tongue of Frog. — Destroy the brain of a frog, and
after a time curarise it and fix it on its back on a frog-plate of cork with a hole
cut in it just in front of the head of the animal, the hole corresponding in
size to that of the tongue. The tongue is attached in front, and it can thus
readily be pulled out of the mouth so as to display its blood-vessels from the
under-surface. Pin out the tongue over the hole in the cork, when the under-
surface of the tongue will be uppermost. Fix the tongue under the microscope
and examine it (L). One can study the circulation in its vessels, and also
observe nerve-fibres in their normal condition.

LESSON XX.

THE LYMPHATIC SYSTEM— SPLEEN— TONSILS—
THYMUS GLAND.

THE lymphatic vessels have thin translucent walls, and in all
essential respects resemble veins in structure. There are three
coats in the larger vessels. The muscular fibres are abundant in
the middle coat, and the epithelium of the inner coat is in some
situations sinuous in outline. Valves are numerous in some
situations. The larger vessels spring from so-called lymph capil-
laries, which are usually wider than blood-vessels, and they unite
with each other and form an irregular plexus. They often present
dilations and constrictions, and consist of a single layer of endothe-
lium with sinuous outlines. They are without valves, and open into
the smallest regular lymphatic vessels.

Lymphatic Glands. — These vary much in shape, size, and colour,
but they are frequently oval or kidney-shaped. At one part is a
depression — the hilum — where the medullary part of the gland
comes to the surface, and where the blood-vessels enter the gland
and the efferent lymphatic vessel leaves it. There is usually only
one efferent vessel and several afferent lymphatics ; the latter per-
forate the capsule and enter the gland on its convex side. On
making a section of a gland, with the naked eye one can see that it
is divided into a cortical and a medullary part. The gland is
invested by a fibrous capsule, which in some animals contains
smooth muscular fibres. It consists of two layers, the outer of
coarser and the inner of finer connective tissue. It sends somewhat
flattened, large, usually unbranched, septa or trabeculse into the
cortex, thus dividing it into a series of compartments or alveoli

XX.]

LYMPHATIC GLANDS.

23$

(fig. 222). These trabeculae are continued into the medulla, where
they branch, become finer, and anastomose to form an irregular net-
work. The lymphoid tissue lies in the meshes of this trabecular
framework, but everywhere separated from it by a lymph sinus or
lymph channel.

In the cortex the alveoli are arranged in a regular manner, and
the greater part of each alveolus is occupied by a mass of adenoid
tissue crowded with leucocytes ; but this follicular substance is
everywhere separated from the capsule and trabeculse by a lymph
sinus, traversed by a network of fine fibrils with flattened cells
lying on them at the points of intersection. The network
is coarser than that of the adenoid tissue, and it contains a few
leucocytes. It, as well as the trabeculse, is covered by a layer of
sinuous endothelium. The medulla is also occupied by adenoid
tissue crowded with leucocytes, but the lymphoid tissue forms

FIG. 222.— L.S. Cervical lymph glands of Dog. c. Capsule ; 8. lymph sinus ; F. Follicle ;
a. Medullary cord ; b. Lymph paths of the medulla ; V, Section of a blood-vessel ;
11 F. Fibrous part at the hilum, x 10.

branching or anastomosing cords — medullary cords — each one sur-
rounded by its lymph sinus. The medullary cords are continuous
with the follicular substance in the cortical alveoli. Near the centre
of the lymphoid tissue of the alveoli are clearer areas, the lymph-
knots or germ-centres. In them mitosis goes on rapidly, but it
requires a high power to discern the stages of the division of the
nuclei. The lymph sinuses are continuous throughout the gland,
and they are the channels through which the lymph moves. Some
glands are pigmented.

LYMPHATIC GLANDS.

1. Lymphatic Glands.- — Harden in 5 per cent, ammonium
bichromate or alcohol the lymphatic glands of the mesentery of a
cat or calf, or those found in the neck or under the lower jaw of a

236 PRACTICAL HISTOLOGY. [XX.

cat. For the germ-centres harden a small gland in Flemming's fluid
and stain with safranm. Make sections including both poles of the
gland and the hiluin. Stain them with logwood and then with
eosin. Mount in balsam.

(a.) (L) Observe the capsule (fig. 222, c) surrounding the gland,
and sending at fairly regular intervals septa or trabeculse into the
substance of the gland. The trabeculae and capsule are stained by
the eosin. The trabeculae are flattened in the outer part or
cortex, and divide ifc into compartments — follicles or alveoli (F).
The trabeculae are continued into the central part or medulla,
where they form a network of smaller, branched, more rounded
trabeculse.

(b.) The compartments in the cortex are nearly filled by leuco-
cytes lying in a meshwork of adenoid tissue constituting the
follicles (cortical nodules) of the cortex (fig. 222, F). Between
the trabeculae of the medulla the leucocytes are equally abundant.
Owing, however, to the arrangement of the trabeculse, they form
medullary cords, but everywhere the adenoid tissue is continuous
throughout the gland.

(c.) The lymph channels (s) exist between the capsule and the
follicles, and between the trabeculae and the lymphoid tissue of the
cortex and medulla (figs, 222, 223). They form an anastomosing
system of paths or channels throughout the gland, and are traversed,
by a fine network of coarse adenoid tissue with comparatively few
leucocytes in its meshes (fig. 223).

(d.) (H) The capsule and trabeculae, chiefly composed of connec-
tive tissue, and in some animals (ox) with smooth muscular fibres.
Continuous with its under surface is a delicate network of adenoid
tissue, which stretches across the lymph channel to the follicle,
where it becomes continuous with the adenoid tissue supporting
the leucocytes (fig. 223). Between the trabeculae and the medul-
lary cords in the cortex similar lymph paths with an adenoid net-
work.

(e.) The follicles and medullary cords, everywhere crowded with
leucocytes.

2. Adenoid Reticulum. — (i.) As this is largely obscured by the
presence of the leucocytes, these must be got rid of. This is readily
done by making sections of the lymph gland of an ox, hardened for
two or three days in 5 per cent, ammonium bichromate, and then
shaking the sections in a test-tube containing water, or the leuco-
cytes may be "pencilled " out by a camel's-hair pencil.

(ii.) A better method, perhaps, is to inject by the puncture
method (p. 237) a J per cent, solution of silver nitrate into the
lymph gland of an ox. The fluid being driven in forcibly, causes
an artificial oedema and forces the parts asunder. Harden in

XX.]

LYMPHATIC GLANDS.

237

Lymph-path; M.C. Medullary cord. Silver
nitrate and hamiatoxylin, x 300.

alcohol, stain in logwood and eosin, and mount in balsam. The
reticulum in the ox has a brownish appearance from the deposition
of a brownish pigment. Sinu-
ous outlines of endothelial
cells may be seen on the tra-
beculae, and branched cells of
the lymph sinus (fig. 223),
and even the follicular sub-
stance. It is continuous with
the endothelial lining of the
lymphatic vessels.

3. Injection of the Lymph
Channels. — Fill a hypoder-
mic syringe with a watery T' i_ M.C. L T

Solution Of Berlin blue, force Fia 223._Lyfnph sinuses from the Medulla of a
the nozzle of the syringe into Lymphatic Gland. T' and T. Trabeculfe ; L.

a small lymph gland of an

ox, and inject the blue fluid

haphazard into the gland. The blue passes into the lymph

channels. Harden in alcohol. Make sections by freezing, stain

them with picro-carmine, and mount in Farrant's solution.

(L) The channels are filled with a blue mass, while the leuco-
cytes are red and the septa yellowish-red. The blue mass lies under
the capsule, and in the lymph paths around the trabecula. Notice
the difference in the distribution of the blue mass in the cortical
and medullary parts of the gland. If it be desired to study the
endothelium covering the trabeculae, inject the gland as above, but
with y1^ per cent, silver nitrate, and harden in alcohol.

4. Central Tendon of Diaphragm. — Lave the central tendon of
the diaphragm of a newly-killed rabbit in distilled water. Place it
for an hour in .2 per cent, silver nitrate in a dark place. Remove
it, wash again, and place it for twenty-four hours in water contain-
ing a little alcoholic solution of thymol (10 percent.), or a drop
of carbolic acid to prevent the formation of fungi. Maceration in
water enables the endothelium on the surface to be readily pencilled
off. Mount it in balsam.

(L) Examine the pleural surface with the naked eye or with a
lens, and a plexus of lymphatic vessels — clear on a dark-brown
ground — will be seen (fig. 224). The vessels anastomose, and lead
into narrow vessels, which run more or less parallel to each other,
and correspond to inter-tendinous spaces.

(H) Observe the dilations and constrictions in the finer lymphatic
vessels, and their sinuous epithelium (fig. 225, L), and the com-
munications between the cell-spaces and the lymphatics. (Lesson
XL 12.)

238 PRACTICAL HISTOLOGY. [XX.

5. Septum Cisternse Magnse Lymphaticse.— Open the abdomen

f IG. 224.— Lymphatic Vessels of the Central Tendon of the Diaphragm of a Rabbit the
lining endothelium shown. Silver nitrate.

of a newly-killed frog and remove the intestines. Turn the frog

f the Central Tendon of
us endothelium ; c. Cell

its belly. Cut through the vertebral column at its lower end,

FlO. 225.— Pleural Surface of the Central Tendon of Diaphragm of Rabbit. L. Lymphatic
vessel lined with sinuous endothcliuiu ; c. Cell-spaces of connective tissue Silver
nitrate, x no.

XX.]

LYMPHATIC GLANDS.

239

raise it, and cut out a square window — about f inch in diameter,
including the whole of the posterior body-wall. This exposes the
dorsal surface of a thin membrane, the septum of the great lymph-
sac. It is attached to the kidneys on each side. Pin the
membrane, by means of hedgehog-spines, to a thin ring of cork
with a hole in it, "Wash the membrane with distilled water and
place it for ten minutes in . 5 per cent, silver nitrate solution. Wash
it — still on its ring of cork — in distilled water, and expose it to
light. After it has become brown, cut it into pieces, and mount
them in Farrant's solution, one with the peritoneal surface upper-
most, the other with its dorsal surface uppermost.

A. (H) Peritoneal Surface. — Observe the slightly sinuous
"silver lines," indicating the existence of

a single layer of endothelium. Here and
there small stomata or openings, which
lead from the peritoneal cavity to the great
lymph-sac (fig. 226). The stomata may
be closed or open, and are recognised by
their brownish appearance. They are sur-
rounded by a few finely-granular brown-
stained cells — germinating epithelium.
The pointed angles of several of the larger
endothelial cells radiate from these aper-
tures.

Focus through the thickness of the
membrane, and note its fibrous character.
( hi the deeper surface another layer of
endothelial cells comes into view. They
are more sinuous and narrower than those
on the peritoneal surface.

B. The Dorsal or Cisternal Surface. — Observe the more sinuous,
polygonal, and broader endothelial cells covering this surface of
the membrane ; and the other openings of the stomata.

The serous cavities communicate by means of stomata with the
lijmphatic system. Stomata occur on the pleura, under-surface of
the diaphragm, and mesentery =,

FIG. 226. — Endothelium and
Stomata of the Peritoneal
Surface of the Septum Cis-
ternse Lymphaticse Magnte

of the Frog, x 200.

THE TONSILS.

6. Tonsils. — Use those of rabbit or cat, as the human tonsil
does not give such good results. Fix them in Kleinenberg's
fluid (12 hours), and then harden in gradually-increasing strengths
of alcohol. Or use mercuric chloride or Flemming's fluid (3-6
hours). Make sections by the paraffin method, and stain them

240

PHACTICAL HISTOLOGY.

[XX.

with logwood or logwood and eosin, and mount in balsam. Sections
may be stained first in acid-hsematoxylin and then counter stain
them with eosin. For the lymph-knots or germ-centres stain with
safranin and counter stain with picric acid.

For fixing the sections on a slide, Gulland 1 uses a modification of
the capillary-attraction method of Gaule. The sections in paraffin
are placed in warm water as recommended by GaskelL2 Float the
sections on to a slide, pour off the surplus water, and expose the
slide for several hours to a temperature under that of the melting-
point of the embedding paraffin. When dry remove the paraffin
by xylol and then stain the sections. They adhere to the slide by
capillary attraction.

(L) Observe on the surface the stratified squamous epithelium,
and under it numerous round or oval aggregations of adenoid tissue
(fig. 227). These form but imperfect nodules. Pit-like recesses
are seen, lined by stratified epithelium, and into them mucous glands
sometimes open.

(H) Trace some of the leucocytes of the adenoid tissue upwards

JlQ. 227.— Single Follicle of Tonsil, x 20. i. Cavity of follicle ; 2. Epithelium infil-
trated with leucocytes; 3. Adenoid tissue ; fi, 2, and 3. Follicles cut in various
directions,/! with a lymph-knot ; 4. Fibrous sheath; 5. Section of duct of mucous
gland ; 6. Blood-vessel.

between the epithelial cells, so that the epithelial layer is at places
infiltrated with leucocytes.

1 Jour, of Anat. and Phys., xxvi., 1891, p. 56.
* Quart. Jour. Micros. Sci., xxxi., 1891, p. 382.

XX.]

THE THYMUS GLAND.

241

THE THYMUS GLAND.

This gland is very large in the embryo and infant, but it begins
a retrograde development about the sixth year, and is eventually
replaced by fat and connective tissue. In the rabbit it retains its
structure. It is composed of a number of lobes, and these again of
smaller lobules. A capsule composed of connective tissue holds
all together, and sends in septa — carrying blood-vessels and lym-
phatics— between the lobes and lobules, and also fine prolongations
into the interior of the latter. There are no smooth muscular fibres
in the septa. Each lobule consists of a cortical and a medullary
part. Within each lobule is a delicate network of reticular con-
nective tissue, finer and more like adenoid tissue in the medulla.
It appears to consist of branching cells, and is coarser in the cortical
part. The meshes are crowded
with leucocytes, which, however,
arc most abundant in the cortex.
The medullary substance contains
the concentric corpuscles, like
nests of concentrically-arranged
flattened epithelial cells (fig. 228).
The blood-vessels run along the
septa and form a capillary plexus
within the lobules.

7. Thymus Gland.— Harden
the thymus of a young animal
or child in Mtiller's fluid (three
weeks), and then in gradually-
increasing strengths of alcohol,
mounted in balsam.

(a.} (L) Observe the capsule sending septa between the larger
lobules, and finer septa into the lobules, thus subdividing them into
smaller secondary lobules. Each such small lobule is about i mm.
in diameter, and as one is exactly like the others, it suffices to
study one.

(b.) A darker, denser peripheral zone, the cortex, and a more
open light central part or medulla, the former surrounding the latter
(fig. 228).

(«.) (H) The septa consist of fibrous tissue with some elastic
fibres, with numerous blood-vessels and slits ; the latter are the
lymphatics. The lobule consists of adenoid tissue — the mesh-work
not visible because it is crowded with leucocytes.

22 Q

FIG

— Section of a Few Lobules of a
Child's Thyraus. C. Cortical, M. Medul-
lary part; c. Concentric corpuscles,

X 20.

Sections stained with logwood are

242 PRACTICAL HISTOLOGY. [XX.

(d.) A variable number of concentrically-striated bodies, con-

FiQ. 229.— In jected Lobules of Thymus
of a Cat. a. Cortex ; b. Medulla ;
c. Blood-vessels ; d. Septum of
connective tissue.

FlG. 230. — Elements of the
Thymus Gland, a. Leu-
cocytes ; b. Concentric
corpuscle, x 300.

centric corpuscles or HassaLTs corpuscles.

amongst the adenoid tissue (fig. 230).

Sections of capillaries

THE SPLEEN.

The spleen, like the thymus, thyroid, and some other glands,
is a " ductless gland," and is invested by a fibrous capsule. The
capsule consists of an outer layer of connective tissue covered by
endothclium — the serous or peritoneal covering — and a deeper
layer of connective tissue with networks of elastic fibres, and in
some animals (dog, cat, pig) smooth muscular fibres. From the
deeper surface of the latter flattened or rounded trabeculse pass into
the organ, and as they do so they branch and nastomose, thus
forming a spongy mesh work with a labyrinth of communicating
spaces. These spaces are filled with a -reddish-purple soft substance,
the splenic pulp. The blood-vessels are ensheathed by connective
tissue, to which the trabeculse are attached. The red colour of the
pulp is due to the large number of blood-corpuscles. The trabe-
cular framework is continuous with the connective tissue entering
the organ along with and covering the blood-vessels — " adventitial
sheath " — at the hilum of the organ. In the splenic pulp are small
spherical whitish bodies — Malpighian corpuscles (0.2-0.7 mm.).
The Malpighian corpuscles are small groups of leucocytes de-
veloped here and there in the adventitia of the splenic artery.

XX.]

THE SPLEEN.

243

In the guinea-pig the lymphoid mass lorms almost a continuous
covering.

The Malpighian corpuscles occur chiefly at the bifurcations of
the artery, so that the artery perforates them usually at one side,
and thus the mass is arranged in a lob-sided manner on the artery.
In structure they resemble the follicular substance of lymphatic
glands, and in the centre of some of them is a lymph-knot, in which
mitosis occurs.

The splenic pulp consists of a mesh-work composed of branched
cells with membranous expan-
sions, and the processes of Ca gule
neighbouring cells anastomose
to form a fine reticulum, which
occupies the irregular chambers
of the trabecular framework, Trabeculw.
and with which it is continu-
ous. The meshes of this fine
network are occupied by cellu-
lar elements in considerable
variety. This reticulum is per-
meated by blood-corpuscles — in
fact, the blood stream of the
spleen seems to bear the same
relation to this reticulum that
the lymph stream bears to a Pulp-
lymph gland. Besides red
blood - corpuscles there are
larger cells called splenic cells ; Trabecula.
some of these often contain
degenerated blood-corpuscles or
pigment. There are also leuco- Arteir-
cytes (fig. 232). The arteries
enter the spleen at the hilum,
and run for a short part of
their course in the trabeculse,
which they soon leave, enter the reticulum, and break up into
pencils or groups of small arteries. Some of these open into true
capillaries in the Malpighian corpuscles; other fine branches open
directly into the reticulum. The endothelial lining, instead of
forming a continuous membrane, leaves apertures between the cells,
through which the blood escapes. The veins arise from the spaces
of the reticulum, and rapidly pass into the trabecula?, in which they
are firmly fixed. Near the hilum, and for part of their course, the
arteries and veins lie in a " common sheath," with the corresponding
nerves. The vein is always much wider than the corresponding

FIG. 231.— T.S. Part of the Human Spleen.
M tiller's fluid, haeniatoxylin, x 10.

244 PRACTICAL HISTOLOGY. [XX.

artery. The lymphatics are not very numerous. For the nerves
see Lesson XVII. 7.

8. Spleen. — Tie the blood-vessels at the hilum of the spleen of a
cat, so as to keep the blood in the spleen. Cut it out and place it
in a large volume of Miiller's fluid (two weeks) or 2 per cent,
bichromate of potash. Wash it thoroughly in running water for an
hour or two. Cut out small pieces and harden them in alcohol.
Make transverse sections, i.e., across the long axis of the organ ;
stain one in logwood, and mount in balsam. Other sections are to
be stained in picro-carmine and mounted in Farrant's solution, and
a thin set placed in i per cent, osmic acid (twenty-four hours), and
mounted in Farrant's solution. This sharpens the outlines of the
elements. Stain other sections in eosin-logwood or safranin (forty-
eight hours).

9. T.S. Spleen (|_) (Cat; li<jematoxylin or eosin and licematoxyliri).
— The Malpighian corpuscles are visible as small blue spots to the
naked eye.

(a.) Externally the capsule, fibrous, thick, firmly adherent and
closely applied to the organ, sends trabeculse into the spleen, where
they branch and anastomose to form a trabecular framework. Some
of them will be cut longitudinally, others obliquely, and some trans-
versely. In the larger trabeculse, sections of large blood-vessels

Fro. 232.— Elements of Human Splenic Fia. 233.— Reticulum of
Pulp. i. Leucocytes ; 2. Epithelial the Splenic Pulp.;

cells; 3. Coloured blood-corpuscles;
4. Cells containing pigment-granules.

(fig. 231). Note that there is no lymph space between the capsule
and the gland substance, as is the case in lymph glands.

(b.) Filling the interstices of this network, the splenic pulp, and
in it oval or rounded bodies — Malpighian or splenic corpuscles —
as blue-stained bodies contrasting with the yellowish brown pulp in
which they lie. In the centre of each is a lighter area, the "germ-
centre" of Flemming. In each corpuscle a section of a small
artery lying excentrically in the mass. The splenic corpuscles are

XX.] THE SPLEEN. 245

small lob-sided aggregations of lymphoid tissue around branches of
the splenic artery. They are relatively more numerous than in the
human spleen. The track of the blood in the pulp is mapped out
by the yellow blood-corpuscles.

(c.) (H) The capsule and trabeculae, composed of fibrous tissue,
with elastic fibres and smooth muscle. The Malpighian corpuscles,
consisting of leucocytes in an adenoid reticulum. The centre is
lighter in tint than the circumference, which is more condensed.
The lighter centre is due to the larger cells present there. They
are undergoing proliferation. The cells formed in the splenic
corpuscles pass into the spaces of the pulp and leave the organ by
the venous blood stream.

(d.) In the pulp irregular rows of coloured blood-corpuscles —
yellow — and between these leucocytes and other cells.

The exact structure of the pulp can only be properly studied in
a section which is very thin, and especially at the edges of the
section, or best of all in a section of a dog's (or cat's) spleen, whose
blood-vessels have been washed out, and cleared of all blood-
corpuscles by a warm stream of normal saline solution. The
vessels are then injected with a 5 per cent, solution of ammonium
bichromate, and the organ hardened in a large quantity of the same
fluid, and subsequently in alcohol.

(e.) In a section prepared in this way, or at the edges of a very
thin section, the fine reticulum of branching cells may be seen
(fig. 233) with the cells of the splenic pulp washed out of it.

10. Human Spleen. — Harden this in the same way as 8. Note
that, as a rule, the Malpighian corpuscles are less numerous. In
other respects the general structure is the same.

11. Injected Spleen. — It is very difficult to inject the finer splenic
blood-vessels. They should be washed out first with normal saline,
and preferably a watery solution of Berlin blue, or Berlin blue with
gelatine, should be used as the injection. Note that an artery and
capillaries exist in the Malpighian corpuscles, but the splenic pulp
seems to be infiltrated with a blue mass. The capillaries open into
this system of labyrinthine blood-passages. These intermediate
blood-passages are merely the spaces amongst the cells of pulp and
arc not lined by epithelium. The terminations of the capillaries
in some situations are surrounded by thick sheaths or collars of
tissue, perhaps derived from the cells of the pulp (Banntcartk).1

12. The varieties of Leucocytes in lymph glands are best studied
by fixing the gland in Hg C10 and staining sections with Ehrlich-
Biondi lltiid (Hoyer).'2 There are at least four varieties, not including
phagocytes.

1 Arcliivf. mile. Anat., xxxviii. p. 345. 2 Ibid., xxxiv.

PRACTICAL HISTOLOGY.

[xxi

LESSON XXL
TONGUE— TASTE-BUDS— SOFT PALATE.

TONGUE.

PLACE small portions in Miiller's fluid or 2 per cent, potassic
bichromate for fourteen days, and complete the hardening in
alcohol, or harden it in mercuric chloride. Make vertical trans-
verse sections. It is well to have the tongue of a small cat or
kitten, and parts of the human tongue also — the former because a
complete transverse section can be put on a slide. The structure
will vary according as the section is made through the anterior or
posterior part of the organ, as the latter contains many lymph
follicles and mucous glands. The sections may be stained in
logwood and mounted in balsam.

1. T.S. Tongue of Cat.— (a.) (|_) Observe the papillae, of various
shapes, on the dorsum of the tongue, and covered by stratified epi-
thelium. Under this the
connective tissue of the
mucous membrane (fig.

234).
(b.) Muscular Fibres. —

Many cut transversely and
arranged in groups under
the dorsal mucous mem-
brane and elsewhere ; others
which run from the vertical
mesial plane or septum hori-
zontally outwards, and some
which pass vertically. The
last may be seen to become

conical and end in the connective tissue of the mucous membrane.

Some of these fibres branch. (The methods of isolating branched

fibres are referred to in Lesson XVI. 3.)

(r,.) Lingual Papillae. — The dorsum of the tongue is beset with

elevations- of the mucous membrane covered by stratified epithelium,

and constituting three varieties of papillae.

(i.) Filiform (.7-3 mm long), by far the most numerous, and are

placed all over the dorsum. They are conical elevations of the

mucous membrane, the upper end of which is beset with five, fifteen,

FIG. 234.— T.S. ot One-halt of the Tongue of a Cat.

XXL]

TONGUE.

247

or thirty secondary papillae. Each papilla is composed of fibrous
tissue with elastic fibres, and covered by many layers of stratified
epithelium, the superficial cells of which are often corneous (fig. 235).

(2.) Fungiform (0.5-1.5 mm. long), are not nearly so numerous
as the foregoing, and are scattered over
the doi'sum. Each papilla is club-shaped
or lenticular, with a constricted base.
The apex is beset with secondary
papillae, but the epithelium covering
them is thinner than in (i) (fig. 236).

(3.) Circumvallate (1-1.5 mm- niSn
and 1-3 mm. broad), are confined to
the posterior part of the tongue, where
they (8-15 in number) are arranged in
the form of a V, the apex of the V
being directed backwards. Each circu-
lar elevation is raised above the level
of the tongue and surrounded by a
circular trench or fossa. Secondary
papillae occur only on their surface.
Taste-bulbs occur in the wall of the
papilla directed toward the fossa. They
are the organs of taste, and are supplied
by the glosso-pharyngeal nerve. It may require several sections to
obtain views of all three forms of papillae.

(d.) (H) The stratified squamous epithelium covering the papillae
and sides of the tongue.
The superficial cells are
very thin.

(e.) Glands, in the
back part mucous
glands with clear con-
tents, and it may be
also serous glands in
which the acini are
more granular (lig.

237)-

(/.) Fat-cdls, like
padding between the
striped muscular fibres
here and there. Near

the lower surface, sections of the lingual artery and nerves,
latter cjunglionic, cells may sometimes be seen.

At the back part of the tongue are little depressions of the
mucous membrane called crypts (fig. 237). In the walls of these

FIG. 235.— Filiform Papillfe, x 30.
i. Primary papilla; 2. Secon-
dary papillae on its summit ; 3.
Epithelial process on papilla;
4. Single process, with en-
tangled loose epithelial cells.

Secondary
papillae.

Fungiform
papilla.

FIG. 236.— Fungiform Papilla, x 30.

In the

248

PRACTICAL HISTOLOGY.

[XXT.

Epithelium.

Crypt.

Adenoid
tissue.

Mucous
glands.

Mucous
glands.

are spherical masses of adenoid tissue, and into some crypts open the
ducts of small mucous glands (fig. 237).

2. The mucous glands occur chiefly at the base of the tongue

and along its edges.
They have the same
structure as the sali-
vary glands of the same
name (fig. 237), i.e.,
their acini are lined by
a single layer of mucous
cell-s, but there are no
demilunes. They are
small compound tubu-
lar mucous glands.

3. The serous glands
occur only near the
circumvallate papillae
and taste-bulbs. Their
acini are granular and
resemble those of the
parotid gland in struc-
ture (fig. 240, d).

4. T.S. Tongue (Double-Stained).— Stain a section from th<?
posterior part of the organ, first with methyl-green and then with

cosin. Mount in balsam. The
connective tissue and papillae are
reddish ; the serous glands are red-
dish also, while the mucous glands
have a purplish-green colour.

5. T.S. Injected Tongue (L).—
This is obtained when the head or
whole body is injected. Observe
the very vascular muscular portion
and the papillae, each with an artery
entering it. If the papillae be com-
pound, i.e., beset with other smaller
secondary papillae, a small capillary
loop passes into each of these
secondary papillae ; sections of large
blood-vessels in the connective tissue
of the mucous coat (fig, 238). If

FIG. 237. —Crypt from Back of Tongue, with Cluster
of Lymphatic Follicles.

FIG. 238.— T.S. Injected Tongue of Cat.

desired, another section may be faintly stained with logwood and
mounted as above.

XXI ] TASTE-BUDS. 249

TASTE-BUDS.

6. Taste-Buds, or the peripheral organs of taste, occur on the
fungiform papilla) and lateral surface of the circumvallate, soft
palate, posterior surface of the epiglottis, and a few amongst the
epithelial cells on the dorsum and sides of the tongue. It is more
convenient, however, to study them in the rabbit. On either side
of the posterior part of the rabbit's tongue are two oval patches
with transverse ridges and intervening furrows, the papillse foliatae

(fig- 239).

(i.) Cut out these parts and harden them for fourteen days or
so in Miiller's fluid and then in spirit. Stain in bulk in borax-
carmine or haematoxylin and cut in paraffin.

(ri.) The excised organ, with as little adherent muscle as possible,
is placed for one hour in i per cent, osmic acid, or pinned on a
cork and exposed for the same time to the vapour
of osmic acid. Fine sections are made across the
laminae, and stained with logwood and mounted in
balsam.

(a.) (L) Observe the sections of the laminae, each
one with a central papilla or projection of connective
tissue (fig. 240, /). This is covered by many layers
of stratified epithelium.

(b.) Between the laminae a furrow, and embedded
in the epithelium, on each side of this furrow, the
taste-buds (#), which are oval in shape, and composed
of epithelial cells, whose bases touch the connective
tissue of the mucous membrane, where they receive FIG.
a branch of the glosso-pharyngeal nerve. The apex
has an open mouth — gustatory pore — which com-
municates with the furrow. The cells composing the bud are
arranged somewhat like the staves in a barrel.

(<?.) Gland-ducts open at the bases of the furrows, and if these
ducts be not seen, sections of their acini — serous gland — are sure
to be seen deep in the corium (fig. 240, d). The corium has what
look like secondary papillae on it, but they are really septa (I'}.

(<L) (H) Study a single taste-bud (80 ^ long and 40 //, broad)
It is composed of two kinds of elongated epithelial cells.

(i.) The sustentacular cells, which are most numerous. They
are elongated, flattened, and either of uniform breadth or narrowed
at their base. They form a protective covering for the true gusta-
tory cells, which lie between and within them.
23

250

PRACTICAL HISTOLOGY.

[XXI.

(2.) The gustatory cells consist of

FIG. 240.— Papillae Foliatae in the Rabbit. I, I. Pri-
mary and secondary septa ; g. Taste-buds ; n.
Medullated nerve ; d. Serous gland ; a. Its duct ;
M. Muscular fibres, x 80.

nerves in the papillae foliatse,

see

narrow fusiform nucleated
cells, whose lower pointed
end is continuous with a
branch of the axial cylinder
of a nerve-fibre, while the
free end is continued into a
line point or ciliuin, which
projects through the gus-
tatory pore.

If it be desired to study
the mode of termination
of the nerves in these
organs, use the lemon-juice
gold chloride method, with
subsequent exposure of
the tissue to sunlight in
water acidulated with
acetic acid, Golgi's rapid
hardening method, or
mcthylene-blue. For an
elaborate research, with
beautiful plates showing
the terminations of the
Drasch.1

SOFT PALATE.

1. T.S. Soft Palate.— Harden the soft palate of a rabbit or dog
in Miiller's fluid, alcohol, or corrosive sublimate. Make transverse
sections by freezing, or stain in bulk in borax-carmine and cut in
paraffin.

(a.) (L and H) One of the most beautiful methods of staining
is that recommended by List, viz., to stain with aniline-green and
eosin, and mount in balsam. Even with the naked eye the thick
layer of mucous glands can be seen.

(b.) The stratified epithelium and connective tissue are rosy-red,
the nuclei blue. The glands are bluish, and are seen to be mucous
in character, lined by a single layer of mucous cells without demi-
lunes. In the borax-carmine section, the cells lining the acini of
the glands are clear and transparent and show no demilunes, a
typical example of a pure mucous gland.

" Ilnters. iiber d. Paj>. fol. et circurnvall. d. Kaninchens," Abhand. d.
math. -phys. Classe d. K. Sachs. Gesell. d. Wisaensch. . Bd. xxiv.

XXII.] TOOTH. 2$I

ADDITIONAL EXERCISES.

Glands of Tongue or Palate.— Harden the tongue or soft palate of a rabbit
in 3 per cent, nitric acid for 1-2 hours. Wash out all the acid and stain the
sections with methylene-blue. Wash out the blue with alcohol until only the
glands remain blue. Mount in xylol-balsam.

Terminations of Nerves in the Lingual Papillae and Glands. —These maybe
studied by staining small pieces of the tongue of mouse or rat by Golgi's silver
nitrate method (p. 78), or by the rapid hardening method (bichromate of potash
and osmic acid, Lesson XXVI. 14). The nerve in the papilke contains nerve-cells
which are in connection with the nerve-fibres. The nerve-fibres form a plexus
of fibres outside the basement membrane of the serous gland acini — epilemmal
plexus — and one within this membrane amongst the secretory cells — hypo-
leminal plexus (Fusari and Pancud).1

LESSON XXII.

TOOTH— (ESOPHAGUS.

TOOTH.

THE chief mass of a tooth consists of dentine. It is capped by
enamel, and the root or fang is invested by a layer of bone, the
crusta petrosa. All three tissues are calcified, and contain calcic
phosphate. The enamel, however, is an epithelial structure, and
consists of modified and calcified epithelial cells, while the dentine
and crusta petrosa belong to the connective tissue group.

Unsoftened Tooth. — This is one of the few preparations which
had better be bought.

1. Longitudinal Section of a Dry Tooth (fig. 241).

(a.) (L) Observe the crown and fang, and, connecting the two,
the neck.

(/>.) The dentine surrounding the pulp cavity, the enamel
covering the dentine of the crown, and the crusta petrosa or
cement covering the dentine of the fang. The wavy black lines in
the dentine or dentinal tubules are really tubules filled with air,
hence they appear black. Note their direction from the pulp cavity
towards the outer margin of the dentine. Quite at the apex of the
crown of the tooth they run vertically ; in the fang they run nearly
horizontally, and in the part of the dentine intermediate between
1 Ardiiv. ital. dc Biol.t xiv. p. 240, 1891.

252

PRACTICAL HISTOLOGY.

[XXII.

...RM.

PC

both they gradually become more and more oblique from the centre
of the crown.

Arched or curved lines — incremental lines or Schreger's lines

— may sometimes be seen crossing
the course of the dentine tubules.

(c.) The enamel covering the
dentine on the crown of the tooth;
somewhat brownish-coloured con-
centric lines may be seen in it.

(d.) (H) The enamel consists
of striated prisms, hexagonal when
seen in transverse section (fig.

243)-

The dentinal tubules lie in a

homogeneous matrix, and are
wavy tubes, which divide di-
chotomously, and give off lateral
branches which anastomose with
other lateral branches from ad-
jacent tubules. At the outer part
of the dentine are irregular inter-
globular spaces, which appear
black when they contain air (fig.
244).

The crusta petrosa consists of
bone — a thin layer — composed of
lamellae and bone-corpuscles, but
no Haversian canals (fig. 244).

FIG. 241. — V.S. Tooth in Jaw. E. Enamel; O Sryftpnpfl Trmth £tnlnr>f fVm
D Dentine; P.M. Periodontal mem- . *> »

brane; P.O. Pulp cavity ; c. Cement ; jaw oi a small mammal, e.g., a cat,

and decalcify a short length — J
inch — of the lower jaw in chromic
acid and nitric acid. It will take two or three weeks to remove
all the bone-salts, and the decalcifying fluid must be frequently
renewed. The tooth is sufficiently soft to be cut when a needle
can be pushed into it. Make vertical sections through the whole
jaw and a tooth in situ.

Stain one section in picro-carmine, another in osmic acid (twenty-
four hours), and mount both in Farrant's solution or in glycerine-

B. Bone of lower jaw ; V. Vein ; a.
Artery ; N. Nerve.

(a.) (L) Observe the tooth in the alveolus or depression of the
jaw in which it is fixed. The enamel has disappeared. The bone
of the jaw with its periosteum, lining the alveolus and forming
there the periodontal membrane (fig. 241, P.M.).

(b.) Next the latter on the fang the cement (C), the dentine,

XXII.]

TOOTH.

253

pulp-cavity and its contents. If the section passes directly through
the middle of the tooth, the orifice in the fang of the tooth may be
seen.

(c.) (H) The dentinal tubules, not so distinct as in the dry tooth.

r

€

FIG. 242.— Enamel
Prisms.

FIG. 243.— Their Hexa-
gonal Ends.

FIG. 244.— T.S. Fang of Tooth, a. Crusta pet-
rosa, with bone-corpuscles ; b. Dentinal
tubules ; c. Granular or interglobular
layer.

If they are cut obliquely, they appear merely as tailed dots in a
homogeneous matrix.

(d.) The pulp-cavity contains blood-vessels and fine connective
tissue, but next the dentine there is a layer of large cubical cells —
odontoblasts — which give off fine processes which enter the den-
tinal tubules — the fibres of Tomes. They are best seen, however,
in a tooth which has not yet cut the gum.

3. Development of Tooth. — Without entering into all the
details of the development of the teeth, the following directions
will suffice as to the method of preparing sections so as to show the
various stages. What may be called the first stage — that shown in
fig. 245 — is to be obtained from the lower jaw of a sheep's embryo
7 cm. in length. At this stage only a very little bony matter exists.
Harden the whole jaw in corrosive sublimate and decalcify in dilute
hydrochloric acid. Stain in bulk in borax-carmine, embed in
paraffin, and make T.S. across both rami of the jaw and the tongue.
( )r harden and decalcify at the same time the jaw of a fretal kitten
by placing small pieces containing embryonic teeth in Flemming's
fluid. This yields excellent results, — the tissues are thereby suffi-
ciently differentiated and may be cut in paraffin.

The second stage, fig. 246, is obtainable from the upper jaw of an
embryo sheep 1 5 cm. long. It is treated in the same way.

The third stage, fig. 247, is obtained from the lower jaw of a dog
six days old or thereabout.

254 PRACTICAL HISTOLOGY. [XXIL

With a high power it is easy to observe the structure of the
.&

Epithelium.

FIG. 245. — Diagram of Early
Stage of the Development
of a Tooth, a. Epithelium
of dental ridge on lower
jaw ; b. Portion of epithe-
lium about to be modified
into enamel (enamel organ);

c. Beginning of germ of den-
tine in the tooth papilla ;

d. Lamination of corium
about to form tooth sac.

FlG. 246. — Lower Jaw of Human Foetus at 4th
Month, x 40. i. Dental ridge; 2* Stalk of
enamel germ ; 3. Enamel organ ; a. Peri-
pheral cells ; b. Germ pulp ; c. Cylindrical
cells of enamel ; 4. Papilla.

several parts, and to see the odontoblasts lining what is to be the

Dental Sac.

(Outer layer

]

(inner lay

Dentine. ~t

f Odontoblasts.
PapittaA Spherical elements __
v. with blood-vessels.

Bone of the lower jaw.-

Peripheral flat^

cells.

Enamel pulp. ,
Enamel cells. (or!'an'

Enamel.

FIG. 247.— T.S. Lower Jaw of New-Born Dog, x 40. The dental sac is shown only in the
left side. The tissues originating from connective tissue are shown on the left, and
those of epithelial origin on the right.

pulp-cavity. If the latter happen to be partially detached, their

XXII.]

(ESOPHAGUS.

255

processes — fibres of Tomes — may be seen partially withdrawn from
the dentiiial tubules in which they lay.

(ESOPHAGUS.

The (Esop-agus consists from within outwards of —

(i.) Mucous coat, composed of stratified squamous epithelium,
into which project small simple papillae from the corium or con-
nective-tissue basis of the membrane. At the. outer part of the
eoriimi is a narrow layer of smooth muscular fibres — muscularis
mucosse — arranged for the most part longitudinally.

(2.) Submucous coat, consisting of connective tissue and the
larger blood-vessels and some nerves. In those animals (e.</., dog)
in which glands occur, the acini of the
glands lie in the submucous coat, so that
their duets have to perforate the muscu-
laris nmcosne and traverse the mucous
membrane before they open on the surface
of the epithelium.

(3.) Muscular coat. — This will vary
with the animal used. In man this coat
in the upper third of the oesophagus is
composed of striated muscular fibre, the
lower two-thirds of smooth muscle. The
outer layer of fibres runs longitudinally,
the inner circularly.

(4.) Fibrous coat, composed of fibrous
tissue.

4. The (Esophagus.— Cut out a piece
of the oesophagus of a dog or cat — 2 cm.
in length — and harden it in equal parts

of chromic acid (i per cent.) and spirit, or FIG. 248. — T.S. Small Part of
Miiller's fluid (fourteen days), or mercuric
chloride, and then in alcohol. Make
transverse sections ; stain one in picro-
carmine and mount it in Warrant's solu-
tion, and another in logwood and mount
it in balsam. Perhaps even more instructive sections are obtained
from a small animal, such as a rat, where the whole oesophagus (with
trachea) can be stained "in bulk" in borax-carmine. Cut sections
of both tubes to show their structure and relations.

(a.) (L and H) The circular tube has several coats, the inner-
most one being thrown into folds. The mucous membrane covered
by stratified sipiamous epithelium, under this the connective tissue

(Esophagus of Dog. E. Epi-
thelium ; M. M. Muscularis
mucosac; G. Glands; C. Cir-
cular, and L. External or
longitudinal muscular coat;
F. Fibrous layer. Miiller's
fluid, picro-carmine.

256 PRACTICAL HISTOLOGY. [XXII.

with small simple papillae. In the deeper part of the mucous mem-
brane are several layers of non-striped muscle, the muscularis
mucostjB (248, M.M).

(b.) Outside this is loose submucous connective tissue with a
few blood-vessels, and in the dog and some other animals the acini
of mucous glands. The ducts of the latter traverse the coats lying
internal to the glands, and open on the inner surface by funnel-
shaped openings.

(c.) Outside this, again, is the muscular coat, which varies in
the upper and lower parts of the tube, and also with the animal
examined. In the upper part there is striped muscle, in the lower
part two layers of non-striped muscle, an inner circular and an outer
longitudinal. Between the two layers may be seen ganglionic cells
of Auerbach's plexus.

(d. ) Outside all is the fibrous coat or adventitia, composed of
coarser connective tissue, with elastic fibres and blood-vessels.

For the epithelial cells lining the oesophagus see Lesson IV. 5.

It is to be remembered that there are very great differences as
regards the presence of glands in the oesophagus. Some animals
have a considerable number — e.g., dog — and others very few.

ADDITIONAL EXERCISE.

5. Other Methods ((Esophagus). —Very good results are obtained by harden-
ing in absolute alcohol containing methyl-green, the gland-cells being thereby
sharply denned (Subeli).1 Also double stain with borax-carmine (extract with
acid-alcohol), then alcohol, and stain again with iodine-green (twenty-four
hours), extract with alcohol, embed, cind cut in paraffin. The mucous mem-
brane of some animals (e.g., pig) contains lymph follicles, which can readily
be detected in a part of the tube stained in bulk in borax-carmine.

LESSON XXIII.

THE SALIVARY GLANDS AND PANCREAS.
THE SALIVARY GLANDS.

ALL these glands have not the same structure, hence it is necessary
to classify them.

Mucous Salivary Glands. — The sub-maxillary and sub-lingual
glands of the dog and sub-lingual of guinea-pig.

1 Zeits.f. mik. Anat., vii. p. 224, 1890.

XXIII.] THE SALIVARY GLANDS. 2 57

Serous Salivary Glands. — The parotid of man and mammals,
and the sub-maxillary of the rabbit.

Mixed or Muco-Salivary. — The human sub-maxillary, retro-
lingual of the dog, or sub-maxillary of the guinea-pig.

The salivary glands are compound tubular glands, i.e., the duct is
branched, while the acini or alveoli — the true secretory parts of
the glands — are tubular in form. Each gland consists of lobes,
held together by connective tissue, which forms a capsule for the
whole gland and gives septa to enclose the lobes and lobules.
Each lobe in turn is made up of numerous smaller lobules also held
together by connective tissue, which carries the blood-vessels,
nerves, lymphatics, and larger ducts. From mutual pressure the
lobes and lobules are usually polygonal in shape. The main duct
is made up by the convergence of ducts from the lobes — lobar
ducts — while from each lobule there is a duct— lobular ducts,
which unite to form lobar ducts. Each lobule is made up of a
number of alveoli or acini. Each alveolus, which has a closed
extremity, leads into or discharges its secretion into a fine duct or
ductule, and these ductules by their union form the intralobular
ducts. Practically the arrangement of the ducts is the same in all
this set of glands ; the diiferences in structure are in the alveoli.
The alveoli consist of a basement membrane, which by appro-
priate means can be shown to consist of branched cells forming a
reticulated or basket-like membrane. This is lined internally by
the secretory epithelium, leaving a larger or smaller lumen in the
centre, \vhich leads into a fine duct or ductule by means of a narrow
functional piece or intermediary part or ductule, in which the epi-
thelium is somewhat flattened. Usually several alveoli open into
one intermediary tubule or ductule. The ducts with a fibrous wall
are lined by a single layer of columnar epithelium, which is striated
or " rodded " in its outer part, and granular towards the lumen of
the tube (fig. 249); a little inwards from the centre of each cell is
a nucleus.

In mucous glands the acini (35 p, in diameter) are lined by a
layer of polyhedral clear cells, whose broader bases rest on the
basement membrane, while their apices abut on the lumen, which
is small (fig. 249). Usually in a transverse section of an acinus
five or six cells are seen. The appearance of these cells varies
according as a gland is at rest or in a state of activity, i.e., whether
the gland is "loaded" or "charged" (resting phase), or "unloaded"
or "discharged" (active phase). In a resting gland the mucous
cells are clear, for the most part, while at the outer part of the cell
is a flattened nucleus surrounded by a very small quantity of
granular protoplasm. The clear part is traversed by a network of
fibrils, which includes in its meshes mucigen. The granular matter

258 PRACTICAL HISTOLOGY. [XXIII.

and nuclei stain readily with the ordinary dyes, while the clear
part does not do so.

In some mucous glands, e.g. dog, but not in all mucous glands,
here and there between the bases of some of these cells, and the
basement membrane, are groups of smal], granular, nucleated cells,
the group having a somewhat crescentic shape ; they are called
demilunes or crescents of Gianuzzi (fig. 250). They stain readily
with dyes, and are darkened by osmic acid, and contain two or
more nuclei.

In the discharged or active gland, the acini are smaller, the
lumen wider, the clear part of the cell diminished in volume, while
the outer part of the cell is wider, and appears to have encroached
on the clear part. The nucleus is usually spherical, and placed
nearer the centre of the cell.

In serous or albuminous glands the chief differences are in the
cells lining the alveoli. In serous alveoli there is but one layer of
cells, and nothing corresponding to the demilunes. The cells are
somewhat smaller than mucous cells ; they are more granular, and
stain more uniformly with dyes. The nucleus is spherical, and
placed nearer the centre of the cell. The differences between
active and passive phases are not so marked as in mucous acini.

During activity the cells become smaller, and the "granules"
disappear from the outer part of the cell ; the cells become more
sharply defined, while the nuclei are large and spherical.

N.B. — In all cases examine the acini of the glands in the fresh
condition.

A. Mucous Salivary Glands. — These must be prepared in
several ways.

Methods. — (i.) For the general structure of salivary glands : —

Harden small piece ; for 2 or 3 days in the following mixture : —
3 parts 90 per cent, alcohol and 2 parts .5 per cent, chromic
acid. To see the finer points — after staining — mount the sections
in glycerine (Langley).

(ii.) Small pieces of a perfectly fresh dog's sub-maxillary gland
are placed for an hour in 75 per cent, alcohol, then for five hours
in absolute alcohol, which is then changed, and the hardening is
completed in fresh absolute alcohol in twenty-four hours. Sections
of the unstained gland are apt to fall to pieces, although the small
pieces show the structure sufficiently well. A part of the alcohol-
hardened gland should be stained " in bulk " in borax-carmine, and
cut in paraffin. In this way the relative position of the parts is
retained.

(iii.) Harden very small pieces in i per cent, osmic acid
(24 hours) ; wash thoroughly, and complete the hardening in
alcohol.

XX II I.] THE SALIVARY GLANDS. 259

(iv.) Harden other pieces in Flemming's mixture, and stain "in
bulk " in borax-carmine as in (ii.).

(v.) Heidenhain's Method.1 — This method is also applicable
to the pancreas. Small pieces of glands — e.g., sub-maxillary of
guinea-pig, dog, and cat — hardened in alcohol (ii.) are placed in 10
cc. of 0.5-1 per cent, watery solution of hsematoxylin (6-8 hours);
and then for an equal period in 0.5-1 per cent, potassic bichromate,
or i per cent, watery solution of alum, or stain with .3 per cent,
hsematoxylin (distilled water to be used), and differentiate with I
per cent, neutral chromate of potash, which forms a steel-grey
compound with haeniatoxyliii (p. 70). The stain does best with
objects hardened in alcohol or picric acid. The pieces are quite
black when removed from the second fluid. In this method, the
union of the reagents takes place in the tissue itself. The nuclei
are bluish-black, the cell-substance a steel-grey, while the demilunes
.stand out distinctly. This is an excellent method for these glands.
Cut in paraffin.

(vi.) Mucous glands harden well in picric acid.

My experience leads me to believe that in studying a mucous
salivary gland, it is best to begin with one whose acini contain only
mucous cells and no demilunes. Such glands are the sub-lingual
of a guinea-pig and sub-maxillary gland of the mole. Then proceed
to the sub-maxillary of a dog, which has demilunes at intervals,
and lastly, take the acini of a cat's sub-maxillary, where the demi-
lunes form a nearly complete layer outside the true mucous cells.

In order to obtain a general view of the origin of the ducts from
the acini of the lobules, and the union of small lobular ducts to
form larger ducts, it is well to examine a preparation of the salivary
glands of the cockroach, which can be removed en masse (p. 265).

1. Sub-Lingual Gland of Guinea-Pig. — This shows acini lined with
mucous cells iviihout demilunes (L and H). Note the acini, each lined
by a single layer of clear transparent mucous cells, resting directly
upon a basement membrane without the intervention of any demi-
lunes. In the arrangement of capsule, septa, and ducts, it resembles
the salivary glands of the dog. The gland is like fig. 249 without
the demilunes.

2. Sub-Maxillary Gland of Dog. — (a.) (L) Observe the capsule,
which sends off thin connective-tissue septa into the gland, mapping
it out in polygonal lobes, which are further subdivided by finer
septa into lobules. In the larger septa, sections of blood-vessels and
gland-ducts (lig. 249).

(/>.) Within each lobule aggregations of acini or alveoli, which
make up the smaller lobules. The shape of the lobule depends on
the way it has been cut. Branches of the finer gland-ducts — few —
1 Archivf. mik. Anat., 1884, p. 468, and 1886, p. 383.

260

PRACTICAL HISTOLOGY

[XXIIL

FlG. 249.— Small Lobule of a Sub-Maxillary Gland, Dog.
L. Lobule ; D. Duct. Osmic acid.

between the lobules. Some of the acini appear to be crowded with
cells inside the basement membrane. The gland-ducts have a
distinct lumen.

(c.) (H) Although numerous acini are visible, only those that

have been cut across
so as to show the
lumen are satisfactory;
the others appear
merely to be filled
with cells, and vary
much in size, accord-
ing to the plane of
section through the
alveolus. Each aci-
nus has a clear, trans-
parent basement mem-
brane, and inside, and
on it, is arranged the
secretory epithelium.
(d.) The mucous cells form a single layer, and are large, clear,
cubical cells. Each has a nucleus which is usually flattened and
placed near the attached end of the cell. In borax-carmine pre-
parations, the nucleus, surrounded by granular protoplasm, is stained
red, while the rest of the cell appears clear, and traversed, it may

be, by fine threads or
fibrils. In reality, the
coil-substance consists of
a network containing a
clear substance — muci-
gen.

(c.) The demilunes lie
singly next the basement
membranes. Two or
three may be seen in the
section of each alveolus
as more granular deeply-
stained bodies, sometimes
with two nuclei. As
their name indicates,
they are somewhat half-
moon shaped, but they
send processes between the mucous cells. As they are deeply
stained by pigments and also darkened by osmic acid, their shape
and distribution are readily recognised.

(/.) The lumen of each acinus is a small more or less regular

FlG. 250.— Sub-Maxillary Gland, Dog, showing Duct
Communicating with an Alveolus by a Narrow
Ductule. The alveoli with mucous cells and dense
demilunes. Osmic acid and hasmatoxylin, x 300.

XXIII.] THE SALIVARY GLANDS. 26 1

space in the centre of the acinus, "but many acini may be so divided
as not to show it. It is difficult to find the connection between the
lumen of an acinus and a duct (fig. 250).

(g.) Duct. — This is best studied in a transverse section of one of
the finer ducts lying within the lobules or the
larger ones in the septa (fig. 251). The wall
consists of circularly disposed connective tissue,
and is lined by a single layer of tall, narrow,
cylindrical epithelium. The outer part of each
cell is distinctly striated or "rodded," and the
spherical nucleus is placed about the middle of
the cell.

N. B. —The student should compare with this FIG 251 -T.S. Salivary

„ 111 i- -i • • ••! Duct showing only

a section of a gland hardened in osmic acid or the "Rodded" Epi-
Flemming's fluid, or one stained by Heidenhain's ^m Linin* **•
method.

3. Sub-Maxillary Gland of Cat. — In some of the acini there
may be a nearly complete layer of demilune cells between the base-
ment membrane of the acinus and the lining layer of mucous cells.

B. Serous Salivary Glands — Methods. — Use the parotid of any
mammal — rabbit, cat, or dog — or the sub-maxillary gland of a
rabbit. Harden small parts of the gland in the same way and by
the same means as for mucous glands. A saturated watery solution
of mercuric chloride is to be pre-
ferred to picric acid for serous
glands. I find that Flemming's
mixture is specially good for the
sub-maxillary gland of the rabbit.
Sections are made and stained — the
hardened gland stained " in bulk "
— just as for mucous glands.

4. T.S. Parotid Gland. — (a.)
(L) Observe the capsule, septa,
lobes, and lobules as in the mucous
glands, but the alveoli or acini are
smaller. More sections of gland-
ducts will also be seen.

// \ /i_i\ r\\~ • TJ. FIG. 252. — Resting Serous Gland, Babbit.

(5.) (H) Observe an acinus. It Alcohol and carmine.

is lined by a layer of polyhedral

cells, leaving a very small lumen. The cells are very granular, with
a spheroidal nucleus placed near the centre of the cell (fig. 252).
Numerous sections of ducts, some cut transversely, others longitudi-
nally. They are like those of mucous glands.

5. Fresh Serous Gland. — Tease a fragment of a parotid gland in
normal saline, and observe how the cells are crowded with granules.

262

PRACTICAL HISTOLOGY.

[XXIII.

j. 253.— Human Sub-Maxillary Gland. On the right
groups of mucous, and on the left of serous
alveoli, x 300

C. Muco-Salivary Glands, e.g., sub-maxillary of man or retro-
lingual of the dog, are treated as the other salivary glands.

6. Human Sub-Maxillary Gland. — (L and H) Observe'that some
of the acini are like those of mucous glands, and others like serous
acini, while some of the acini contain both mucous and serous

cells. Acini, serous and
mucous, may be found
lying side by side (fig.

253).
7. Dog's Sub-Maxillary

Gland (Double-Staining).

— (i.) Stain alcohol-hard-
ened sections, first in a
watery solution of aniline
green and subsequently in
eosin. An easier plan is
to stain first in aniline
green, ' rapidly dehydrate
the section in alcohol,
taking care that all the
green is not washed out
of the gland-cells, and clarify with clove-oil in which is dissolved
some eosin. Mount in balsam. The mucous cells are green, the
demilunes pinkish, and the nuclei generally are green. The cells
of the ducts are green, and the interlobular connective tissue
pinkish,

(ii ) Stain sections in aniline blue, to which is added a saturated
watery solution of picric acid. Mount in balsam. The cells of the
acini are blue, while the ducts are yellowish -green.

(iii.) Schiefferdecker's method is also to be recommended. Add
a few drops of a 5 per cent, alkaline alcoholic solution of eosin to
a watch-glassful of alcohol. Allow the sections to stain in this for
half an hour or so, and place them for a few minutes in i per cent,
watery solution of aniline green, and mount in balsam.

(iv.) The sub-maxillary gland of a dog or guinea-pig hardened in
picric acid or HgCl2, if stained with aniline blue and safranin,
shows the demilunes red and the mucous cells blue in balsam
preparations.

THE PANCREAS.

The pancreas is a compound tubular gland, and resembles the
serous salivary glands in the arrangement of its capsule, lobes,
lobules, and duct, with its branches. The epithelium of its ducts,
however, is not so distinctly striated, and in a section, as a rule,

XXIII.]

THE PANCREAS.

263

not many ducts are visible. Curious groups of cells, "inter-tubular
cell clumps," each supplied by a glomerulus-like tuft of capillaries,
lie in the interlobular septa or amongst the acini. The alveoli,
tubular or flask-shaped, with a very small lumen, consist of a base-
ment membrane lined by a single layer of columnar or pyramidal
cells, each showing two zones ; an outer zone nearly homogeneous,
and staining with logwood and some other dyes, and an inner zone
crowded with "granules." The spherical nucleus lies about the
middle of the cell. Sometimes sections of Pacinian corpuscles and
groups of nerve-cells are found in the pancreas.

This gland, like other glands, should be examined in different
phases of physiological activity. One, the active state ("dis-
charged "), when the gland is removed from the body (rabbit) two
or three or four hours after a full meal ; and the other, the passive,
or better, the resting state ("charged" or "loaded"), when the
pancreas is not secreting actively, which can be secured by allowing
an animal to fast for fourteen hours (dog or rabbit). The human
pancreas is rarely satisfactory.

Methods. — (i.) One of the best methods of fixing the pancreas is
i per cent, osmic acid or Flemming's mixture (24 hours). It is
then to be thoroughly washed and hardened, first in 75 per cent.,
and afterwards in absolute alcohol. In such a preparation the
" granules " are usually
well preserved, and they
stain deeply with saf-
ranin.

(ii.) Apiece hardened
in absolute alcohol and
stained in bulk in borax-
carmine or Heidenhain's
logwood (p. 70), and
afterwards cut in paraf-
fin, shows well the
general arrangement.

(iii.) Fix a small piece
in corrosive sublimate,

and, after the Usual pro- FIG. 254.— T.S. Pancreas, Dog. A. Acinus ; C. Capsule;
cautions, Stain the sec- D- Duct- Corrosive sublimate and picro-carmine,
. \ . . X 300.

tions with picro-carmine.

Mount the osmic acid sections in glycerine, and the stained ones
in Farrant's solution or balsam.

8. Resting Pancreas.— (a.) (L) Observe the capsule (thinner),
septa (thinner), lobes, and lobules, as in salivary glands. This
arrangement is well seen in the carmine specimen (fig. 254, C).

(b.) (H) In the osmic acid preparation observe the alveoli (fig.

264 PRACTICAL HISTOLOGY. [XXIII.

254, A), with a basement membrane lined by a single layer of
columnar cells, tapering somewhat at their central ends, leaving a
small irregular central lumen.

(c.) In each cell a crowd of dark "granules," occupying about
the inner two-thirds of each cell, while the outer third or zone is
comparatively homogeneous and free from granules (tig. 254). The
nuclei of the cells are apt to be obscured by the presence of the
granules. In the borax-carmine preparation the nuclei and granules
are stained red ; the outer zone, about one-third, is also stained red,
the inner two-thirds being either not stained or only faintly so, and
granular, but the granules are not so sharply denned as in the osmic
acid preparation.

(d.) The ducts (few), lined by a single layer of columnar epithe-
lium, with very faint longitudinal striation (fig. 254, D).

9. Active Gland (H). — The cells of the alveoli are less granular,
so that each cell shows an outer zone with no granules, occupy-
ing about one-half of the cell, and an inner granular zone with
granules, which, however, are not nearly so numerous as in the
resting alveoli. The nucleus (sometimes with
a nucleolus and accessory nucleoli) is distinct, and
near the centre of the cell. It is to be noted,
however, that all the alveoli are not in the same
phase.

pj HH9^ 10- Fresh Pancreas. — A very good view of the

I : J% granular character of the inner zone of the pan-

Vj ^j' Iv-^ pf creatic cells is obtained by examining a piece of

fresh pancreas — e.g. ox — teased in normal saline.

FlG'Acini~of a °Fre&h It is easy to observe the difference between the

Pancreas outer homogeneous zone and the inner granular

one. Many of the granules are liberated in the

process (fig. 255).

11. Injected Pancreas (L). — Study a section with its blood-
vessels injected. It is very vascular, and in the inter-tubular cell
clumps are groups of capillaries. It is best injected with a Berlin-
blue gelatine mass from the thoracic aorta.

C

ADDITIONAL EXERCISES.

12. Active Mucous Gland. — The student should examine a section of a sub-
maxillary gland (dog), which has been in action for several hours. He cannot
without a license prepare such a gland for himself. It is prepared, however,
by stimulating at intervals for several hours the chorda tympani of a dog. In
this way the gland is kept secreting, or it may be stimulated by injecting

XXIIL] THE! PANCREAS. 265

pilocarpin. It is then removed from the body and hardened in one of the
ways stated on p. 258. It is essential that the active and non-active glands
be hardened in the same way, so as to show that such differences as exist are
not due to the method of hardening.

(H) Observe that the mucous cells are not so clear as in the passive gland,
but are more granular and smaller, while in stained sections part of their cell-
substance is stained by the pigments, and thus there is less difference between
the demilunes and the mucous cells. The nuclei become more spheroidal. All
the acini in the section are not necessarily in the same phase of activity, so
that the appearances in any two acini may not be identical.

13. Isolated Mucous and Demilune Cells. — (i.) Place fragments of a fresh
dog's sub-maxillary gland in 5 per cent, ammonium chromate (4-6 days).
Tease a small piece in the same fluid. Note the isolated mucous cells, each
with its fibrillar network, spherical nucleus embedded in protoplasm, and
what was the attached end of the cell prolonged into a process.

(ii. ) The cells, membranes, &c., are readily isolated in 33 per cent, caustic
potash.

14. Mucous Granules. — The mucous granules are readily seen by "fixing"
a small piece of the gland with the vapour of osmic acid (Langley}.*

15. Salivary Glands of Cockroach. — It is better to use the species Peri-
plancta amcricana. Kill the animal with chloroform. Pin it out on its back
on a cork plate and make the dissection in normal saline. On cutting open
the thorax longitudinally one sees the intestinal tract, and on each side of
this, lying on the wall of the latter, are the flattened salivary glands. They
can readily be removed. They may be examined fresh, or stained in picro-
carmine, or exposed to the vapour of osmic acid, and mounted in glycerine.
They show well the general arrangement of ducts and lobules. Each duct is
lined by a spiral chitinous fibre like the tracheae.

16. Terminations of Nerves in Serous Glands. — The rapid hardening
method of Golgi (Lesson XXX.) as directed for the pancreas has been used by
Fusari and Panasci '2 for the nerve terminations in the serous glands of the
tongue of the rat, rabbit, and cat. The terminal fibrils form an epilemmal
plexus, i.e. , outside the basement membrane, and other fibrils pass between the
gland cells, i.e., are hypolemmal. Fusari's paper is accompanied by a plate.

17. Gland-Ducts. — Sometimes on using Golgi's method one gets the lumen
of the ducts black. If this happens, then an excellent view is obtained of
their course and connections.

18. Fresh Pancreas. — In the rat and rabbit the pancreas is spread out in
lobules in the mesentery, and if a piece of this containing a thin part of the
pancreas be stretched on a ring of cork the granules can be seen in the fresh
alvi-olar cells. Osmic acid does not alter the granules much, but alcohol does.

19. Changes in Pancreas Cells may be seen in frogs— one fed say three or
four days previously, and the other a few hours before it is required.

20. Rodded Structure in Cells. — Macerate a small piece of fresh pancreas in
5 per cent, ammonium chromate for 2-3 days ; tease and examine in the same
fluid. The outer part of some of the cells will be found to be " rodded."

21. Outer and Inner Zones of Pancreas Cells. — (a.) Use the pancreas of a
starving animal. To stain the outer zone, use ammoniacal or borax-carmine,
and for the granules of the inner zone stain a section (fixed on a slide) with
metliyl-green-fuchsin-S solution made by mixing methyl -green (i per cent.)
60 cc. with fuchsin-S solution (i per cent. ) 20 cc. Stain lor 10 minutes. Wash
quickly in water. Mount in balsam. The granules are red, and the outer
zone clear. [Fuchsin-S is acid-fuchsin. ] For preparations from Flemming's

1 Journal of PJt ?/.s?Ww/?/, vol. x. p. 433.

2 Arcliiv. ital. dc J! /'<>/., xiv. p. 240, 1891.
24

266 PRACTICAL HISTOLOGY. [XXIV,

fluid, stain with safranin and wash with alcohol containing picric acid. The
safranin stains the granules.

(b.) A pancreas fixed in Flemming's fluid or sublimate is beautifully stained
by means of the following modification by Oppel of Bioudi's fluid : — •

Meth yl -green ( I per cent.) . . . 120 cc.
Eosin(i percent.) .... 2 ,,

Acid-fuchsin (i per cent.) . . . 40 ,,
Absolute alcohol 3 . . . 40 ,,

The granules are red and the nuclei green.

22. Terminations of Nerves in Pancreas. — By means of Golgi's rapid
hardening method (Lesson XXX.) — i.e., osmico-bichromate fluid and then
silver nitrate — Ramon y Cayal and Sala,1 and more recently Erik Miiller,2
have traced the terminations of nerve-fibres in the pancreas (dog, rabbit).
Numerous nerve-fibres enter the pancreas along its ducts, and terminate in a
rich plexus of fibrils around the individual acini. The nerve-fibres which
surround the acini are derived from two sources, some fibre fibrils are branches
of Remak's fibres, and others are processes of very characteristic cells, which
R. y Cayal calls "visceral sympathetic ganglion cells." These cells send off
processes which are arranged as part of the "peri-acinous" plexus, and some
of them penetrate between the gland-cells. Miiller finds that the fibrils lie in
relation with the secretory cells.

LESSON XXIV.
THE STOMACH.

THE walls of the stomach, like the intestine, are composed of four
coats, named from within outwards —

(i.) Mucous coat, i.e., the glandular coat.

(2.) Submucous, composed of loosely-arranged connective

tissue, the larger blood-vessels, lymphatics, and nerves.
(3.) Muscular, composed of three layers of non-striped muscle

— (a.) longitudinal, (b.) circular, (c.) oblique. In some

situations only two layers exist.
(4.) Serous, from the peritoneum.

The mucous coat is lined by a single layer of tall, narrow, cylin-
drical mucous cells — in reality mucus-secreting goblet-cells — for
they have open mouths and contain mucigen. The cardiac portion
of the mucous membrane is composed of tubular glands — fundus
glands — placed side by side. Several gland-tubes may open into

1 Termination de los nervios y tubos glandulares del pancreas de los verte-
Irados, Barcelona, 1891.

a Archivf. mik. Anat., xl. p. 405, 1892.

XXIV.] THE STOMACH. 267

one common duct. The duct is short, and is lined by cells like
those covering the surface of the stomach. The secretory part of
each gland is lined throughout by a layer of polyhedral or short
columnar, granular, nucleated cells, called chief, principal, inner,
or adelomorphous cells. At intervals between these and the base-
ment membrane of the gland are large, ovoid, conspicuous, granular
cells — outer, parietal, delomorphous, or oxyntic. The lumen of
the gland is small and ill-defined.

The pyloric mucous membrane is beset with pyloric glands,
which have a long duct or neck, and are usually branched at their
lower ends. The duct is lined by a layer of cells like those lining
the stomach, and the secretory part by a single layer of short, finely
granular, short columnar cells. The lumen is well-defined. There
is more connective tissue between the glands than in the cardiac
portion. Masses of adenoid tissue are not unfrequently seen
between the bases of the pyloric glands.

Methods. — (i.) Select a cat or dog that has hungered for two
days. Kill the animal, open the stomach, and place small parts of
the cardiac and pyloric ends (all the coats) in equal parts of chromic
acid (J per cent.) and spirit (7-10 days). Change this fluid writhin
twelve hours. Complete the hardening in alcohol.

(ii.) Place small pieces of the cardiac and pyloric mucous mem-
brane (J inch cubes) in i per cent, osmic acid (24 hours). Wash
well and harden in alcohol.

(iii.) Fix pieces of the pyloric and cardiac mucous membrane
in mercuric chloride (2-3 hours). Take care to remove all the salt
by prolonged washing in alcohol. Perhaps this is one of the best
methods to use.

(iv.) Absolute alcohol is also a good hardening medium.

To bring out all the chief characters of the glands, some sections
are to be stained in logwood and mounted in balsam. The sections
may be cut by freezing or in paraffin, after staining in bulk. Stain
otkers in picro-carmine, and mount in Farrant's solution ; others in
dilute carmine (24 hours). Stain others with i p.c. watery
solution of aniline-blue (Nicholson's No. i) for twenty minutes.
AVash in glycerine and water, and mount in Farrant's solution.
For other methods (p. 271).

1. V.S. Cardiac End. — (a.) (L) Observe the relations, relative
thickness, and structure of the several coats. The mucous coat,
with its gastric or cardiac glands, or glands of the fundus, set
vertically like so many tubes in a rack (fig. 256). The glands are
simple tubular glands, and some of them have several secretory
parts opening into one duct ; at their bases is delicate connective
tissue, adenoid tissue, and blood-vessels. Below the closed ends
of the glands, in the cat, a clear homogeneous layer of condensed

268

PRACTICAL HISTOLOGY.

[xxiv.

connective tissue, and under this two or three thin layers of non-
striped muscle — the muscularis mucosse. The clear layer of con-
densed tissue is not present in the rabbit, dog, or man.

(6.) The submucous coat, composed of loose connective tissue,
with large blood-vessels and a few fat-cells. If the mucous

membrane be folded and rugaB are
present, the connective tissue will
be seen to run up into the folds.

(c.) The muscular coat consists
of two or three layers of smooth
muscular fibres. The appearance
varies according to the manner in
which the plane of the section
cuts them, for the stomach has
an outer longitudinal and an inner
circular, and in some places an
oblique muscular coat.

(d.) Outside all a thin layer —
peritoneum or serous coat — con-
sisting of fibrous tissue covered by
a layer-of endothelium.

(e.) (H) Study specially the
mucous coat. Observe the
columnar epithelium lining the
stomach, and dipping into the
mouths and lining the ducts,
which are slightly funnel-shaped.
The cells are tall and narrow, and
if the section be not too thin, the
ends of them may be seen as

111 i i i G i

very small, clear, sharply-defined,
polygonal areas. As they secrete
mucus, they have been called mucous cells. The upper two-thirds
or so of the cell is much clearer than the lowest third, which tapers
somewhat, and is more granular. Each cell contains an oval nucleus
in its lowest third, i.e., near its attached end.

(/.) Select a fundus gland (fig. 257). Trace its duct down-
wards, and note that perhaps it is common to two secretory
portions. In the true secretory part, note the large ovoid, border,
parietal, or outer cells, lying next the basement membrane, — ovoid
in shape, granular in appearance, and containing an ovoid nucleus.
They do not form a continuous layer, but bulge out the basement
membrane here and there. At the upper part of the gland-tube the
parietal cells are smaller, and placed nearer each other, while
towards the fundus or base of the gland they are larger and further

Serosa. !_ _____ / ^, — -^I____

FIG. 256.— V.S. Wall of Human Stomach.
E. Epithelium ; G. Glands ; Mm. Mus-
X is-

XXIV.]

THE STOMACH.

269

apart, the interval between any two being occupied by the inner or
central cells. The parietal cells are deeply stained by aniline-blue
and carmine, and osmic acid, while in a logwood-stained section the
inner cells are usually more deeply stained.

Observe a continuous layer of cells — inner, central, or chief —
lying internal to the parietal cells. They are smaller, and belong
more to the columnar type of cell, but they are not of uniform height
throughout ; thus they are shorter over a parietal cell, and larger
between two parietal cells. Each cell contains a spherical nucleus.

The lumen of the gland-tube is very
narrow. The secretory part is much longer
than the duct.

(fj.) Arrange the preparation so that the
lower ends of the glands come into view.
Note their closed extremities, and if the
granular epithelium have shrunk somewhat,
the basement membrane of each gland-tube
may be seen ; very probably transverse or
oblique sections of the lower part of the
gland will be found (lig. 257).

(It.) At the bases of the gland-tubes, but
outside their basement membrane, note the
delicate connective tissue or adenoid tissue,
with a number of leucocytes. If the section
be from a cat's stomach, a clear homogene-
ous layer runs outside this.

(i.) Outside this the muscularis mucosaB
composed of at least two layers of non-striped
muscle arranged in opposite directions.
Especially in a balsam specimen, fine strands
of muscular fibres are always to be seen
passing from the muscularis mucosa3 in-
wards towards and between the glands,
t'sually these fine strands pass inwards
between groups of glands. In the osmic
acid preparations, the parietal cells are much
darker than the central cells.

(./.) There is nothing particular to note in the structure of the
other coats.

2. T.S. Fundus Glands.— Make a section parallel to the surface
of the mucous membrane, stain with logwood, or picro-carmine and
aldehyde-green, or use an osmic acid preparation.

(H) Observe that all the gland-tubes are not cut at the same
level ; some are divided through the duct, others through the
secretory part of the tube. In the latter observe the large parietal

FlO. 257. — V.S. Mucous Mem-
brane of the Stomach, Cat.
Osmic acid.

2/0

PRACTICAL HISTOLOGY.

[xxiv.

FIG. 258.— T.S. Duct of Gland of Fundus. a
Chief, h. Parietal cells ; r. Adenoid tissue
c. Capillaries.

cells — few in number — and inside these a complete layer of inner
cells bounding the small lumen of the tube (fig. 258). The glands

are arranged in groups as shown
by the connective tissue sur-
rounding several tubes. This
is very marked in the pig.

3. Fresh Fundus Glands. —
From the
m u c o u s
membrane
of the sto-
mach of a
newly-
k i 1 led
guinea-pig
make a
thin verti-
cal section
with scis-
sors, and tease it in normal saline to isolate
some of the glands. This animal is selected
because its gland-tubes are short, but a rabbit
does very well. The elevations of the base-
ment membrane due to the bulging of the
parietal cells are usually well seen.

4. V.S. Pyloric Mucous Membrane (fig.
259). — (a.) (L) Observe the same arrangement
of coats as in the cardiac end ; but the muscu-
lar coats are thicket- ; the mouths of the gland-
tubes are wider and longer, the secretory part
more branched and shorter than in the cardiac
portion. There is also much more connective
tissue between the glands.

(&.) (H) The wide mouth of the glands, lined
by narrow columnar epithelium, the secretory
part consists of several tubes opening into one
gland-duct. The secretory part lined by a
single layer of cells, somewhat cubical, but
there are no parietal cells.

(c.) There is much more connective tissue
between the tubes. A mass of adenoid tissue
— solitary follicle — may be seen in the deeper
part of the mucous coat. In some cases its pointed apex may be
seen reaching nearly to the surface of the mucous membrane.

5. Osmic Acid Preparations. — Few reagents are so good for

259. — V.S. Pyloric
Mucous Membrane. D.
Duct ; S. Secretory part
of gland ; C. Connective

tissue.

XXIV.] THE STOMACH.

fixing the cells of the glands of the mucous membrane (mount in
Farrant's solution). In sections of the fundus, the outer cells are
more deeply stained, and so are readily distinguished. The columnar
cells lining the ducts of the cardiac and pylofic glands may be
blackened by the osmic acid where they contain mucigen.

6. Blood- Vessels of the Stomach.— Make Y.S. of an injected
stomach embedded in paraffin. Mount in balsam. They must not
be too thin. Note the large vessels in the submucous coat, and
from them smaller vessels proceeding vertically upwards, splitting
up into capillaries between the tubules, and forming a capillary
network round the mouths of the glands. Beautiful plates by Mall.1

7. Pyloro-Duodenal Mucous Membrane. — It is well to have a
section through the pyloric valve to include the mucous membrane
on its gastric and duodenal boundaries. To ensure this, the mucous
membrane or entire thickness of the stomach must be pinned out
on cork before it is hardened by any of the methods, e.y.t sublimate
(p. 267). It is treated like the sections of the stomach, and does
best when stained with eosin-hsematoxylin. On one side of the
thickened pyloric valve — the increased thickness being due chiefly
to an increase of the circular muscular fibres — one sees the pyloric
structure, and on the other that of the duodenum. The tubular
glands of the stomach are confined to the mucous membrane, but
the acini of Brunner's glands lie in the submucous coat of the
duodenum.

8. Junction of (Esophagus and Stomach.— Similar preparations
may be made. The transition from the cesophageal mucous mem-
brane with stratified epithelium and few glands in the oesophagus
to that of the stomach with its columnar epithelium and mucous
glands is sudden and abrupt.

ADDITIONAL EXERCISES.

9. Double-Staining the Stomach.— Harden the stomach in Miiller's fluid,
and stain (24 hours) the sections in indigo-carmine (p. 67) ; afterwards extract1
the excess of pigment by steeping them for half an liour in a saturated solution
of oxalic acid. Mount in Farrant's solution or balsam. The parietal cells aro
grey or blue, the central ones coloured, but with red nuclei, and the smooth
muscle blue with red nuclei.

10. Ehrlich-Biondi Fluid. — Stain in this fluid sections of the mucous meni'
brane fixed in sublimate (saturated in .6 per cent. NaCl). If the fluid be kept
for some time, an additional quantity of acid-fuchsin must be added to it.
The parietal cells are red and their nuclei bine ; the chief cells are scarce!)
stained at all, but their nuclei are faintly blue. Yacuoles may be seen in some
of the outer colls.

11. Aniline-blue and Safranin. — Sections of the cardiac end fixed in

J Vessels and Walls of Dog's Stomach, John* 7/^>/.-///.v Ifosp. Rrp., vol. i. 1893.

PRACTICAL HISTOLOGY.

mercuric chloride are stained with aniline-blue and then with safranin. In
balsam preparations the parietal cells are pale blue, the inner cells red.

12. Isolated Cells of Gastric Glands. — Macerate fragments of the gastric
mucous membrane of a newt in 5 per cent, ammonium chromate (24-48 hours).
Stain in picro-carmine, and tease in glycerine. Numerous isolated cells from
the ducts and secretory parts of the glands are obtained.

LESSON XXV,
THE SMALL AND THE LARGE INTESTINE.

SMALL INTESTINE.

IT has four coats — mucous, submucous, muscular, and serous.

Study specially the mucous coak In man, in certain parts, there
are permanent folds of the mucous membrane — valvulse con-
niventes — and everywhere the surface is beset with small conical
elevations — villi. At the bases of the villi is a layer of simple
tubular glands — Lieberkiihn's glands — embedded in an adenoid
tissue matrix. Underneath this is the muscularis mucosse, usually
consisting of three thin layers of smooth muscle.

A villus (.5-3 mm. long) consists of a central core, enclosing a
lacteal, and covered by a single layer of columnar epithelium with
goblet-cells (Lesson V.). The body of a villus consists of a tissue
like adenoid tissue with leucocytes and other cells. The central
lacteal is really a lymphatic, and begins by a closed extremity.
Several strands of smooth muscle pass from the muscularis mucossB
into the villus, and reach its upper extremity. It is very vascular,
and the blood-vessels are distributed immediately under the epi-
thelium.

The mucous membrane also contains solitary follicles, and in
some situations Peyer's patches, the latter are most abundant in
»he ileum.

Methods. — Make transverse sections of the small intestine of a
cat or dog hardened in a mixture of potassic bichromate and
chromic acid ; Klein's fluid ; i per cent, chromic acid ; Kleinen-
berg's fluid ; or mercuric chloride.

(i.) Stain a section in hsematoxylin and mount it in balsam, or
stain another in picro-carmine and mount it in Farrant's solution.

(ii.) All the parts and their relations are best preserved by
staining in bulk in borax-carmine and cutting in paraffin, or embed
in paraffin, cut, fix on a slide, and then stain.

XXV.] SMALL INTESTINE. 273

(iii.) Cut sections by freezing and place some in i per cent, osmic
acid (24 hours). This sharpens the outlines of many of the structures.

(iv.) It is convenient in teaching to give a complete transverse
section of the small intestine of a small animal, e.g., mouse or
kitten. In herbivora the wall of the gut is very thin. Stain in
bulk and cut in paraffin. Flemming's fluid is an excellent " fixing "
reagent both for the small and large intestine. Stain the sections
in safranin.

1. T.S. Small Intestine (L). — Observe the serous, muscular,
submucous, and mucous coats (fig. 260).

(a.) In the mucous coat, the surface beset with small conical
projections — villi — which, if they are contracted, exhibit wrinkles
on their surface. At the bases of the villi a single layer of simple
test-tube-like glands — glands of Lieberkunn — or intestinal glands,
embedded in an adenoid tissue matrix. Outside this the muscu-
laris mucosae.

(b.) The submucous coat, composed of fibrous tissue with nerves
and blood-vessels.

(c.) The muscular coat, composed of two layers, an outer longi-
tudinal and an inner circular layer. In the cat, the latter is much
thicker than the former.

('/.) The serous coat.

(e.) (H) Study a villus. Observe the single layer of granular
nucleated columnar epithelium covering it, each cell with its free
end covered by a clear disc, with vertical strise (fig. 260). The
succession of these free clear borders looks like a clear hem round
the circumference of the villus. Occasionally leucocytes may be
seen between the cells.

(/'.) The goblet cells, chalice, or calictform cells scattered among
the former (fig. 260). They may be seen from the side, or their
open rounded mouths may be directed toward the observer. When
seen from the side, they are ovoid, with a larger and clearer upper
part containing mucigen, and a smaller, lower, granular, nucleated
part (Lesson V.). Sometimes a plug of mucus may be seen protruding
from the mouth of a cell. It is stained blue with logwood and brown
with Bismarck brown. If the mucous glands be active and the fresh
tissue be fixed in osmic acid, then the plug of mucus is black.

(g.) In the centre of the villus a vertical space, the radicle of a
lacteal, with a thin nucleated wall. The substance of the villus
consists of adenoid tissue beset with leucocytes. Close under the
epithelium, perhaps, sections of capillaries, and a little farther in
one or more strands of non-striped muscle, which can be traced to
the apex of the villus, and downwards to the muscularis mucosse
(fig. 260, MM).

(h.) The glands of Lieberkuhn lined by short columnar nucleated
25 S

274

PKACTICAL HISTOLOGY.

[XXV.

cells, the nucleus near the attached end of the cell. There is
a gradual transition from these cells to those covering the villi.
A clear border may be seen on their free ends. Bizzozero has
called these "protoplasm cells" to dis-
tinguish them from the goblet-cells which
lie amongst them. Goblet-cells, however,
are far more abundant in the large intestine.
The lumen of each gland is distinct, and,
especially if the lining cells be raised
slightly, the basement membrane of the
gland-tube may be seen. Between the
gland-tubes numerous leucocytes and
adenoid tissue ; in fact, the glands are set
in a mesh work of this tissue.

(i.) The muscnlaris mucosse sends deli-
cate processes into the villi. This is best
recognised in balsam specimens, by the
arrangement of the fusiform nuclei of the
smooth muscle cells.

(/.) A solitary gland or a Peyer's patch
may be cut, but it is better to have special
preparations for these.

2. Peyer's Patches or Agminated
Lymph Follicles. — (i.) Make V.S. through
a hardened Peyer's patch (sublimate or
alcohol). Stain with eosin and hsemato-
xylin and mount in balsam.

(ii.) Fix a Peyer's patch of a rabbit or
guinea-pig in Flemming's fluid. Fix a sec-
tion on a slide and stain it first in i per cent,
aniline-blue (watery). Wash out fn i per
cent, ammonia, then in .5 per cent. HC1,
and stain in safranin (Garbini).
in balsam.

(L) Observe a group of oval or roundish
masses of adenoid tissue crowded with

Mount

FIG. 260.— T.S. Small Intestine
(Cat). V. Villi ; LG. Lieber-
kiilm's glands; MM. Mus-
cularis mucosse ; C and L.
Circular and longitudinal

fibres of muscular coat; s. leucocytes confined to one side of the gut.
The conical apices of some of them may be

seen projecting upwards quite to the mucous surface, covered only
by a layer of columnar epithelium. Between the epithelial cells
may be seen colourless corpuscles which have wandered from the
adenoid mass. The lower ends of the masses usually pass down
into the submucous coat. No villi exist over the apices of these
masses of adenoid tissue if they project well into the mucous layer.
If, however, they do not, but exist merely as rounded masses of

XXV.]

SMALL INTESTINE.

275

Villi

with epi-
thelium.

adenoid tissue in the submucous coat, then they present the appear-
ance seen in fig. 261.

A solitary follicle — exactly like one of the numerous follicles
which compose a Peyer's patch — may be seen (fig. 262).

3. Blood Vessels of
the Small Intestine.—
Make a pretty thick
transverse section of a
well-injected small in-
testine (cat). Cut in
paraffin and mount in
balsam.

(L) The mucous coat
is the most vascular
part ; the larger vessels
lie in the submucous
coat, and few vessels in
the muscular coat.

(H) An artery runs
to the upper part of
each villus and gives
off a plexus of capil-
laries ; a vein on the
opposite side. A .rich ^.
plexus of capillaries <
between Lieberkiihn's 2
glands. If the section §
pass through a solitary J
follicle, note that the -~
capillaries pass into it
and form loops (fig.
262).

The general distribu-
tion of the blood-vessels
is shown in fig. 263,
which shows how the

FlG. 261. — Longitudinal Section through a Peyer's Patch
OUS COats. of the Small Intestine of a Dog.

4. Surface View of

Injected Villi. — To see the general arrangement of the blood-vessels
in the mucous membrane, inject the blood-vessels of a rabbit with
a red gelatine mass. Mount in balsam a part of the wall of the
small intestine, placing the mucous surface uppermost.

(L) Xote the villi, and trace the artery to its origin from a larger
artery in the submucous coat (fig. 264) The artery runs on one

Circular
muscle.

Longi-
tudinal
muscle.

2/6

PRACTICAL HISTOLOGY.

[XXV.

side of the villus quite to the apex of the latter, and the vein — a
wider vessel — descends on the opposite side. There is a dense
plexus of capillaries (C) placed close under the epithelial covering.
The best figures of the blood-vessels of the intestine are to be found
in Mall's paper.1

5. Nerve-Plexuses in Intestine.— (a.) Gold Chloride Method.—
Wash the small intestine of a rabbit with normal saline, fill it with

Villi with Solitary

blood-vessels. follicle.

- — Muscular coat.

Fia. 262.— Mucous Membrane of Small Intestine (Kabbit), Injected, showing
villi and a solitary follicle, x 50.

lemon-juice or 5 per cent, arsenic acid, and leave it in lemon-juice
or acid for five minutes. Allow the juice to escape, wash the gut
in water, fill it with i per cent, gold chloride, and place it in J per
cent, gold chloride solution (30 mins.). Wash it in water, and
keep it in the dark in 25 per cent, formic acid (48 hours). It is
now easy to separate the coats of the intestine from each other.
Wash in water to remove all the acid, and with a pair of forceps
strip off the longitudinal muscular coat (Auerbach's plexus adheres
to this) ; it separates quite easily from the circular coat. Preserve
what remains. Mount a small part of the longitudinal muscular
coat in Farrant's solution.

(1.) Methylene-Uue Method (p. 284).

1 " Die Blut- u. Lymph-wege im Diinndarm des Hundes," AWiand. d. math.-
phys. Classe d. K. Sachs, Gesell. d. Wissens., Bd. xxiv., 1887.

XXV.]

SMALL INTESTINE.

2/7

Villi.

Glands.

6. Auerbach's Plexus lies between the muscular coats, but when
they are separated it usually adheres

to the longitudinal coat. The general
arrangement of the plexus can be
seen with the naked eye (fig. 265).

(L) The polygonal mesh work of
purplish stained fibres, with slight
swellings at the points of intersection
(fig. 266).

(H) The meshes are so large that
only a part of them comes into the
field of view at once. Note the non-
medullated nerve-fibres, and at the
nodes, groups of nerve-cells. From
the plexus numerous fibres are given
off to supply the smooth muscle of
the intestine.

7. Ganglionic Cells. — In a vertical
section of the gut (prepared as in 1)
look for groups of ganglionic cells in
the submucous coat and others be-
tween the t\vo layers of muscular
fibres (fig. 267).

8. Meissner's Plexus. — Spread
what remains after removal of the

longitudinal muscular coat on a slip of glass, mucous surface

FIG. 263.— Scheme of the Distribution
of Blood-Vessels in the Small In-
testine of a Dog. 3/.3f . Muscularis
mucossc.

Fio. 264.— Injected Villi of Small Intestine of Rabbit, seen from above and laterally.
A. Artery; V. Vein; C. Capillary network.

uppermost. With a knife gradually scrape away the mucous coat.

2/8

PRACTICAL HISTOLOGY.

[xxv.

With care — observing the preparation from time to time under a
low power of the microscope — the progress of the process of denu-
dation can be easily observed. By and
by the plexif orm arrangement of Meissner's
submucous plexus will be seen. Mount
in Farrant's solution (fig. 268).

(L) Observe the large wide meshes of
the plexus. Compare the general arrange-
ment of the plexus with that of Auer-
bach's plexus. The fibres are finer, and
the groups of nerve-cells smaller.

(H) Note the ganglionic nerve-cells at
the nodes (fig. 268).

9. Brunner's Glands. — These glands
are confined to the duodenum. Proceed
exactly as recommended for the small in-
testine. Stain a section with logwood and
use eosin as a counter stain. This gives
excellent results.

Perhaps the best method is to slit up the duodenum longitudi-

FlG. 265. — Auerbach's Plexus
(Rabbit). Lemon-juice and
gold chloride, x 8.

PIG. 266.— Auerbach's Plexus (Dog).

nally, pin it out on a cork plate, and fix it in mercuric chloride (an
hour at 40° C. in a thermostat, or 24 hours at ordinary tempera-

XXV.]

SMALL INTESTINE.

279

ture). The surplus HgCl2 is extracted with water at 30° and
afterwards with alcohol at 40° C. It is then removed from the
cork plate and hardened in alcohol. Embed and cut in paraffin.
Kuczynski1 has investigated these glands in a great number of

FlQ. 267. — Awerbach's Plexus in Section, a. Ganglionic cells ; b. Nerve-fibres ; c. Circular,
ami (2. Longitudinal muscular fibres.

animals and man, and tried a great variety of stains. Kleinenberg's
fluid is also very good as a fixing reagent.

(L and H) Note villi, Lieberkiihn's glands and the usual intes-
tinal coats. In the submucous coat, which is thick, are the acini

Fio. 268.— Meissner's Plexus Intestine (Rabbit). Lemon-juice and pold chloride.

of Brunner's glands (fig. 269), each composed of a basement mem-
brane lined by cubical or short columnar cells. A duct from each

1 Internat. Monats. f. Anat. u. Phys., vii., 1890, 419.

2 80

PRACTICAL HISTOLOGY.

[xxv.

gland perforates the muscularis mucosse, passes up between the

intestinal glands, and opens
on the free surface at the
bases of the villi. It has a
distinct lumen, and is lined
by low cubical epithelium.

' 10. Fresh Villus.— Pre-
ferably that of a mouse.
Examine in normal saline.

LARGE INTESTINE.

Methods. — Prepare it in
the same way as the small
intestine, .and make vertical
transverse sections. The
details of the structure will
necessarily vary with the
animal used, but perhaps the
cat is as convenient an
animal as any to employ.
I can strongly recommend
Flemming's fluid. Sections
to be stained with safranin.

11. T.S. Large Intestine (fig. 270).— As the large intestine is
wide, it is necessary in making the section to select a portion of the
great gut which shows the longitudinal coat. In man, the coat
is not a continuous one, the longitudinal fibres being grouped for
the most part into three flat bands of fibres.

(L) Beginning from without inwards, observe four coats. In
the muscular coat, note the peculiarities of the longitudinal coat
and the thick well-marked continuous circular coat. There is
nothing special about the .submucous coat, unless it be the exist-
ence of solitary follicles, which, however, arc not confined to
the large intestine. The mucous coat is characterised by negative
characters. There are no villi. It may exhibit folds, into each
of which there runs a projection of the submucous coat.
Lioherkiilm's glands, cut vertically, and some of them obliquely or
horizontally.

(H) In the mucous coat the glands of Lieberkiihn, larger than
those of the small intestine, and lined by cells — protoplasm cells —
with nuclei near their attached ends. Amongst them are very
many goblet-cells (fig. 271).

Serous coat.

FIG. 269.— V.S. Duodenum of Cat. c, I. Circular
and longitudinal layers of muscle; Lg. Lieber-
kiihn's glands ; JSg. Bruuner's glands ; g.
Ganglion-cells ; v, Villi.

Muscular
coat.

LonjritiKli-
Jial fibres.

FIG. 270.— L.S. Large Intestine.

12. Vermiform Appendix (Rabbit). — This is
instructive on account of the lymphatic follicles
it contains and the micro-organisms contained
in these follicles (p. 284). Fix it in mercuric
chloride and treat it as recommended for the
other parts of the intestine. The lymph-sinuses
are readily injected by the "puncture" method
with watery solution of Berlin-blue. Fix in
Muller's fluid and harden in alcohol.

The lymph follicles are arranged in two or
three rows, the one inside the other, but the
innermost layer does not project beyond the level
of the mucous membrane. The apices of the
inner row are covered only by cylindrical epi-
thelium.

let-ceils.

2S2

PRACTICAL HISTOLOGY.

[xxv

ADDITIONAL EXERCISES.

13. T.S. Villus. — Stain a hardened mucous membrane (with villi) of a dog
or cat "in bulk" in borax-carmine or Kleinenberg's logwood, embed and cut
T.S. in paraffin, fix on a slide, remove the paraffin with turpentine, clarify
with clove-oil, and mount in balsam. Many of the villi will be cut obliquely.
(H) Observe the lacteal (L) in the centre, and round it the structure of the
stroma of the villus, with several groups of non-striped muscle-cells (m) close
to and surrounding the lacteal. In some animals (dog) there is a double row
of these smooth muscles (fig. 272). The capillaries immediately under the

epithelium (c), the smooth muscular
fibres parallel to the lacteal, and the
stroma — composed of anastomosing
fine trabeculae with parenchymatous
cells and leucocytes — make up the
elements present in the core of a
villus.

Great differences exist in the rela-
tive size of the stroma and lacteal in
villi. In the dog and cat the lacteal
is relatively small, and the stroma
abundant ; in the rabbit the lacteal
is very large, and the stroma scanty.
14. Non-Striped Muscle in Villi. —
The method of Kultschitzky enables
the course of the fibres to be more
clearly traced. Harden (for twenty-
four hours) a piece of dog's small in-
testine in the following fluid : — A
saturated solution of potassic bichro-
mate and copper sulphate in 50

FIG. 272.— T.S. Villus (Dog). L. Lacteal
m. Muscle ; c. Capillaries, x 400.

cent, alcohol (in the dark), to which, immediately before using, is added
5-6 drops (to 100 cc.) of acetic acid. The preparation and fluid must be kept
in the dark. Complete the hardening in absolute alcohol. Make sections,
and stain them in acid chloral-hydrate carmine, which is made as follows : —
Chloral hydrate, 10 grams; hydrochloric acid (2 per cent.), 100 cc. Add to
this dry carmine (.75 to 1.5 gram), according to the strength of stain desired.
Boil for one and a half hours, preventing evaporation by means of a cooling
apparatus. Allow it to cool and filter. If the preparations stained with this
dye be washed in 2 per cent, alum, the nuclei and other tissues become violet.

Sections stained thus show the course of the smooth muscle -from the
muscularis mucoscs obliquely between Lieberkiihn's glands into the villi, where
they are arranged in several bundles near the lacteal. They curve as they
ascend in the villus, the concavity looking outwards, and are fixed or inserted
close under the epithelium. They pass quite to the apex of the villus, becom-
ing thinner as they go, where they each split up into a pencil of fibres, the
fibres being inserted close under the epithelium.

15. Heidenhain's Method.— Harden small pieces of the small intestine,
e.g., dog or cat (24 hours), in a .5 solution of common salt saturated with
mercuric chloride. Place it for twenty-four hours in alcohol 80, 90, and 95
per cent., and finally in absolute alcohol. Saturate with xylol, embed in
paraffin, and cut thin sections, which are fixed on a slide with a "fixative."
After the paraffin has been got rid of by turpentine or xylol, and the turpen-
tine displaced by alcohol, the sections are stained on the slide with Ehrlich-

XXV.]

LARGE INTESTINE.

283

Biondi's fluid (p. 81), diluted with 40 or 50 volumes of water. It requires
10— 12 hours to stain the sections, which are then mounted in balsam. This
preparation is particularly valuable for studying the various forms of cells that
occur in the stroma of a villus. 1

16. Absorption of Fat. — (i.) Feed a frog on fat bacon ; after two days kill
it, and tease a portion of the mucous membrane of the intestine in normal
saline, or dissocinte it in dilute alcohol. Observe the isolated columnar cells
crowded with fine granules of oil, which are blackened on the addition of
osmic acid.

(ii.) A better plan is to stain the mucous membrane in osmic acid (24 hours),
and embed it in paraffin. It is to be remembered,, however, that steeping the

FIG. 273. -Villus of Dog's In-
testine, with Lacteal and
Non- Striped Muscle. L.
Lacteal, x 250.

FIG. 274. — Section of Intestine of
Frog. Absorption of fat. Osmic
acid.

mass or sections of it in paraffin discharges in part the black colour of the fatty
granules (p. 33).

(iii. ) Feed a rat on bread and fat of bacon ; kill it four hours afterwards by
means of curare. Harden the small intestine in Os04.

(H) Observe the projections like folds of the mucous membrane. The
columnar cells covering them are crowded with blackened particles — fatty
granules blackened by osmic acid (fig. 274).

17. Tubular Glands of the Intestine. —In studying these, different fixing
fluids are used according to the animal selected (Bizzozero).2 Amongst the
best fixing reagents are saturated watery solution of picric acid or picro-
siiljilinric acid (2 days), and wash in water (i day), subsequently hardening in
alcohol. This is suitable for the rectum of the mouse and dog. For the
duodenum, either alcohol or Flemming's fluid, or Hermann's fluid (Lesson
XXXV.). They are stained in safranin (1-2 hours), washed (10-15 sees.) in
absolute alcohol, and then stained in luematoxylin. Cleared in bergamot
oil and mounted in balsam. Safranin stains the nuclei of the cells, but not
the mucin. The latter is stained by the logwood, and one can trace it from
the goblet-cells passing into the lumen of the tubule. Mitosis may be
observed both in the protoplasmic and goblet cells.

1 Pfliigers Archiv, Supp. Bd., 1888.

2 Archivf. mik. Anat., p. 325, 1892.

284 PRACTICAL HISTOLOGY. [XXV.

The mucus in the fresh goblet-cells occurs in the form of granules. This is
best seen in a fresh preparation teased without the addition of any fluid, or at
most in Miiller's fluid. In sections fixed in Flemming's fluid and stained with
safranin, the goblet-cells are reddish. The network in these cells may be
stained with Bismarck-brown.

According to Bizzozero, the epithelial investment of the villi is not renewed
by the proliferation of the cells covering it ; but by the proliferation and
upward growth of the cells lining Lieberkuhn's follicles.

18. Terminations of Nerves in Stomach and Intestine. — Besides the gold-
chloride method (Drasch,1 nerves of the duodenum), and the methylene-blue
methods as used by Aronstein,2 Golgi's silver method has been recently applied
by Erik M tiller3 for this purpose. The method used was Golgi's "rapid
method" (Lesson XXX.), i.e., osmico-bichromate solution and subsequent
staining with silver nitrate. The nerve- fibres become black, and numerous
communications are found to exist between the plexuses of Auerbach and
Meissner. A very large number of nerve-fibrils enter the villi, and are dis-
tributed in them, reaching to the cylindrical epithelium covering them. They
end free and have not b.een seen to end in or between the cylindrical cells.
Some end in the smooth muscular fibres of the villi. Other fibrils surround
the gland tubes. It is said that branched cells, like nerve-cells, lie in the villi
(R. y CayaT).

19. Methylene-Blue. — Immediately after death inject into the thoracic
aorta of a guinea-pig or rabbit the following fluid: — I gram methylene-blue
BX in 300 cc. normal saline. Open the abdomen and expose the gut to the
air for 2-3 hours. Place a thin piece of the wall of the small intestine in
picrin-glycerine of S. Mayer (p. 192) and search for the red dish- stained nerve
plexus (S. Mayer).

If a piece of the gut be teased in picrin-glycerine, it is easy to isolate blood-
vessels with their nerves. It is to be noted that methylene-blue not only
stains the axis-cylinders of nerves, but also the cement of epithelial cells, so
that in some respects it acts like silver nitrate.

20. Vermiform Appendix (Rabbit}. — Fix in Flemming's fluid, Fol's solution,
or absolute alcohol.

A. For Mitosis. — (i.) Stain the sections (5-10 mins. ) in

Gentian-violet ..... i gram.

Absolute alcohol . . . . 15 cc.

Aniline oil . . . . . 3 ,,

Water 80 ,,

(2.) Wash in absolute alcohol. (3.) Immerse (30-40 sees.) in i per cent, chromic
acid. (4.) Again absolute alcohol (30-40 sees.); and (5.) in chromic acid.
Then in absolute alcohol to remove all surplus dye. Balsam. In this way
the chromatin of the nuclei is stained.

B. For Micro- Organisms. — Stain as above in (i). (2.) Absolute alcohol
(5 sees.). (3.) Weak iodine solution, i.e., Gram's method (p. 105) (2 mins.).
(4.) Then alternately chromic acid and alcohol as above. Balsam (Bizzozero).
The secret of getting good preparations is to wash them well in absolute
alcohol until all surplus dye is removed.

21. 'Peyer's Patches and Phagocytosis.— (a.) Harden a Peyer's patch
(rabbit) in absolute alcohol, cut sections in paraffin, and stain them in alum-
carmine, or in addition with gentian-violet by Gram's method (p. 284)

1 Sitzb. d. k. Akad. d. Wissenscli., Bd. 81, iii. Abth., Wien, 1880.

2 Aunt. Anzeigcr, Bd. ii., 1887.

3 Archivf. mik. Anat., Bd. xl., 1892.

XXVI.] LIVER. 285

(Ruffer1}. (H)- In the sections numerous small leucocytes will be found to
have wandered from the lymph follicles between the epithelial cells. There
are always to be seen several much larger cells — mono-nucleated — in the lymph
follicles. The smaller forms — which may be mono-nucleated or poly -nucleated
— have been called microphages, and the largest macrophages because both
are capable of taking up bacteria — dead and alive — into their protoplasm and
changing them by a process of intnicellular digestion. Transition forms
between microphages and macrophages of leucocytes may be found. In a
preparation stained by Gram's method the bacilli — which are found in the
lymph-cells, but not in the epithelial-cells — are stained deep blue-violet.

(b.} Sections of glands fixed in sublimate may be stained with Ehrlich-
Biondi lluid and the same kinds of lymph-cells as occur in lymph-glands are
found.2 The most numerous are (i) small lymph-cells with a nucleus (stained
green) surrounded by a small quantity of rose-coloured protoplasm. (2.)
Larger cells with rose-red protoplasm. (3.) Granular cells which seem to
correspond to eosinophilous cells. (4.) Cells undergoing degeneration ; and
(5.) Phagocytes.

LESSON XXVI.
LIVER.

IT is composed of a large number of lobules (i mm. (^ inch) in
diameter), held together by a greater or less amount of connective
tissue. Each lobule practically resembles its neighbour, and is
composed of a mass of polyhedral or cubical liver-cells which have
in relation with them blood-vessels and bile-ducts. The liver is
covered by a capsule, which sends processes into the organ at the
portal fissure, forming Glisson's capsule, which lies between the
lobules in the portal canals, and surrounds the portal vein, bile-duct,
and hepatic artery. The liver is supplied with blood by the vena
portse and hepatic artery ; they enter at the portal fissure, and the
former divides into branches v/hich ramify between the lobules,
constituting the interlobular veins. From these veins capillaries
pass into and traverse the substance of the lobules, and converge to
a veinlot in the centre of each lobule — the central or intralobular
veins or rootlets of the hepatic vein, which form sublobular veins,
and these in their turn form the hepatic vein which carries the
blood from the liver to the inferior vena cava. The substance of
each lobule between the capillaries is composed of liver-cells (20 /*,
Yo-Vtf inch in diameter), which form anastomosing columns, being
more radiate next the centre of the lobule. Between the liver-cells,

1 Quart. Jour. Micr. Sci., xxx. p. 481.

2 Hoyer, Archivf, mik. Aiiat., xxxiv., 1889.

286 PRACTICAL HISTOLOGY. [XXVI.

but always separated from the blood-capillaries by hepatic cells or
part of a cell, is a fine polygonal plexus of channels — the bile
capillaries, which become continuous with interlobular bile-ducts
at the margin of the lobule. The smaller interlobular bile-ducts
unite to form larger bile-ducts, which are lined by columnar epi-
thelium, and in the walls of the largest of them are mucous glands.
The hepatic artery supplies chiefly the connective tissue between
the lobules, and accompanies the branches of the bile-duct and
portal veins, so that these three structures lie together in portal
canals.

Methods. — Harden portions of the liver of a, e.g., pig, rabbit, cat,
and man in Miiller's fluid or 2 per cent, potassic bichromate (10-14
days). If it be desired to retain the chromatin fibrils in the liver-
cells avoid the chromic acid salts. Use sublimate, and then
gradually increasing strengths of alcohol. Cut sections parallel to the
surface of the organ, and others at right angles to it, the latter to
include the capsule. The sections can be stained in haematoxylin
and mounted in balsam ; or picro-carmine, or picro-lithium carmine,

and Farrant's solution
may be used. Staining
in bulk and cutting in
paraffin is also good.

If unstained sections
are mounted, the out-
lines of the tissues will
be much better defined
if they be soaked in i
per cent, osmic acid (24
hours) previous to being
mounted in Farrant's
solution.

1. Liver of Pig
275.— Liver of Pig, showing Lobules. P.O. Portal (Hcematoxylin and .Ba/-

canal, containing Me-duct, hepatic artery, and por- sam or PicrolitUum
tal vein (P. V.) • S. Septa ; S. V. Sublobular vein ; /. V . .

intraiobuiar vein. Carmine, or Methylene-

Blue).

(a.) (L) Observe the polygonal lobules (fig. 275) mapped out from
each other by a network of septa of connective tissue, or Glisson's
capsule (fig. 275, S). In the centre of each lobule a small thin-
walled vein, a rootlet of the intraiobuiar or hepatic vein
(I.V.).

(b.) At the periphery of each lobule sections of branches of the
portal or interlobular vein ; if possible, find a transverse section of
the latter, and it will be found to be accompanied by similar sec-
tions of the bile-duct (one or more) and hepatic artery (fig. 277,

XXVI.]

LIVER.

28;

P.V.). Branches of all three structures always run together in the
portal canals, and are surrounded by Glisson's capsule.

(c.) Capillaries — for the most part empty — running from without
inwards in each lobule. Radiating from the hepatic veinlet columns
of liver-cells, forming a network of secretory cells intertwining with
the capillary plexus within the lobule.

(d.) (H) The columns of hepatic cells radiating from the hepatic
veinlet, composed of polygonal or cubical cells, with granular con-
tents and an excentrically-placed spherical nucleus. At the outer
part of the lobule the network of cells is more polygonal, correspond-
ing to the arrangement of the capillaries. Between the columns of
cells, capillaries, sometimes with a few blood-corpuscles. The
however, are not as distended as normal. Transverse

FIG. 276. — Columns of Liver-Cells
from a Starving Dog.

FIG. 277.— T.S. Portal Canal with Glisson's Capsule
enclosing Portal Vein (P.V.), Bile-duct (B.D.), and
Hepatic Artery (H.A.). The slit is a lymphatic.
C. Capillaries. I.e. Liver-cells.

branches running between adjoining capillaries, and the whole
interwoven with the cellular network.

In a hsematoxylin-stained section, note that the nuclei of the
liver-cells are stained to a less degree than the other nuclei. The
nuclei of the cells of the connective-tissue, bile-ducts, and capillaries
are more deeply stained. In the picro-lithium preparations, the
cells are yellowish with nuclei red, but of different shades of the
same.

With the low power find a portal canal with its contents ; fix
them under the microscope and examine with (H).

(e.) One or more sections of interlobular bile-ducts, lined by a
single layer of shorter or taller columnar cells; outside them con-
nective tissue, disposed circularly and continuous with that of
Glisson's capsule (fig. 277). A section of a branch of the hepatic
artery and — the largest opening of all — of the portal vein. Some
slits may be seen in Glisson's capsule ; they are lymphatics.

288 PRACTICAL HISTOLOGY. [XXVI.

2. Liver of Rabbit. — It is convenient to study this liver, because
in some respects it shows a transition between that of the pig and
man, as it occupies an intermediate place with regard to the demar-
cation of its lobules. In it the lobules are not nearly so well
defined by connective tissue as in the pig or camel, while their
separation from each other is more definite than in man.

3. Human Liver. — Hsematoxylin, balsam.

(L) Observe the greater fusion of the lobules. Practically, the
arrangements of blood-vessels and cells in other respects is the same
as in the rabbit's liver (fig. 278). If the liver is anaemic, the intra-

Intralobular
vein.

Bile ducts.

Interlobular
veins.

FlG. 278. — Section of Human Liver, showing Liver Lobules and the Radiate Arrange-
ment of the Hepatic Cells from the Centre of each Lobule, x 20.

lobular blood-capillaries are narrow, and the liver-cells appear to
compress and narrow them.

4. Liver of Frog or Newt. — Harden a small piece in absolute
alcohol or J per cent, osmic acid (24 hours). Stain the mass "in
bulk" in Kleinenberg's logwood or borax-carmine, and mount in
balsam. Mount the osmic acid sections in Farrant's solution with-
out staining, or stain them in safranin (48 hours).

(a.) (L) Observe the anastomosing system of gland-tubes made
up of hepatic cells. Between them the narrower blood-capillaries,
many of them filled with blood-corpuscles. Here and there black
patches of pigment — melanin — especially in winter frogs (fig. 279).

(/;.) (H) Each cell is polygonal, with a large spherical nucleus ;
the contents may be more or less granular, according to the phase

XXVI.]

LIVER.

28g

FIG. 279.— Liver of Frog. C. Capillaries.
Osniic acid, x 300.

of secretory activity of the cells. In some conditions the granules
within the cells may be arranged next the capillary ; in others they
are more regularly scattered throughout the cell substance. Tha
cells are arranged round the bile-capillaries. This is best seen in
transverse sections of the latter, which appear as very small circular
apertures bounded by four or five cells (tig. 279). When the tubes
are cut longitudinally, the bile
capillaries are seen to pursue a
zigzag course between the cells,
but this will be better seen in
injected specimens.

('•.) The blood-capillaries with
their nucleated blood-corpuscles,
and note that there is always a
cell or part of a cell between
the blood-stream with its wide
lumen, and the bile-channels
with very narrow lumina. Any
fatty granules present in the
osmic acid section are black.

5. Blood- Vessels of the

Liver — Opaque Injections. — Mount in balsam a section of a pig's
liver, with the P.V. injected with a red opaque mass, and the H.V.
with a similar yellow mass. The light from the reflector must be
turned off, and light focussed on the preparation by a condenser
(fig. 1 6).

(a.) (L) Observe the polygonal lobules, the branches of the
portal veins around the periphery of the lobules, and sending fine
branches into the latter. In the centre of the lobules the hepatic
veinlets yellow, with capillaries converging to them. The capil-
laries within the lobules partly filled with red and partly with
yellow mass, connecting the portal and hepatic venous systems.

(I>.) Sometimes a longitudinal section of a lobule may be seen.
Trace the veinlet to a larger branch under the lobule, i.e., to a
sublobular vein. The sublobular veinlets lie between the lobules,
but they differ from branches of the portal vein in not being accom-
panied by a branch of the hepatic artery and bile-duct.

6. Transparent Injections— Liver of Pig.— Mount in the same
way a transparent injection of the liver of a pig. P.Y. blue, and
II. V. red.

(".) (L) The hepatic veinlet in the centre of the lobule. If cut
transversely, it is circular ; if obliquely, oval ; and if the lobule be
cut longitudinally, it appears as a central channel joining a sub-
lobular vein. Trace outwards from this a radially-arranged capillary
network, with its cross-branches, right out to the outer part of each
26 T

290

PRACTICAL HISTOLOGY.

[XXVI.

lobule. Notice that the shape of the mesh work of capillaries is
different at the centre and periphery. Between the lobules and
outside each lobule branches of the portal vein.

7. Liver of Rabbit, Injected (fig. 280), e.g., a section with its
blood-vessels injected with different colours, e.g., P.V. red (carmine
gelatine) and H.Y. blue (Berlin-blue gelatine). Also a section
where all the blood-vessels are injected with a mass of one colour.
In a double injection the red and blue masses do not always occupy
the area corresponding to the blood-vessel into which they were
injected, but with care such a piece can be found in an injected
liver. The arrangement of the blood-vessels corresponds to that
seen in the uninjected specimens, only in the former the blood-
vessels are more prominent than the cells.

(a.) (L) Observe the in terlobular* veins round the periphery of
the lobule, the central or intralobular vein, and the plexus of

Fibrous
wall.

FIG 281.— .uiterlobular Bile-Duct.

FIG. 280.— Injected Blood- Vessels of a Lobule
of a Rabbit's Liver.

capillaries connecting the two. The capillaries converge towards
the centre of the lobule, where the meshes are more elongated.
At the periphery of the lobule the capillary mesh work is more
polygonal.

Very instructive also are those with only one set of vessels
injected, e.g., portal or hepatic. They serve to show the capillaries
belonging to each area within a lobule.

8. Injected and Stained Section. — A beautiful preparation is
obtained by staining a section (blue injection) with picro-lithium
carmine. In it the vessels are blue, the cells yellow, and the nuclei
of the latter red.

9. Bile-Ducts. — Mount in balsam a section of a rabbit's, or, better
still, a guinea-pig's Iryer in which the bile-ducts have been injected
with a concentrated watery solution of Berlin-blue. It is very

XXVI.]

LIVER.

29I

difficult to get a perfect injection, but it is easy in the guinea-pig to
inject the large bile-ducts. They are injected from the common
bile-duct after ligature of the cystic duct. Sometimes it is better
not to clamp the cystic duct, as after the gall-bladder is full it acts
as a reservoir, and prevents too great pressure, causing extravasations
of the injection fluid.

(a.) (L) Large Bile-Ducts. — Numerous sections of these lying in
portal canals between the lobules. Each is provided with a fibrous
coat containing circular, smooth, muscular fibres, lined by columnar
epithelium. In the walls
of the largest ducts are
mucous glands, which
open into the bile-duct.

(?>.) (H) The bile-
capillaries within the
lobules appear as a fine
hexagonal network of ,

blue lines between the "* — »«B»MBMBMfl«MW» *«
surfaces of the hepatic
cells (fig. 282). If cut
transversely, they appear
as mere blue specks
between adjacent cells.
They are not to be con-
founded with the blood-
capillaries, which are
much wider, and are
arranged in a different
way.

(c.) The columnar epi-
thelium lining the interlobular bile-ducts and their fibrous walls
(fig. 281).

10. Auto-Injection of Bile-Ducts. — Place a piece of indigo-
carmine, about the size of a split pea, under the skin of the fore-
arm of a pithed (brain-destroyed) frog. Tie the slit to prevent its
escape. After twenty-four hours the whole frog will appear quite
blue. Kill it, rapidly remove the liver, cut it in small pieces, and
place it at once in absolute alcohol, which fixes the blue colour.
After it is hardened, cut sections and mount in balsam.

(a.) (H) The blood-vessels are yellow, with gland-tubes between,
but the bile-capillaries are bine. They can be seen as blue zigzag
fine streaks between the cells when the tubes are cut longitudinally,
and as very small dots when cut transversely. A thin section gives
a clear view of the relation of blood-capillaries, cells, and bile-
capillaries.

-B.C.

FIG. 282.— Lobule of Rabbit's Liver. Vessels aixt bile-
ducts injected. P.V. Portal, and H.V. Hepatic
vein ; B.D. Bile-duct, and B.C. Bile-capillaries.

292 PRACTICAL HISTOLOGY. [XXVI.

A good plan is to clarify an unstained section with clove-oil con-
taining eosin. The cells are stained red, and form a sharp contrast
to the blue. A watery solution such as picro-carmine cannot be
used to stain the cells, as it rapidly extracts the blue from the bile-
channels.

11. Fresh Liver-Cells. — Scrape the surface of a fresh liver, and
.observe in normal saline.

(H) Observe the pale, nucleated, polygonal, or cubical faintly-
granular cells, of ten containing small refractive oil globules. Often
the cells are broken up. There are always many blood-corpuscles
in the field (fig. 94).

ADDITIONAL EXERCISES.

- 12. Connective-Tissue Stroma. — With a camel's-hair brush pencil away as
many as possible of the hepatic cells from a thin section of any properly
hardened liver. The connective-tissue network may be afterwards stained
with picro-lithium carmine or eosin.

13. Intralobular Connective Tissue. — Various methods have been adopted
to show the existence of connective tissue within the liver lobules and between
its cells. The following methods reveal a network of black fibrils continuous
with the interlobular tissue, and traversing as a network the substance of the
lobule. This method is also useful for showing the trabeculse of the spleen,
and the fibrillar structure in the splenic corpuscles and lymph-glands generally.

(a.) Bohm's Method.

(1. ) Harden small pieces (.5 cm. cube) in 0.5 per cent, chromic acid (2

days).

(2.) Silver nitrate .75 per cent. (3 days).
(3.) Wash in distilled water, harden, cut, and mount in balsam.

(&.) Oppel's Method is available for liver hardened in alcohol.

(1.) Yellow chromate of potash 10 per cent, (i day).

(2.) Silver nitrate, large volume, .75 per cent. (2 days). Much silver

chromate is formed, so that the silver must be frequently

renewed.
(3.) Wash in water, harden in alcohol, sections, balsam.

By either method the liver cuts readily on freezing, but hand sections, I
think, are best. Paraffin embedding is not to be recommended.

14. Bile-Capillaries (Bohm). Golgi's method.

(1.) Harden (3-4 days) small pieces of the liver (.5 cm. cube) in

Bichromate potash solution (3 per cent.) . 4 vols.
Osmic acid (i per cent.) .... I vol.

(2.) Place in 0.75 per cent, silver nitrate (2 days).
(3.) Wash in distilled water, harden in alcohol.
(4.) Sections. Balsam.

XXVI.] LIVER. 293

The bile-capillaries appear as a black polygonal network on a yellow ground.
It is rare, I find, to have the whole thickness of the tissue equally well
stained. Only a thin layer on the surface shows the capillaries well, but the
result is excellent. I find that the sections keep for a long time if they are
mounted under a cover-glass.

15. Glycogen in Liver-Cells.— (a.) The animal must be well fed, e.g., rabbit,
with carrots, and six hours or so thereafter it is killed. As glycogen is soluble
in, or is at least extracted from, the liver by water, small pieces of the liver
must be hardened in alcohol. In a section placed in a weak iodine solution
or Lugol's solution (p. 93), the glycogen granules in the cell protoplasm
are stained of a port-wine colour. In sections of liver sometimes one sees
vacuoles from which the glycogen granules have been washed out.

(b. ) Harden the liver of a well-fed frog in osmic acid, make thin sections,
and irrigate with iodine. The granules of glycogen in the hepatic protoplasm
are stained brownish.

16. Reactionfor Iron( Tizzoni's). — («.) Select the liver of a young animal, and
harden it in alcohol. Place sections in the following fluid, which should be
freshly prepared •:—

Water 90 cc.

aer

Hydrochloric acid (25 per cent.)
Ferricyanide of potash (i : 12)

Mount in balsam. Particles of free iron are coloured blue. Particles of free
iron are seen in the spleen, liver, and kidney by this reaction.

(b.) Zaleski.1 — Harden the liver in 65 per cent, alcohol, then in 96 per cent,
alcohol to which a few drops of sulphuretted hydrogen ara added. After 24
hours the iron granules assume a green colour.

17. Injected Human Liver.— (a.) For a double injection, and in order to
save injection-mass, the plan recommended by Orth is excellent, viz., to pass
an elastic catheter as far as it will go into one of the branches of the portal
vein, and through it to make the injection. The hepatic vein is treated in
the same way. Sections of a well-injected part may be afterwards stained
with picro-lithium-carmine.

(?>.) In a way a natural injection of the liver is obtained by hardening a
human liver which is congested, and contains a large amount of blood. Such
livers are apt to show pigmentation of the liver-cells and other changes due to
disease.

18. Pigment in Liver.— The presence of pigment in the liver-cells or capil-
laries is a matter of considerable importance in regard to the question of the
destruction of blood-pigment in this organ, or whether the liver acts as a trap
for pigment already altered by its passage through the spleen or gastro-
mesenteric capillaries.2

In winter frogs the liver contains a large amount of black pigment, which
lies in the blood-capillaries.

19. Granular Cells of Ehrlich.— Harden the liver (ox, pig) in absolute
alcohol. Place sections for 24 hours in Westphal's fluid (p. 67), wash in
absolute alcohol (4-6 hours) until all is clear, only the granular cells remaining
stained. Balsam. The granular cells ( ' ; Mastzellen ") are reddish violet,
and lie in the interlobular connective tissue.

1 Zcitsch./. Phys. Chem., xiv. p. 274, 1890.

2 W. Hunter, Brit. Mcd. Jour., Nov., Dec. 1892.

2 §4 PRACTICAL HISTOLOGY. [XXVII.

LESSON XXVII.
TRACHEA— LUNGS— THYROID GLAND.

THE trachea, a fibro-muscular tube, the wall of which contains
16-20 C-shaped pieces of hyaline cartilage held together by a
fibrous membrane. Behind, the rings are deficient, and the trachea
is membranous, and there it is strengthened by smooth muscle —
tracJiealis muscle — which stretches between the ends of the carti-
lages. It is lined by a mucous membrane, which is united to the
outer fibrous coat by a submucous coat. The mucous coat consists
from within outwards of — (i.) Stratified, columnar, ciliated epithe-
lium; (2.) Basement membrane; (3.) A basis of connective tissue
with capillaries, and infiltrated with adenoid tissue ; (4.) A layer of
elastic fibres arranged longitudinally. Outside this is a loose sub-
mucous coat, in which lie the acini of the glands.

The intra-pulmonary bronchi are lined by stratified ciliated epi-
thelium resting on a basement membrane. Outside this is a basis
of fibrous tissue, with numerous longitudinally-arranged elastic
fibres, and some adenoid tissue. Outside this, again, is a com-
pletely circular layer of smooth muscle — bronchial muscle. Then
follows the submucous coat with its vessels, glands, and in some
animals (cat) masses of adenoid tissue. Most externally is the
fibrous coat, in which are embedded several pieces of hyaline
cartilage of irregular shape. As the bronchi pass into the lung,
they divide and form smaller and smaller tubes, until they end
in terminal bronchi or bronchioles. Each bronchiole, with thin
walls, no glands or cartilage, and the epithelium cubical and non-
ciliated, opens into several wider expanded parts — infundibula or
alveolar passages — which are beset with air-cells or alveoli. The
alveoli are spherical or polygonal vesicles, which open by a wide
opening into the infundibula ; the air-vesicles, however, do not open
into each other. The air-cells are lined by a layer of squames —
large, flattened, irregular plates — with small granular cells here and
there between them.

Blood- Vessels. — The branches of the pulmonary artery accom-
pany the bronchial tubes, and finally terminate in a rich capillary
plexus over and outside the basement membrane of the air-vesicles.
The blood is returned by the pulmonary veins. The bronchial
vessels seem to supply chiefly the connective tissue along the
bronchi and in the septa. Numerous ganglia exist in the intra-
pulmonary nerves. Lymphatics are numerous.

XXVII.]

THE RESPIRATORY ORGANS.

29$

THE RESPIRATORY ORGANS.

Methods.— (i.) Place small portions of the trachea of a cut and the
human trachea in .2 per cent, chromic acid (10-14 days) or sub-
limate, and harden afterwards in gradually increasing strengths of
alcohol.

(ii.) Fill the lungs of a cat with .2 per cent, chromic acid, and
after closing the trachea suspend them in a large quantity of the
same fluid. After two days cut them into small portions and keep
them (14-20 days) in fresh chromic acid solution and complete the
hardening in alcohol.

(iii.) Harden small parts of the human lung — as fresh as possible
— in the same fluids.

(iv.) Fix: the trachea in Flemming's fluid, harden in alcohol, and
stain with safranin. Admirable for epithelium.

Trachea. — Make transverse sections across the trachea of a cat,
and also longitudinal vertical sections, so as to include the cartilage
of two or three rings. This
is best done by staining in
•bulk and cutting in paraffin.
If done by freezing, stain
T.S. and L.S. sections in
hsematoxylin, and mount in
balsam ; others in picro-car-
mine, and mount in Farrant's
solution.

1. T.S. Trachea (fig. 283).

(a.) (L) Observe internally
the mucous coat, outside it
the submucous coat, and
external to this an incom-
plete ring of hyaline cartilage
embedded in the outer or
fibrous coat. The fibrous coat becomes continuous with the sub-
mucous coat.

(h.) (H) The fibrous coat of connective tissue, and embedded
in it an incomplete ring of hyaline cartilage. Notice the arrange-
ment of the cartilage cells in the latter. The ring is deficient
posteriorly, but bridging ovef the gap and extending between the
ends of the cartilage there is a transverse band of smooth muscle,
the frachealis muscle. Outside it are muscular fibres cut trans-
versely. The coats just inside the muscle are apt to be thrown
into folds in a trachea detached from all its surroundings. Sections
of nerves — perhaps with ganglionic cells — may be seen near the
muscle.

83__TA ^^ ot KiUen> x 15.

295

PRACTICAL HISTOLOGY.

[XXVII.

(c.) The submucous coat, composed of more open connective
tissue, and continuous with the former. In it are the acini of

mucous glands ; but as
the glands are more
abundant in the spaces
between the cartilages,
their distribution is
better seen in a longi-
tudinal section. It is
rare to find a duct, as
they pierce the mucous
coat obliquely, and
open on its inner sur-
face.

(d.) The mucous
coat, composed of
fibrous tissue covered

FIG. 284. — L.S. Mucous Membrane of Trachea of Cat.

by stratified ciliated epithelium. Under the epithelium is a longi-
tudinal layer of elastic fibres, which are therefore cut across trans-
versely. Within this, a basis of connective tissue infiltrated with
adenoid tissue, and internal to this again a structureless basement

Bronchia! Muse/e.

Bronchia/ flrfery.

Garfitcge

Gfaiicf acini &. c/ucf:

FlG. 285.— T.S. Intra-Pulmonary Bronchus of Cat. PA and PV. Pulmonary artery and
vein ; bv. Bronchial vein; V. Air-vesicles.

membrane, best seen in very thin sections, and best of all in a
human trachea. Besting on the basement membrane is the ciliated
epithelium. It occurs in several layers, but only the superficial
layer of cells is ciliated. In the lower layers of cells — three or four

A XVI I.]

THE RESPIRATORY ORGANS.

297

layers — -some are pear-shaped and the lowest more oval. Very
frequently a thin layer of mucus is adherent to the cilia. The
arrangement of the cells of the mucous layer is the same as in the
bronchus (fig. 286).

2. L.S. Trachea (fig. 284) (L and H).— Compare these with the
previous section. The coats are the same, but several oval pieces
of hyaline cartilage are seen one after the other embedded in the
fibrous coat. The elastic fibres are now seen arranged longitudi-
nally. The acini of the mucous glands are most numerous in the
interspaces between the cartilages, and amongst the acini may be
seen leucocytes and some adenoid tissue. If a gland-duct be found,
it opens by a funnel-shaped expansion on the free surface of the
mucous membrane. If it be desired to study the glands, use
hcematoxylin and eosin as stains.

3. T.S. Human Trachea (L and H). — Observe the general
similarity to the previous prepara-
tion. Here, however, the glands

are well-developed ; the basement
membrane is well marked, and in
picro-oarmine specimens is stained
red. Some of the superficial epi-
thelial cells are apt to be detached.

4. T.S. Intra-Pulmonary Bron-
chus.— (a.) (L) Observe the fibrous
coat, and outside it the vesicular
tissue of the lung. In the fibrous
coat — two or three — pieces of
hyaline cartilage. The submucous
coat, with its glands (fig. 285).

(b.) Inside this a complete ring
of smooth muscle — bronchial muscle
— perforated here and there by the
ducts Of .the glands. Immediately Fw
inside this, in the mucous coat,
several layers of longitudinal elastic
fibres cut transversely. Most in-
ternal, ciliated epithelium, like
that lining the trachea, and resting

on a basement membrane. The mucous membrane is frequently
thrown into ridges or folds.

(c.) In the fibrous coat, external to the cartilages, search for
sections of two large vessels — the pulmonary artery and vein — and
of small branches — the bronchial vessels. Also several sections of
nerves; in the course of some of them may be found ganglionic
cells.

27

Membrane of
Human Bronchus, a. Mucus ; b.
Ciliated cells ; c. Deep cells; d. Base-
ment membrane ; e. Longitudinal
elastic fibres ; /. Bronchial muscle ;
g. Connective tissue, with leucocytes
and pigment.

298

PRACTICAL HISTOLOGY.

[XXVII.

(d.) (H) Study specially the mucous membrane. Note the
epithelium, basement membrane, and the longitudinal layer of
elastic fibres under it (fig. 2 86).

5. Vesicular Structure of the Lungs. — Make sections of a
lung hardened by the freezing method. Let the sections be cut so
as to include the pleura and subjacent lung. Stain in haematoxylin,
and mount in balsam.

(a.) (L) Observe the pleura, with its two layers, the deeper

Fro. 287.— V.S. Human Luuff. p. Pleura; a. Epithelium of a bronchus ; b. Blood-vessel;
c. Pulmonary vein ; *. Interlobular septum, continuous with deep layer of pleura;
v. Air-vesicles ( x 50 and reduced |).

layer sending fine septa — interlobular septa — into the lung between
its alveoli (fig. 287).

(b.) The alveoli or air-cells cut in every direction; some appear
as an open network, and others with a base. The outline of the
alveoli may be somewhat irregular, according to the extent of dis-
tension of the lung. Here and there, between groups of alveoli,
may be seen a wide passage — the infundibulum.

XXVII ]

THE RESPIRATORY ORGANS.

299

(c.) (H) In each alveolus stained oval nuclei, belonging to the
squamous epithelium lining it, but the outline of the squames them-
selves cannot be seen. Sections of the capillaries on the wall of the
alveolus and their nuclei stained.
In the thin walls, separating ad-
joining alveoli, fine elastic fibres,

6. Respiratory Epithelium
of the Alveoli. — (i.) Remove
the lungs from a young kitten,
and fill them with a \ per cent,
solution of silver nitrate. Run
in the fluid from a pipette pro-
vided with a bulb on its stem.
Pump the lungs from time to
time to remove as much air as
possible. After half an hour
replace as much as possible of
the silver solution by means of
alcohol. Tie the trachea, and
suspend the lungs in alcohol till
they are hardened. Cut sections
by freezing, mount in Farrant's
solution, and expose the sections

to light. They become brown. Fia
Sections can be stained in
hsematoxylin or picro-oarmine.

(ii.) Fill the lungs with .25 per cent. AgN03, and suspend them
in .5 per cent. AgN03 (1-2 hours). Diffusion takes place. Cut
into pieces and place in alcohol (80 per cent.).

(H) Select an alveolus which is so divided as to have a base, and
observe the silver lines showing the boundary-lines of the squamous
epithelium which lines it. At the junction-points of some of these
are groups of two or three small, polyhedral, granular cells stained
deep brown (fig. 288).

7. Blood-Vessels of a Mammalian
Lung. — Mount in balsam sections of an
injected lung.

(L and H) Observe the alveoli, each
beset with a dense plexus of capillaries
(fig. 289), and especially where the edge
of an alveolus is seen, the wavy course
of the capillaries passing from one side
to the other of the inter-vesicular septa.
Search for the termination of a branch of
the pulmonary artery and the commencement of the pulmonary vein.

288. — Alveoli of Lung of Kitten,
Silvered, a, b. Squames ; d. Granular
cells ; c. Young epithelium-cell ; e.
Alveolar wall.

FlG. 289. — Capillaries of Human
Lung, Injected, x 90.

300

PRACTICAL HISTOLOGY.

[XXVII.

8. Fresh Lung. — Tease a small piece of lung in normal saline.
It is difficult to get rid of all the air, but this may be done by
beating the tissue with a needle.

(H) Observe the large number of fine elastic fibres, which
branch and anastomose. They can be rendered more evident by
running in dilute caustic potash (2 percent.) under the cover. This

destroys to a large extent the other
elements.

9. Dried Lung. — (i.) With a dry
razor make thin sections of a dried
and distended lung. Examine the
section in water, taking care that
it does not curl up, which it readily
does. Get rid of the air-bubbles by
pressing on the section with a needle,
(ii.) Make rather thick sections
parallel to the pleura, and examine
them by reflected light. A very good
idea of the air-vesicles is thus ob-
tained.

(a.) (L) Observe the air-vesicles,
and the thin partitions between them. Also sections of the
infundibula or alveolar passages. Connective-tissue septa may
be seen. Of course the finer details of structure cannot be made
out (fig. 290).

10. Foetal Lung. — This serves very well for showing that a lung

FIG. 290.— T.S. Dried Lung. a. Vesi-
cles ; 1. Infundibula.

FIG. 291.— T.S. Foetal Lung, showing a Bronchus Terminating in Air- Vesicles, x 75
B. Epithelium lining an alveolus, x 300. Miiller's fluid and hwmatoxylin.

is made up of lobules. Harden in Miiller's fluid (14 days) the
lungs from a human foetus (12 cm. in length). Cut sections and

XXVII.]

THYROID GLAND.

301

stain them either in hasmatoxylin or picro-carmine. Stain in bulk
and cut in paraffin.

(«.) (L) Observe the pleura sending well-marked septa into the
lung, thus denning the lobules, which are polygonal, about i mm.
in diameter, and separated from each other by connective tissue.

(/;.) In each lobule sections of bronchi, which can be seen
occasionally to terminate in several vesicles, thus presenting a very
gland-like arrangement (fig. 291). Many of the alveoli are cut
across, and appear like sections of tubes lined by columnar epithe-
lium. There is much embryonic connective tissue between the
alveoli.

(H) Select an alveolus, and note that it is lined by a layer of
low, columnar, granular epithelium, while individual alveoli are
separated from each other by much embryonic connective tissue,
with numerous cells, and as yet few or no elastic fibres.

THE THYROID GLAND.

Methods. — (i.) Harden pieces of the human thyroid, or the
complete thyroid of a cat or dog, in M tiller's fluid (3 weeks) and
then in alcohol. Sections are stained with hsematoxylin and
mounted in balsam.

Or stain in bulk and ^ - . -V^ • • • w >'

cut in paraffin.

(ii.) Fix in Flem-
ming's fluid (1-3
hours), and stain —
wash thoroughly,
alcohol — with Ehr-
lich-Biondi fluid, or
stain with Heiden-
hain's logwood.

11. T.S. Thyroid
Gland. —(«.) (L)
Composed of small
polygonal lobules
united to each other
by loose connective
tissue. In each lob-
ule a iarije number

FIG. 292.— T.S. Thyroid Gland, a. Closed vesicle.

of completely closed acini held together by loose connective tissue.
(/;.) (H) The spherical acini (tig. 292), bounded by a basement
membrane, are lined by a single layer of low cubical epithelium, and
contain a homogeneous fluid. This fluid, however, is often of a

3O2 PRACTICAL HISTOLOGY. [xxvu.

colloid nature, and is then a pathological product. It is stained hy
logwood. Numerous sections of blood- and lymph-vessels outside
the basement membrane of the acini.

12. Injected Thyroid (thickish section in balsam). — (L) Numer-
ous large vessels in the connective tissue, with a plexus of capil-
laries over the acini, but outside their basement membrane.

ADDITIONAL EXERCISES.

13. Lung of Newt. — (i.) This is an elongated sac, and is of comparatively
simple structure. Fill a lung with gold chloride (.5 per cent.), and suspend
it in a few cc. of the same Huid for twenty minutes. Reduce the gold by
exposure to sunlight in water feebly acidulated with acetic acid. Mount a
portion of the thin wall in glycerine.

(a.) (L) Observe islands or small groups of epithelial cells. They lie in the
intercapillary spaces. The capillaries are wide, and form an anastomosing
network. Outside this capillary layer is a layer of smooth muscle, and one of
fibrous tissue.

(ii.) If the gold chloride be reduced by formic acid (25 per cent, in the dark),
the epithelium lining the lung is shed, and then the nerves to the lung — with
many ganglia in their course — can be seen.

14. Lung of Frog. — (i.) Fill a lung with dilute alcohol, suspend it in the
same fluid (twenty-four hours), lay open the lung and pencil away the inner
lining epithelium and mount in Farrant's solution.

(a.) (L.) Observe the large, coarse, but short primary septa, which project
inwards from the wall of the lung towards the large central cavity. From
them secondary septa pass to form a trabecular arrangement, thus giving the
interior of the lung a honeycomb-looking appearance. The trabeciilae consist
chiefly of smooth muscle.

(ii.) The nerves of the frog's lung are readily demonstrated by the gold
chloride formic acid method and the methylene-blue method. The numerous
ganglionic cells in the course of the nerves have a straight and a spiral
process.

15. Elastic Fibres in Trachea and Lung.— (i.) Stain a longitudinal section
of the hardened human trachea according to the method described in Lesson
X. 10. The elastic fibres become black.

(ii.) Use the safranin method (Lesson X. 9). This shows beautifully the
arrangement of the fibres, now of a purplish or black tint.

16. Elastic Fibres in the Lung. — (i.) I have devised the following two
methods, which give good results: — Make sections of a dried and distended
lung, stain, a section in dilute magenta, and allow the section to dry com-
pletely on a slide ; add balsam and cover. The elastic fibres are red, and their
arrangement can be seen with the utmost distinctness.

(ii. ) Or. stain a section in methyl-violet, and clarify it wit i the aniline-oil
and xylol mixture (p. 123).

XXVIII.]

KIDNEY,

303

Cortex.

LESSON XXVIII.

KIDNEY— URETER— BLADDER
KIDNEY.

IF a kidney be divided longitudinally, one distinguishes a cortical
and a medullary part, the latter consisting in different animals of
one or more pyramidal

6

portions — the pyramids
of Malpighi — whose
apices project into the
pelvis of the kidney,
while their bases are
surrounded by cortical
substances. The medul-
lary portion is subdi-
vided into the boun-
dary or intermediate
zone and the papillary
portion (iig. 293). The
kidney is covered by a
loosely adherent cap-
sule. .It is a compound
tubular gland, consist-
ing of numerous urini-
ferous tubules closely
packed together with
very little connective
tissue between them,
the connective tissue
carrying the blood-ves-
sels, lymphatics, and
nerves. The urini -
ferous tubules pursue a
straight course in the
medulla, but they ex-
hibit a contorted or
convoluted arrangement
straight tubules — the

Boundary or
Omarginal

zone.

Fid. zy i.— L.S. of n Pyramid of MalplgbL /'/•'. Pyramids
of Kerrein ; HA. Branch of renal artery with an in-
terlobular artery; R\'. Lumen of a renal vein re-
ceiving an interlubular vein ; VR. Vasu recta; PA.
Apex of a renal papilla; l>b. Embrace the bases of
the lobules.

in the cortex, although bundles of
medullary rays or pyramids of Ferrein
pass into the cortex from the medulla (fig. 293). Each uriniferous

3°4

PRACTICAL HISTOLOGY.

[XXVIII.

tubule consists of a basement membrane lined by a single layer of
epithelium. The tubules alter their character and course in dif-
ferent parts of the organ. The tubules begin in the cortex in a

Sub-cap-

sular layer.

12. Junc-
tional tu-
bule.

ii. Convolu-
ted tubule.
10. Irregu-
lar tubule.

A. CORTEX.

3. Proximal
convoluted
tubule.

p. Ascend-
ing tubule.
2. Neck.

4. Spiral
".ubule.

* ^ i. Capsule

and glome-
rulus.

7 and 8. Ascending
part of Henle's loop.

5. Descending limb
of Heule's tubule.

6. Henle's loop.

15. Discharging
tubule.

C. PAPILLAKY ZONK.
FIG. 294.— Diagram of the Course of Two T'rmiferous Tubules.

globular dilatation — the Malpighian capsule — which encloses a
tuft of vessels— the glomerulus (fig. 294). The capsule leads into
a narrow neck, which passes into the first or proximal convoluted

XXVIII.]

KIDNEY.

305

tubule, which in turn is continued into the spiral tubule. After
this it narrows suddenly and runs into the medulla as the descend-
ing tubule of Henle, where it forms a narrow loop — loop of Henle —
and passes in the reverse direction towards the cortex as the ascend-
ing tubule of Henle. In the cortex it has a zigzag course — irregular
tubule — and again becomes wider and convoluted — second or distal
convoluted tubule — which leads into a straight tube— junctional
tubule — which joins a straight or collecting tubule. This passes

Tabular View (after Schdfer) of tie Parts, Situation and Epithelium
of a Uriniferous Tubule.

Portion of Tubule.

Kind of Epithelium (always in a
single layer).

Situation of Tubule.

I. Capsule . . .

Flattened, reflected over

Labyrinth of cortex.

glomerulus

2. First convoluted

Cubical, rodded cells, which

» ) i

tube

interlock

3. Spiral tube . .

Like last, but "rods "very

Medullary ray of

distinct

cortex.

4. Descending limb
of Henle's tube

Clear flattened cells

Boundary zone and
partly in papillary

zone.

5. Loop of Henle .

» »

Papillary zone of

medulla.

6. Ascending tube

Cubical, rodded, sometimes

Medulla and medul-

of Henle

imbricated

lary ray of cortex.

7. Irregular zigzag

Cells cubical, very "rodded,"

Labyrinth of cortex.

tube

lumen small

8. Second convoluted

Like those of (2), but longer

»> »

tube

and more refractive

9. Junctional tube .

Clear, flattened, cubical cells

Labyrinth passing to

medullary ray.

10. Straight or col-

Clear cubical and columnar

Medullary ray and

lecting ttibe

cells

medulla.

ii. Duct of Bellini .

Clear columnar cells

Opens at apex of

papilla.

straight through the cortex and medulla, receiving other similar
tubes as it goes; and becoming wider, it opens as a discharging
tubule (duct of Bellini) en the apex of a Malpighian pyramid. The
tubes are lined throughout by a single layer of epithelium, which,
however, changes its characters in the different parts of the tube.
The Malpighian capsule is a globular expansion (200 /*.), composed of
a basement membrane lined by a single layer of squamous epi-
thelium. In the convoluted tubules, proximal and distal, the epithe-

U

30(5 HlACTlCAL HISTOLOGY. [XXVIII.

lial cells are somewhat cubical, but their outlines are not well
denned. They each contain a spherical nucleus, and their proto-
plasm is "rodded," especially at the outer part. In the spiral
tubule the cells are not unlike those of the convoluted tubule, but
they are not so tall, and therefore leave a more distinct lumen, and
they are not so markedly "rodded." In the descending part of the
loop and the loop itself — very narrow (10-15 /x,) — the cells are
clear and flattened, with a bulging opposite the nucleus, and
these projections alternate with those on the opposite side of
the tubule. The ascending limb (30 p wide) has somewhat
cubical cells, which leave a regular lumen. They are striated,
and often present an imbricate arrangement. The irregular or
zigzag tubule bends on itself with sharp angles, and is wide,
with an irregular lumen. Its cells stain deeply with staining
reagents, and are conspicuously striated in their outer part. The
second or distal convoluted tubule is like the proximal. The junc-
tional and collecting tubules are lined by low, columnar, clear, trans-
parent cells with small nuclei. The cells do nob stain readily.
In the discharging tubules the cells have the same general character,
but they are taller and more columnar. Fig. 294 shows the general
arrangement of the tubules, and from it it is easy to see in what
part of the kidney each kind of tubule is placed.

Blood- Vessels. — The renal artery enters the kidney, splits into
branches which run towards the cortex, and at the junction of the
cortex and medulla form incomplete arterial arches (fig. 293).
From these arches arise the radiate or interlobular arteries, run-
ning in the cortex between two medullary rays in a radial direction
towards the surface. They give off at intervals on all sides short,
slightly-curved vessels — vasa afferentia — which run without branch-
ing to end within the Malpighian capsules, and there form the
glomeruli. The vas efferens comes out of the Malpighian capsule
close to where the afferent vessel enters it and at the pole opposite
to the origin of the uriniferous tubule. The efferent vessel splits
up into capillaries, which ramify amongst the tubules of the cortex.
The blood is returned from the cortex by interlobular veins (fig.
293), which run alongside of the corresponding arteries. The
medulla is supplied by leashes of vessels — vasa recta — which for
the most part proceed from the arterial arches. The vasa recta are
pencils of arterioles (10-15), splitting up into capillaries which
ramify between the tubules of the medulla ; the medulla, however,
is not so vascular as the cortex. The blood is returned by corre-
sponding veins. The connective tissue is very scanty in the cortex,
but abundant in and near the apices of the Malpighian pyramids.

Methods. — (i.) Harden small pieces in a 2 per cent, solution of
potassic bichromate (18—20 days) or Miiller's fluid. Corrosive

XXVIII.] KIDNEY. 3O7

sublimate is also very good, and so is alcohol. The pieces should
not be large, and should be cut according as a longitudinal or trans-
verse section is desired. Stain and cut in bulk in paraffin. In all
cases the pieces should include both the cortical and medullary
portions, and should not be more than half-an-inch in thickness.

(ii.) Place small pieces in boiling water and then complete the
hardening in Miiller's fluid or alcohol.

(iii.) To fix the epithelium use Flemming's fluid, e.g., for mouse's
kidney.

Make radial sections from the cortex to the apex of a Malpighian
pyramid. For a general view stain a section in haematoxylin and
mount it in balsam. Another one should be stained in picro-
carmine or picro-lithio-carmine and mounted in Farrant's solution.
For unstained sections, steeping them for twenty-four hours in
i per cent, osmic acid before mounting them in Farrant's solution
is excellent. The best way is to begin with a section of the entire
kidney of a small animal, such as- a mouse, rat, or guinea-pig. In
this way a good view is got of the entire organ in section.

1. Radial Section of Kidney of a small mammal.

(a.) (L) Observe the capsule ; it is thin and apt to fall off; the
cortical and medullary portions of the parenchyma. The medulla,
composed of straight tubules of different sizes, and running radially
from the pelvis of the kidney outwards.

(b.) Trace some of the straight tubules outwards through the
intermediate layer, in bundles — the pyramids of Ferrein or medul-
lary rays — into the cortical layer (fig. 293, PF). Many medullary
rays pass from a Malpighian pyramid, and they run radially out-
wards in the cortex nearly to its outer part — although they do not
reach the surface — becoming narrower as they are traced outwards.

(c.) In the cortex, between every two medullary rays, are con-
voluted tubes, twisted and cut in every direction, and two rows of
glorneruli, enclosed in their capsules — the labyrinth. Here and
there a glomerulus may have fallen out, and the space it occupied
be left as a round aperture. The regular arrangement of the
glorneruli will only be seen provided the section runs parallel to the
course of the medullary rays. The glomeruli are confined to the
cortex, but none of them reach quite to the capsule.

(<l.) (H) In the cortex, the Malpighian capsules, each enclosing
a tuft* of capillaries or glomerulus. They consist of a structureless
membrane lined by a single layer of squames. The oval, flattened
nuclei of the latter are seen lying on the inner surface of the cap-
sule. Within each capsule a cluster of capillaries — glomerulus —
arranged, in several groups. Although the squamous lining of the
capsule is reflected over the capillaries, it is not easy to distinguish
the nuclei of these cells from the very numerous nuclei of the

308

PRACTICAL HISTOLOGY.

[XXVIIL

capillaries (fig. 295). The basement membrane of the capsule is
continuous by a narrow neck with the basement membrane of a
convoluted tubule, but it is only in the rare case where the section

cuts the capsule at this level that
this connection is seen. This con-
nection must usually be made out
on isolated tubules, although some-
times it is seen in sections of a
mouse's kidney.

(e.) The convoluted tubules,
with a sinuous or twisted course,
cut some longitudinally and others
transversely. Each tube is lined
by a single layer of cells, the indi-
vidual cells not sharply mapped off
from each other, and leaving a
small lumen in the centre. The
outer part of each cell is striated
or " rodded," and near its centre
it contains a spheroidal nucleus

FIG, 295.— Glomenilus and Sections of Con- ic.a 9r> A\

voluted Tubules of a Kidney, x 300. ^ ° /, Vr , m , ,

(/..) Irregular Tubules. — Here

and there in the cortex may be seen zigzag portions of tubules
of unequal width, and usually more deeply stained than the rest,

and the epithelium dis-
tinctly rodded (fig. 297).
(<).} Note the small
amount of connective
tissue between the tub-
ules of the cortex. It
is best seen just outside
the Malpighian capsules.
It is far more abundant
in the medulla,

(h.) In the medulla, the straight tubes, which are largest and
widest where they are about to open on the apex of a Malpighian

pyramid — discharging tubules — but
it is not so easy to get a section
showing this, as from their radial
arrangement they are apt to be cut
obliquely. The collecting and dis-
charging tubes have a wide lumen,
and are lined by a layer of clear
columnar cells with ovoid "tmdei. Trace the straight tubules out-
wards towards the surface ; they become smaller, still their lumen

FlG0 296.— Rodded Epithelium of a Convoluted Tubule.
Ammonium ehromate.

FlG. 207.— Irregular Tubule, Kidney of
Dog. Muller'B fluid.

XXVIII.]

KIDNEY.

309

remains distinct, and they are lined by clear nucleated short
columnar or cubical epithelium.

(i.) In the medullary rays in the intermediate layer very narrow
tubes — the descending portion of the looped tubule of Henle (fig.
300), not unlike fine capillaries — may be seen, and also the wider,
more deeply stained ascending portion of the same tubule.

2. T.S. of the Apex of a Malpighian Pyramid (Hcematoxylin
and Balsam}.

(a.) (L and H) Observe the large amount of connective tissue
between the tubules. The large collecting tubules are cut across,
so that their large lumina and the
clear columnar epithelial cells lining
them are distinctly seen (fig. 298).
Not infrequently the epithelium
falls out, and then the connective
tissue appears as a network with
round or oval holes.

3. T.S. Medullary Ray. — Sec-
tions should be cut in various direc-
tions and at different levels in the
cortex. One of the most instructive
is to cut a section across the direc-
tion of the medullary rays. In it
will be seen groups of transverse
sections of the various tubes — col-
lecting, ascending, and descending
portions of the looped tubule of
Henle — which make up a medul-
lary ray. Between the rays there
are sections of glomeruli and con-
voluted tubules.

Blood- Vessels of the Kidney.— The sections should be cut
from a kidney injected with carmine gelatine or Berlin-blue gela-
tine ; they should be radial, not too thin ; best from the kidney of
a small mammal, and mounted in balsam. In injecting a kidney
do not use too great pressure, as otherwise the glomerular capillaries
are apt to burst, and the injection-mass passes into the tubules.
When the stellate veins on the surface are seen to be well injected,
one may infer that the mass has traversed the glomerular capillaries.
In a small animal inject from the aorta, in a large one from the
renal artery.

4. T.S. Injected Kidney. — (a.) (L) Between the cortex and
the medulla, i.e., in the boundary or intermediate layer, sections
of the larger branches of the renal artery and vein will be seen
(fig. 293, KA), i.e., along the "line of vascular supply." From

FIG. 298.— T.S. Apex of a Malpighian
Pyramid. A. Large collecting tub-
ules; B, C, D. Wide and narrow
rts of Henle' a tubule; E, F.
-vessels.

310

PRACTICAL HISTOLOGY.

[XXVIII.

these the interlobular arteries and
veins (tig. 299, a), running out-
wards in the middle between every
two medullary rays (fig. 293).
From these are given off on all
sides, at short intervals along the
course of the vessels, short arteries
- — the vasa afferentia. Each vas
afferens, after a very short course,
runs to a Malpighian capsule, and
splits up into capillaries to form
the glomerulus.

(b.) If the capsule is cut at the
level where the artery enters, the
short and narrower efferent vein or
vas efferens will be seen coming
out at the same pole, and after a
similar short course breaking into
a network of capillaries, which sur-
rounds the tubules of the cortex.
Around the convoluted tubules the
meshes of the network are some-
what polygonal, but in the medul-
lary rays in the cortex they are
more elongated.

(c.) From this capillary network
arise' short veins, which join the
interlobular veins (fig. 299). Quite
at the upper end of the inter-
lobular artery a few branches are
given off, which end directly in
this capillary network without the
intervention of glomeruli. Under
the capsule the small veins are
arranged in a stellate manner, con-
stituting the venae stellatte (fig.
299, S).

(d.) Passing again to the "line
of vascular supply," the short
vessels which break up into leashes
Fia. 299.— Blood-Vessels of the Kidney. Or bundles of small blood-vessels

med^nal'^Inteflobufar^rtefy ; °i. (fig- 299, r), the Vasa recta (arteriffi
Vas afferens;. 2 Vas efferens; r e. Tecfa venaS rectae), which rim be-
Vasa recta; FF. Interlobular vein ;

s. Origin of a stellate vein ; i, i. BOW- twecn the medullary rays into the

man's capsule and glomerulus; P. ^n.lnWi WI-.OT-G fViov fnrrn nn

Apex of papilla ; c. Capsule of kid- medulla, where they lorm an
ney; e. Vasa recta from lowest vas elongated capillary meshwork be-

efterens. J

XXVIII.] KIDNEY. 3 1 i

tween the straight tubules of the medulla. The medulla is not so
vascular as the cortex, and it has no glomeruli.

5. Injected and Stained T.S. Kidney.— A not too thin injected
section — say injected with a blue mass — may be stained with picro-
carraine, which makes the tubular structures more distinct by
staining their nuclei.

6. Fresh Kidney — Glomeruli and Basement Membranes of
Tubules. — Expose a fresh kidney to the air for a day or two
according to the temperature. Cover it to prevent evaporation.
Tease part of the medulla and cortex in normal saline.

(a.) (H) Observe the long, partly empty, structureless, basement
membranes of the tubules, often exhibiting folds ;
also isolated cells of the tubules.

(b.) Isolated glomeruli, consisting of several
tufts of capillaries. The nuclei of their cells are
revealed by dilute acetic acid.

(c.) Narrow tubules, not unlike blood-capil-
laries, but they possess a wall lined by a layer of
squames, the nucleated part of .the squames alter-
nating on opposite sides of the tubule. These are
the descending part of the looped tubule (fig. FIG. 3oo.— Part of De-

scending Looped
n T i j. i /* n * ^ -^'0- , m -L , Tubule of Heule.

7. Isolated Cells of the Different Tubules.—

Place very small pieces (size of a -split-pea) 24-48 hours in a 5 per
cent, solution of neutral ammonium chromate. Wash in water, and
tease small fragments in a 50 per cent, solution of potassic acetate,
or, without washing, tease a fragment in the
chromate solution.

(a.) (H) Note specially the cells of the con-
voluted tubules and those of the ascending limb
of Henle's loop. They show the " rodded " char-
acter of the outer part of the protoplasm. Ad-
joining cells tend to interlock with each other

(% 3° 0- FIG. ooi.- Isolated

8. Isolated Tubules. — Place small pieces (size ceils from COM vo-

c \ n ,i i • j n . , ^ luted Tubules, i.

or a pea) 01 the kidney ot a mouse, tortoise, or on the flat with
guinea-pig (3-4 hours) in pure hydrochloric acid cesses°-C2 "on e^ige
or 40 per cent, nitric acid (2-4 hours), wash in and '" i-odded."
water and leave them in water for 18-24 hours. ^™ui
They swell up, and their constituents readily fall
asunder. Place a fragment in water slightly tinged with iodine and
gently tap the glass slide, or stain with dilute acid-fuchsin. This
is sufficient to cause the tubules to fall asunder.

(L and H) In a part from the cortex search for a convoluted
tubule still connected with its capsule, the twists on the tube itself,

3I2

PRACTICAL HISTOLOGY.

[XXVIII.

and the transition to the narrow part of the looped tubule of Henle.
This is perhaps easiest to obtain from a mouse's kidney. Isolated
straight tubules from the medullary part. To preserve this prepara-
tion, suck away the fluid and gently replace it with glycerine.

Ureter and Bladder. — Harden small pieces in Mtiller's fluid
(14 days) or in corrosive sublimate (5-6 hours), and then in
gradually increasing strengths of alcohol. Make transverse sections
of the one and vertical sections of the other. Flemming's fluid for
epithelium. Stain in haematoxylin and mount in balsam ; and
others in picro-carmine and mount in Farrant's solution. Or stain in
bulk in borax-cai-mine and cut in paraffin. The ureter of a cat or
monkey does very well, and it is well to use the contracted bladder
of a small mammal.

9. T.S. Ureter (fig. 302).— (a.) (L) Externally is a thin fibrous

"Adventitia.

FIG. 302.— T.S. Lower Part of Human Ureter, e. Transitional epithelium ; s. Sub-
mucosa ; I and r. Longitudinal and circular smooth muscular fibres ; t. Tunica
propria. Miiller's fluid, x 15=

coat or adventitia, consisting of connective tissue with the large

vessels and nerves.

(b.) The muscular coat consists of an outer layer of smooth

muscle arranged circularly, and inside this a longitudinal coat

arranged in bundles, the latter, of course, divided transversely. In

the lower part of the ureter there is an incomplete longitudinal

muscular coat outside the circular coat.

(c.) The submucous coat is thin, and passes gradually into

(d.) The mucous coat, which is thrown into ridges or folds, and

is lined by transitional epithelium.

XXVIII.]

KIDNEY.

313

Cylindrical
cells.

Leucocyte.

FlG. 303.— V.S. Epithelium of the Mucous Membrane
of a Human Bladder. Muller's fluid, x 560.

(e.) (H) The transitional epithelium, and the variation in the
shape of the cells arranged in several layers, from the free mucous
surface outwards.

10. V.S. Bladder (fig.
303).— (a.) (L) Most ex-
ternally a thin fibrous coat,
in some places covered by
a serous coat.

(b.) The muscular coat,
composed of longitudinal
and circular smooth fibres.
Usually there is an outer
and an inner longitudinal
layer with a circular layer
between. The appearance
of these layers will depend
on the plane of the sec-
tion.

The submucous and mucous (H) coats are like those of the
ureter.

(c.) (H) The transitional epithelium and the great variety in the

shape of the cells from below

upwards. Occasionally amongst
the epithelial cells are leucocytes
(tig. 303). It is important to
observe that the thickness and
shape of the lining transitional
epithelium will necessarily vary
with the state of distension or
contraction of the bladder.

11. T.S. Penis, e.g., of a
monkey or other small mammal.
Harden it in alcohol or fix in
Flemming's fluid. Make T.S.
and stain with safranin.

(a.) (L) It consists of the two
corpora cavernosa, placed dor-
sally, one on each side of the
middle line, and inferiorly the
cor/tuft i<ixiji(jiosum. In the
centre of the latter is the
urethra as a transverse slit.
In the prostatic part the mucous membrane is lined by transitional
epithelium, but in the body of the penis it is lined by the columnar
variety, except at the meatus, where it is stratified.
28

sum; s. Septum; it. I'lvthra : J)V.

&™Vv<Jil1 ; LA' Duml urU'rie" : *'

314

PRACTICAL HISTOLOGY.

[XXVIII

(6.) Note the trabeculse of connective tissue — bounding wide
spaces — in the cavernous part. The whole is surrounded by a
tough capsule, in which, dorsally, are sections of blood-vessels — one
vein (DV) and two arteries (DA) — and nerves (fig. 304, N).

SUPRARENAL CAPSULE.

It is well to remember that there are great variations in the
structure of this gland in different animals. The suprarenal
capsule is a ductless gland, consisting of a cortical zone and a

Zona glomerulosa.

Z. fasciculata.

Z. reticularis.

Strands of cells of
the medulla.

T.S. of a nerve.

Ganglion ic cells.

T.S. bundles of
smooth muscle.

T.S, vein.

N Medulla.

FIG. 305. — T.S. Human Suprarenal Capsule, x 50.

medulla. It is invested by a fibrous capsule which sends septa
into the gland. Especially in the cortex, these septa run so as to
give a columnar arrangement to the cells which lie between them.
The parenchyma of the organ consists of cells which vary in their
characters in different regions. The cells of the cortex (15 /x) are
polyhedral, nucleated, granular, yellowish-coloured cells, arranged
under the capsule in rounded groups — zona glomerulosa. Next
this is the widest zone — the zona fasciculata. Next the medulla

XXVIII.] SUPRARENAL CAPSULE. 31$

is the zona reticularis. In the medulla the cells are often irregular
or polygonal with a clearer protoplasm, which is often tinged of a
yellowish or brownish colour. There are numerous vessels and
nerves, the latter with ganglionic cells.

Harden the suprarenal capsules of a guinea-pig in Klein's fluid
(5-7 days), and then in alcohol, or fix in Flemming's fluid. Harden
a human suprarenal in Kleinenberg's fluid (24 hours), and then in
alcohol. Make radial sections, and stain some in hsematoxylin, and
others in picro-carmine. Or stain and cut in paraffin.

12. V.S. Suprarenal Capsule. — (L) Observe the arrangement
already described. It is to be noted, however, that there are great
variations in the structure of these organs in different species of
animals.

(|-|) Examine the cells in the various zones (fig. 305).

ADDITIONAL EXERCISE.

Termination of Nerves in Suprarenal Capsules. — The literature and most
recent results will be found in Fusari's paper1 (with a plate). He used
capsules of the mouse, rabbit, pig, cat, and new-born infant. The method
employed was the quick method of Gdlgi (Lesson XXX.), i.e., small fresh pieces
are placed in the osniico-biehromate fluid (3-10 days), and afterwards in I per
cent, silver nitrate solution (1-2 days).

LESSON XXIX.
SKIN AND EPIDERMAL APPENDAGES.

THE SKIN.

THE skin consists of the epidermis and cutis vera, dermis, or
corium. The epidermis consists of many layers of stratified
squamous epithelium (p. 317). The corium is composed of a basis
of fibrous connective tissue — white and yellow fibres — and its sur-
face is thrown into a number of papillae, which differ in size,
number, arrangement, and form in different parts of the body.
Undivided conical elevations are called simple p<tpill<r, but when
these are beset with smaller papillae, they are called Htnt/tnun'f
papilla'. The epidermis completely covers in the apices of the

1 Ardiivitttl. tie 7'/W., xvi. p. 262, 1891.

316 PRACTICAL HISTOLOGY. [XXIX.

papillae, and also dips down into the furrows between adjoining
rows of papillae, so that the surface of the skin is smooth, although
the arrangement of the papillae is readily detected by the lines on
the palmar aspect of the hand and foot. The fibrous tissue of the
cutis, next the epidermis, forms a very thin modified layer with
scarcely any fibrils and no corpuscles. This layer acts the part of
a basement membrane, and is continuous with the basement
membrane of a sweat-gland. In the dermis, the bundles of white
fibres interweave with each other, and form a dense tissue ; at the
lower part of the skin it becomes more open in texture, and gradu-
ally passes into the subcutaneous tissue. Elastic fibres in the form
of networks exist in large numbers in the cutis ; they are finer in
the papillae, and coarser lower down.

The subcutaneous tissue consists of a complex system of
trabeculae of fibrous tissue, and in some of the meshes are lobules
of fatty tissue forming a fatty layer, constituting the stratum
adiposum.

The arrangement of the Hood-vessels is stated at p. 325. There
are also numerous lymphatics and nerves — some of the latter with
peculiar terminations — glands (sweat and sebaceous), and, in some
situations, hairs with their hair-foil ides.

It is important to distinguish between the hairy skin and the
parts of the skin without hairs. The non-liairy parts are the volar
surfaces of the hands, feet, fingers, and toes, nails, lips, mammary
papillae, certain parts of the external genitals, and the inner part of
the external auditory meatus. The hairy parts are the remainder
of the skin. The non-hairy parts are concerned with direct tactile
sensations, the hairy parts with indirect tactile sensations, the hairs
themselves being the chief tactile organs (Blaschko1). This
observer has shown that the epidermis projects into the cutis vera
in the form of septa, varying in form and distribution in different
parts of the skin (p. 325).

Methods. — Tlie skin must be prepared in various ways according
to the particular part which it is desired to study. For a general
view proceed as follow :— (a.) Procure a fresh portion of human
skin from the palm of the hand or sole of the foot, cut it into
pieces about i cm. square, and remove most of the subcutaneous fat ;
pin it, epithelial surface downwards, on a piece of cork, and harden
it in absolute alcohol (12 hours). Renew the alcohol for another
twenty-four hours. Sections may be cut by freezing, and then
stained with hsematoxylin or picro-carmine (the latter to be mounted
in Farrant's solution). Better still, stain the whole "in bulk " in
borax carmine or haematem, and embed and cut it in paraffin.

1 Arckivf. mik. Anat., xxx. p. 495.

XXtX.]

THE SKIX.

Mount in balsam. Or skin so hardened may be double stained in
bulk, first in borax-carmine and then in haemateiin.

(b.} Haidi-n the skin in Miiller's fluid.

(c.) For the layers of the epidermis fix say small pieces of the
skin in i per cent, osmic acid or Flemming's fluid, and harden in
alcohol. Stain sections in safranin.

1. V.S. Skin, Palm of Hand.

(a.) (L) The epidermis, consisting of many layers of stratified
squamous epithelium, resting on the cutis vera, dermis, corium, or
true skin. The latter consists of connective tissue, and is provided
with finger-shaped elevations or papillae, which project into the
deeper layers of the epidermis, the latter in the form of septa, dip-
ping in between the papillae (fig.
306).

(/>.) The epidermis, composed
entirely of stratified epithelial cells.
Proceeding from the outside (fig.
307), observe — •

(i.) The stratum corneum, of
variable thickness, consisting of
many layers of flattened or slightly
fusiform, clear, non-nucleated cells
united to each other. As the cells
are seen on edge, they are very thin.
Those on the surface are about to
be shed, and consist of keratin.

(ii.) The stratum lucidum, a
thin, narrow, clear, homogeneous
layer, composed of two or more
layers of flattened cells, containing
sometimes a rod - shaped nucleus.
The cells do not stain well with
dyes. The eleidin granules seem to
become fused together and form the basis for cornification, as the
cells are changed and become corneous.

(iii.) The stratum granulosum, a somewhat thicker layer, com-
posed of ovoid cells two or three rows deep. Each cell is distinctly
granular, and usually this layer stands out deeply stained, because
its granules of eleidin or keratohyalin are stained with the car-
mine. The cells, like the foregoing, are devoid of "prickles."

(iv.) The stratum Malpighii, several layers of more plastic cells.
At the lowest part, where they rest on the papillae of the true skin,
the cells — prickle-cells — are smaller and columnar in shape (with
oval, vertically-placed nuclei), but above this they become more
spheroidal or polygonal, and each one is distinctly nucleated.

FIG.

. 306.— V.S. Skin of Palmar Surface
of Finger. F. Fat ; P. T.S. Pacinian
corpuscle.

PRACTICAL HISTOLOGY.

[xxix.

Stratum
corneum.

(c.) The cutis vera. The papillae, conical elevations projecting
into the Malpighian layer. Tliey consist of compact fibrous tissue.
The rest of the skin consists of bundles of white fibrous tissue
interwoven with networks of elastic fibres, and at its lower part
masses of fat-cells. The connective tissue and fat-cells below
become continuous with the subcutaneous tissue, which is of a
more open texture ; but there is a gradual transition from the one

to the other. The nuclei of the
connective tissue corpuscles appear
as red oval dots.

In sections of the sudoriferous
glands, their coils (in the deeper
layers of the corium), their ducts
running vertically through the
skin, and a corkscrew passage in
the epidermis may be seen. In
some of the papillae observe a
touch-corpuscle (p. 320), and in
the subcutaneous tissue occasion-
ally sections of Pacinian bodies
(p. 320).

(<#.) (H) Observe in the epi-
dermis the shape and characters
of the successive layers of epi-
thelium. In the Malpighian
layers, "prickle-cells," i.e., cells
connected with each other by
fine "inter-cellular bridges," are
better studied in an osmic acid
section (fig. 97). (See also
Lesson IV.)

(i. ) In the stratum Malpicjliii
the lowest cells are arranged in
a single layer of elongated, some-

wi.ni- PnllirnTinT, r(A\~ If. T<7 ,.\
WJiat C ells V0"12 J*/»

with large OVal nuclei SlUTOlinded

by granular protoplasm. The
lower ends of the cells frequently exhibit processes which fit into
the derm is. The remainder of the cells of this layer are irregularly
cubical, and exhibit prickles (Lesson IV., and p. 127). In the dark
races the particles of melanin, which give the dark colour to the
skin, are present in the cells of this layer, especially in the deepest
layer of cells. Nuclei are sometimes seen in process of division.

(ii.) The cells of the stratum yranulosum are arranged in two or
more layers, and are flattened horizontally, so that they are lozenge-

FlG. 307.— V.S. Human Epidermis with
Terminations of Nerve - Fibrils. n.
Nerve; d. Dennis; 6. Branches of

XXIX.] THE SKIN. 319

shaped, devoid of prickles, and are crowded with granules of eleidin.
a substance, apparently one of the stages on the way to the hotly
keratin. These cells stain deeply with picro-carmine, haematoxylin,
and osmic acid. They are soluble in caustic potash, and in this
respect differ from keratin.

(iii.) The stratum Iwidum is composed of two or more layers of
flattened transparent cells, with no prickles, and free from granules,
but with a horizontally-placed, rod-shaped nucleus. They do not
stain readily.

(iv.) The stratum corneum or horny layer consists of horny
sijuames composed of keratin (Lesson IV.).

(e.) The sweat-glands are most numerous in the palm of the
hand and sole of the foot. Each gland is a simple tube coiled up
at its lower extremity into a coil -^ inch in diameter. To see their
whole course — coil and duct— the sections must not he too thin,
and should be parallel to the course of the gland. The coil of the
gland lies in the subcutaneous tissue. The secretory part of the
tube consists of a basement membrane lined by a single layer of
nucleated transparent cubical or columnar cells surrounding a small
but distinct lumen. Between the epithelium and the basement
membrane is a longitudinally-
disposed layer of smooth
muscular fibres. The coil
also contains a part of the
sudoriferous canal or duct
(fig. 308). The latter is
narrower than the secretory
part, and consists of a base-
ment membrane lined by
several layers of polyhedral
cells. There is no muscular
layer, but internal to the

.,, ,. if.'. • FIG. ^08.— Section of Part of Coil of a Sweat-

opithelial lining there is a Glar?d. ». Duct; s. Secretory part, x 3oo.
delicate membrane or cuticle.

Trace the coil into its duct, which runs vertically through the cutis
vera with a slightly wavy course. It has a basement membrane
lined by two or three layers of short cubical cells, which, if traced
upwards, become continuous with those of the Malpighian layer of
the epidermis, looking like a funnel-shaped expansion. The lumen
of the duct is distinct. The bwrnent membrane becomes continuous
with the altered superficial layer of the corium just under the epi-
dermis. The lumen of the duct is continued upwards in a corkscrew
spiral through the epidermis. A complete view of its course is only
obtained in a thickish section. In a thin section the twistings are
of course divided.

32O PRACTICAL HISTOLOGY. fxXIX.

2. V.S. Skin of Negro (H). — Harden in alcohol, and stain the
sections slightly in eosin. Mount in balsam. Observe the granules
of melanin in the deepest layers of the epidermis.

3. V.S. Skin of Finger (Double Stained).— Stain a section first
in methyl-green iodide and clarify it with clove-oil coloured with
eosin. Wash out the clove-oil with xylol and mount in balsam.
The stratum corneum is green, and so are the nuclei of the other
epidermic and connective-tissue cells.

4. Prickle-Cells and Touch-Corpuscles. — Place in i per cent,
osmic acid or Flemming's fluid (24 hours) a very small piece of
fresh skin from the palmar surface of a finger. Wash it well in
water and complete the hardening in alcohol. Make V.S. and
mount in Farrant's solution. Or cut in paraffin, stain in safranin,
and mount in balsam.

(a.) (H) Observe the various layers of the epidermis, but in the
Malpighian layer note the prickle-cells. The cells appear to bo
joined by their edges by fine striae, the striae leaving small spaces
between them (fig. 97). The striae are fine " intercellular bridges,"
stretching from one cell to another, and it is only when the epi-
dermis is dissociated and the bridges broken that these cells appear
as cells beset with fine spines, and hence they were called " prickle-
cells."

(b.) In a papilla search for a Wagner's touch-corpuscle (fig. 351).
It is an oval body, with its long axis in the long axis of the papilla,
but they are not present in all papillae (fig. 350). They consist of
a fibrous-looking material, with flattened nuclei arranged trans-
versely. To their lower end passes a medullated nerve-fibre, which
usually twists round the corpuscle before it enters it. The ultimate
distribution of the nerve is best seen in a gold chloride preparation
(Lesson XXXIV.).

5. V.S. Foetal Skin for Sweat-Glands and Pacinian Corpuscles.
— Harden the skin of the tips of the ringers of an infant in alcohol
and make V.S. Stain with hgematoxylin and eosin.

(«.) (L) Observe the general arrangement already described, but
the sweat-glands are much more closely placed than in adult skin,
and there is less intervening connective tissue. A child at birth
has its full complement of sweat-glands, and hence they must be
more crowded together than in the adult.

(b.) In the subcutaneous tissue are lobules of fat and sections of
Pacinian corpuscles (Lesson XXXIV. 5). The latter appear to con-
sist of concentric laminae surrounding a central core.

(c.) (H) Observe two or three layers of more or less cubical cells
lining the duct of the gland, while the true secretory portion is
lined by a single layer only of low, clear, columnar cells.

Hair Follicles. — To see their structure, harden the scalp in

XXIX.J THE SKIN. 321

alcohol. V.S. must be made parallel to the course of the hair-
follicles, which requires some care. Others are made across the
follicles at different levels ; but in this case care must be taken not
to make the section parallel to the surface of the skin, but at right
angles to the course of the hair-follicle. If an oblique section be
cut, the hair-follicles are cut at different levels.

Harden a small piece of the human scalp (2 cm. square) in
Miiller's fluid and afterwards in alcohol. Stain a section in log-
wood, or first with logwood and then with picro-carmine. Mount
in balsam.

6. V.S. Hair-Follicle.— (a.) (L) and (H) Observe the very thin
epidermis, the thick cutis vera, and deep down the subcutaneous
masses of fat; the hair- follicles, running obliquely through the
skin, each one with a lair in it. At the lower part the hair has a
bulbous end implanted on a papilla ; the various coats of the hair-
follicle, some continuous with the corium, and others with the
epidermis. The following scheme shows the layers of the hair-
follicle ;—

Coverings of a Hair- Follicle from Without Inwards.

„., , f (a.) Longitudinally-arranged fibrous tissue.

1. Fibrous layers . | ^ CircSlarly-arringed sgindle-cells.

2. Glass-like or hyaline membrane.

( (a.) Outer root-sheath. ( Henle's layer.

3. Epitheliallayers < (b.} I Inner root-sheath, -j Huxley's layer.

( (c.) Cuticle of the hair. ( Cuticle of root-sheath.

4. The hair.

(b.) Dermic Coverings.— (i.) (a.) The outer fibrous sheath
denser than and continuous with the corium. The fibres run for
the most part longitudinally, (b.) The inner fibrous sheath of
fibrous tissue has a more circular arrangement and is seen as fibres
cut across transversely, with a few nuclei interspersed.

(2.) The hyaline or basement membrane, clear, structureless, and
well marked. It separates the dermic from the epidermic coverings
of the hair.

(3.) The Epidermic Coverings. — (a.) The outer root-sheath — the
most obvious part of the covering — consisting of several layers of
nucleated cubical cells, continuous with and resembling those of the
Malpighian layer.

(b.) The inner root-sheath, much narrower and paler, consisting
of three layers of cells of different characters, is present only in the
lower part of the follicle, i.e., below the sebaceous gland.

(4.) The hair with its cuticle.

(a.) At its lowest part the bulbous enlargement of the hair, with
29 X

322

PRACTICAL HISTOLOGY.

[XXIX.

the papilla of the hair follicle (fig. 309) projecting into it, and con-
tinuous with the corium.

(b.) The sebaceous gland or glands. In a balsam preparation

FIG. 309.— V.S. Hair-Follicle of Human Scalp, i and 2. Outer and inner fibrous sheaths
of hair-follicle ; 3. Hyaline layer ; 4. Outer, and 5 and 6. Inner root-sheaths ; p. Root
of hair, with its papilla; A. Arrector pili muscle; C. Ciitis vera,- a. Subcutaneous
tissue, with fat lobules; b. Epidermis (horny layer); d. Rete Malpighii ; g. Blood-
vessels ; v. Lymphatics of papilla ; h. Fibrous part, i. Medulla, k. Cuticle of hair ;
K. Sweat-gland and its coil.

its acini are yellowish and clear, opening by a duct into the hair-
follicle at about its upper third (fig. 309, T).

(c.) The arrector pili muscle (smooth muscle) stretching ob-

XXIX.] THE SKIN. 323

liquely from the deeper part of the hair-follicle to the upper part of
the corium (fig. 309).

7. T.S. of Hair-Follicles (Scalp).

(a.) (L) In the human scalp the hair-follicles are arranged in
groups of three or four, with interweaving strands of connective
tissue between them. The various coverings — dermic and epi-
dermic— can now be distinctly seen, especially if the section be
through the lower half of the hair-follicle.

(6.) (H) Observe both V.S. and T.S. to see the structural ele-
ments forming the outer coverings of a hair-follicle.

(i.) The inner root-sheath consists of an outer layer of cells, clear
and non-nucleated — Henle's layer — and an inner nucleated layer —
Huxley's layer. Both are best seen in T.S.

(c.) The hair has a cuticle, while the hair itself may or may not
have a medulla.

Carefully compare the structures of the hair-follicle in the T. and
V. sections.

(d.) The sebaceous gland, its acini lined by cubical cells, contain-
ing fat, and rendered clear by the balsam.

The epithelium of the duct continuous with that of the outer
root-sheath.

8. Sebaceous Glands. — (a.) Harden the alae of the nose of a
new-born child in corrosive sublimate. (6.) Or the ala of nose or
adult scrotum in picro-sulphuric acid (2-3 hours). Stain all
with haematoxylin. Large sebaceous glands opening free on the
surface without any hair-follicle are found. In other situations
they open into the neck of a hair follicle. They are saccular
glands with oval alveoli, which lead into a short duct. The alveoli
are lined by a layer of polyhedral cells, and internal to this are
larger cells containing fatty matter. The sebaceous secretion is
formed by these cells undergoing disintegration, and liberating the
fatty matter they have formed. They are developed from the outer
root-sheath. In balsam they are clear, but in water they appear
dark and granular.

9. Human Hair (H). — Place it in water, cover, and examine.
A rod-shaped body covered by a single layer of thin, non-nucleated
transparent imbricate scales arranged transversely — cuticle. In
some hairs it is seen merely as fine, more or less transverse,
irregular, or wavy lines joining each other. These indicate where
the one cell overlaps the other. The substance or cortex of the
hair, composed of horny, fibrous substance — hair fibres — finely
striated longitudinally, with, in some hairs, fine pigment-granule?
scattered along the course of the hair between the hair-fibres. In
some hairs a darker central core or medulla composed of polyhedral
cells.

324

PRACTICAL HISTOLOGY.

[XXIX.

10. Elements of a Hair (H). — Place a small piece of a hair in
a drop of strong sulphuric acid. Cover and press lightly with a
needle. Be careful to avoid letting a drop of the acid fall on the
brasswork of the microscope. The hair splits up longitudinally
into what look like fibres, but by gentle tapping on the cover they
split into cells, so that a hair is composed of epithelial cells joined
together, having previously undergone conversion into keratin.

11. Babbit's Hair (H). — Mount in balsam. This hair contains
one or more rows of cubical cavities containing air. The cavities
appear black, and are surrounded by a small quantity of cortex.

12. Wool (Lesson I. 10).

FIG. 310.— T.S. One-half of a Hair in its
Follicle ; a. Outer, c. Inner fibrous
sheath ; b. Blood-vessels ; d. Hya-
line layer; e. Outer, /, g. Inner
root-sheath (/. Henle's layer, g.
Huxley's layer) : h. Cuticle ; I.
Hair.

FIG. sii.-V.S. Injected Skin, Palmar
Surface of Finger.

Blood- Vessels of the Skin. — V.S. of a piece of skin cut from a
limb injected with a gelatine mass. They must not be too thin.
After injection the skin is hardened in Miiller's fluid and afterwards
in alcohol (three weeks). Mount (balsam) a section of the palmar
surface of a finger, and one from the general surface of the body.

A good injection-mass is a watery solution of china-ink. It is
rubbed down on a hone until a moderately thick black solution is
obtained, so that when a drop is placed on blotting-paper it holds
together, and no grey ring is formed round the drop. It has this

XXIX.] THE SKIN. $25

advantage, that it is not changed by exposure to light, hut the
tissue must be hardened before sections are cut.

13. V.S. Injected Skin, e.y., Palm. — The section should be cut
in paraffin, and include the subcutaneous tissue (fig. 311).

(a.) (L) The arteries of the skin are branches of the larger
arteries in the subcutaneous tissue. A branch may be seen running
towards the surface. In its course it gives off three independent
sets of branches, which end in capillaries : —

(i.) The lowest to the groups of fat-cells, where it forms a net-
work of capillaries around and between the fat-cells.

(ii.) The short branch to the coil of a sweat-gland, forming a rich
network of capillaries between the coils of the tube.

(iii.) The highest is from the terminal branches of the artery,
and splits up into capillaries, which form a network chiefly in the
upper part of the corium, and from this branches pass which form
capillary loops in the papilla? of the skin. From it also proceed
branches to the hair-follicle and its sebaceous gland.

(l>.) The vein arises from the capillaries of the papillae and the
branches of the arteries to the upper part of the cutis, and in its
course — running near the corresponding artery — it collects the
veinlets from the coil and masses of fat. For blood-vessels of skin
see W. Spatleholz.1

14. Under-Surface of Epidermis.— Separate by one of the follow-
ing methods the epidermis from the cutis, most easily done in the
foetus: — (a.) Use a foetus that has died and been macerated in utero.

(b.) Place pieces of skin in J-J per cent, acetic acid (1-3 days^,
adding a drop or two of chloroform to prevent putrefaction
(Phttippson).

(r.) Macerate at 40° C. — preferably foetal or young — skin pinned
out on cork in 6 per cent, (or weaker) wood vinegar for 1-2 days.
The epidermis separates rapidly (Loewy).2

After the epidermis peels off, in all cases turn its deeper surface
upwards, and stain it for 3-4 minutes with a watery solution of
Pxx'hmer's logwood (p. 68). Wash and mount in balsam, pre-
ferably without a cover-glass.

(L) Observe a system of septa crossing each other, and forming
longitudinal and transverse ridges, which project into the cutis.
They form, as it were, the negative picture ; the papillae of tho
cutis vera represent the positive image. Part of the cells lining
the sweat-glands and hair-follicles may also be pulled out, and are
turned toward the observer. Figures of the arrangement of these
septa are given in the papers of Blaschko and Loewy (pp. 316, 325)-

1 Archiv f. Anat. v. Phys., Anat. Abth., 1893.

2 Arckwf, ntik. Anat., xxxvii. p. 159, 1891.

326

PRACTICAL HISTOLOGY.

[XXIX.

THE NAILS.

The body of tlie nail rests on the nail-bed, the root of the nail
on the matrix, and the part at the root and sides from which the
nail springs is the nail-groove. The body of the nail is made up of
numerous clear horny cells, each containing a rod-shaped nucleus.
The nails are really the stratum lucidum, the stratum corneum
being absent, and this rests on the Malpighian layer like that of

the epidermis. The
corium or nail-bed, on
which the nail rests,
is beset with very
vascular longitudinal
ridges, papillae being
absent.

Harden the nail of
a child and its sub-
jacent bed in alcohol.
Make T. and L. sec-
tions. Stain in hse-
matoxylin (balsam) or
picro - carmine (Far-

FlG. 312.— T.S. Through Half the Nail. Injected, a. Nail *.««<-' a onln+irm\
substance; b. More open layer of cells; c. Stratum railA? S
Malpighii; d. Transverse sections of ridges; e. Nail- 15. T.S. Nail (L

projecting over the nail; and H).— Observe the
substance of the nail
(fig. 312), and under it a series of transverse sections of the ridges
of the corium of the nail-bed projecting into the epidermis. Under
this the dense fibrous matrix.

16. L.S. Nail. — Observe the same general arrangement, but note
that no papilla-like sections of ridges are to be seen.

ADDITIONAL EXERCISES.

17. Elastic Fibres in the Skin.— (i.) Those resist gastric digestion ; hence,
add some pepsin to .2 per cent, of hydrochloric acid. If small pieces of skin
he partially digested in artificial gastric juice at 4O°C., part of the connective
tissue is dissolved and the elastic networks remain.

(ii.) Herxheimer's Method.

(1.) Harden in Miiller's fluid.
(2.) Stain 3-5 minutes in

Hfematoxylin. i grnm.

Absolute alcohol ... 20 cc.

Water . . . 20 ,,

Sat. sol. lithic carb. . i ,

XXIX.] THE NAILS. 327

(3.) Extract (5-15 sees.) with official perchloride of iron solution.
(4. ) Wash in water.
(5.) Alcohol, oil, balsam.

The elastic fibres are bluish-black or black, and the surrounding tissue light
bine. What Herxheimer described as "spirals" in the lower layer of the
epidermis are spiral fibrils proceeding from the lowest layer of the cells of the
epidermis, as shown by Kronmayer (p. 327).

(iii.) Unna's Method.— Place sections of the skin for 12-24 hours in the
following mixture : —

Orcein . . . .0.5 gram.

Absolute alcohol . . . 40 cc.

Water . . . . 20 ,,

Hydrochloric acid . . .20 drops.

The sections arc afterwards decolorised in very dilute I1C1 containing some
alcohol. A full account of the arrangement of the elastic fibres is given by
Lenthoefer.1

18. Sweat-Glands of Axilla. — Make V.S. of the skin of the axilla hardened
in alcohol. Stain in hrematoxylin, or stain in bulk and cut in paraffin. The
sweat-glands, and particularly the coils, are very large. In these glands it is
easy to see the smooth muscular fibres outside the lining

epithelium of the secretory part of the coil (fig. 313).

19. Development of Hairs. — Make V.S. of the skin of
a foetus at the fourth to the fifth month, after being
hardened in Miiller's fluid (12-15 days) and then in
alcohol. Stain a small piece " in bulk " in borax-car-
mine, and cut in paraffin. The sections may also be
stained subsequently with methyl-green. Mount in
balsam.

20. Double-Staining of Hair-Follicles.— («.) Make V. FIG. 313.— T.S. Socre-
and T. sections, and stain some with eosin and hfemato- tory Part of Sweat-
xylin, and others first with picro-carmine (12 hours) and Nuclei ° of smooth
then with methyl-green iodide. The latter preparation muscle.

is specially beautiful, and both T.S. and L.S. should be

stained by this method. The scalp is best hardened in potassic bichromate.

Harden other pieces in alcohol. Methyl -green stains the inner root-sheath

green.

(b.) Stain with aniline-blue and safranin. Henle's sheath is rosy, Huxley's
blue.

(c.) Or stain in safranin in a weak alcoholic solution of picric acid.

21. Tactile Hairs. — Harden the skin containing the large tactile hairs of
a rabbit or cat in alcohol or Miiller's fluid. Make T.S. and L.S. Outside the
sheaths of the hair-follicle, already described, there is a large blood-space
traversed by tnibeculse, and thus presenting the characters of cavernous
tissue.

22. Nail (double-stained}. — Picro-carmine and methyl-green.

23. Blood-Pigment in Hair (H).— Examine in normal saline one of the
large " feelers" from the lip of an albino rabbit (S. Mmjer}. At some part of
the hair in its centre a red pigment — 'hremoglobm — may be seen.

24. Fibrillation of Protoplasm of Epithelial Cells (Kronmayer}.* By means
of Weigert's fibrin-staining method (Lesson III. 20) Kronmayer has demon-

1 Topographic d. Elastisclicn Geuv.be, Leipzig, 1892.

- "Die Protoplasmafaserung d. Epithelzelle," Archiv f. mik. Anat., xxxix.
p. 141, 1892.

328 PRACTICAL HISTOLOGY. [XXIX.

strated the passage of the fibrils of one epithelial cell into adjacent epithelial
cells, thus giving rise in the intercellular spaces to the appearances known as
"intercellular bridges " (p. 128). The fresh skin of the sole of the foot or
palm of the hand is hardened in absolute alcohol. Failing this, use part of an
epithelionia. It may be stained with alum-carmine or borax-carmine, and then
embedded and cut in paraffin. The sections must be very thin (at least 0.005
mm.), and in order to obtain these the knife must be placed obliquely and the
sections cut from the epithelial surface towards the cutis vera.

The sections are placed in a watch-glass and xylol added to dissolve the
paraffin. Add fresh xylol, pour it off', and then slowly add absolute alcohol.
Gradually add water until the sections float flat on the surface. They are then
carefully transferred to a slide, and by the aid of a pad of filter-paper gently
fixed on the same. The sections so fixed are stained on the slide.

The best staining reagent is methyl- violet-6B, prepared as follows : — Mix
equal parts of aniline- water and a saturated watery solution of methyl -violet.
Pour a few drops of this fluid on the section fixed on the slide, and in five
minutes lave the section in water.

Decolorise (few seconds) in iodine solution (p. 93). Wash in water. Remove
the water by pressing blotting-paper on the section, and then flood it with
aniline-xylol (aniline oil, I : xylol, 2). This mixture extracts the surplus
dye very rapidly, so that the preparations must remain but a few seconds
therein.

The march of events, supposing one wishes to stain the cell nuclei before
staining the protoplasm fibrils, is as follows — the cells being previously
hardened : —

(i.) Alum-carmine.

(2.) Wash in water (HCl-alcohol,

absolute alcohol).
(3. ) Methyl-violet-aniline-water.
(4. ) Wash' in water.

(5.) lodo-potassic-iodide fluid.

(6.) Wash in water.

(7.) Aniline-xylol.

(8.) Xylol-balsam.

LESSON XXX.
SPINAL CORD.

THE spinal cord, like the brain, is invested by three membranes,
named, from without inwards, dura, arachnoid, and pia mater.
The pia mater closely invests the cord, and sends processes into its
substance as well as into its fissures. The cord itself is composed
of white matter externally and grey matter internally. Running
along the cord anteriorly and posteriorly are the anterior and
posterior median fissures; the former is the wider, the latter
rather a groove than a fissure. The two fissures do not meet in the
middle line, but they serve to divide the cord incompletely into two
lateral halves, which are united across the middle line by a com-

XXX.] SPINAL CORD. 329

missure, composed anteriorly of white fibres crossing from one side
of the cord to the other — white commissure ; and posteriorly of
grey matter — posterior commissure. In the middle of the latter
is the minute central canal, lined by columnar ciliated epithelium.
In each half of the cord is a crescent-shaped mass of grey matter
as seen in transverse section, the two masses connected across the
middle line, and presenting more or less the form of an H, with the
extremities of its vertical limbs turned outwards. Its extremities
are the anterior and posterior cornua. The anterior cornua are
generally wider and shorter than the posterior, which are narrow,
and come nearer the surface of the cord. The nerve-roots arise
from the cornua, the anterior root by several bundles from the
anterior cornu, and the posterior root by a single bundle from the
posterior one. In this way, and by the existence of the fissures,
each half of the white matter of the cord may be conveniently
described as divided into an anterior, lateral, and posterior
column, or more correctly into an antero-lateral and a posterior.
The anterior cornu contains numerous large multipolar nerve-cells
arranged in groups. Each cell is continuous, through its axis-
cylinder process, with a nerve-fibre. The arrangement and number
of cells, however, vary in different parts of the cord. There are no
large nerve-cells — only small fusiform ones and some small isolated
or " solitary " cells — in the posterior cornu, which is capped by a
peculiar greyish matter — the substantia gelatinosa of Rolando.
The white matter is composed of medullated nerve-fibres — small
and large — arranged for the most part longitudinally, so that in a
transverse section, when stained with carmine, they appear like
clear rings with a central, red-stained spot — the axis-cylinder. In
Pal's method the myelin is stained, and so they appear as blackish
circles with a clear centre. The nerve-fibres, and grey matter as
well, arc supported by a peculiar sustentacular tissue — neuroglia —
composed of glia-cells (p. 343), and both are supplied by blood-
vessels, the grey matter, however, being far more vascular than the
white. The grey matter, besides blood-vessels, lymphatics, glia-cells,
nerve-cells, and their numerous processes, also contains nerve-fibres.

The nerve-cells vary in size, shape, and other characters in the
different parts of the cord. Golgi speaks of two types of nerve-cells
in the spinal cord.

Type i, or Motor Type, corresponding to the multipolar cells of the
anterior cornu. In these one process retains its individuality, and
passes directly to become an axis-cylinder of a medullated motor
nerve-fibre in the anterior root. This axis-cylinder process gives off
a few secondary lateral processes or collaterals, which divide and
enter into the formation of the nerve-complex in the grey matter.
The protoplasmic processes subdivide, and also form part of the

33O PRACTICAL HISTOLOGY. [XXX.

same grey complex, but they do not unite with fibres from adjoining
cells or with nerve-fibres.

Type 2, or Sensory Type. Also multipolar, smaller cells, with no
axis-cylinder process. All the processes subdivide into finer pro-
cesses, lose their individuality, and pass in toto into the nervous
complex, or diffuse nervous network in all strata of the grey matter
of the cord. These cells are therefore connected only indirectly
with nerve-fibres, i.e., through the intervention of the grey nervous
network ; and it is only in this indirect way that they come into
relation with — contact, not actual union with — the branches of
axis-cylinders of the nerve-fibres of the posterior root.

Arrangement of Nerve-Cells. — In the grey matter of the anterior
cornu, the large multipolar nerve-cells — cells of the anterior cornu —
are arranged in groups varying in different parts of the cord. Many
of their axis-cylinder processes pass out as axis-cylinders into the
nerves of the anterior root on the same side of the cord. These
cells are in relation with the fibrillar nerve-endings of the fibres of
the pyramidal tracts, and also with the collateral fibres of the pos-
terior root-fibres. In the upper dorsal and lower cervical regions
is a group of nerve-cells — intermedio-lateral tract — lying well for-
ward in a projecting part of the grey matter known as the lateral
cornu (fig. 317, Til). In the middle of the crescent is the middle
cell group. At the base of the posterior cornu, on its inner aspect,
is a group of large cells — best marked in the thoracic region —
Clarke's column (figs. 318, 319, CO/). The axis-cylinder pro-
cesses of the cells of Clarke's column pass into and become the
nerve-fibres of the direct cerebellar tract (p. 331). The cells of the
posterior cornu are small, and for the most part isolated.

Diffuse Nervous Grey Network. — The network or complex of
nervous fibrils in the grey matter of the cord is formed by — (i.)
the fibres and their branches of the 2nd type of nerve-cells (p. 330) ;
(2.) the fibrils and prolongations of the protoplasmic fibres of the
ist type; (3.) the lateral branches of the axis-cylinder processes of
the ist type ; (4.) the fibrils produced by the terminal arborisations of
the axis-cylinders entering the cord by the posterior roots.

Thus it is evident that some nerve-fibres spring from the cord
directly from nerve-cells, and others indirectly from the nervous
complex in the grey matter. Thus they are different, both morpho-
logically and physiologically.

By several lines of research- — including the facts of development,
experimental and pathological evidence — the anatomical columns of
the cord can be shown to be further subdivided. By these methods
it is found that the posterior column consists of a narrower internal
part, the postero-internal or postero-mesial column, or funiculus
gradlis or column of Goll, and an outer, postero-lateral tract or

XXX.] SPINAL CORD. 331

ftifucttltis cuneatuSj or column of Bur dad i. In the upper part of the
cord these two tracts are mapped off from each other by a septum
of connective tissue (fig. 317).

Tlic postero-wtfrnal tract is composed chiefly of rather small fibres,
derived from the fibres of the posterior roots and fibre of the pos-
tero-lateral column. They end above in grey matter in the nucleus
gracilis of the bulb.

The postero-lateral trad is chiefly composed of nerve-fibres of the
posterior roots which run in it a certain distance before they pass
into the grey matter and Goll's column. They end in grey matter
in the cord and in the nucleus cuneatus of the bulb.

In the postero-lateral column is a small zone which undergoes
descending degeneration, but only for a short distance, after section
of the cord — the so-called comma trad.

LixMuer's Tract, or " marginal bundle" lies near the entrance of
the posterior roots, either in the lateral column or postero-external
column. It is derived directly from the fibres of the posterior roots. ^
It undergoes ascending degeneration.

The antero-lateral column contains a large tract — the crossed
pyramidal tract — which lies external to the posterior half of the
grey matter, and in the greater part of its course is separated from
the surface of the cord by the direct cerebellar tract. It consists
of fibres descending from the central areas (motor,) of the cerebral
cortex, which have crossed at the decussation of the pyramids in
the bulb. It consists of moderately large and some small fibres.
Its fibres end by breaking up into fibrils, which form arborisations
around the nerve-cells in the anterior cornu. It gradually
diminishes in size as it passes down the cord, and can be traced as
far as the origin of the 3rd or 4th sacral nerve, where it reaches the
surface.

The direct pyramidal tract or column of Turck bounds the
anterior median fissure, and consists of fibres coming from the
motor areas of the cerebral cortex, which do not cross at the bulb.
This tract gets smaller, and gradually disappears about the mid-
dorsal region. Its fibres perhaps cross in the cord. These two arc
descending tracts.

Beginning at the lower dorsal region, and increasing in size as it
passes upwards, is a tract lying external to the crossed pyramidal
tract — the direct cerebellar or dorso-lateral tract — which con-
sists of large fibres derived from the cells of Clarke's column,
which pass up to the cerebellum, and enter it on the same side by
its inferior peduncle or restiform body. Near the surface of the
cord, and lying more anteriorly, is a tract which extends ventrally
into the anterior column — the antero-lateral awndinrj tract of
Gowers. Sometimes the term ventro-lateral is applied instead of

332 PRACTICAL HISTOLOGY. [XXX.

antero-laterul. It enters the cerebellum by the superior cerebellar
peduncle.

In the dog, as a result of excision of one-half of the cerebellum,
a circumferential tract, occupying three-fourths of the surface of the
antero-lateral tract — the antero-lateral descending cerebellar tract —
has been mapped out. The remainder of the antero-lateral column
is spoken of as the antero-lateral ground bundle.

As regards the results of degeneration following section or lesion
of the cord, those parts that undergo degeneration above the lesion
are called "ascending tracts"; and "descending tracts," are those
nerve-fibres that degenerate below the lesion or seat of section.
The parts which may undergo these respective degenerations are : —

( Direct pyramidal tract.
Descending \ Crossed pyramidal tract.

degeneration 1 Autero-lateral descending cerebellar tract (limited).
I Com ma tract.
( Goll's column.

Ascending \ Direct cerebellar tract.

degeneration, j Tract of Gowers.
L Tract of Lissauer.

It will thus be seen that in the antero-lateral columns there are
some nerve-fibres which do not undergo degeneration.

Course of Fibres of Nerve-Roots. — The fibres of the anterior
roots arise in several bundles from the axis-cylinder processes of the
multipolar nerve-cells in the anterior cornu.

The fibres of the posterior roots enter the cord by a single bundle,
but each one is an axial cylinder process of a nerve-cell in the
corresponding spinal ganglion. They pass into the postero-lateral
column, some pass into the posterior cornu, and a few small fibres
form the marginal bundle (p. 331). Many fibres pass up in Goll's
column, and the postero-external tract to end in terminal arborisa-
tions of fibrils around nerve-cells in the bulb, the fibres of Goll's
column in arborisations around the cells of the nucleus gracilis, and
those of the postero-external tract in the nucleus cuneatus. After
entering the cord, the fibres in the posterior columns bifurcate, one
branch passes upwards and one downwards (fig. 322). Collateral
fibres are given off from the original fibre, and also from its
branches,- which enter the grey matter and end by terminal arbori-
sations of fine fibrils which come into relation — but not direct union
— with the nerve-cells of the grey matter, notably with the colls
of Clarke's column.

THE SPINAL CORD.

It is convenient to begin with the cord of a small animal, e.//., a
cat or dog, but the student must also be provided with sections of

XXX.] SPINAL CORD. 333

the human cord. The same methods are applicable to both.
Remove the whole length of the spinal cord from a cat, taking care
not to squeeze or crush it in the process. Make transverse cuts
into it about f of an inch apart, and suspend it in a tall vessel in a
large quantity of Miiller's fluid, or 2 per cent, ammonium or
potassium bichromate. Keep it in a cool place in the dark.
Bichromate of ammonium hardens the cord very slowly indeed.
In fact, to get a properly hardened cord months are required. To
test if the cord is properly hardened, cut a section of the cord taken
from the ammonium bichromate fluid, place it in water, and if it
curls up, it is not properly hardened. It ought to remain flat.
The process may be expedited by hardening first for 4 or 5 weeks
in the bichromate, and completing the hardening for 2-3 weeks in
J per cent, chromic acid. Change the hardening fluid on the
second day, and repeatedly thereafter. After 4-5 weeks, when it
becomes tough, wash it, and harden in the various strengths of
alcohol, beginning with 50 per cent. If, however, the spinal cord
is to be used for Weigert's haematoxylin stain, it must not be
washed in water, but placed in alcohol direct from the Miiller's
fluid or potassic bichromate.

T.S. are made from the cervical, dorsal, and lumbar regions.
They may be made by means of the freezing microtome, the cord
having been previously saturated in the sugar and gum mixture ;
or small pieces of the cord may be stained in borax-carmine
(i week), and then cut in paraffin. Or they may be embedded and
cut in celloidin, and afterwards stained (p. 60). The paraffin'
sections are fixed to a slide by a "fixative/' the paraffin removed
by turpentine or xylol, the sections clarified by clove-oil, and
mounted in balsam. Sections, if made by freezing, may be stained
with carmine, hsematoxylin, aniline-blue-black, safranin, methylene-
blue, or by other methods.

1. T.S. Spinal Cord of Cat (L) and (H).— Speaking broadly,
the same general arrangement obtains as in fig. 314. Suppose it to
be the dorsal region, and the cord to be stained in carmine and
mounted in balsam, observe : —

(a.) (L) The nearly circular outline of the section, covered
externally by the pia mater, composed of two layers. From its
under surface septa run into the white matter of the cord, and some
of them carry blood-vessels, and processes pass into the fissures of
the cord.

(/>.) The anterior and posterior median fissures. The
anterior fissure is wider and better marked. In it run both
layers of the pia mater. The posterior fissure appears as a
septum due to the prolongation of the inner layer of the pia mater
into it, the outer layer of the pia runs over the fissure. The

334

PRACTICAL HISTOLOGY.

[XXX.

cord is thus almost completely divided into two symmetrical halves

(fig- 314).

(c.) If the section is made at the level of the origin of the
nerve-roots, these may be seen. The mode of origin of some of the
fibres of these roots can always be seen. The anterior root passes

A.M.F

P.M.F

FlG. 314.— T.S. Lower Dorsal Cord, Human. A. L, P. Anterior, lateral, and posterior
columns; A.M.F , P.M.F. Anterior and posterior median fissures; a, b, c. Nerve-
cells of the anterior horn; d Posterior cornu and substantia gelatinosa; e. Central
canal ; /. Veins , g. Origin of anterior nerve-root ; h. Posterior nerve-root ; i. White,
and j. Grey commissures ; if. Reticular formation.

out of the cord in several bundles, while the posterior root enters
the cord in one compact bundle.

(d.) The white matter externally, and the grey matter internally,
the latter deeply stained. The origin of the nerve-roots dividing
the white matter into the anterior, lateral, and posterior columns,
but owing to the anterior root leaving the cord in several bundles,
there is not an exact anatomical limitation of the anterior from the
lateral column.

(e.) The grey matter, deeply stained — a crescentic mass in each
half of the cord, with a broader anterior cornu, with numerous

XXX.]

SPINAL CORD.

335

large multipolar nerve-cells, arranged in groups — which does not
reach the surface of the cord, and a posterior cornu — with a few
small nerve-cells — which conies to the surface, and is prolonged into
the posterior root. At the hinder part of the posterior cornu, an oval,
deeply stained part, the substantia gelatinosa. A neck connecting
the two cornua. There is also a
group of nerve-cells placed laterally
in the grey matter, the vesicular
column of Clarice (fig. 318). In
the anterior horn, note especially
the entrance of the fibres of the
anterior nerve-root and the large
multipolar nerve-cells (fig. 315).

(/.) Connecting the two halves
of the cord, the anterior and
posterior commissures, running
between the grey matter of
opposite sides, and in the middle
between them the central canal,
which is surrounded externally
by a deeper stained layer of
neuroglia.

(g.) In front of the anterior
grey commissure is the anterior
white commissure, with large medullated fibres crossing each other
at an angle.

(h.) (H) The pia mater surrounding the white matter. The cut
ends of the medullated nerve-fibres of various sizes. In the centre
of each circular area the stained
axis-cylinder, surrounded by a
concentric clear area — the white
substance of Schwann (fig. 316).
In a preparation hardened in
chromic acid not infrequently a
number of concentric lines are
seen in the myelin. Between
the fibres here and there the
neuroglia, composed of glia cells.
On the surface of the cord there
is a thin layer, in which the fibrils
of the neuroglia can readily be seen.

tions, as fine septa, of the deeper part of the pia into the
siibstance of the cord. The larger septa carry blood-vessels.
The nerve-fibres of the antero-lateral columns are generally
larger, i.e., broader, than those of the posterior column, while those

FIG. 315.— Entrance of Anterior Root into
the Anterior Cornu of Lumbar Region,
i. Part of anterior white column ; 2.
Anterior grey matter with four multi-
polar cells ; a, a'. Two rootlets, x 30.

FIG. 316.— T.S. White Matter of Cord.
a. Peripheral layer ; b. Septum ; c.
Branched glia-cell ; the remainder
nerve-fibres, small and large, x 150.

Note also the prolonga-

336

PEACTICAL HISTOLOGY.

[XXX.

of the column of Goll are remarkable for tlieir small size. Measure
the sizes.

(i.) The anterior cornu, with large multipolar nerve-cells arranged

FIG. 317.— T.S. of Human Spinal Cord, Level of
Sixth Cervical Nerve. Prm. Middle cervical
process of the anterior cornu ; Til. Lateral
horn.

FIG. 318.— At the Level of the
Third Dorsal Nerve.

in groups, chiefly in the anterior and lateral parts of the cornu.
Each cell has numerous processes, a spherical well-defined nucleus
with a membrane and a nucleolus (fig. 202). Owing to the method

319.— At the Twelfth
Dorsal Nerve. CCl. Clarke's
column.

FlG. 320.— Level of Fifth Lumbar
Nerve. m. Median group of
nerve-cells of anterior-cornu ;
Iv, Id, and c. Latero- ventral,
latero-dorsal, and central groups
of cells, x 5.

of hardening, all the cells are somewhat retracted, each cell ap-
parently lying in a cavity.

(j.) Trace medullated nerve-fibres across the white commissure

XXX.] SPINAL CORD. 337

from the grey matter of one side to the white matter of the opposite
side.

(A-.) The posterior cornu. Note the absence of large cells.
Only a few small fusiform nerve-cells are seen. Some of the fibres
of the posterior root pass into the posterior cornu; some pass
directly through the substantia gelatinosa, and others sweep with a
curve through the posterior column before they enter the grey
matter. The grey matter generally is traversed by fine fibrils, and
has a somewhat finely granular appearance.

(I.) The central canal, lined by a single layer of columnar
ciliated epithelial cells. The cilia may be wanting. Near it sec-
tions of blood-vessels. In the grey matter observe the plexus of
fine fibrils and numerous axis-cylinders. (Multipolar nerve-cells,
Lesson XVIII. 9.)

2. T.S. Human Spinal Cord in several Regions. — Stain sec-
tions with carmine or lithium-carmine, hsematoxylin, or aniline
blue-black, and mount in balsam. Mount sections from the cervical
enlargement (fifth nerve) and lumbar enlargement, and compare
them with the dorsal section. With the naked eye (and L)
observe : —

(a.) The cervical and lumbar sections (figs. 317, 320) are not
only larger, but the amount of grey matter is greater than in the
dorsal region. Note the large expanded anterior cornu with
numerous nerve-cells. In the lumbar region the grey matter is
large in amount, the white matter smallest. The white matter is
more abundant in the douiftl region and most abundant in the
cervical region. This can, to a certain extent, be determined by
the amount of white matter lying between the grey matter and
the pia mater. There is a gradual transition from the one region
of the cord to the other.

(b.) In the cervical region note a thin septum, which dips into
the white matter and divides its posterior column into a smaller
internal part — the postero-intemal column or column or fasciculus
of GoU, and a larger external or lateral part — the postero-external
column, or fasciculus cuneatns. The grey matter, containing some
nerve-cells, also projects into the white matter about midway
between the anterior and posterior cornua, forming the inter mcdio-
lateral tract.

(c.) In the dorsal region, observe a group of nerve-cells at the
inner part of the neck of the grey matter. It lies behind the plane
of the central canal— column of Clarke, or posterior vesicular column

(ii-. 319, cat).

In sections of the cord at different levels it is important to note
the sectional area of the different columns. Thus, Coil's column is
largest in the upper cervical region, and diminishes from above
30 Y

338 PRACTICAL HISTOLOGY. [xxx-

downwards. The increase or decrease in the sectional area of some
other columns can only be made out by a study of degenerated or
developing cords.

3. L.S. of Cord (Antero-posterior direction). — Stain as for
T.S. (L and H.)

(a.) Observe the longitudinally-arranged nerve-fibres in the white
matter of the anterior and posterior columns. In the anterior
cornu the rows of multipolar nerve-cells.

(b.) The anterior roots passing obliquely through the anterior
column.

(r,.) The posterior cornu, with its gelatinous substance and nerve-
fibres passing into and through it.

4. Weigert's Hsematoxylin Method.1 — The myelin of medul-
lated fibres is stained of a deep blue-black tint, while degenerated
parts are not so stained; they are lighter just in proportion to
the disappearance of medullated fibres. The spinal co^d, or the
brain, is hardened for four to six weeks in Miiller's fluid, and the
hardened pieces are placed direct into 70 per cent, alcohol, and
must not be washed with water. Keep in the dark. Next day
add 90 per cent, alcohol, and harden completely in 95 per cent, or
absolute alcohol. Instead of Miiller's fluid, Erlicki's fluid may be
used. This is best done by keeping the fluid at the temperature
of the body in a warm chamber, when the hardening process is
completed in a few days ; but the results are not so satisfactory as
in the case of a cord hardened in the ordinary way. Small pieces
of the hardened tissue are placed in^a half-saturated solution of
neutral acetate of copper (i.e., a saturated solution is mixed with
its own volume of distilled water), where they remain for three to
five days. They are then transferred direct to 90 per cent, alcohol,
and can then be cut in alcohol, or the pieces can be embedded in
celloidin and then cut (p. 47).

(A. ) The sections are placed for two or three hours in a few cc. of
Weigert's heematoxylin, in which they become black.

Weigert's Hsematoxylin.

Hrematoxylin . . . . . I pram.

Alcohol absolute . . . . 10 cc.

Cold saturated solution of lithium carbonate I ,,

Distilled water . . . 90 „

Dissolve the haematoxylin in the absolute alcohol, add the water
and boil. After it is cool, add the lithium carbonate. The time
which the sections remain in this fluid depends on what it is

1 Fortschrit. d. Mcd., 1884 and 1885; Zeits. f. wissensch. MLTc., 1884 and
1885.

XXX.] SPINAL CORD. 339

desired to show. Two hours or so are enough for the cord, but
if the fine plexuses of medullated fibres — described by Exner — in
the cerebral cortex are to be well seen, let them stain for twenty-
four hours. After they are sufficiently stained, throw the watch-
glass and the stained sections into a large basin of distilled water.
Remove the sections from the water at once, and place them, for
about half an hour or more, in the following mixture : —

Potassic ferricyanide . . . 2.5 grams.

Borax . . . . . 2 ,,

Water ..... 100 cc.

This fluid decolorises and differentiates the black sections. The
grey matter becomes of a brown or light-yellow tint, and should
remain so while the white matter becomes violet. The sections
must remain in the decolorising fluid until the deep blue or violet-
coloured medullated nerve-fibres are seen. This can readily be
determined after a little practice. The sections are then washed
in water— in which they can be kept for a considerable time —
transferred to 90 per cent., and finally to absolute alcohol, clari-
fied by xylol or origanum oil, and mounted in xylol-balsam. This
method stains the medulla or myelin of the nerves — especially of
the central nervous system — of a deep blue or violet tint, while the
nerve-cells, neuroglia, and axis-cylinders are not stained. The
method, however, may be combined with staining methods. After
the organ is hardened in the chromium salt and alcohol, small pieces
are embedded in celloidin and cut in a microtome. The sections
are placed in 80 per cent, spirit — not water. The celloidin enables
the sections to be readily handled and transferred to a slide. They
are then stained in the special haematoxylin fluid already described.
If a series of sections is to be mounted on the same slide, see the
method described at p. 60. The sections are clarified in origanum
oil or a mixture of xylol and carbolic acid (p. 83). Mounted in
balsam. This method is also applicable to the spinal ganglia.

(B.) Freezing Method.— The piece of cord or brain, after harden-
ing in M tiller's fluid, is placed in spirit and transferred to a mixture
of equal parts of absolute alcohol and ether, and then embedded in
celloidin. The embedded tissue is placed for forty-eight hours in
Erlicki's fluid, to get rid of the spirit, and they are then placed in
the following mixture, and kept in stoppered bottles in a warm
chamber at 38° C. for two or three days : —

Cupric sulphate . .... 0.5 gram.

Potassic bichromate .... 2.5 grams.
Mucilage of syrup and gum . . . 100 cc.

Sections are cut in a freezing microtome and received into Krlicki's
fluid, washed in methylated spirit, stained in the hamiatoxyliii fluid,

340 PRACTICAL HISTOLOGY. [XXX.

decolorised by the ferricyanide mixture, clarified, and mounted in
balsam (Hamilton).

Stages of Weigert's Method.

(1.) Harden nervous system in Miiller's fluid.

(2. ) Harden in alcohol without previous washing in water.

(3. ) Embed in celloidin.

(4.) Place celloidin block in a half-saturated solution of copper acetate

(24-48 hours).

(5.) Alcohol, 70 per cent. (24 hours).

(6.) Cut sections and stain them in Weigert's hfematoxylin (24 hours).
(7.) Wash in water.
(8.) Partially decolorise in ferricyanide fluid until grey matter becomes

yellow.
(9.) Wash in water, dehydrate in absolute alcohol, clarify in xylol, and

mount in xylol-balsam.

Pal's method is referred to on p. 343, but the following combina-
tion of the Pal and Weigert method gives good results : —

5. Modified Weigert-Pal Method.

(i.) Harden in Miiller's fluid and then in alcohol, without wash-
ing pieces in water.

(ii.) Embed and cut sections in celloidin.

(iii.) Wash in water and transfer to Marchi's fluid (5-10 hours),
(iv.) Wash and transfer for 10-16 hours to

Kultschitzky's Hsematoxylin.

Hfematoxylin ..... i gram.
Absolute alcohol .... 2-5 cc.

Dissolve, and to the fluid add

Acetic acid (2 per cent.) . . . 100 cc.

The sections become black.

(v.) Bleaching process — (a.) Wash in water and bleach for 5
mins. in .2 per cent, permanganate of potash. Wash
again, and transfer to

(b.) Pal's solution (p. 344), in which they are rapidly (5-10
mins.) bleached, the medullated fibres alone remaining
black.

(vi.) Wash in water, dehydrate in alcohol, balsam, oil (xylol),
balsam (Schafer).

6. Weigert-Stained Cord.— (a.) (Land H) Observe the medulla
of the medullated nerves, stained purplish. There are so many fine
medullated nerve-fibres revealed in the grey matter that it is impos-
sible to reproduce their complexity in a woodcut. The nerve-cells
are not specially in evidence, although they can be stained with
picro-carmine.

XXX.] SPINAL CORD. 34!

Formerly Weigert used acid-fuchsin for staining the medullated
nerve-fibres of the central nervous system, but this method is now
given up in favour of the haematoxylin copper method.

7. Neuroglia (H). — Stain in safranin (24 hours) thin sections of
the white matter of the cord hardened in M tiller's fluid, partially
decolorise in absolute alcohol, and mount in balsam.

(a.) Observe the branching neuroglia-cells between the white
nerve-fibres (fig. 316, c). The connective-tissue elements have a
tint more towards the violet, and are thus differentiated from the
nervous elements. The network of neuroglia-fibres is readily seen
near the surface of the cord.

8. Blood- Vessels of the Spinal Cord. — Mount a transverse sec-
tion of the cord, with its blood-vessels injected. Cut the cord in
paraffin. The sections must be rather thick (L and H). Inject the
animal, e.g., cat or rabbit, from the aorta with a blue or red mass.
Harden in alcohol.

(a.) Observe the greater vascularity of the grey matter as com-
pared with the white. A blood-vessel may be seen running into the
anterior median fissure, and at the bottom of it dividing, and giving
a branch to each mass of grey matter.

(b.) The dense plexus of capillaries in the grey matter. Branches
of blood-vessels passing into the cord along the roots of the nerves
and along the larger septa, which pass from the pia mater into the
cord.

9. Nerve-Fibres of the Spinal Cord (H).— Crush a piece of the
white matter of the cord, either fresh, or after maceration in ^ per
cent, bichromate of potash, between a cover-glass and a slide.

(a.) Observe the nerve-fibres, many of them with lateral bulgings,
or presenting a beaded or moniliform appearance. This is due to
the fact that these nerve-fibres are devoid of a primitive sheath.

(6.) Droplets of "myelin" with concentric markings are seen in
the field.

10. Nerve-Fibres of the Spinal Cord (H).— By means of a
hypodermic syringe make an interstitial injection of I per cent,
osmic acid into the white matter of the antero-lateral column of the
spinal cord of an ox. Tease a piece in glycerine.

(a.) Observe tubes of different sizes, many varicose, with incisures
and cylinder- cones, but no primitive sheath0

11. Staining the Cord. — To stain sections the following dyes
may be used : —

(i.) Ordinary carmine, picro-carmine, or acid-carmine. In using
the last, use a very dilute (scarcely coloured) solution, and let the
sections remain in the solution for 1-2 weeks. (2.) These stains
may be combined with hsematoxylin. (3.) Benzo-azurin. (4.)
Aniline-blue soluble in water but insoluble in spirit. (5.) The same

342 PRACTICAL HISTOLOGY. [xxx.

blue with eosin or Magdala-red. If (5) be used the axis-cylinders
are blue, the myelin rose, and there is also a sharp distinction in
colour between the nerve-cells and glia-cells. (6.) AVatery solution
of Congo-red. The sections are dipped for a moment into very
dilute sulphuric or hydrochloric acid (i drop to 10 cc. water).
They become blue. (7.) Weigert's hamiatoxylin-copper. (8.)
Golgi's silver and mercuric-chloride method. The M tiller's fluid
should contain 3—3.5 grams of potassic bichromate to i gram of
sodic sulphate. (9.) Golgi's silver method and sections stained by
Magdala- red (Lavdowsky).

12. Tracts in the Spinal Cord. — These are made out histo-
logically by studying (a.) embryonic cords in mammals from the 5th
to the Qth month. These sections are stained either by Weigert's
method (p. 338) or by the modified Pal method. A T.S. of the
cord of a human foetus just before birth shows the nerve-fibres in
the pyramidal tracts still devoid of myelin, and thus they are easily
mapped out from the other parts of the cord which are already
medullated.

(b.) The degeneration changes resulting after hemi-section or
section of the cord. Section of the cord is practised say 6-10 days
before the cord is required. Parts of the cord above and below the
seat of injury are hardened for 10 days or so in Miiller's fluid.
Thin pieces are then placed for several days in Marchi's fluid

(P- 347)-

Sections are mounted in balsam. All the nerve-fibres which
have undergone degeneration are stained black ; healthy .nerve-
fibres are yellowish (p. 347). Or instead, Weigert's process may be
used, as then the degenerated tracts remain unstained owing to the
absence of myelin.

ADDITIONAL EXERCISES.

13. Dry Preparation. — Stain a T.S. with metliylene-blue (i per cent).
Wash it, and allow it to dry on a slide. Add a drop of balsam. This shows
very well the general characters of the cord ; the multipolar nerve-cells arc
somewhat shrunken, but still they and their processes are well stained.

14. Sections in Ehrlich-Biondi's Fluid. — Place sections in tins fluid (p. 81)
well diluted (i : 40), and heat them in a watch-glass until vapour is just given
off. Mount thern in balsam, clarifying either with xylol or aniline-oil and
xylol (Lesson III. 16). The glia-fibrils are violet in tint, and so is the con-
nective tissue generally ; the nuclei of the glia-cells bluish, the myelin orange,
the axis-cylinder somewhat violet The cells in the grey matter have a
pleasant violet tint. Benzo-azurin can be used in the same way.

15. T.S. Cord. Eosin and Logwood. — The neuroglia-cells, connective-
tissue, and epithelium of the central canal have a logwood tint, the other parts
are rosy.

XXX.] SPINAL CORD. 343

16. Staining the Cord in Bulk in Aniline Blue-Black. — Small pieces of the
cord are placed iu the following fluid for a day or two : —

Aniline blue-black . . .2 grams.

Water. . . . 60 cc.

Alcohol . . . . 40 ,,

Mount the sections in balsam.

17. Transverse Markings on Axis-Cylinders and Nerve-Cells. — Place small
pieces of a perfectly fresh cord in I per cent, silver nitrate solution, and keep
them in the dark for forty-eight hours, renewing the fluid several times.
Wash the pieces and place them, exposed to light, in the following mixture : —
Formic acid ( I part), amylic alcohol (i part), and water (100 parts), for 5-7
days. Tease a fragment in glycerine and observe the alternate brown and
clear markings on the axis-cylinders (Fronimaim's lines) and on the nerve-cells
(JaJcimovitch).1

18. Isolated Neuroglia-Cells. — (a.) These are obtained by the interstitial
injection of osmic acid into the white matter of the cord (Lesson XXX. 10.)
A small piece is teased and stained with picro-carmine.

(H) Observe the branched cell, with a granu-
lar body and long processes (fig. 321). It re-
quires considerable care to dissociate such a
cell, and it must usually be looked for and
isolated with the aid of a dissecting microscope
(p. 22).

(b.) In sections of the cord prepared by
Golgi's method, neuroglia-cells may be stained
along with the nerve-cells, and on other occa-
sions they may be the only elements on which
the silver or mercury takes effect. Note that
each cell gives off many very fine processes. FIG> 32I.— isolated Neuroglia-Oll
Some of the latter may be seen to become of Spinal Cord of Ox. n. Nu-

(c.) Isolated glia-cells may be found after

maceration in Landois' fluid (p. 26), and subsequent staining with Magdala-
red.

19. Isolated Nerve-Cells of the Cord. — The best dissociating reagents are
dilute alcohol or Landois' fluid (3-4 days, p. 26). A staining fluid may be
added to the dilute alcohol, and thus dissociation and staining go on simul-
taneously. With cells — either nerve or glia — isolated by Landois' fluid, it is
better to stain after maceration. The best stains are Magdala-red, methyl-
blue II. (0.5-1 per cent., 5-10, drops added to 10 cc. of the macerating fluid).
Lavdowsky'2 uses a "semidesiccation method" like that used for connective
tissue ; the isolated cells are allowed to become nearly dry, and then alcohol
is slowly added to remove the remainder of the water (see also Lesson XVIII.).
Or after maceration shake the tissue in a very small quantity of water in a test-
tube, and place it in a watch-glass, add 6-10 drops of glycerine and a little
picro-carmine, and dry the whole over a sulphuric acid desiccator. This
removes all the water, and one has then the cells stained in glycerine ready to
be mounted.

20. Pal's Method of Staining Nerve-Fibres. — This method requires consider-
able care. Harden the cord or brain in Miiller's fluid, makeT.S., and place
them in alcohol. Stain iu .75 per cent, watery solution of hnematoxylin con-

1 Journ. de TAnat. ct de la Phys., xxiv. p. 142, 1888.

2 Archivf. mik. Anat., xxxviii. p. 264.

344 PRACTICAL HISTOLOGY. [xxx.

taining some alcohol and a few drops of a saturated solution of lithium
carbonate (6-10 hours). They are then "differentiated" in £ per cent, solu-
tion of permanganate of potash (10-15 seconds), and subsequently in the
following mixture : —

Oxalic acid . . . I gram.

Potassic sulphite , . i ,,

Distilled water . „ . 200 cc.

When the grey and white matter are differentiated, the sections maybe stained
in carmine, safranin. or alum -carmine, and mounted in balsam.

Stages of Pal's Method.

(1.) Harden in Miiller's fluid.

(•2.) Cut sections and stain in Weigert's hsematoxylin (24-48 hours).

(3.) Wash in water to which 1-2 per cent, lithium carbonate is added. Thi
sections must be deep blue.

(4.) Differentiate the sections in 0.25 per cent, potassic permanganate solu-
tion (20-30 sees. ) till grey matter becomes yellow.

(5.) Transfer to oxalic acid solution (few sees.).

(6.) Wash in water, and dehydrate in alcohol, xylol, balsam.

This method can be done rapidly. Only the nerve-fibres are stained ; the
intervening parts may be stained subsequently with picro-carmine.

21. Vessale's Modification of Weigert's Method. l

(1.) Harden in Miiller's fluid and afterwards in alcohol.
(2.) Stain celloidiu sections (3-5 mins.) in I per cent, hfematoxylin
dissolved in warm water. Allow it to cool. The sections
become black.
(3. ) Place in saturated filtered solution of neutral copper acetate (3-5

mins.) and then lave in water.
(4.) Differentiate in a solution —

Borax ... 2 grams.

Potassic ferridcyanide . 2.5 ,,

Water , . . 300 cc.

Ganglion-cells, glia-cells, and degenerated parts soon become decolorised,
only the medullated fibres remain dark-violet.

(5.) Wash thoroughly in water. They may be stained with alum-
carmine as a contrast stain.

(6 ) Absolute alcohol. Xylol-carbolic acid (3 : i), remove surplus with
bibulous paper. Balsam.

22. Kulschitzky's Method for Medullated Fibres.2

(1.) Harden in Miiller's or Erlicki's fluid (1-2 mins.), then wash in

water (1-2 days). Alcohol. Make sections in celloidin.
(2.) Stain (1-3 hours) in his acid haernatoxylin (p. 342).
(3.) Differentiate (2-3 hours) in

Sat. sol. lithium carb. . . 100 cc.

Ferridcyanide of potash ( i per cent.) 10 ,,
(4. ) Wash thoroughly in water. Balsam.

23. Golgi's Silver Methods3— (a. ) Slow Method.— Small parts of the cord or

1 Zeit. wiss. Mikros., vii. p. 517, and Archiv. itol. de Biol.t xv. p. 158,
1891.

2 Annt. Anzeig., iv. p. 519, 1890.

3 Sulla, fina anatomia degli organi centrali del sistema iiervoso, Milano, 1886.

XXX.] SPIXAL CORD. 345

brain, after being hardened in 2 per cent, potassium bichromate for 3-4 days,
are then transferred to a stronger solution, say 3 per cent., for 4 days. In-
crease successively the quantity of the bichromate until 4-6 per cent, is
reached. It takes 30-50 days, according to temperature or other circum-
stances, to get the tissues properly hardened. The hardened parts are then
transferred for 24-48 hours to .5-. 7 5 per cent, silver nitrate. It is better to
place them first of all in a small quantity of silver nitrate, and to wash them
in this fluid. The fluid becomes of a dark orange tint from silver chromate.
Place the pieces in fresh silver nitrate. Harden in alcohol. Make hand
sections, clarify them in the usual way, and mount in balsam, but do not apply
a cover-glass.

(b.) Rapid Method. — Make the following mixture : — •

Osmico- Bichromate Mixture.

Potassic bichromate (3 per cent.) . . 20 cc.

Osmic acid (i per cent.) . . . 5 ,,

Place small pieces of the fresh organ in 15 cc. of this fluid for 2-3 days, and
then place them in the silver solution (.5-1 per cent.; for 1-2 days. I have
found the cells of the cerebral cortex stained black two or three hours after
immersion of the organ in silver nitrate.

This is an excellent method, specially useful for young or embryonic nervous
system, as the fluid penetrates more readily where the myelin is scanty. It is
very capricious in its action. Sometimes only small parts are stained. The
nerve-cells and their processes, and nerve-fibres without myelin, and axis
cylinders are stained black.

(c. ) Medium Method. — Small pieces of the fresh tissue are placed for 3-5 days
in 3 per cent, potassic bichromate ; then 3-4 days in the osmico-bichromate
mixture as above, and then in silver nitrate.

It is not a matter of indifference which method is used. Golgi's method
only stains certain elements — nerve-cells, neuroglia-cells, and sometimes
blood-vessels. Thus, by the rapid method, in the case of the cerebellum, the
parallel fibres and the granular layer are the chief parts stained; Purkinje's
cells stain best by the slow method. As a general rule, the axis cylinders and
their processes stain best by means of the rapid and medium methods, the
slow method, as a rule, staining best the protoplasmic processes.

The rapid silver method has been extensively used by Golgi and his pupils,
by Kb'lliker, but above all by Ramon, y Cayal. The black deposit Golgi calls
a " black reaction."

24. Golgi and Mondino's Sublimate Method. — Small pieces of the central
nervous system, after hardening in bichromate of potash, are placed in a
watery solution (.25 per cent.) of corrosive sublimate. The volume of fluid
must be large, and renewed frequently, j,.e., as often as it is yellow. After
10-15 days the reaction has occurred in small pieces ; but it is better to expose
the pieces longer than this to the action of the salt ; in fact, prolonged im-
mersion rather improves it. The tissues may remain for months in the fluid
without disadvantage. The sections must be very thoroughly washed, else,
after being mounted, needle-shaped crystals of the sublimate are apt to form.
The sections are mounted in balsam or glycerin ; only they are mounted with-
out a cover-glass.

In Golgi's silver method the cells, and in some preparations the blood-
vessels as well, are opaque and black. Sometimes the body of the cell and its
finest ramified processes can be seen with the utmost sharpness. The silver is
deposited only on the cells and their processes, not on the nerve-fibres. The
sections, however, are often dotted over with a black metallic deposit. In the
mercury preparations, if the cells do not appear to be very black, they may be
darkened by washing them in sodic sulphite.
31

346 PRACTICAL HISTOLOGY. [XXX.

The sublimate may act on (a.) the ganglionic cells, (5.) the neuroglia cells,
and (c. ) the blood-vessels. The best results with these methods are obtained
with the cerebral cortex. The sections can be stained afterwards by the usual
methods, and particularly by Magdala-red.

The parts acted on by the sublimate are white by reflected light, and appear
black by transmitted light, because they are opaque. Golgi1 finds that for the
study of the diffuse network in the central nervous system, by this method the
best results are obtained when the " metallic white " is converted into a
metallic black. For this purpose he uses the "fixer" employed for. fixing
positive photographs on aristotypic paper.

(A.) Water. . ' . . . I litre.

Hyposulphite of soda . . . 175 grains.

Alum . . . . 20 ,,

Ammonium sulphocyanide . .10,,

Chloride of sodium . . . 40 „

Leave the mixture for 8 days and then filter.

(B.) Water, . . . . 100 cc.

Gold chloride. . . i gram.

For the toning and fixing fluid, mix of

A . . . . 60 cc.

B 7 „

Procedure.

(1.) Wash in distilled water.

(2.) Immerse sections (1-2 mins.) in the above mixture. The sections

become black.

(3.) Prolonged washing in water.

(4.) (Optional.) Faint coloration of the sections with acid carmine.
(5.) Wash again, then mount in balsam.

25. Double Impregnation Method of Eamon y Cayal.2 — Morsels of tissue
are placed in the dark in the osmico-bichromate mixture as for Golgi's rapid
method (2-3 days), and then they are gently washed and placed in .$-.75
per cent, silver nitrate (1-2 days). They are retransferred for 3-4 days to the
osmico-bichromate fluid (3-4-5 days), and then again to silver. They are then
hardened in alcohol and cut. This method certainly gives good results in
some cases, and is very useful for the brain and embryo preparations.

26. Embryo Cords. — (a.) Harden the spinal cord of an embryo chick at the
9th day as above. Remove as much as possible of the surrounding vertebral
column before placing it in the osmico-bichromate mixture.

(L) Observe the axis-cylinders of the fibres of the anterior roots springing
from the axis-cylinder processes of the multipolar branch cells of the anterior
cornu, themselves black. Note that the fibres of the posterior root enter the
cord and split up into fibrils.

(b.) Collateral Fibres. — Harden the spinal cord of an embryo mammal (e.g.,
sheep, 20-25 cm. long) or embryo of chick in the above fluid, and make L.S.
in the line of entrance of the posterior roots. The sections need not be very
thin, but one may have to make several preparations before one gets a satisfac-
tory result.

(H) Note that when a fibre of the posterior root enters the white matter
of the cord it divides, sending one branch upwards and one dowmvards (fig.
322), which run for a distance more or less longitudinally, but many enter the

1 Archiv. ital. de Biologic, xv. p. 462, 1891.

* Internat. Monatsch. f. Anat. u. Phys., vi. p. 170.

XXX.]

SPINAL CORD.

347

grey matter, and end free in fine branches without forming connections with

nerve-cells. The fibres give off at

right angles to their course fine fibrils

— collaterals — which enter the grey

matter, divide into fibrils, and end

free.

27. Degeneration of the Cord —
(a. ) Method of March! for degenera-
tion, in central nervous system or in
nerves (Lesson XVII.) : —

(1.) Harden very small pieces of a
nerve or spinal cord — either of
which is undergoing degenera-
tion— in Miiller's fluid for at
least 8 days.

(2.) Place for 6 days in the follow-
ing :—

Miiller's fluid . . 2 parts.

Osmic acid (i per cent.) I part.

(3.) Wash in water, harden in

alcohol.
(4.) Embed and cut in celloidiu.

All degenerated parts appear black,
all the others light grey or yellowish
(see p. 212).

(6.) Wcigert's hsematoxylin method FIG. 322.— L.S. of the Cord of the Cervical

may be used for the same purpose. Region of an Embryo Sheep (22 cm long),

rp, J i i C1 to show division of posterior roots after

The degenerated fibres are unstained, entering the spinal cord.
i.e., on differentiating the section
the degenerated purls rapidly give up their stain, and thus appear unstained.

LESSON XXXI.

MEDULLA OBLONGATA— CEREBELLUM-
CEREBRUM.

MEDULLA OBLONGATA OE BULB.

THE medulla oblongata is hardened in the same way as the cord.
T.S. are made at different levels, and stained in the same way
as the cord (L and H).

1. T.S. Decussation of Pyramids.

348

PRACTICAL HISTOLOGY.

[XXXI.

(a.) Observe the shape of the cord, the decussation of the anterior
pyramids, i.e., bundles of fibres are seen coming from the lateral
column of one side, and running inwards and towards the middle
line, thus separating somewhat the anterior from the posterior

corima (fig. 323). They actually
pass to the anterior pyramid of
the opposite side of the medulla.

(b.) At the anterior part, on
each side of the anterior median
fissure, sections of the anterior
pyramid.

((•.) At the posterior part, a
small part of the posterior cornu
extending backwards as the
nucleus of the clava in the
FIG. 323.-T.S. Medulla Obiongata through funiculus gracilis. Another mass

Decussation of Pyramids. D.Py. An- OI grey matter in the IliniCUlus
terior pyramid ; Fa. Anterior Cornu ;
Ng. Nucleus of the funiculus gracilis ;
g. Substantia gelatinosa ; XI. Spinal

2. T.S. Of Oil Vary Bodies (L

and H). — Observe the folded mass
of grey matter, with many multipolar nerve-cells, constituting the
olivary nucleus (fig. 324); the complex of fibres, horizontal, vertical,

and those cut longitudinally,
constituting the formatio
reticularis ; the much altered
arrangement of the grey
matter, which appears in the
floor of the fourth ventricle ;
and, according to the level
at which the section is made,
there may be met with the
origin of certain of the cranial
nerves (fig. 324).

Of course sections should
be made from higher levels
in the medulla, and also

through the pons. If a student has the requisite time, it is best to
make a series of sections from below upwards, fixing them in order
on slides.

The two following tables show, the one the fissures, areas, and
mouldings to be noted on the medulla oblongata, and the other the
grey matter of the cord and medial a oblongata.

.FIG. 324.— T.S. Medulla Oblongata at the Level
of the Olivary Body ; partly Diagrammatic.

XXXI.]

MEDULLA OBLONGATA.

349

Objects seen on the Surface of the Medulla Oblongata.

( Anterior median.
A. Fissures. -I Posterior median.
iTwo lateral.

I. Anterior area
(between A

B. Areas.

rior area |Anterior pyramids.

.M. F. and |Decussat£i of 1)yramids.

^^2?.« iQUroyboay.

and

fillet.

3. Posterior-
area.

C. External Arcifonn Fibres.

( Restiform body.

Lower l Fuuiculus of Rolando and its tubercle.
Part. ) Funiculus cuneatus and its tubercle.

( Fuuiculus gnicilis and its clava.

F1°°r °f f°Urth ventricle'

Table of Grey Matter of the Medulla Oblongata.

CORD. MEDULLA.

, Nucleus lateralis.

Anterior part of formatio reticularis.
Nucleus of the fasciculus teres.

Nucleus of Rolando.

Posterior part of formatio reticularis.
( Nucleus of the funiculus gracilis (clava).
-I Nucleus cuneatus.
1 Nucleus on floor of fourth ventricle.

^Hieel!

Cornu' (Base

Grey Matter

of the

fHead

Spinal Cord. Po8terior Neck

1 Comu- 'iBase

I I

Grey
in the Medulla.

Nucleus of external arciform fibres.

CEREBELLUM.

Methods. — The cerebellum is hardened in the same way as the
cord. It is advantageous, however, to wash out the hlood-vessels
of the whole brain with Miiller's fluid, and afterwards to distend
them with the same fluid.

If ammonium bichromate be used (2 percent.), the pieces harden
quicker than in the case of the cord.

For the application of Golgi's method, see p. 345-

3. V.S. Cerebellum, i.e., including the grey and white mattr-

350

PRACTICAL HISTOLOGY.

[XXXI.

cut at right angles to the direction of the folds. Stain it with
carmine, aniline blue-black, or, better still, first with logwood and
then with eosin. Mount in balsam. The best way for the student
is to stain a small piece of the cerebellum in bulk in borax-
carmine for two or three days, or longer, and then embed and
cut in paraffin. In this way there is no fear of the sections break-
ing up, and the relative positions of the several parts are accurately
maintained.

In making sections of the cerebellum, it is important, if one
wishes to see the wide expanse of the protoplasmic processes of
Purkinje's cells, to make sections across the direction of the laminae.
If made in the direction of the laminae the leash of protoplasmic
processes appear quite narrow (fig. 330, B). The protoplasmic
processes, or dendrites, as they have been called by His, spread out
in planes transverse to the direction of the lamella?, hence the
necessity for the above precaution.

Suppose a hsematoxylin-eosin specimen to be prepared.

(a.) (L) Observe the primary and secondary convolutions (fig.
325). In each leaflet from within outwards, the white matter,

FIG. 325.— Leaflet of the
Human Cerebellum,
x 10.

FIG. 326. — Cortex of the Cerebellum, x 90.
«. Outer; b. Inner or granular layer:
p. Cells of Purkinje.

composed of medullated nerve-fibres, and outside this the grey
matter, composed of two layers, viz. :— (i.) The nuclear layer,
composed of many • layers of small nuclei — stained blue — eacli
surrounded by a very small quantity of protoplasm. (ii.) The
outer layer of the cortex, thicker than (i.), with a somewhat
granular appearance, and containing branches of Purkinje's cells
and some small branched angular nucleated cells (fig. 326).

XXXI.] CEREBELLUM. 351

Numerous blood-vessels enter the surface of the cerebellum from
the pia mater covering it.

(b.) At the boundary-line between (i.) and (ii.) a row of large
cells — Purkinje's cells — each with a somewhat oval or globular
body, with a single central process, which becomes continuous with
a nerve-fibre, although this is not seen in this preparation. Each
cell gives off a peripheral process, which immediately branches, the
larger branches running laterally for a short distance, arid each
branch divides again and again, the fine branches — protoplasmic
processes or dendrites — running vertically through the outer layer
of the cortex nearly to its free surface. The branched arrangement
of these fibres has been compared to the antlers of a stag.

By means of the rapid Golgi method, a basket-shaped complex
of fibrils can be seen round the basis of Purkinje's cells.

(c.) (H) The granular or nuclear layer, with two kinds of nuclei
or rather cells. The most numerous are small and granular, and
arranged in groups ; they are stained violet by the haematoxylin.
The others (larger and spherical, with a nucleolus) are the nuclei
of small ganglionic nerve-cells. They are stained reddish. Amongst
the granules may be seen medullated fibres stained reddish.

The more recent methods of Golgi and Ramon y Cayal show
that these two kinds of cells correspond to nerve-cells. The
small cells have an axis-cylinder and several protoplasmic pro-
cesses, while the large cells in some respects resemble small
1'urkinje's cells.

4. Blood- Vessels of the Cerebellum. — These are injected when
the blood-vessels of an animal are injected from the aorta with a
Berlin-blue or carmine-gelatine mass. Mount an unstained sectin
in balsam (L and H).

(a.) Observe the vascular pia mater sending at intervals long or
medullary branches through the cortex to the medulla, and short
or cortical branches which break up into a rich plexus of capil-
laries in the cortex, so that the latter is far more vascular than the
white matter. Each vessel is surrounded by a perivascular lymph-
space.

CEREBRUM.

Methods. — Harden it in the same way as the cord and cerebellum.
For Golgi's methods (p. 345).

If Weigert's method is used, the sections must remain for twenty-
four hours in the hamiatoxylin solution in order to see the plexus of
medullated fibres in the superficial layers of the cortex, and three,
hours for the fibres which ascend between the pyramidal cells of
the cortex cerebri.

5. V.S. Cerebrum.— Make V.S. through the central part of the

352

PRACTICAL HISTOLOGY.

[XXXI.

brain of a small animal. In the case of a human brain, select the
ascending parietal or ascending frontal convolution. Make V.S.
either by freezing or in paraffin after staining in bulk in borax-
carmine. Other sections may be stained in aniline blue-black, and
all are mounted with balsam.

"With the naked eye observe the shape of the convolution, the
grey matter outside, of a certain thickness (y^-i in. thick), and
deeply stained, and inside it the white matter less deeply stained.

(L and H) (a.) Observe the white matter, composed of medul-
lated fibres, with leucocytes here and there between them.

(b.) Outside this the grey matter, composed of several layers,
recognised by the arrangement and shape of the cells present in it.

FlG. 327- — y.S. of Cortex of the
Ascending Frontal Convolution,
i.e., a Motor Area. Carmine,

FlQ. 328.— V.S. Middle Frontal
Convolution. Carmine, x 20.

It will depend very greatly upon the plane of the section whether
the student sees all the layers in any single section. They are
usually in a five-layer type, but the relative thickness of the layers
varies in different parts of the cerebrum.

A. Arrangement of Nerve-Cells.

(c.) The layers from the surface inwards are : —

(i.) The narrow outer or first layer (or finely-granular or mole-
cular layer) consists of a network of fibrils with a very few small
cells. Chiefly neuroglia cells, mostly vertical to the surface.

By Golgi's method, it can be shown to contain a layer of medul-
lated fibres just under and parallel to the pia, and also some
branched non-medullated fibres. It also contains a few small
nerve-cells with two or more axis-cylinder processes. The latter
details can only be detected in a preparation made by Golgi's

XXXI.] CEREBRUM. 353

method. In it may be seen sections of blood-vessels passing from
the pia.

(ii.) The second layer, or layer of small pyramidal cells, is usually
narrow, with several rows of small pyramidal cells, the peripheral
processes of the latter pointing towards the surface of the convolu-
tion. It passes gradually into

(iii.) The third layer, or layer of large pyramidal cells, which is
much thicker than the others, and contains large pyramidal cells,
each with a peripheral process, which can sometimes be traced
outwards for a considerable distance. The axis-cylinder process
gives off several collaterals, and can be traced into a medullated
fibre of the white matter. The cells, however, may be cut obliquely,
and then they appear triangular. Usually the cells are larger in
the deeper layers, and become smaller in the outer layers.

(iv.) Tk& fourth layer, or layer of irregular or polymorphous cells.
This is a narrow layer of small, usually angular but irregularly-
shaped cells. They lie between the nerve-fibres which pass into
the cortex. In the motor areas of the frontal and parietal convolu-
tions large pyramids arranged in groups or nests may be found
between these cells.

(v.) T\\Q, fifth layer, or layer of fusiform cells, also a narrow layer,
with a few fusiform cells with nerve-fibres between them. This
layer abuts on the white fibres of the medulla, and may in some
regions be fused with the previous layer. In the grey matter
superficial to the island of Reil, it exists as a separate layer consti-
tuting the clanstrum.

Not unfrequently in bichromate and chromic acid preparations,
the place of the pyramidal cells is partly represented by clear
spaces, produced by vacuolation of the cells. It is by no means
easy to distinguish a nerve-cell from a neuroglia-cell in the cere-
brum. Usually the glia-cell nuclei are smaller. Between the rows
of cells may be seen fine longitudinal striation, indicating the exist-
ence of nerve-fibres in the grey matter.

The structure of the cortex is not identical, although similar,
throughout. In the motor areas especially, i.e., in the ascending
frontal, ascending parietal, and part of the marginal convolutions,
the pyramidal cells are usually larger than in those areas which are
described as sensory — e.g., the occipital, temporo-sphenoidal, or even
in the anterior part of the frontal.

It is advisable, therefore, to provide the student with sections
from these different areas. There is no abrupt transition between
oiK1, type of cortical structure and another.

B. Course of White Fibres in the Cortex Cerebri. — This cannot
be made out in sections prepared as above. All that can be seen is
that fine strands — radii — of medullated fibres pass from the white

Z

354

PRACTICAL HISTOLOGY.

[xxxi.

centre at intervals into the grey matter, and run outwards as
medullary rays between the nerve-cells. By other methods it can
be shown that some of them become continuous with axis-cylinder
processes of the pyramidal and polymorphous cells, while, as shown
in fig. 331, others end in free arborisations. Tracing the fibres the
other way, i.e., as axial-cylinder processes of pyramidal cells, it has
been shown by other methods that some of them — chiefly from the
large pyramids of the motor areas — pass into the white matter,
enter the corona radiata, and pass through the inner and anterior
two-thirds of the posterior division of the internal capsule to enter
and form the pyramidal tracts. They constitute the projection
fibres, and end in free arborisations in relation with the multi-
polar nerve-cells of the anterior cornu of the grey matter of the
cord.

The so-called commissural or callosal fibres, which join opposite
halves of the brain, pass into the corpus callosum either directly or
by collateral fibres (fig. 331) and pass to the opposite side to end as

FIG. 329. — Injected Cerebral Cortex of Dog. i. Layer with few vessels ; 2. Layer of large
pyramidal cells ; 3. Deepest layers of cortex ; 4. Medulla.

free arborisations in the grey matter there (fig. 331, /). Others are
said to join the antero-posterior fibres (fig. 331, B) or association
fibres, which run between grey matter in different parts of the same
hemisphere.

6. V.S. Middle Frontal Convolution (fig. 328). — Compare this
with that from a motor area, and note the absence of the very large
pyramidal cells. In the gyms hippocampi or uncinate gyrus there
are other peculiarities, its grey matter consisting of numerous
conical cells with very long processes. It is hardened in the same

XXXI.] CEREBRUM. 355

way. The peculiar shape of the cells in this region is best shown
by the "double-impregnation" method of Ramon y Cayal (p. 222).

7. Blood- Vessels of the Cerebrum (L and H). — Make rather
thick sections of an injected brain, and mount them in balsam.
They are best embedded and cut in paraffin. The whole head
should be injected from the aorta.

(a.) (L) The larger vessels in the pia mater send into the cerebral
cortex two sets of arteries : those that perforate the grey matter
and proceed to supply the medulla— the long or medullary arteries ;
and a more numerous, shorter set, that ramify chiefly in the grey
matter — the short or cortical arteries. The grey matter is much
more vascular than the white, and the vessels are surrounded by
pori vascular sheaths (fig. 329).

(/A) Study the arrangement and relative vascularity of the capil-
lary plexus in the cortex. At the surface it is less dense, and it is
most dense in the region of the large pyramidal cells.

The best resume of the researches of Golgi, R. y Cayal, and
Kolliker is given by Waldeyer,1 whose papers contain numerous
woodcuts showing not only the histological results, but their bearing
on physiological problems.

ADDITIONAL EXERCISES.

8. Cerebellum in Osmic Acid. — Place very small pieces of the grey matter
(i unn. cubes) in i per cent, osmic acid (24-48 hours) as recommended for the
cerebrum (Lesson XXXI. 13). Wash in water and harden in alcohol. In the
sections note the medullated fibres in the granular layer, but none of them
pass into the outer layer of the cortex.

9. Cerebellum in Ehrlich-Biondi's Fluid.— Stain sections in this fluid as
directed under Spinal Cord (Lesson XXX. 13) and mount in balsam. The
outer grey layer and the medulla are red, the granular layer violet. In the
latter can be seen the two kinds of cells, one stained red, the other violet.

10. Purkinje's Cells by Golgi's Method (p. 344).— (a.) The easiest method
is the slow silver nitrate one (p. 344). If the preparation is successful, one is
rewarded by the beauty of the preparation. It is almost impossible to
reproduce the complexity of the processes of Purkinje's cells (fig. 330). They
are stained black, the nerve-fibres may or may not be stained. The sections
are mounted in balsam without a cover-glass. The much-branched proto-
plasmic processes, or dendritcs, are shown in fig. 330, A, as they appear when
a section is made across the lamina?, and in B when the section is in the
direction of the laminae. The dendrites do not anastomose with each other,
and all lie in one plane in the transverse direction of the leaflet.

(b.) Fibres in Cerebellar Grey Matter. — By using the rapid hardening
method of Golgi and Cayal many other details may be made out, but not
all necessarily in one section. Round the body of each cell of 1'urkinje is
a plexus of fibrils — basket-work of fibres — produced by the division or arbori-

1 Deutsche med. WocJwnsch., Nos 44, 45, 46, 47, 1891.

356

PRACTICAL HISTOLOGY.

[XXXI.

sation of the axis-cylinder processes of nerve-cells lying in the molecular

layer of the grey matter.
The dendrites are in part
also invested by a basket-
work due to the arborisa-
tion of fibres proceeding
from the medulla — i,c.,
fibres which do not ter-
minate in the cells of
Purkinje. Some of the
cells of the granular
layer are nerve-cells with
protoplasmic and axis-
cylinder processes. The
small nerve-cells, by far
the most numerous, give
axis - cylinder processes
which run out into the
molecular layer, where
they divide and become
continuous with a fibre
running at right angles
to it. In the molecular
layer there is an arrange-
ment of nerve-fibres run-

Fia. 330.— Cell of Purkinje. Corrosive sublimate. A. Seen wing more or less parallel
on the flat, and B, from the side, x 120. to the surface of the

leaflets, and joined here

and there by the axis-cylinder processes of the nerve-cells of the granular

Kia. 331.— Scheme of T.S. of Cerebrum of New-born Rat, prepared by Cayal's Double-
impregnation Method. ^.Corpus callosum ; e. Lateral ventricle ; B. antero-posterior,
or association fibres arising from large pyramidal cells ; a. Large pyramidal cells whose
axis-cylinder processes pass into the antero-posterior layer; b. Fibre of corpus cal-
losum bifurcating ; c. Callosal fibre ; d. Callosal fibre arising from a pyramidal cell ;
e. Axis-cylinder process descending obliquely to enter the corpus callosum ; /. Final
ramification of a callosal fibre in the grey matter of the cortex ; h. Collateral fibre
from a large pyramidal cell ; i. Fusiform cells with axis-cylinder processes passing into
outer layer of cortex ; j. Final ramification of a callosal fibre arising in opposite side
of cortex.

XXXI.]

CEREBRUM.

357

layer already described. If the cerebellar lamina be cut in a direction trans-
verse to the course of these fibres, then they merely appear as black dots.
My experience leads me to believe that this system is best demonstrated by
the rapid Gclgi method.

11. Cerebrum by Golgi's Method (see Lesson XXX. 19).— Do the same as
for the cord and cerebellum. Either the AgN03 or HgCl.2 methods may be
adopted. In the latter case the best results are obtained by keeping the por-
tions of the brain for months in the mercuric-chloride

solution. I have usually got good results with the
brain of the rat or rabbit. In all cases mount the sec-
tions in balsam without a cover-glass. In new-born
animals better results are obtained — especially with the
rapid method (osmico-bichromate method, p. 345) — than
with adult brains. Fig. 331 shows some of the results
which may be obtained in a T. S. of the brain of a new-
born rat. The results have been pieced together from
a study of many sections.

By the rapid Golgi method it is easy to obtain beauti-
ful preparations showing the pyramidal cells of the
cortex with their apical, lateral, and axis-cylinder pro-
cesses ; in fact, I have often obtained such in the
superficial sections after a few hours' immersion in
silver n itrate. For the cornu ammonis cells the double-
impregnation method of K. y Cayal is excellent. Some-
times, however, one gets the blood-vessels stained.

12. Cerebrum by Weigert or Weigert-Pal's Method
(see Lesson XXX. 4). — This method reveals the exist-
ence of medullated fibres in the cerebral cortex arranged
according to the scheme shown in fig. 332.

-At the lower part of the grey matter a large number
of fine medullated nerve-fibres enter it — radii — which
run outwards in the cortex as medullary rays. Between
the latter is an inter-radial plexus, and above them is
a supra-radial plexus, and quite under the pia are tan- F
gential fibres. On the boundary-line between the supra-
radial plexus and the inter-radial plexus is a white
stripe, visible to the naked eye, especially well marked
in the cuneus. It has been called the stripe of Genuari,
Baillarger, and also of Vico d'Azyr.

13. Medullated Nerve-Fibres in the Cortex Cerebri.
—Place very small pieces of the outer part of the cortex
in i per cent, osmic acid (24 hours). The pieces must
be black throughout. Make thin sections, place one
on a slide, add a drop of ammonia ; the section swells
up and the medullated fibres become distinct. Expose
the section to the vapour of osmic acid, i.e., place the
section on a slide over a glass thimble filled with osmic

acid and cover the whole with a bell-jar. After half an hour or less the
"fixation" is complete. This is Exner's method as modified by Ranvier.
Preserve in glycerine. Observe the narrow medullated nerve-fibres between
the nerve- cells"; the nuclei only of the latter are distinctly seen.

Hypophysis Cerebri or Pituitary Body.

14. Hypophysis Cerebri. —This consists of two parts, the one derived from
the brain, a continuation of the infundibulum ; the lower part is derived from

332.— V.S. Cortex
Cerebri. The right
side drawii from a
Weigert's lijema-
toxylin preparation,
and the left from a
Golgi's sublimate
one. On the ri^ht
the mednllated

fibres, and on the
left only the nerve-
cells are shown.
There are really
more cells than
shown in the draw-
ing.

358 PRACTICAL HISTOLOGY. [XXXII.

an inflexion of the embryonic mucous membrane of the mouth. Harden in
Miiller's fluid, stain sections in picro-carmine, and mount in balsam. Observe
sections of closed gland-tubes or alveoli almost completely filled with cubical,
somewhat granular, or clear cells. A lumen is rarely visible. The communica-
tion between the mouth and the lower part of the gland is cut off in the pro-
cess of development.

LESSON XXXII.
THE BYE.

Methods. — (i.) Enucleate the eyeball of a rabbit or cat ; remove
any adhering fat and muscles. With a sharp razor make a single
cut at the equator of the eyeball, and suspend it in 200 cc. of .25
per cent, chromic acid. After twenty-four hours cut the eye into
an anterior and a posterior half, and place them for several days in
new fluid. Wash and harden in the dark in gradually increasing
strengths of alcohol. The eye hardened in this way may be used
for preparing sections of some of its parts.

(ii.) Harden the other eyeball for two or three weeks in Miiller's
fluid, after cutting into it in the same way as above. Complete the
hardening in alcohol. It is well to have sections of the cornea of
several animals, e.g., the pig and ox.

THE CORNEA.

Make Y.S. from a cornea hardened in Miiller's fluid or Flemm ing's
fluid. Stain with picro-carmine and mount in Farrant's solution,
or in haematoxylin and mount in balsam. Stain the one fixed in
Flemming's fluid with safranin. A good method is to stain it in
bulk in borax-carmine. Eosin-hsematoxylin is a good stain.

1. V.S. Cornea (L. K).

(a.) Observe the anterior or conjunctival epithelium, consisting
of several layers of stratified epithelium. The most external cells
are flattened ; those of the middle layers are more oval or rounded,
many of them with finger-shaped processes dipping down between
the deeper rows of cells. The cells in the lowest layer are columnar,
and placed perpendicularly upon the cornea, resting on (&)

(%• 333)-

(b.) The narrow, clear, transparent layer — anterior elastic
lamina ; this is best marked in the human eyeball.

(c.) The substantia propria, or body of the cornea, composed of

XXXII.]

THE EYE.

359

layers of transparent fibrous tissue arranged in laminae. The
laminae, while arranged in the main parallel to each other, have
connecting processes joining adjacent laminae. Between the laminae
oval spaces, and in each space a nucleated cornea corpuscle (fig.
333> <"), which, as seen on edge, is thin and flattened. The processes
of these corpuscles are best seen in gold specimens.

(fl. ) The posterior elastic lamina, or membrane of Descemet,
a well-marked, thin, transparent, hyaline lamina, with sharply
defined outlines, and covered posteriorly by

(e.) The posterior epithelium, consisting of a single layer of
large flattened nucleated cells, seen in profile. They are apt to be
displaced if the section be roughly handled.

If the posterior elastic lamina be broken across, it is apt to curl
up. It stains readily with carmine.

FlQ. 333.— V.S. Cornea, a. Epi-
thelium ; h. Anterior elastic
lamina; c. Cornea! corpuscles;
I. Lamclhc of cornea ; ft-rf,
Siilistantia propria; d. Des-
cemet's membrane; and e. Its
epithelium.

FIG.

1*10. 334 — Corneal Corpuscles, and a few
Nerve-Fibrils of Frog. Gold chloride.

2. Nerves of the Frog's Cornea. — In a pithed frog, squeeze the
head to make the eyeball project, and with a sharp razor cut off the
cornea. Wash it in normal saline, treat it with gold chloride by
the lemon-juice method (p. 79).

After impregnation with the gold solution, with scissors make
three snips into its margin, dividing it into three sections, so that
it may He flat on a slide. Mount in Farrant's solution.

(a.) (L and H) Observe the branching cornea corpuscles,
arranged in many layers, one under the other, readily seen by
raising or depressing the lens (fig. 334). The numerous processes
given off from ,the cells anastomose with similar processes from

360

PKACTICAL HISTOLOGY.

[XXXII.

adjoining cells, not only in the same plane, but with cells lying in

planes above and below them.

(6.) The Nerves. — If a part of the sclerotic is adherent to the

cornea, the fine medullatcd nerve-fibres, deeply stained, are seen in

the sclerotic and passing
into the cornea, where
they lose their myelin
and become non-medul-
lated. The larger

non-

medullated fibres have
nuclei in them, and unite
with other fibres to form
a coarse plexus — the
ground or primary nerve
plexus.

(c.) From this plexus
finer bundles of fibrils
proceed to form a finer

jftG. 335.— Sub-Epithelial Nerve-Flexus, Cornea of plexus, and from the
Frog. n. Non-medullated nerve with nerve- i0ffnr T1,irnoT>rma fiVmilo
fibrils ; a. Nerve-fibrils, x 300.

often with varicose swel-
lings— running chiefly in the planes of the laminae are seen.^

(d.) (H) Fine fibrils are seen in the larger branches of the non-
medullated nerves (fig. 335).

If the cornea of a rabbit be used, then tangential thin sections

must be made and mounted
in Farrant's solution or
balsam.

3. Inter-Epithelial Ter-
mination of the Nerve-
Fibres. — The cornea of a
rabbit is stained in i per
cent, gold chloride (25-30
minutes) and reduced in
slightly - acidulated water
(acetic acid) by exposure to
light (p. 79). The lemon-
Fia. 336.— v.s. Cornea of Frog. n. Nerve-fibres ; juice and formic-acid method

a. Perforating fibrils; r. Nucleus ;#&. Inter- , i i , 4i

epithelial plexus of fibrils. Gold chloride. Dltlfit not be used, as the

formic acid removes the

epithelium. Make V.S. either by freezing or free hand and mount
them in glycerine (L and H).

(a.) Observe sections of the lamellae and corneal corpuscles, and
between these, parts of the primary nerve-plexus cut across, more
or less obliquely, the branches being finer towards the anterior

XXXII.]

THE EYE.

36:

elastic lamina, Bundles of fine fibrils — the rami perforantes —
perforate this membrane and spread out under the anterior epi-
thelium to form the sub-epithelial
plexus, from which fibrils proceed to
form a plexus of fine varicose fibrils —
the epithelial plexus — between the
epithelial cells, where they terminate
in fine points, forming no connection
with special end-organs (fig. 336).

4. Cell-Spaces in the Cornea (Silver
Method) (L and H). — Pith a frog, scrape
off the corneal epithelium, and rub the
surface of the cornea with solid silver
nitrate. After 20-30 minutes, excise
the cornea, mount it in glycerine, and
expose to light.

(a.) The general substance of the
section is stained brown, and in it are
clear branching spaces, each one corresponding in shape to that of

FIG

337 —Cornea of Frog with Cell-
Spaces, s Cell-spaces. Solid
silver nitrate, x 300.

Sclerotic.

Lam. supra-choroidea.

Layer of large vessels.

Z...^L.:_.~ .Limiting layer.

-i^v'S .Chorio-capillaris.

gnSr — "Hyaline membrane.

Pigment layer of retina.

FlO. 338. — V.S. Part of Sclerotic ami the Choroid. g. Large vessels; p. Pigment-cells;
c. T.S. of capillaries, x 100.

a cornea corpuscle (fig. 337). The branches of adjoining spaces
anastomose, so as to form a system of juice-canals, not only with
32

PRACTICAL HISTOLOGY.

[XXXII.

spaces in the same plane, but also with the spaces in lower and
deeper planes.

5. Iron Sulphate Method (Lesson XI. 13). — The matrix is blue
and the spaces are clear and not stained.

THE SCLEROTIC, CHOROID, AND CILIARY REGION.

6. Sclerotic and Choroid (L.H.). — From an eye hardened in
Miiller's fluid and stained in bulk in borax-carmine cut sections in
paraffin to include the sclerotic and choroid. Observe —

(i.) The sclerotic (fig. 338). (a.) Composed mainly of bundles
of white fibrous tissue crossing each other in several directions.

PIG. 3r,9.- Horizontal Section of Anterior Quadrant of Eyeball. /. Conjivn
Sclerotic; h. Iris; ?. Llfj. ixjct.inat.iini iri«lis; k. Canal of Schlernm ; I. Long
m. Circular fibres of ciliary muscle ; n. Ciliary process ; o. Ciliary part of reti

f. Conjunctiva; s.
\\ ; 1. Longitudinal ;
part of retina.

(/>.) A narrow layer, the lamina supra-choroidea.
(2 ) Inside this a section of the choroid composed of the follow-
ing layers from without inwards —

(a.) Layer with large vessels and numerous branched pigment-

cdls. This forms by far the thickest layer.
(b.) Limiting layer.

XXXII.] THE EYE. 363

(c.) The chorio-capillaris, containing numerous sections of capil-
laries.

(d.) A basement or hyaline membrane.
(e.) Pigment-cells containing melanin.

7. Pigment-Cells of the Choroid (L.H.). — From the inner cho-
roidal surface of an eye hardened in Miiller's fluid or spirit, scrape
off a little of the black pigment layer and mount it in balsam.

(a.) Observe the branched pigmcnted cells, filled with brown or
black granules of melanin. The nucleus contains no pigment
granules. If the granules be discharged from the cells and float in
a watery fluid, they exhibit Brownian movement. The nucleus
stains readily with hsematoxylin.

8. Ciliary Region, Ciliary Muscle, and Iris.— Make a meri-
dional section through the corneo-scleral junction of a hardened eye
(Miiller's fluid), e.g., of an ox, cat, or rabbit, so as to include the
ciliary muscle and the iris Stain the section in haematoxylin or
eosin-hsematoxylin, and mount in balsam ; or it may be stained in
picro-carmine and mounted in Farrant's solution. To preserve all
the parts exactly in position cut it in celloidin, or stain in bulk and
cut in paraffin. Fix the sections on a slide by a fixative.

(a.) Observe the cornea passing into the sclerotic, and near it an
opening, the canal of Schlemm (fig. 339).

(b.) From the angle of the iris and cornea, running in a fan-
shaped expansion, the radiating fibres of the ciliary muscle, con-
sisting of non-striped muscle. The circular fibres cut transversely
inside the radiating fibres are not so well marked as in the human eye.

(c.) The membrane of Descemet splits up into fibres, some of
which curve into the iris, others spread into the ciliary processes.
This region constitutes the ligamentum pectinatum iridis.

((?.) The pigmented folds or ciliary processes. A layer of black
pigment-cells continuous with those of the retina continued over
these processes, and inside the pigment a clear unpigmented layer
of columnar epithelium.

(e.) The iris, composed of five layers from before backwards.

(i.) A layer of emloilidiuin continuous with that covering the
posterior surface of the cornea (fig. 339, r).

(2.) The anterior boundary layer, chiefly consisting of branched
connective tissue-cells.

(3.) The vascular layer, or body of the iris, composed of a stroma
of connective tissue with numerous sections of blood-vessels and
branched pigment-cells, and at its deeper part transverse sections of
the circular smooth muscular fibres, which constitute the circular
muscle or sphincter ]>upilhB of this organ. It is doubtful if a
dilator pupillae exists in man.

(4.) The posterior liyaline layer, of an elastic nature.

3^4

PRACTICAL HISTOLOGY.

[XXXII.

(5.) Two layers of pigment-cells — the uvea — the outlines of the
cells difficult to define, and continuous with the epithelium of the
pars ciliaris retinae.

(6.) Blood- Vessels. — A large number of blood-vessels and nerves
pass into the iris, but their distribution must be studied by the
methods already described for such purpose*.

THE LENS.

The lens is developed by the inflection of part of the epiblast, so
that it consists of modified epiblastic epithelial cells invested by a
transparent capsule. The capsule of the lens is a clear trans-
parent elastic membrane, not unlike a basement membrane, but is
less resistant to acids than the latter. Immediately on the inner
surface of its anterior part is a layer of clear flattened epithelial
cells — hexagonal and nucleated — the remains of the original cells.
Tracing these cells towards the margin of the lens they become
narrower and more cylindrical, until further on they gradually pass
into lens fibres. Posteriorly there are no epithelial cells between
the lens and its capsule, the lens fibres resting directly on the
capsule itself. The lens fibres are modified epithelial cells, the
fibre constituting the principal part of the cell. The cells are long,
soft, hexagonal prisms united to each other by more or less toothed
edges. The superficial fibres are longer and larger than the deeper
ones, and contain, or have on their surface, nuclei lying on the part
that corresponds to the equatorial (nuclear) zone of the fibre. No
nucleus is visible in the central fibres.

9. Methods. — Harden in its capsule, the lens of a cat or rabbit,
in Miiller's fluid (2-3 weeks) ; do not place it in spirit, but cut it

in a freezing microtome, or cut in cel-
loidin. Paraffin is not suitable. Stain
the sections in eosin and mount in
Farrant's solution. Make meridional
and equatorial sections.

T.S. Lens— (a.) (LH.). — Observe
the hyaline capsule — if present — and
just under its front part a layer of
epithelial cells, which, when traced
outwards, are seen to rapidly elongate
into lens-fibres (p. 364). Nuclei may
be seen on some of the fibres.

(I.) T.S. of the fibres are hexagonal
(fig. 340, B).

10. Isolated Lens Fibres — Methods. — (a.) Place the lens of a
frog or fish in a 10 per cent, solution of sulphocyanide of potas-

FlG. 340. — A. Fibres of the lens :
B. T.S. of the same.

XXX II.] THE EYE. 365

slum (24—36 hours). Tease out a fragment of the softened lens in
glycerine. (/>.) Or use dilute sulphuric acid (4-5 drops to 5 cc. of
water for 24-36 hours). Wash in water to remove the acid. Stain
in carmine or liseinatoxylin. (c.) Methyl-green feebly acid, does well
for fresh-teased fibres, but the preparations do not keep. The
nuclei on the fibres may be seen in this preparation.

(H) (a.) Observe the flattened bands with serrated edges; the
teeth dove-tail into each other. The teeth may be seen from the
side or directed to the observer.

RETINA.

Methods. — (i.) With a sharp razor cut the eye of a cat, rabbit,
pig, or other animal into an anterior and posterior half. Place the
posterior half in 2 per cent, potassic bichromate, or in a mixture of
Miiller's fluid and spirit (two weeks), and complete the hardening
in spirit.

(ii.) Stain "in bulk" in. borax-carmine for several days the
whole posterior segment of the hardened eyeball, then place it for
a day in acid alcohol, and embed in paraffin and cut sections. Per-
haps celloidin is to be preferred if the sclerotic be thick. Fix the
sections on a slide and mount in balsam.* In this way the whole
structure of the retina is preserved in its relations to other struc-
tures.

(iii.) The same process may bo adopted with the posterior seg-
ment of the eyeball hardened in i per cent, osmic acid.

(iv.) Harden the retina in a 3 per cent, solution of nitric acid
(i 5-20 mins.). Wash out the acid, stain in bulk, and make sections.
Stain with eosin and methylene-blue.

(v.) For small eyes, such as those of a lizard, it is sufficient to
expose the whole unopened eyeball to the vapour of osmic acid for
several hours, and then to stain the eyeball in bulk in borax-carmine.
Embed in paraffin and cut sections of the whole eyeball. Or, expose
the eye of a frog or triton to osmic acid vapour (10 mins.). Place
in 40 per cent, alcohol (4-6 hours) ; divide eyeball by incision
parallel to edge of cornea ; bichromate of ammonia (3 per cent.) for
5-10 hours; wash in water; alcohol; borax-carmine; embed and
cut in paraffin.

(vi.) Make sections of an eyeball hardened in Muller's fluid and
stain them in eosin-hsematoxylin. The latter stains the granular
layers of the retina, while the other parts are rosy in tint.

(vii.) Methylene-Blue Method. — This is done cither by injecting
methylene-blue (i in 300 normal saline) into the blood-vessels of an
animal just killed, and waiting two or three hours thereafter, or by
placing the fresh retina in the same fluid and observing with the

366

PRACTICAL HISTOLOGY.

[xxxii

microscope the coloration of the nervous elements, especially the
cells in the retina. Mount in picrate of ammonia and glycerine (p.
192).

11. V.S. Retina. — (L) Beginning from within, i.e., next the
vitreous, observe (fig. 341) —

(a.) The internal limiting membrane,
and springing from it by wing-shaped
expansions the fibres of Miiller, which
run vertically outwards through the
layers of the retina to the outer limiting
membrane.

(b.) The fibrous layer, composed of
the non-medullated fibres of the optic
nerve. The fibres are medullated in the
optic nerve, but they lose their medulla
as they enter the retina. The layer is
thinner in the anterior part of the
retina. Some of the nerve-fibres end
in the ganglionic cells of the next layer,
but others pass outwards to the inner
molecular layer to end by terminal
arborisations there (shown by Golgi's
method).

(v.) The ganglionic or nerve-cell
layer, consisting of a single row of large
multipolar nerve-cells, each cell with a
large conspicuous nucleus. Each cell
givos on? (i-) one thick process, which

is and cones; le, li. Exter- may divide into several processes which
' pass towards and ramify by terminal

pb Basal plexus ; cb. Bipolar arborisations in the layer external to this,

cells; CM. Unipolar cells ; bi. ... . J.

Internal basal cells ; pc. Cere- and (ll.) OI1C 11116 axis-Cylinder process

towards the optic nerve -fibre layer,
where it becomes continuous with a
nerve-fibre. Between the nerve-cells, or
in close relation to them, sections of the
ami'granular layers; C. Cellular larger blood-vessels.

layer; ^.Fibrous layer. (d.)The internal molecular layer

appears to consist of fine fibrils or

granules. It is not unlike the grey matrix of the cerebrum, and is
traversed by the fibres of Miiller and also by fibres of the optic nerve,
and processes of the cells of the ganglionic and inner granular layers*

(e.) The internal nuclear layer,- deeply stained, and consisting
of several rows of large spherical oval nuclei.

Some of the granules, however, by other methods can be shown

FIG.

bral plexus ;
nerve-cells ; fo. Fibres of optic
nerve. On.the left the initials
of the usual names for these
layers ; R C. Rods and cones ;
EN., EG., and IN., IG. Ex-
ternal and internal nuclear

XXXII.] THE EYE. 367

to be bipolar cells with large nuclei. The processes extend into the
inner molecular layer and end in arborisation, and by Golgi's method
the opposite processes have been traced outwards as far as the
external limiting layer. Other branched cells supposed to resemble
neuroglia cells lie amongst these cells.

(/.) The outer molecular layer is thin, and is composed of the
terminal arborisations' of processes from the rods and cones and
inner granular layer.

(g.) The external nuclear layer consists of many more rows of
nuclei than in (*). They are the nuclei of the rod and cone fibres.

(/>.) The external limiting membrane, cut across, and therefore
appearing as a thin clean-cut line.

(/.) The layer of rods and cones. The rods are more numerous
than the cones, and the latter are shorter than the former. Each
rod and cone consists of an outer and an inner segment.

(/) A layer of hexagonal pigment-cells, somewhat flattened,
which sends pigmented processes or filaments between the rods and
cones. The length and condition of these processes depends upon
the influences to which the eye has been exposed before death
(P- 37°)- The pigment granules, some of which are crystalline,
exist chiefly in the inner part of the cells, and may extend into the
cell processes which pass between the rods and cones.

(H) Study the successive layers. Although the layer of rods
and cones and external nuclear layer have been described as
separate layers, in reality they should be studied together. Each
rod is continued inwards by a tapering fibre — the rod-fibre ; in the
course of the latter is an oval nucleus, the fibre itself ending in an
arborisation in the outer molecular layer. Each cone is similarly
prolonged into a cone-fibre — thicker than the rod-fibre — which also
has an enlargement in it containing a nucleus. The cone-fibre ends
like the rod-fibre in the outer molecular layer. These two kinds of
nuclei intercalated in the course of the rod and cone fibres make up
the external nuclear layer, which as the rods and cones are merely
modified epithelial cells — must be regarded as epithelial in their
origin. In a freshly -teased retina the nuclei of the rod-fibres are
marked by alternate transverse light and dim stripes.

Each rod arid cone consists of two segments. The outer segment
of the rods is clear hyaline and transparent, and striated trans-
versely, and readily breaks up into transverse discs. During life
the external segments of the rods contain the visual purple. The
inner segment is wider, granular and striated longitudinally. The
cones also consist of a shorter tapering outer segment transversely
striated, and an inner bulging larger, longitudinally striated, segment
which is continued through the external limiting membrane into
the cone-fibre.

3^8 PRACTICAL HISTOLOGY. f XXXII.

The outer segments are stained black by osmic acid, and the
inner segments red by carmine.

Study successively the other layers to make out some of the
details already described on p. 366.

12. Ketina of Frog.— (i.) Place the posterior segment of the
eyeball, with its vitreous humour removed, in i per cent, osmic
acid (24 hours) ; wash well, and tease a fragment of the now
blackened retina in glycerine. Observe

(a.) The rods, each with an outer and inner segment; the outer
segment, however, is blackened by the osmic acid, and usually
shows a tendency to split transversely.

(b.) In the cones there is a small refractive oil globule between
the outer and inner segments. It first becomes brown, and then
black, in osmic acid. In some animals, e.g., some birds and
reptiles, the oil globule is pigmented, the pigment being held in
solution by the fat of the globule.

(c.) Numerous pigment cells, each consisting of a rather thick
body, part of which contains the nucleus and is non-pigmented,
while the other half is pigmented, and sends numerous fine pro-
cesses between the outer segments of the rods.

(d.) Other parts of the retina are also isolated, e.g., the nuclei of
the nuclear layer and the limiting membranes.

(ii.) Fix the whole eyeball in osmic acid, and make V.S. of all
the coats after embedding in paraffin or celloidin.

13. Cones. — To see large cones, place the fresh eye of a cod in 2
per cent, potassic bichromate (2-3 days) or osmic acid (24 hours),
or isolate them by dilute alcohol.

14. T.S. Optic Nerve (L and H). — Place the optic nerve in 2 per
cent, ammonium bichromate (2-3 weeks) or in Flemming's mixture
(10-20 hours). Make T.S. Stain in hsematoxylin, picro-carmine,
or safranin, or by Weigert's method. Mount in balsam. Double-
stain some in picro-carmine and haematoxylin.

(a.) Observe the sheaths, thick and well-marked, sending septa
into the nerve, and thus breaking it up into small bundles of nerve-
fibres, each surrounded by a fibrous sheath. In the larger septa
are blood-vessels. The anastomosing septa form a kind of alveolar
system, so that a section of this nerve is readily distinguished.

(b.) The nerve-fibres are all medullated, but they possess certain
peculiarities. They are without sheath of Schwann, and the
fibrous tissue runs parallel with the nerve-fibres.

(c.) As to the sheaths, one is continuous with the dura mater;
under it is a space, the subdural space ; inside is a prolongation of
the arachnoid with subarachnoid space, and a thin continuation of
the pia mater.

If desired, make L.S. This enables one to note the structure of

XXXIT.] THE EYE. 369

the nerve-fibres and the arrangement of the connective tissue. An
interstitial injection of osmic acid is a good method for isolating the
structural elements.

15. Blood- Vessels of Eyeball. — It is not difficult to inject from
the aorta with a carmine-gelatine mass the blood-vessels of the eye-
ball of an animal. Select an albino rabbit. Sections are mounted
in balsam. One-half of the injected eyeball of an albino rat
mounted in balsam shows the connections, distribution, and
arrangement of the blood-vessels.

16. The eyelids. — Harden the eyelids of an infant in corrosive
sublimate or .25 per cent, chromic acid (3 or 4 days) and then in
alcohol, or in alcohol alone. Make sections, vertical to its surface
and transverse to its long axis, which must be rather thick, and
mounted in balsam to show the general arrangement of the parts.
Finer sections are stained in haematoxylin or picro-carmine. Be-
sides the sections of hair-follicles, connective tissue, and orbicularis
muscle, note the Meibomian glands.

ADDITIONAL EXERCISES.

17. Fibrils of Cornea. — (a.) Macerate the cornea in lime-water, baryta-
water, or dilute potassic permanganate, (b.) A better method is to dry the
cornea, make T.S., allow them to swell up in water, and stain with picro-
carmine.

18. Epithelium of Lens.— Use a small lens (frog or rat), ami place it for a
few minutes in AgN03 (i : 300). Expose to light in glycerine, and examine.
Note on the anterior surface («.) silver lines bounding polygonal areas cor-
responding to the inter-epithelial cement. (J.) Black granular converging lines
corresponding to the cement between the crystalline fibres (best seen on
posterior surface).

19. Other Methods for the Retina.— By the usual methods of hardening the
retina, it is impossible to make out the connections between its several elements.
The two methods which have quite recently yielded the best results— apart
from the direct use of osmic acid — are the methods of Golgi and Ehrlich's-
mel.hylene-blue method.

(a.) Golgi's Methods. —Ramon y Cayal has made a large number of prepara-
tions both by the silver method (p. 344) and the rapid osmico-bichromat",
method (p. 345), especially on the retina of birds. In this way he lias shown
that the protoplasmic processes of the nerve-cells of the ganglioiiic layer
ramify in the inner molecular layer, and end in terminal arborisations in several
planes in this layer. Several varieties of nerve-cells with extensive terminal
arborisations in the other layers of the retina are also described by him.
The following medium method gives good results : —

(1.) Place the retina and sclerotic in weak osmico-biahromate fluid
(osmic acid, i per cent.), 2 cc., bichromate of potash (3 per
cent.), 20 cc., for 24 hours.

(2.) Transfer to i per cent, silver nitrate (24 hours).
(3.) Auain for 24 hours, as in (i).
(4.) Again for 24 hours in silver.
33 2 A

3/0 PRACTICAL HISTOLOGY. [XXXII.

(b.) Methylene-Blue Method. — Dogiel1 by means of this method finds
that in man there are more nervous elements present than one has been in
the habit of supposing. He arranges the nervous elements (in three layers)
and other layers as follows : —

Membrana limitans Pigment epithelium (I).
externa. > Neuro-epithelial layer (II).

External reticular) g!1 ij6?1 ie ^ cells ("fe^ > ^" ^u^er ganglionio

Bipolar nerve-cells (c).

Inner reticular
layer.

. Layer of spon gio-
Middle ganglionic layer. blasts of W.

Miiller.

Inner ganglionic layer. C.

Layer of nerve-fibres. D.

Membrana limitans
intern a. >

The neuro-epithelial layer embraces the layer of rods and cones and subjacent
external nuclear layer. What he calls the outer ganglionic layer corresponds
to the inner nuclear layer, and contains three varieties of nerve-cells. The
middle ganglionic layer corresponds to a layer of cells— of which there are
several varieties lying on the confines between the internal granular (reticular)
layer and the internal nuclear layer. These cells were called spongioblasts
by W. Miiller, but according to Dogiel, they are nerve-cells. The inner
ganglionic layer corresponds to the cellular layer.

20. Epithelium of the Eetina. — Harden the eye of a frog in picro-sulphuric
acid (24 hours), stain in bulk in borax -carmine, and make vertical sections
after embedding in paraffin.

21. Isolated Elements of the Eetina. —(a. ) Macerate the retina in Schieffer-
decker's fluid as described for the isolation of the cells of the cord ( Lesson XXX. ),
using the sulphuric acid method to remove the water.

(b.) For M tiller's fibres 10 per cent, chloral hydrate is good.

22. Retina of Frog in Light and Darkness. — (a.) Keep one frog in abso-
lute darkness for thirty-six hours. Kill it in the dark, and harden the eye in
alcohol, (b.) Place another frog in direct sunlight for a few hours ; kill it,
and harden the retina in alcohol. Sections are made and stained with picro-
carmine. The pigment-cells covering the rods of the retina in (a) jire
retracted, while those in (b) are pushed out between the segments of the rods.

23. V.S. Macula Lutea. — Secure a human eye as fresh as possible. Harden
it, and make sections through the macula lutea, and observe the peculiarities
of its structure. There are no rods in the fovea centralis, while the cones are
long and narrow. The other layers are reduced to a minimum at the fovea
centralis, but at the margins of the fovea they are thicker. There are a large
number of ganglionic bipolar cells.

24. V.S. Entrance of Optic Nerve. — Make from an eyeball stained in
bulk in borax-carmine a section longitudinally through the optic nerve to
include its entrance into the eyeball. Observe the lamina cribrosa, i.e., the
felt- work of fibrous tissue of the sclerotic, perforated by the fibres of the optic
nerve.

25. Eye of Triton Cristatus. — Excise one eyeball, pin it to the under surface
of a cork, and fix the cork in a glass thimble containing a little osmic acid.
In ten minutes or so the retina is "fixed," owing to the tenuity of the
sclerotic. Divide the eyeball into two by a cut at the equator, place the
posterior half in dilute alcohol (6 to 10 hours), and then in picro-carmine for

1 Archivf. inik. Anat., xxxviii. p. 317, 1891.

XXXIII.] EAR— NOSE. 3/1

the same period. Fix it finally in osmic acid, embed and cut it in soft paraffin.
The retina of this animal is particularly serviceable, because its structural
elements are so large.

26. Lachrymal gland is treated precisely in the same way as the serous
salivary glands (Lesson XXIII. ).

LESSON XXXIIL
EAR— NOSE.

EAR.

1. Membrana Tympani (H). — Fix it in osmic acid and mount
in Farrant's solution. Observe the radiate yellow-looking fibres,
and also fibres disposed circularly, the latter best developed near
the periphery of the membrane. The thin epithelial coverings on
the two surfaces of the membrane can be seen, and so can a few
fine blood-vessels.

2. Ceruminous Glands of the Meatus. — Harden in absolute
alcohol a portion of skin from the external auditory meatus, pre-
ferably from a new-born infant. Make rather thick sections, stain
in haematoxylin and mount in balsam. Observe the Ceruminous
glands, which in some respects are like the sweat-glands. They
have a narrow duct and a coil, the latter lined by a single layer of
cubical cells, and the former by several layers of cells, as in a sweat-
gland. In the secretory part smooth muscular fibres lie between
the epithelium and the basement membrane. The lumen of the
secretory part is very wide, and the lining cells have a clear cuti-
cular disc, and contain a yellowish pigment and fatty granules.

THE COCHLEA.

Methods. — (i.) In a freshly-killed rabbit or guinea-pig, inject,
by means of a hypodermic syringe, osmic acid (i p. c.) through the
membrana tympani into the middle ear. Cut away the lower jaw,
so as to expose the large spherical osseous bulla. Open the bulla,
cut away its walls, and expose the middle ear. On the inner wall
of the latter are seen the turns of the cochlea. Cut away the
surrounding parts from the osseous cochlea, and place the latter in
Miiller's fluid (a week), after opening one of the turns, to allow the
hardening fluid to penetrate. It is better to do this under fluid, to
prevent air entering, as the perilymph escapes. Decalcify in chromic

372

PRACTICAL HISTOLOGY.

[XXXIII.

and nitric acid fluid, and harden in the dark in alcohol. Stain the
softened cochlea in bulk in borax-carmine. Embed in paraffin, and
cut sections from base to apex of the coiled tube. Fix on a slide,
and mount in balsam. Use the other decalcified cochlea, stained as
above, and cut it in celloidin.

(ii.) The following is a better method (Ranvier), and preserves
the finer structures in situ. Open the bulla of a guinea-pig, and
cut out the inner ear. Make an opening or two in the turns of the
cochlear tube, and place it in 1-2 cc. of i per cent, gold chloride.
Add to the gold solution from time to time a few cc. of the follow-
ing mixture : — i per cent, gold chloride and a fourth part of formic
acid, boil, and allow the mixture to cool. Leave the cochlea in the
gold (20-30 minutes), remove it, wash, and expose it to light in

FIG. 342.— V.S. Cochlea of Guinea-Pig. V. Scala vestibuli ; T. Scala tympani ; VT. There
the two communicate ; C. Cochlear canal or scala media ; R. Reissner's membrane ;
t. Membrana tectoria.

water slightly acidulated with acetic acid (1-2 drops to 20 cc. water).
After 2-3 days harden it in alcohol and decalcify it in picric acid.
Embed in gum and harden in alcohol, or embed it in celloidin
and make sections parallel to the axis of the cochlea.

(iii.) After removing the inner CT, open its osseous canal under
osmic acid .2 per cent, and leave it in this fluid for 10-12 hours.
Wash in water and decalcify in chromic acid, 2 per 1000.

(iv.) After treating the cochlea with osmic acid and hardening it
in alcohol, the following decalcifying fluid may be used : — i cc. of
a i per cent, solution of chloride of palladium, 16 cc. of hydrochloric
acid, and 1000 cc. of water.

3. V.S. Cochlea, parallel to its axis.

(a.) (L) The turns of the cochlear tube cut across (fig. 342), each
tube divided by a transverse partition — the lamina spiralis — into

XXXIII.]

THE COCHLEA.

373

an upper — scala vestibuli — and a lower compartment — scala tym-
pani ; the central column or modiolus ; the last is osseous and
contains channels for nerves and vessels. The lamina spiralis is
partly osseous, viz., that part next the modiolus, and partly mem-
branous, viz., that part next the wall of the cochlea; the latter
part is the membrana basilaris. The osseous portion terminates
in a crest — the crista spiralis — and its free end is scooped out into
a groove — sulcus spiralis.

(!>.) From the upper surface of the lamina spiralis ossea there
runs to the wall of the cochlea a thin membrane — membrane oi

FIG. 343. — Scheme of the Canalis Cochlearis and the Organ of Corti. N. Cochlear nerve ;
t. Inner row, and p. Outer rows of hair-cells; n. Nerve-fibrils terminating in p\
a. Supporting cells for p\ d Cells in the sulcus spiralis ; G aud //. Epithelial cells ;
o. Membrana reticularis ; z. Inner, and y. Outer Rod of Corti.

Reissner — which completely shuts off a small three-sided cavity
from the scala vestibuli. This is the ductus cochlearis, canalis
cochlearis, or scala media. It is bounded by the wall of the cochlea,
the membrane of Reissner — and its base is formed by the outer
part of the osseous and the whole of the membranous spiral lamina.
The latter is called the basilar membrane. This cavity contains
a fluid, the endolymph, and into it pass the cochlear branches of the
auditory nerve to end in a complex structure — organ of Corti —
which rests on the basilar membrane.

(c.) Note in the scala media a membrane — membrana tectoria

374 PRACTICAL HISTOLOGY. [XXXIII.

— which arises from the lamina spiralis ossea, and spreads over so
as partially to cover the organ of Corti. The organ of Corti con-
sists of two rows of pillars — inner and outer — the rods of Corti —
which meet above, forming arches, and leave a three-sided tunnel
between them. Internal and external to these are rows of cells,
some of them provided with fine bristles — hair-cells — constituting
the cells of Corti and Deiters. Some of these cells are the actual
terminal end-organs of the cochlear nerve, and others are susten-
tacular in function. The scala vestibuli and scala tympani com-
municate at the apex of the cochlea, as shown in fig. 342, V.T.

(H) Observe the rods of Corti, the inner more numerous than
the outer, and how the head of the one lies over the head of the
other. The rods vary in length and span, in different parts of
the cochlea.- Internal to the inner rods is a single row of hair-
cells, and external to the outer rods are several rows of hair-cells.
Between the latter are supporting cells, and beyond them are
columnar epithelial cells (fig. 343). The rods and hair-cells are
covered by a special membrane, seen in section, which is perforated
by the upper ends of the hair-cells — membrana reticularis — the
basilar membrane terminating towards the wall of the cochlea in a
fibrous expansion, the spiral ligament.

The student must give considerable time and attention to the
subject if he wishes to get preparations showing the structure of all
the parts. It is difficult to keep the parts from falling asunder, and
if the ear be not properly decalcified, bubbles of gas are discharged
within the cochlea, which rupture the finer parts ; hence arises the
necessity for opening the cochlea and fixing the parts with a " fix-
ing" medium previous to decalcifying it.

4. Semicircular Canals.— Fix the membranous semicircular
canals, and their ampullae, of a skate in Flemming's or Fol's fluid.
Harden in alcohol. In the ampullae are the terminations of the audi-
tory nerve in the crista acustica. Make T.S. of the canals and V.S.
of the crista acustica ; in the latter case take care to include the
entrance of the nerve- fibres. The preparation of suitable specimens
to stain the termination of the nerves in the crista acustica presents
very considerable difficulties. The student may have to repeat the
process several times if he wishes to get typical specimens.

THE NOSE.

5. Olfactory Mucous Membrane. — Divide longitudinally the
head of a freshly-killed rabbit. Place small pieces of the olfactory
mucous membrane — readily recognised by its brownish colour —
in dilute alcohol (2 hours), and then in i per cent, osniic acid

XXXIIL]

THE NOSE.

375

(24 hours). Harden in alcohol, make V.S., stain with haematoxylin,
and mount in balsam.

(a.) (L) Observe on the surface the row of columnar epithelial
cells with oval nuclei, and under these numerous rows of cells with
spherical nuclei. At the base of the latter a row of more granular-
looking basal cells (fig. 344). The basis of the mucous membrane
composed of connective tissue with sections of numerous glands —
Bowman's Glands. Sections of blood-vessels and branches of the
non-medullated olfactory nerve may also be seen.

6. Isolated Olfactory Cells. — Place small pieces of the olfactory

FIG. 344.— V.S. Olfactory Region (Rabbit), s. Disc
on cells ; zo and zr. Zones of oval and round
nuclei ; b. Basal cells ; dr. Part of Bowman's
glands ; n. Branch of olfactory nerve.

.FIG. 345. — Olfactory
Cells. N. Ilumau;
n. Frog ; E. Sup«
porting cell.

mucous- membrane in dilute alcohol (24 hours). Fix the membrane
in i per cent, osmic acid (5 minutes). Stain the pieces in bulk in
picro-carmine (24 hours). Scrape off a little of the stained and
softened epithelial covering and mount it in glycerine.

(H) (a.) Observe the olfactory cells as very narrow, cylindrical,
elongated cells with a large spherical nucleus, much broader than
the body of the cell. The free surface carries fine cilia, but they
are apt to be displaced in the process of teasing the tissue (fig. 345).

(/>.) Supporting cells, not unlike columnar epithelium, but they
have a large oval nucleus (fig. 345, E). They have a long central

376 PRACTICAL HISTOLOGY. [XXXIII.

process. The free surface of the cell is covered by a clear hem, but
its exact constitution and significance are unknown.

(c.) There may also be found large granular polygonal cells
derived from Bowman's glands— their acini and ducts.

ADDITIONAL EXERCISES.
Olfactory Bulb.

1. This is a very complicated structure, and has been investigated recently
by Golgi and K. y Cayal. The bulb of a new-born animal is hardened by
Golgi's rapid method and sections made. It contains a layer of white ncrve-
fibres, and under this large nerve-cells — mitral cells— and one remarkable
arrangement known as olfactory glomeruli, which are nests of fibrils, formed
partly by the terminal arborisations of the processes of mitral cells and by non-
medullated nerve-fibres of the olfactory nerve, which are passing onwards to
terminate in tlie olfactory epithelium.

2. Olfactory Eegion by Golgi's Method.— The mucous membrane of the
ol factory region is placed for 7 days in Golgi's osmico-bichromate fluid, then in
silver nitrate, and hardened in alcohol. One often fails, but if a successful
preparation be obtained the ends of the olfactory nerve are seen passing into
the olfactory cells.

3. T.S. Nose. — Harden the whole nose of a mouse in Miiller's fluid, stain in
bulk in borax-carmine, and make T.S. across the whole organ to show its
walls, septum, turbinated bones, respiratory and olfactory regions. In such
animals as possess a well-developed organ of Jacobson study it. In it also,
if a young animal, will be found beautiful sections of developing tooth.

LESSON XXXIV.

TERMINATION OF NERVES IN SKIN AND SOME
MUCOUS MEMBRANES.

Sensory nerve-fibres terminate in the skin and mucous mem-
branes in three ways : —

(I.) Free nerve-endings, i.e., by intra-epidermic fibrils.
(II.) In special terminal corpuscles.

(III.) In neuro-epithelial cells, i.e., specially modified epithe-
lial cells, as the rods arid cones of the retina, the
auditory hairs in the ear, the inner cells of taste-buds
and olfactory cells.

I; Free nerve-endings occur especially in stratified epithelium,
e.g., in the skin, mouth, and cornea (Lesson XXXII.). The norve-
fibres lose their myelin and primitive sheath, and the axis-cylinder

XXXIV.] NERVES OF SKIN. 377

splits up into fine fibrils, which penetrate into the epithelial layer
and run between the epithelial cells, and sometimes anastomose
with each other, to end in free points which do not form connec-
tions with any structure in the epidermis. In the skin these
fibrils are confined to the rete Malpighii, and do not reach the
stratum granulosum.

I. V.S. Skin (gold). — Use boiled gold chloride and formic acid
(p. 372). Take very small pieces (2 mm. cubes) of the palmar
skin of the fingers or toes, preferably from a new-born child or a
young infant; cut away all the adipose tissue, and place the pieces
in the gold mixture for at least one hour. Then place the tissue in
slightly acidulated water, and expose to sunlight until the gold is
reduced. Harden in alcohol, and mount the sections in formic
glycerine.

(H) Observe the blackened fine nerve-fibrils. Some of them
beaded, running between the epithelial cells (fig. 336).

II. Terminal Corpuscles. — They are very varied in their form,
and include the following : —

A. Simple tactile cells.

B. Compound tactile cells.

C. End-bulbs with many modifications.

D. Touch-corpuscles.

A. Simple Tactile Cells— V.S. Skin (gold) (H).— Treat very
small pieces of the human skin

(volar surface of finger or toe)
or the snout of a pig by the
boiled gold formic acid method.
(a.) Observe a nerve-fibre
((ig. 346, n) passing towards
and entering the epidermis,
where it is non-medullated and
splits into fibrils, which ter-
minate in oval, nucleated, tactile
discs or menisci (m), each of
which lies under a tactile cell
(a). The cells are 6-12 /x in

length, and lie in the deeper FIG. ^.-Tactile Cells Snout of Pig «. Xcrve-

. , . fibre ; a. Tactile cell ; m. Tactile disc.

part of the epidermis.

B. Compound Tactile Cells, called also Grandry's and Merkcl's
Corpuscles, consist of two or more cells (15 p. x 50 //.) piled one on
the other ; between each two cells is a disc or plate — tactile disc —
which is connected with the axis-cylinder of a nerve. The cells
have a large pale nucleus, and the whole is surrounded by a fibrous
capsule. The nerve loses its myelin after it passes into the organ,

378

PRACTICAL HISTOLOGY.

[XXXIV.

FIG. 347.— Tactile Corpuscles, Bill of Duck,
A. With three, and B. With two tactile
cells. The axis-cylinder terminates in the
tactile disc (black).

and its sheath becomes continuous with the fibrous cupsule. So

far these structures have been
found only in the bill and tongue
of birds, e.g., duck. They lie in
the corium, close under the epi-
dermis (fig. 347).

3. T.S. Tongue or Bill of
Duck (G-randry's Corpuscles)
(L and H). — Harden in i per
cent, osmic acid small pieces of
the marginal part of the tongue
or the sieve-like structure on
the edges of the mandibles of a

duck. Treat other small pieces by the boiled gold chloride method.
(a.) Observe the tactile cells and discs as in fig. 347.
C. End-bulbs are small, oval, or cylindrical bodies of various
shapes. A nerve-fibre enters one pole, and as it does so it loses its
myelin and terminates in a softer granular inner core. The bulb—-
which may be cylindrical in shape
— consists of a tough fibrous cap-
sule continuous with the sheath of
eg, the nerve ; and a softer inner core
in which the axis-cylinder ter-
minates. Such bodies are found
in the connective tissue of the
mucous membrane of the mouth
and conjunctiva. It is not difficult
to isolate them from the conjunc-
tiva of a calf (fig. 348). They lie
in the sub - epithelial connective
tissue. Certain special forms of
them occur in the genital organs,
e.g., clitoris, and in connection with
joints. A special form is known
as Pacinian corpuscles.

4. Pacinian Corpuscles (Cat).

— These are elliptical transparent bodies (2-3 mm. long and i mm.
thick), readily found in the meso-colon and mesentery of a cat.
They consist of many concentric fibrous laminae, arranged like the
coats of an onion, surrounding a central core, which is continuous
with the axis-cylinder of a nerve. The lamella are lined on both
surfaces by endothelium, which can be stained with silver nitrate
(fig. 350). The capsules are closer together near the centre than
they are at the periphery. Examine one fresh in normal saline,
and "fix" another with osmic ocid and mount it in Farrant's

FIG. 348. — End-Bulb from Human Con-
junctiva, a. Nucleated capsule ; l>.
Core; c and d. Nerve terminating
ind.

XXXIV.]

NERVES OF SKIN.

379

solution. A medullated nerve-fibre surrounded by perineurium
passes to each corpuscle. The lamellae of the perineurium become

FIG. 349.— Pacini's Corpuscle, from Mesen-
tery of Cat. c. Capsules ; d. Endothe-
lial lining separating the latter; n.
Nerve ; /. Funicular sheath of nerve ;
m. Central mass ; n'. Terminal fibre ;
and a. Where it splits up into finer
fibrils. Examined in fresh normal
saline.

FIG. 350. — Endothelium of
Lamellae of a Pacinian
Corpuscle. Silver nitrate.

continuous with the lamellae of the corpuscle, and the nerve — still
provided with its my el in and sur-
rounded by its endoneurium — pierces
the lamellae and reaches the central
part of the corpuscle. The endoneurium
seems to form a soft core round the axis-
cylinder, which usually splits up at the
further end into a tuft of fibrils (fig.

349)-

5. T.S. Pacinian Corpuscles. — They

lie in the subcutaneous tissue. In a
V.S. of foetal skin from the palmar
surface of a digit (Lesson XXIX. 5) it FIG. 35i.—T.s. Pacinian Corpuscle,

» . i , i IT . i T 1 Foutal Skin.

is easy to find these bodies cut obliquely

or transversely. Fig. 351 shows their appearance when cut trans-
versely. Harden the skin in alcohol or osmic acid. Stain in picro-
carmino or hsematoxylin.

6. Herbst's Corpuscles (H) are modified Pacinian corpuscles,
occurring in the mucous membrane of the tongue of the duck. They
lie in the corium, just under the epidermis. Harden the tongue or
bill as for tactile corpuscles (p. 378), or use absolute alcohol, and
stain the tissue in bulk with borax-carmine. Mount in balsam.

380

PRACTICAL HISTOLOGY.

[XXXIV.

(a.)

FIG. 352

H

Observe the same general arrangement as in Pacinian cor-
puscles. These bodies, however, are more
elongated, and the axis-cylinder in the
centre is bordered by a row of nuclei.

D. Touch-Corpuscles of Wagner or
Meissner. — These are elliptical bodies
(40-1 50 fji long and 30-60 p. broad), which
occur in the papillae of the skin, especially
in the volar side of the fingers and toes.
A nerve-fibre passes to each corpuscle,
which has a fibrous sheath. Owing to the
way in which the divisions of the nerve
twist round the corpuscle, they have an
irregular transversely striated appearance.
The nerve-fibre enters the corpuscle, and
its branches take a coiled course round the
j corpuscle.

7. V.S. Skin (Touch-Corpuscles).— (i.)
Make vertical sections of skin (palmar
1 surface of digit), harden in alcohol, stain
them in haematoxylin or safranin, and
mount in balsam (fig. 352).

(ii.) Treat skin by the boiled gold
chloride method to see the terminal
branches and distribution of the nerve

T

In some
situations tlie corpuscles are compound.

The following Table shows the modes
of termination of nerve-fibres in sensory
surfaces : —

V.S. Skin, Palm of .,, . ,, , tc. x

6. Papilla of cutis ; within the corpuscle (fig. 353).

and

S
touch-corpuscle ; g. Cells of

I.

II.

III.

IV.

Free nerve-end-

Simple tictile-

Cylindrical end-bulbs

Neuro - epithe-

ings (cornea,

cells (human

(conjunctiva).

lium (retina,

skin).

skin).

Herbst's corpuscles

internal ear,

Compound tac-

(tongue of duck).

olfactory re-

ti 1 e-c ells

Pacinian or Vater's

gion of nose,

(birds, e.g.,

corpuscles(deep pail

and organ of

duck's bill

of skin, mesentery

taste).

and tongue .

of cat, and near

joints).

Genital corpuscles

(clitoris).

Wagner's touch -cor-

puscles (papillae of

skin).

XXXIV.]

NE11VES OF SKIN.

38l

ADDITIONAL EXERCISES.

8. Tactile Hairs and Nerves to Hair- Follicles. — These are to b« studied
by the boiled gold chloride method.1

FIG. 353.— Wagner's Touch-Corpuscle,
Skin of Hand. n. Nerve ; a. Ter-
minations of n. Gold chloride.

FIG. 354. — Organ of Eimer, Nose
of Mole. n. Nerve ; e. Epi-
thelium. Gold chloride.

9. Organ of Eimer. — Prepare the nose of the mole by the gold chloride
method. In sections study the termination of the nerve-fibrils in the structure
known as the organ of Eimer (fig. 354).

LESSON XXXV.
THE TESTIS.

THE framework of the testis (fig. 355) consists of a strong, tough,
fibrous capsule — the tunica albuginea — which is covered externally
by endothelium reflected from the serous membrane — the tunica
vaginalis. At the back part of the organ, the tunica albuginea is
prolonged for some distance (8 mm.) into the gland to form the
corpus Highmori or mediastinum testis. From the under surface
of the tunica albuginea septa or trabeculse pass towards the corpus
Highmori, and thus subdivide the gland into compartments or lobules
(about 120 in number), with their bases directed outwards and
1 Hoggan, Jour, of Anat. and Phys., 1892.

382

PRACTICAL HISTOLOGY.

[XXXV.

Septa.

Tunica albuginea.

Cavity of tunica
vaginalis.

Outer layer of tunica
vaginalis.

Vas deferens cut.
Spermatic vein.

FIG. 355.— T.S. of Testis.

their apices towards the corpus Highmori. The tunica albuginea
consists of dense fibrous tissue, but it is of looser texture internally,
where it has numerous vessels and lymphatics. This inner layer is

sometimes called the tunica
vasculosa, and is continuous
with the septa. Each lobule
contains 2-8 seminal tubules.
These convoluted tubules (800)
begin by a blind extremity,
and are of considerable length
(30-50 cm.) and of nearly
uniform diameter (.3 mm. or
140 JJL). The tubules of any
one compartment unite at
acute angles to form a small

Arteries. Corpus Epididy-

Highmori mis. number ot much narrower
straight tubules (20-25 //,) —
tubuli recti— which pass into
the mediastinum and there form a network of inter-communicating
tubules of irregular diameter (24-180 //,) — rete testis (fig. 356).

From this proceed 12—15 wider
ducts — vasa efferentia — which
are straight at first, but some
become convoluted and form a
series of conical eminences —
coni vasculosi— which together
form the head of the epididy-
mis. The epididymis, consist-
ing of a single convoluted tube
(600 cm., or 20 feet long), is
continued into a thick-walled
muscular tube — vas deferens
(60 cm., or 2 feet long) — which
conducts the secretion to the
urethra, and is, in fact, the
excretory duct of the testis.

The seminiferous tubules
consist of a thick membrane
composed of flattened nucleated
cells, arranged like membranes
and lined internally by a base-
ment membrane. The latter is
lined by several rows of

cubical cells, which differ much in appearance, according to
the functional activity of the organ. In the resting condition,

End of con-
voluted tube.

Rete Testis.

FIG. 356.— Convoluted and Straight Tube of
Testis.

XXXV.] THE TESTIS. 383

the tubes are lined by several layers of more or less cubical cells,
whose nuclei stain more or less deeply with staining reagents. In
an active gland in some tubules some of the clear cubical cells of
the outer row of cells — lining epithelium — contain nuclei under-
going mitotic division. The rows of cubical cells internal to these
have a radiate arrangement, and are separated into groups by larger
structures, sometimes called spermatoblasts, and by other observers
sustentacular cells, which grow up between the smaller cells,
and at their upper ends are connected with the spermatozoa. Inside
the layer of lining epithelial cells are several rows of larger cells —
spermatogenic cells — with nuclei showing different stages of
mitosis. Next the lumen, which is always well defined, in some of
the tubules are spermatozoa in different stages of development. In
others, however, not so far advanced, the inner row of cells consists
of small protoplasmic cells — the true spermatoblasts — as from these
cells the spermatozoa are developed. The developing spermatozoa
rest by means of their heads on the sustentacular cells. The sper-
matozoa are arranged in tufts or groups, with their tails towards
the lumen. The spermatozoa gradually develop from the true
spermatoblasts and pass towards the lumen, and as they are set
free new spermatoblasts are formed by the mitotic division of the
spermatogenic cells.

The straight tubules are lined by a single layer of flattened
epithelium ; the rete testis has no basement membrane, but it
also is lined by a single layer of flattened epithelium. The vasa
efferentia and the tube of the epididymis have smooth muscular
fibres in their walls, and are lined by columnar ciliated epithelium,
the cilia being very long.

The interstitial tissue of the testis between the tubules is very
loose in texture and laminated, and has numerous lymphatic slits.
In some animals (rat, boar) are numerous polyhedral, nucleated,
sometimes pigmented, cells, the remains of the cells of the Wolman
bodies.

The vas deferens consists of a fibrous coat investing an outer
thick layer of smooth muscle arranged longitudinally ; inside this
is a thick layer of smooth muscle arranged circularly ; inside this
again is a submucous coat of connective tissue. In some parts of the
tube there is a layer of smooth muscle arranged longitudinally just
internal to the circular coat. Then follows the mucosa lined by
columnar non-ciliated epithelium.

Methods. — (i.) Harden the fresh testis in M tiller's fluid (2
weeks). If it be large, cut it into small pieces. The capsule exerts
considerable pressure on the gland substance, so that when the testis
is cut into, the latter projects somewhat. Complete the hardening
in alcohol. Corrosive sublimate is a good hardening reagent. Stain

384 PRACTICAL HISTOLOGY. [XXXV.

sections in hsematoxylin or picro-carmine, and mount in balsam.
The hardened testis of a small animal, e.g., rat, should also be
st'ained in bulk in borax-carmine or Kleinenberg's logwood, and cut
in paraffin, so as to include the body of the testis and the epididymis.
Mount in balsam.

(ii.) Harden very small pieces of the testis of a freshly-killed
rat or mouse in Flemming's mixture and stain the sections in
safranin. A very small testis is taken, as Flemming's fluid only
penetrates a few mm. into the tissue. This is for the study of
spermatogenesis.

(iii.) Harden the testis of a frog in absolute alcohol and stain
it in bulk in Kleinenberg's logwood.

(iv.) Harden the testis of the dogfish in Flemming's fluid and
stain the sections in hsematoxylin or safranin. This, as shown by
Swaen, is an excellent object for the study of spermatogenesis.

(v.) Harden the testis of a mouse, salamander, or frog in the
following fluid1 in which platinic chloride is substituted for the
chromic acid in Flemming's chromo-aceto-osmic acid mixture : —

Hermann's Fluid,

Platinic chloride (i per cent.) , 15 parts.

Osmic acid (2 per cent. ) . . 4 ,,

Glacial acetic acid I part.

The above is used for the tissues of mammals ; but if a salamander
testis is used, then take only 2 parts of the osmic acid.

After 2-3 days the details of the cells are better brought out
than with Flemming's solution. Complete the hardening in alcohol.
Make sections in paraffin, fix them on a slide with albumin fixative,
and stain for 24-48 hours in the following safranin fluid : —

Safranin I gram.

Alcohol . . . 10 cc.

Water . , , 90 ,,

After staining, wash in water, acid-alcohol and alcohol, but do
not remove all the surplus safranin. Then stain for 3-5 minutes
in a gentian-violet solution. Decolorise the sections by means of
Gram's method (p. 105), i.e., after washing in alcohol, place them
for 1—3 hours in the following fluid —

Iodine I gram.

Potassic iodide . . .2 grams.

W uter .... 300 cc.

until they are quite black, and then differentiate them in alcohol.
Mount in balsam.

1. T.S. Testis (Rat}.— (a.} Observe the thick, tough, fibrous
tunica albuginea with sections of large blood-vessels and lymphatics

1 Archiv f. mik. Anat., xxxiv. p. 58.

xxx v.j THE TESTIS. 385

in its deeper part. From its under surface septa pass into the
gland at fairly regular intervals, thus dividing it into a series of
compartments or lobules. At the upper and back part is the
nitrous septum— corpus Highmorianum or mediastinum testis.
Many of the septa are connected with it.

(k,) In the lobules lie twisted or convoluted tubules — the semi-
niferous tubules — which converge towards the mediastinum arid
form near it a number of straight tubes — the tubuli recti — which
in their turn unite and form the rete testis in the mediastinum,
and from this proceed the vasa efferentia, which run to join the
canal of the epididymis (fig. 356).

(c.) The tubules in a state of activity are distinguished from
the resting ones by the intensely stained heads of the young
spermatozoa. An active tubule is lined by several rows of poly-
gonal cells, some of which are larger than the others. Embedded
amongst the cells, near the lumen of the tube, are bunches or
tufts of spermatozoa, best seen in longitudinal sections of the
tubes. The heads are directed towards the wall, and the tails
towards the lumen of the tube.

(d.) In some animals the interstitial tissue between the tubules
is chiefly formed by thin flattened membranes of connective tissue ;
but in others, e.g., boar, the matrix consists of numerous pigmented,
polygonal, very granular cells — interstitial cells.

2. Spermatogenesis (Rat}.— In order to see the structure of
the seminiferous tubules to the best advantage, harden small
pieces of the testis of a rat, or a guinea-pig, as directed under
v. and ii. (p. 384). To keep the parts together, cut in paraffin.
The best stain is safranin. Fix a thin T S. of a tubule under a
high power.

(a.) Observe the rather thick wall — wembrana propria — of the
tubule, composed of flattened cells, perhaps of a connective tissue
nature. This is lined by three or more layers of glandular cells,
which vary in appearance according to their condition of physio-
logical activity. In a state of rest each tube is lined by several
layers of large polygonal cells placed one inside the other.

(/>.) In an active gland, known by the evidences of division of
cells and by the development of spermatozoa (fig. 357), there is an
outer layer of cubical-looking cells, and internal to it several layers
of round or ovoid cells, which are called spermatogenic cells. In
the latter may be seen nuclei undergoing mitotic division. The
lining epithelial cells seem to divide ; one part of each cell passes
into the second layer of cells, and becomes a sperm atogen or sper-
matogenic cell, while the remainder of the original cell enlarges and
grows up as a sustentacular cell. The spermatogenic cells divide
and redivide by mitosis, and yield the small daughter-cells or
34 2 B

386

PRACTICAL HISTOLOGY.

[xxxv.

spennatoblasts of the innermost layer. The latter, arranged in
groups, gradually elongate, and from them the groups of spermatozoa
are formed. Each group rests on and is connected with a susten-
tacular cell ; hence arose the old view that these sustentacular
cells produced the spermatozoa, and for this reason they were
formerly called spermatoblasts. The nucleus of a daughter-cell
forms the head and body of the spermatozoon, while the tail is
formed within the protoplasm, but it is connected with the nucleus,
and in its development grows out and forms a cilium.

3. Isolated Cells from the Tubules. — Macerate small frag-
ments of the testis in dilute alcohol 10-12 hours. Tease a small
fragment in glycerine. Observe the various forms of cells iso-
lated.

4. Spermatozoa (H). — Make a cut into the epididymis of a testis
removed from a rabbit as soon as possible after death. A milky

FIG- 357.— Tubules of Testis of Rat, showing Spermatogenesis. A. Less advanced stage:
B and C. More advanced stages. A and £=T.S.; C=L.S. Memmiug's fluid and
safranin, x 300.

juice exudes. Place a little of this on a slide and dilute it with
normal saline.

(a.) The spermatozoa, each one with a head and a long vibratile
tail or cilium. By the side-to-side movement of the tail the whole
spermatozoon is moved onwards in a zigzag course.

(b.) Add a drop of distilled water. The movements are arrested.
The spermatozoa are slowly killed.

They may be readily preserved by smearing a little of the milky
juice from the epididymis upon a slide and allowing it to dry. It
is then covered and sealed.

5. Spermatozoa of Newt and Frog (H). — The testis of a newt
is teased in a mixture of alcohol and glycerine.

(H) Observe the pointed head, body, and long tail of each sper-
matozoon. There is an intermediate part, which is best seen in a
stained preparation. From it springs a filament which appears to
be prolonged as a spiral filament around the cilium or tail. The

XXXV.]

THE TESTtS.

3*7

spiral filament is in reality the optical expression of a thin membrane
attached in a spiral direction to the cilium. Mount
the spermatozoa of a frog. The male frog is easily
known by the wart on his thumb. Open the
abdomen, and low down in the hollow on each
side of the vertebral column is an oval, kidney-
shaped, whitish body — the testis. It is to be
treated like the testis of other animals. At
certain seasons of the year no spermatozoa are to
be found. The cells are in a resting phase.

6. Human Spermatozoa. — Mount in glycerine
some spermatozoa obtained from a spermatocele
(tig- 358). Observe the head (&), long tail (/),
and middle piece (ra).

7. Cover-Glass Preparations (H).— Compress
a small piece of the testis of a newt or frog
between two cover-glasses. Separate them, and
allow the film on them to dry. Stain one in
methylene-blue, and the other for twenty- four hours
in Ehiiich-Biondi's fluid. Dry and mount in balsam.

(a.) In the methylene-blue specimen only the
heads of the spermatozoa are stained. In the
other, the head of the spermatozoon is stained
one colour, while the tail and the intermediary or Fl(i
junction piece is of a different hue.

8. Testis of Salamander.— Fix the testis (in September or
October) in Hermann's

fluid, and stain as directed Blood-vessel,

at p. 384. The bundles of
ripe spermatozoa are very
marked. The body of
each spermatozoon is red-
dish, its head bluish-violet,
the middle piece also of the
same colour, while the tail
is brownish - violet. In
Hermann's paper figures
will be found showing the
remarkable stages of de-
velopment of the bodies
of the spermatozoon from
the nucleus of the sperm a-
tids. Numerous mitotic
figures may be seen.

9. Epididymis (L and H). — Prepare like the testis.
T.S., stain in hajmatoxylin and mount in balsam.

338.-Spernia-
to/oa. i 2. Human.

T.S. Tubule.

Ciliated

•',! epithelium.

FlQ 359-— T.S. Tubules of Epididymis.

Make

388 PRACTICAL HISTOLOGY. [xxxvi.

(a.) Observe numerous sections of the twisted tube of the
epididymis (fig. 359); each tubule has a fibrous wall, and is lined
by a single layer of tall, slender, columnar ciliated epithelial cells.
Each cell is provided with a fringe of long cilia. The lumen is
wide and distinct} and may contain a confused mass of spermatozoa.
At the bases of the cells are sometimes seen small cells, and in
pigmented animals pigment granules are not unfrequently seen in
the connective-tissue stroma supporting the tubule. There is a
fair amount of connective-tissue stroma between the tubules, and
in it are blood-vessels, lymphatics, and nerves.

10. T.S. Vas Deferens. — Harden it like the epididymis, make
T.S., stain, and observe the arrangement of its parts as described
at p. 383.

LESSON XXXYI.
THE OVARY— FALLOPIAN TUBE— UTERUS.

THE OVARY.

THE ovary consists of a stroma, covered on the surface with a
single layer of short columnar or germinal epithelium. Embedded
in the stroma are numerous Graafian follicles in all stages of ripe-
ness. Each Graafian follicle contains one ovum. The unripe
follicles, with primitive ova, form a superficial layer close under
the surface, while the more mature ova and follicles lie deeper.
The coverings of the follicle and the structure of a ripe ovum are
given at p. 389.

Methods. — Select the ovaries of small animals, e.g.) mouse, rat,
as they can be better "fixed" than large ones.

(i.) Harden the ovary of small animals in toto in Mtiller's fluid
(2 weeks), and then in alcohol. Make two or three transverse
cuts in the human ovary before hardening it in the same fluid.

(ii.) Picro-sulphuric acid, for a day, is also a good hardening
reagent. Complete the hardening in alcohol. The pieces had
better be stained in bulk in borax-carmine, or Kleinenberg's
logwood, and embedded and cut in paraffin, or embedded and cut
in celloidin.

(iii.) For small ovaries use Flemming's fluid, embed and cut in
paraffin. Stain with safranin.

For obtaining a general view, the sections must be pretty thick,

xxxvi.]

THE OVARY\

FIQ.

. 360.— T.S. Ovary.
Graaflan follicle ;

e. Germ epithelium; i. Large
2. Middle-sized, and 3. Small
(/. Membrana granulosa ; 8.

otherwise the follicles are cut -into, and the ova are apt to fall out.
Thin sections must also be made.

1. T.S. Ovary (fig. 360).— (a.) (L) The body of the ovary,
covered on its surface by
a single layer of short,
columnar, nucleated cells
- the germinal epi-
thelium.

(/>.) The substance or
stroma, composed of con-
nective tissue with numer-
ous blood-vessels, and in
some places smooth muscu-
lar fibres. The connective
tissue is denser, and is
arranged in several layers,
near the surface.

(c.) Lying in the stroma
—the Graaflan follicles.
Near the surface is a layer
of smaller unripe ova — <>ya; *. Ovum

. ... *f Stroma.

primitive ova (40 /*) —

while the riper and larger follicles are situated deeper in the stroma.
Each follicle contains an ovum, but if the section be thin the ovum
may drop out and leave only the follicle and its coverings.

(d.) If the ovary be from an adult animal, a corpus luteum may
be found. Its appearance will depend upon whether it has been
recently formed or not, and whether the animal was recently preg-
nant. If it is of some standing, it is large and more or less spherical
in outline, occupying a considerable part of the stroma, with an
umbilicus-like centre and radiating lines of connective tissue. Be-
tween these are numerous large granular cells. In a recent one
there may be a yellow pigment — lutein — staining the scar.

(e.) (H) The germinal epithelium, and under it the tunica
albuginea, composed of two or more layers of connective-tissue
lamellae — with fusiform cells — crossing each other. It gradually
passes into the stroma.

(/.) The Coverings of the Follicle.— Select a well-developed or
nearly ripe ovum (.5-5 mm. in diameter). Outside is the theca
folliculi, composed of two layers, an outer one fibrous — the tunica
fibrosa — and inside it a layer with blood-vessels — the tunica
propria. Inside this several layers of cubical cells, constituting
the membrana granulosa. From this there projects a mass of
cells at one part, forming the cumulus or discus proligerus. The
cells are continued as the tunica granulosa around the ovum.

39O PRACTICAL HISTOLOGY. [XXXVI.

Between the tunica and membrana granulosa is a space which
encloses a fluid in the large follicles — the liquor folliculi.

(<?.) The ovum, composed externally of a hyaline membrane —
the zona pellucida — enclosing more or less granular cell-contents ;
the vitellus or yelk ; and placed excentrically in this the germi-
nal vesicle (corresponding to a nucleus), \vith its small excentric
germinal spot (corresponding to a nucleolus).

(h.) Small Unripe Ova. — Besides the difference in size, the
follicles show no separation between the tunica and membrana
granulosa. They form a layer of cells in which the small ovum
appears to be embedded.

2. The Ovum (fig. 361). — A fresh ovum may be obtained thus.
Take the ovary of a cow or sheep, which on its surface shows clear

Cells of discus proligerus.

Yelk.

FIG. 361.— Ripe Ovum of Ilabbit highly magnified.

elevations about the size of peas, filled with a watery-looking fluid ;
these are ripe follicles. Prick the follicle and receive its contents
upon a slide. The liquor folliculi escapes, and with it the ovum
surrounded by the cells of the tunica granulosa. With (L) search
for the ovum in the fluid on the slide. Do not cover the prepara-
tion while doing so. If a cover-glass be applied, two strips of paper
must be placed under the cover-glass to avoid pressure upon the
delicate ovum. Observe the ovum, and in it will be seen the parts
already described and shown in fig. 361.

3. T.S. Fallopian Tube (L and H).— This is hardened in
Miiller's fluid or Flemmiug's fluid. Treat it in the same way as

XXXVI.]

THE OVARY.

391

the intestine. Make transverse sections, and stain Ihem either
with piero-carmine or logwood.

Circular mus-
cular fibres.

Muscular fibres,
"•cut across.

FIG. 362.— T.S. of Fallopian Tube.

The Fallopian tubes in animals often pursue a curved course.
The peritoneum should be cut off
as close to the tube as possible,
and the latter stretched if complete
T.S. are required.

(a.) Observe, most external, the
thin serous coat.

(b.) Inside this the muscular
coat, composed of non-striped
muscle, a very thin longitudinal
layer of fibres, and a much stronger
circular layer.

(c.) A thin submucous coat.

(d.) The mucous coat, thrown
into numerous ridges or folds, i.e.,
sections of longitudinal folds, so
that the lumen of the tube appears
somewhat star-shaped or branched
(fig. 362). Sometimes the pro-
cesses of the mucous membrane
are very complex in their arrange-
ment, and give rise to arborescent-
looking folds in transverse sections (fig. 363).

(e.) (H) The thick mucous coat is covered by a single layer of

FIG. 363 —T.S. of the Fimbriated Extre-
mity of the Fallopian Tube of Tig.

392

PRACTICAL HISTOLOGY.

[xxxvi.

low cylindrical ciliated cells, but there are no glands, although the
depressions act the part of glands. Under this is connective tissue
with a very thin muscularis mucosce.

4. T.S. Fimbriated End of Fallopian Tube stained in borax-
carmine and cut in paraffin. Observe the very complex folds of
the mucous membrane, each with secondary folds and covered by
ciliated epithelium. The folds are much higher and far more com-
plex than they are in the Fallopian tube (fig. 363).

UTEETIS.

The wall of the uterus is composed of smooth muscle lined by
a mucous membrane. As a rule, two layers of muscular fibre —
imperfectly separated from each other — can be distinguished in the
muscular coat • the external layer having
an irregular course ; then follow blood-
vessels and connective tissue, which sepa-
rate it incompletely from the inner one
arranged more circularly. This thick
inner layer of smooth muscle is said to
represent a much hypertrophied muscu-
laris mucosae. The mucous membrane
(i mm. thick) consists of connective tissue
containing a very large number of cells
and nuclei, and embedded in it are
tubular glands — utricular glands — some
of them simple, and others branched (fig.
364), especially at their lower ends.
These glands seem to be devoid of a
membrana propria, but the surrounding
connective tissue is arranged around them
in a concentric manner. The body of the
uterus, upper half of the cervix, and the
uterine glands are lined by a single layer
of short columnar ciliated epithelium.
The vaginal portion of the uterus is lined
by stratified squamous epithelium, and
carries small vascular papillae covered in
by the epithelium. The mucous mem-
brane rests directly on the muscular coat,
there being no proper submucosa. Small
processes of smooth muscle from the
muscular coat are prolonged inwards between the bases of the
glands. The mucous coat is very vascular and contains many
lymphatics.

FlQ. 364.— V.S. Uterus of a
Bitch.

XXXVII.] MAMMARY GLAND. 393

Methods. — It is rare to obtain the human uterus sufficiently
fresh for microscopical preparations. Harden the uterus of a
bitch, cat, or rabbit, in chromic and spirit fluid, alcohol, or Mailer's
fluid. Make T.S., and treat them as the Fallopian tube.

T.S. Uterus. — (a.) Observe the serous, muscular, and mucous
coats as in the tube, but here the muscular coat is very thick, and
is composed of numerous fibres arranged in bundles and running in
all directions. The arrangement of these bundles is much simpler
in animals than in the human uterus.

(I.) The mucosa is very thick, and is covered by a single layer
of cylindrical nucleated cells, and has numerous glands, which are
lined by similar cells. Not un frequently the gland-tubes branch,
especially near their lower extremities. It is difficult to retain the
cilia on the epithelium lining the cavity of the uterus and its glands.
Between the tubules is a relatively large amount of connective
tissue containing many nucleated corpuscles and blood-vessels. As
the glands pursue a curved course, and do not always run at right
angles to the mucous surface, it is difficult to obtain a section,
through the entire length of a gland (fig. 364).

LESSON XXXVII.

MAMMARY GLAND, UMBILICAL CORD, AND
PLACENTA.

THE mammary gland is a compound racemose gland, but it has
about twenty galactoferous ducts which open on the nipple, each
duct being dilated into a small reservoir just before it ends on the
surface. The ducts, when traced backwards, branch and end in
acini or saccular alveoli. The alveoli — as in all glands — vary in
appearance according as the gland is or is not active. The walls of
the ducts and acini consist of a basement membrane, said to be
composed of branched cells, which in the acini is lined by a single
layer of somewhat flattened polyhedral secretory cells. A cluster
of acini gives origin to one of the larger ducts, and a considerable
amount of connective tissue lies between groups of acini. In fact,
the connective tissue greatly preponderates. During lactation the
secretory cells are taller and larger, and in their interior — probably
formed from and by the protoplasm of the cells themselves — are
formed the fatty granules which are discharged to form the milk
35

394

PRACTICAL HISTOLOGY.

[xxxvii.

globules. The active gland presents some resemblance to a salivary
gland, so closely are the alveoli pressed together, with only a small
quantity of interstitial connective tissue between them. The acini
are in groups and separated from each other by fibrous imperfect

septa. Numerous cor-
puscles, including granu-
lar cells, occur in the
alveolar connective tis-
sue. The ducts are
lined by columnar epi-
thelium, and in a section
of a human gland they
appear large.

(i.) Harden small
parts of the mammary
gland in absolute al-
cohol. Select, when
possible, the gland of
a recently pregnant
woman (or animal).

Fia. 365.— T.S. Mammary Gland. D. Duct; A. Group Qfnir, fk0 rw.fi'™
of acini with much connective tissue between, x 20 btaln the . sectlOns m

hsematoxylin and mount-
in balsam, i.e., to get a general view of the gland structure. Stain
in bulk, embed and cut in paraffin.

(ii.) Harden very small pieces of a fresh gland, e.<j., from a

FIG. 366.— T.S. Secreting Mammary Gland, Mouse.
•Pol's solution and safranin, x 300.

'o " " " o-^-aff ••• QV • r~*&

Fia. 367.— Colostrum.

pregnant cat or rabbit, in Flemming's mixture and stain the
sections (very thin) in safranin.

1. V.S. Mammary Gland (Hcematoxytin) (L and H) (fig. 365).

(a.) Observe the groups of acini, separated by a relatively large

XXXVII.] UMBILICAL CORD AND PLACENTA.

395

amount of somewhat loose connective tissue, and sections of the
ducts (D). The sections should be made so as to include the
nipple, when the larger ducts with their dilations will be seen.
The ducts are large between the lobules, and within the latter the
course of the finer ducts can readily be traced.

(b.) The globular acini, with a basement membrane lined by a
single layer of somewhat flattened or cubical epithelium. In the
inter-alveolar tissue many leucocytes and granular cells.

2. Active Mammary Gland (Safranin) (H).

(a.) Study specially the acini Observe the large and tall
columnar cells lining the acini, and in some of the cells clear
refractive granules of fat. The lumen is wide, and is usually
partially filled with the debris of the secretion-milk. Osmic acid is
a good agent for showing the presence of fatty granules (fig. 366).

3. Colostrum (H), i.e., the first milk secreted after delivery.
If this can be obtained, examine it, and note, in addition to the
ordinary milk-globules (Lesson I. 3), large coarsely granular nucle-
ated refractile cells — colostrum corpuscles. The granules are
sometimes pigmeiited, and are fatty (fig. 367).

UMBILICAL COED AND PLACENTA.

4. T.S. Umbilical Cord.— Harden this in Muller's fluid or
alcohol. Make T.S. by freezing, and stain them with hsematoxylin
or ] )icro-carmine. Methyl-violet is also a good stain.

(a.) Note on the outside of
the circular mass of tissue a
thin layer of flattened cells
derived from the amnion.

(b.) The cord itself, composed
of Wharton's jelly, enclosing
usually two umbilical arteries
and a single vein with very
thick muscular coats. They
are completely surrounded by
AVharton's jelly, which, how-
ever, in a cord at full time is
very largely composed of fibrous
tissue. Still numerous branched
connective tissue corpuscles exist
in the meshes, and there are
also present numerous lymphoiJ-looking cells (Lesson XII. 10).

5. Fresh Placenta (H). — Tease a fragment of a placenta in
normal saline. Note the vitli, each long, tapering, and branched.
In the interior capillary loops which occupy the greater part of the

FIG. 368.— Human Flaceiita Villi.
vessels black.

Blood-

396 PRACTICAL HISTOLOGY. [XXXVIII

villus, so that only a small amount of connective tissue intervenes
between the vessels. Each villus is covered on its surface by a
layer of epithelium, which, however, is thin at one part and thick at
another. Especially at the ends of the villi are large granular masses
of protoplasm containing many nuclei, but one cannot make out a
separation of these masses into cells. They often contain vacuoles.
The arangement of the blood-vessels may be followed from the
distribution of the blood-corpuscles (fig. 368).

Small portions of a placenta are also to be hardened in Miiller's
fluid and stained in bulk in borax-carmine. Individual villi may
be isolated in dilute alcohol or osmic acid.

6. Injected Placenta. — Examine a vertical section of a placenta
with the fo3tal blood-vessels injected, say blue, and the maternal
vessels red. Observe how the one set interlocks with the other, yet
both systems are closed and do not communicate with each other.

LESSON XXXVIIL

TO MAKE PREPARATIONS RAPIDLY PROM
FRESH TISSUES.

IT is of the utmost importance that the student should be acquainted
with the methods of making preparations from fresh tissues placed
in his hands. The following is an outline of the work that each
one can readily do for himself if supplied with a pithed frog, or
other suitable material.

A. From a Frog.

1. Corneal Corpuscles. — With a sharp pair of scissors cut out
the cornea. Divide it into two parts.

(a.) Treat one by the lemon-juice method (p. 79).

(/>.) Treat the other part by placing it direct into .5 per cent.

AuCl3 (half an hour) ; wash in distilled water ; place in

a saturated solution of tartaric acid at 50° C. until the

gold becomes reduced (p. 79).
('•.) A cornea may be placed fresh in dilute methylene-blue

(i : 300 normal saline). Mount in picrate of ammonia

glycerine (p. 192).

2. Corneal Lymph-Spaces. — Remove the eyelids, expose the
surface of the other cornea, scrape olf the epithelium, and rub it

XXXVIII.] FRESH TISSUES. 397

with solid silver nitrate. Cut out the cornea and expose it to light
in water (p. 77).

3. Tendons. — These are hest made from the tarsal tendons,
which can readily be snipped off in considerable lengths.

(a.) Fibrils. — Tease a piece in baryta-water and mount in
glycerine.

(b.) Tendon Cells. — (i.) Add dilute acetic acid to bring into
view the rows of cells, then wash with water, and after
all the acid is removed stain with logwood or picro-
carmine. (ii.) Also tease a piece in normal saline con-
taining a trace of methyl-violet.

(c.) Silver one of the tendons to show the endothelium cover-
ing its surface (p. [66).

(7.) Place a fresh tendon in ammoniacal carmine (10-15 nains.),
wash and place in very dilute Delafield's logwood
(10-15 niins.). Wash, tease; and mount in balsam.
The tendon cells are red, their nuclei blue, and the
tendon fibres rosy.

4. Aponeurosis.— The best is the femoral.

(a.) Kernove the membrane and stretch it on a slide by the
"semi-desiccation" method (p. 159), and after it is
fixed to the slide apply a drop of acid methyl-green,
or normal saline with methyl-violet. The nuclei are
thereby stained, and the crests and ridges of the cells
are made visible.

(?>.) It may be fixed rapidly on a slide with absolute alcohol and
stained with logwood. Or osmic acid may be used to fix it.

(c.) Show the effect of acetic acid.

5. Areolar Tissue. — (a.) Dissect out some from the inter-
muscular septa of the leg muscles. Stain with methyl-violet in
normal saline. This stains the cells. Or use acetic-fuchsin (p. 92).

6. Yellow Elastic Fibres. —These are found in the septa between
the lymph-sacs. Cut out a septum, fix it on a slide by "semi-
desiccation," and then add acetic acid. Or make another prepara-
tion and stain it with a weak solution of methyl-violet- 56 (p. 93).

7. Pigment-Cells. — (a.) These are found in the web of the frog's
foot. Stretch the web between the toes, harden it in absolute
alcohol for an hour or so, peel off the skin, and mount it in balsam

(P- I73)-

(b.) Or use the mesentery, or almost any blood-vessel ; add
dilute acetic acid and mount in glycerine.

8. Hyaline Cartilage. — (a.) Use either the episternum, scraping
off the perichondrium, or make a section of the articular cartilage
on the femur or tibia. Stain in hamiatoxylin. Or, before cutting,
the cartilage may be hardened for an hour in absolute alcohol

39^ . PRACTICAL HISTOLOGY. fxxxvin.

(b.) A silver nitrate preparation may also be made (p. 151).

9. Endothelium of Mesentery. — Place pieces of mesentery in
AgX03 (.25 per cent.) for half an hour, wash in distilled water and
expose to light in 50 per cent, alcohol.

Part may be afterwards stained in hsematoxylin.

10. Endothelium of Great Lymph-Sac. — Open the abdomen
from the front along the middle line, turn aside the intestines,
and note the kidney. A thin membrane or septum stretches from
this to the abdominal wall. With a fine pipette filled with silver
nitrate solution perforate this membrane and allow silver nitrate to
flow into the great lymph-sac. Expose the membrane to light, and
then examine in glycerine to see endothelium and stomata (p. 239).
One-ha^f may be stained with hsematoxylin to show the nuclei. Or
expose the septum from behind as directed at p. 238.

11. Adipose Tissue. — Use the yellow-coloured fat bodies found
in the abdominal cavity.

(a,) Tease a piece in glycerine.
(b.) Use osmic acid (p. 169).
See also other methods in Lesson XII.

12. Striped Muscle. — In this one must demonstrate —
(a.) Sarcolemma (p. 193).

(/>.) Nuclei, e.g., by acetic acid (p. 194).

(c.) Sarcous substance with its cross stripes. Harden for half
an hour in alcohol and stain with hasmatoxylin or picro-
carmine, or both. Mount in glycerine. Osmic acid
also "fixes" the stria tion.

(d.) Fibres may be isolated by means of 33 per cent, caustic
potash, but they must be examined in the same solu-
tion.

13. Cardiac Muscle. — Isolated cells are obtained by the 33 per
cent, caustic potash method. The fresh tissue teased, stains well in
picro-carmine.

14. Smooth Muscle. — Use

(a.) Frog's bladder (p. 190). In addition, spread out the
bladder on a slide, expose it to the vapour of glacial
acetic acid, wash away the epithelium, stain with
violet-B, and mount in picrate-glycerine (S. Mayer).

(6.) Intestine. The muscular coat alone is to be used, after
scraping away the mucous coat. Treat it as above.

15. Epithelium. — Scrape any epithelial surface, diffuse the
scrapings in normal saline and examine fresh, and seal up with
paraffin wax.

Squamous. — Use cornea.

Columnar. — Use intestine.

Ciliated. — Mucous membrane of palate.

xxxviii.] FRESH TISSUES. 399

. Stain others with acid methyl-green or picro-carmine.

Make cover-glass preparations (p. 140).

Before staining pass the cover-glass three times through the flame
of a Bunsen-burner.

16. Medullated Nerve-Fibres. — Expose the sciatic nerve.

(a.) Tease out a few fibres and show them fresh in normal
saline. Seal up the preparation with paraffin wax.

(b.) Tease a piece in i per cent, osmic acid, cover with a watch-
glass, and after half an hour mount in glycerine. This
blackens the myeline. A part of this may be stained
in picro-carmine (p. 206).

(c.) Tease a piece in .5 per cent, silver nitrate for Ranvier's
crosses (p. 207).

(<?.) Harden a piece of nerve in alcohol for a quarter of an
hour. Tease, place in ether for 10 minutes, transfer to
alcohol. Stain with logwood and mount in glycerine.
This shows the axis-cylinder and nuclei of sheath.

(e.) Place a piece of fresh nerve in collodion to show axis-
cylinder. This preparation only lasts for a short time
(p. 211).

(/.) Show with silver nitrate the endothelial sheath on one of
the small nerves to be found in the dorsal lymph-sac,
stretching between the back muscles and the skin
(p. 207).

17. Peripheral Nerve-Cells. — Use the rapid gold chloride
(p. 79) or methylene-blue method (p. 222).

(a.) Cells along course of aorta. Cut out the abdominal part

of the aorta.

(/>.) Cells of sympathetic (p. 216) or
(f.) Cells of spinal ganglia, or other ganglia (p. 215).
(<l.) Interauricular septum of the heart. The heart must be

distended and kept fixed in this position (p. 233).

18. Retina. — Carefully dissect out the eyeball, remove retina.
(a.) Place a part in i per cent, osmic acid and then make

teased preparations. One should show the pigmented

epithelium, rods with outer segments blackened, pieces

of the several layers, the glistening fatty globule

rendered brownish.
(b.) Harden a piece in absolute alcohol and tease in very

dilute eosin. Mount in glycerine.
(c.) Place small pieces in very dilute methylene-blue (p. 222)

to show the nervous elements,, Mount it in picro-

glycerine (p. 222).

19. Blood- Vessels. — A new frog will be required. Proceed as
directed at p. 230.

400 PKACTICAL HISTOLOGY. [XXXVIIL

(a.) Inject AgN03 (.5 per cent.), and isolate arteries, veins,

and capillaries ; best from the intestine. Mount in

balsam.
(b.) Show the effect of acetic acid on one of the larger vessels

of the mesentery.
(c.) Methylene-blue injected into the vessels shows the lining

endothelium and nerve-fibres.

20. Blood and Blood-Corpuscles.

(a.) Osmic acid and picro-carmine (p. no).

(b.) Cover-glass preparations stained by eosin in glycerine and
then in logwood. Or other dyes or double stain
(p. 114).

(c.) Cover-glass preparations stained by methylene-blue for the
nuclei. Pass the cover-glass three times through the
flame of a Bunsen-burner before staining.

(</.) The colourless corpuscles may be stained in the methylene-
blue specimens, but they may be specially stained by
eosin-glycerine or indulin-glycerine, which stain certain
granules in their protoplasm (p. 402).

21. Fibrin (p. 119).

22. Marrow. — Squeeze out same.

Examine fresh in NaCl (.6 per cent.) with methyl-violet.

Cover-glass preparations stained with eosin-glycerine and
logwood (p. 1 88). The easiest way is to pass the cover-
glass with its adherent film of marrow three times
through the flame of a Bunsen-burner before staining.

23. Motor Nerves to Muscles.— Gold method or methylene-
blue (p. 219).

B. From a Mammal.

If a mammal, e.g., rat or guinea-pig, or part thereof, be given
from which to prepare specimens, the methods are much the same
as those described above.

24. Areolar Tissue.— (a.) To show its histological elements the
best plan is to inject under the skin, by means of a hypodermic
syringe, some fluid which will form an artificial oedema, e.g., methyl-
violet in normal saline, osmic acid (i per cent.), silver nitrate
i : 300. The first of these fluids does not alter the tissues. Excise
a piece and examine it in the same fluid.

(b.) Use the " semi-desiccation " method with a small piece snipped
off" and spread out on a slide.

(c.) Cell-spaces by means of AgN03 (p. 162).

In addition to the methods described at p. 161 and p. 162, use
acetic acid to show elastic fibres ; magenta to stain the latter.

25. Tendons, e.g., rat (p. 168), and methods at p. 397.

XXXVIII.] FRESH TISSUES. 40 1

26. Diaphragm. — ('(.) Show its endothelial covering by means
of AgX03, and (h) its lymphatics (p. 237).

27. Adipose Tissue. — One must demonstrate the cells (Lesson
XII.).

(a.) Fresh, unaltered in normal saline, using either part of

omen turn or fat-cells from under skin. Seal up the

preparation with paraffin wax.
(ft.) Action of osmic acid.
(c.) Action of alcohol and ether to remove fats, and show

empty envelopes.
(<L) Harden fat-cells in alcohol and stain in logwood to show

nuclei.
(e.) Subcutaneous injection of silver nitrate (i : 500) to show

general characters of cells.

28. Granular Cells (" Mastzellen "). — They occur in large
numbers in the omentum of the rat. Place a small part of the
omentum in a watch-glass containing aniline- water and 20—30 drops
of a concentrated alcoholic solution of dahlia or gentian. Heat for
short time as directed in Lesson X. 14. Wash in distilled water
and then in acid-alcohol until nearly everything is decolorised
except the granular cells. The tissue may also be stained with
lithium-carmine. Mount in balsam. The nuclei of the cells are
red, and only the granules in the protoplasm of the granular cells
are blue.

The mucous membrane of a dog's tongue hardened in alcohol and
treated in the same way shows numerous granular cells.

29. Bed Marrow of Bone.— Methods (p. 188). Use the ribs
and heads of long bones of guinea-pig.

The dry cover-glass method is excellent. Dry them in flame of
spirit-lamp, or pass them three times through the flame of a Bunsen-
burner. Stain with eosin-glycerine and then with methylene-blue
or logwood. Many of the smaller cells will show eosinophile
granules.

For studying the formation of the elements of the blood in red
marrow or other situations, the following method of Fok is good.1
The red marrow or blood is "fixed" in the following fluid after it
cools: — 100 cc. Miiller's fluid is heated with 2 grams of mercuric
chloride. Keep in a thermostat (2-3 hours) at 35° C. Harden in
alcohol, cut sections, and stain (1-3 minutes) with the following : —

Haematoxylin solution (Bohmers] ... 25 cc.
Safranin I per cent, watery-alcoholic solution . 20 ,,
Water loo ,,

Then stain in weak picric acid. Xylol-balsam.

1 Zeits.f. wiss. Mikrosk., ix., 1892, p. 227.

2c

402 PRACTICAL HISTOLOGY. [xxxvut

30. Blood Crystals (p. 120). — If it be a guinea-pig or rat use
the defibrinated blood to obtain blood crystals.

(a. ) Add water = haemoglobin crystals.

(b. ) Add a small quantity of ether = haemoglobin crystals.

(c.) Add amyl nitrite = methaemoglobin crystals.

31. Blood— Ehrlich's Granules.— Perhaps it might be well to
give here a short resume of some of the results of Ehrlich and his
pupils.1 Cover-glass preparations of the blood of different animals
are made, and they are either exposed to the air to dry (or they
may be carefully heated for several hours at 120° C. or passed
several times through the flame of a Bunsen-burner). On being
dried rapidly in the air, there is no coagulation of the cell-proteids,
and thus the cells retain their natural tendency to stain with dyes.
As haemoglobin is soluble in water it is better to use the dyes dis-
solved in glycerine.

Leucocytes. — The "granules" present in the protoplasm in the
varieties of white blood-corpuscles vary in their reaction to staining
reagents. Thus some are stained by what Ehrlich calls acidophile
dyes, of which eosin is one. It is not enough that the granules are
stained by one of these dyes. As a general rule, granules which are
stained by all the following solutions belong to his a-granulation
class and are " eosinophilous granules."

'1.

(3.

Eosin in glycerine.

Glycerine saturated with indulin.

Concentrated watery solution of orange.

The eosinophile cells (a granules) are always present in the leuco-
cytes of frog's blood, marrow of frog (numerous) — very few in
spleen— numerous also in the mesentery. In the rabbit they occur
in the blood, marrow (very numerous), spleen (few).

Make a cover-glass preparation and dry it either at 120° C.
(several hours) or rapidly in the flame of a Bunsen. Stain for an
hour (or longer) with eosin-glycerine, wash in water, dry and mount
in balsam. Or stain cover-glass preparations in glycerine (30 cc.)
containing 2 grams each of aurantia, indulin, and eosin. Or a
saturated alcoholic solution of bluish-eosin may be used.

If an eosin-indulin glycerine solution be used, the a-granulations
are purplish-red and the nuclei well stained bluish-black by the
indulin.

The granular cells (" Mastzellen "), which occur so abundantly
in the connective tissue of the frog and some other animals, also
occur in the blood of the frog, triton, and tortoise. In man, accord-
ing to Ehrlich, they are found only pathologically. The granules

1 Farbenanalyt. Unters. z. Histol. u. Klinik des Mutes, by P. Ehrlich,
Berlin, 1891.

xxxviii.] FRESH TISSUES. 403

in these cells are stained by a fluid composed of 100 cc. water, 50
cc. absolute alcohol saturated with dahlia, 10-12.5 cc. glacial acetic
acid. The leucocytes are stained blue, while the granules have
what Ehrlich calls a metachromic red-violet tint. They correspond
to Ehrlich's y-granulations, and have been specially investigated by
Westphal (loc. cit., p. i 7).

The 8-granulations occur especially in the mononuclear leucocytes
of human blood. They are stained by basic dyes.

The e-granulations, or neutrophile granules, occur in the poly-
nucleated elements of human blood. They are stained only by
neutral dyes, e.g., acid-fuchsin and methyl-blue. Ehrlich classifies
dyes as acidophile, e.g., eosin, aurantia, and indulin ; neutrophile^
e.g., acid-fuchsin or fuchsin-S, methyl-blue ; lasophile, e.g., dahlia,
gentian-violet, fuchsin.

Ehrlich also calls granules which attract acid dyes " oxyphile " —
a term adopted by Wright and Bruce, * whose method is described
below. According to the latter observers, the nucleus of the leuco-
cyte is invariably basophile, while the granules of normal leucocytes
are oxyphile.

Staining of Oxyphilous or Eosinophilous Granules. — Cover-
glass preparations are fixed either by dry heat (Ehrlich's method)
or by chemical reagents (osmic acid, HgCl2).

Eloat the cover-glass on a i per cent, watery solution of eosin
(J— i min.). If it be desired to stain even more rapidly, add a trace
of acetic acid to the fluid, when the preparation rapidly becomes
over-stained. The surplus dye can be removed from all parts of the
cells, except the oxyphile or eosinophilous granules, by dipping the
cover-glass into a very dilute solution of sodic carbonate.

Basophilous Granules. — These are best stained with Loeffler's
methylene-blue, which stains all basophilous elements, e.g., nuclei
and basophile granules. It may dissolve out oxyphilous granules.
If the specimen has been already stained with eosin (the excess
extracted by weak alkali), then only a second or so is required to
stain with methylene-blue. Thus with care it is possible to stain
the oxyphile and basophile elements of the leucocyte.

These observers deny the existence of so-called neutrophile
granules. They believe them to be really oxyphilous in their
behaviour to stains.

32. Stained Leucocytes. — Either one's own blood or the blood of
an animal, or the leucocytes of lymph-glands, may be used. Use the
dry cover-glass method, passing the cover-glass three times through
the flame of a Bunsen-burner before staining. Excellent prepara-
tions of the nuclei stained blue are obtained by methylene-blue

1 Brit. Mcd. Jour., Feb. 1893.

404 PRACTICAL HISTOLOGY. [XXXVIII.

alone, or first stain in eosin and then in methylene-blue or
haematoxylin. For leucocytes the blood of the horse is specially
valuable, as the white cells are so large (Sherringtoa).

33. Fibrin, Hsemin, Cartilage, Muscle, Nerve, and the other
tissues are treated as recommended for frog's tissues, and the same
is the case with organs.

34. Salivary Glands and Pancreas.

Tease a small part of the parotid, or other salivary gland, or
the pancreas, in aqueous humour to see the fresh condition of these
glands. In the guinea-pig's glands note the zymogen granules.

A permanent preparation may be made by exposing small pieces
to the vapour of osmic acid and mounting in glycerine. In this
way the zymogen granules are preserved (Lesson XXIII. p. 265).

N.B. — In this Lesson the methods stated expressly exclude com-
plicated methods of hardening and section-cutting.

ADDENDA.

A. Altmann's Researches on " Granula " in Cells.

For those who wish to study Altmann's views on the constitution
of the protoplasm and nuclei of cells, and the methods of displaying
what he calls his "granula," we must refer to his monograph,
entitled Die Elementarorganismen und Hire Beziehung zu den Zdlvn,
Leipsig, 1 890, which contains twenty-one beautiful plates.

B. Obreggia's Method for Paraffin Sections.

The method of Obreggia (N enrol ogisches CentralUatt, 1890) has
been applied by Gulland (Journal of Pathology, 1893) to parafttn
sections. The hardened tissues are embedded in paraffin, and
ribbons of sections are cut with a rocking microtome or Minot's
form. The sections are at once transferred to glass plates coated
with the following solution : —

Syrupy solution of powdered candy-sugar made with boiling

distilled water . . . . . . . . 30 cc.

Absolute alcohol . . . . . . . 20 ,,

Transparent syrupy solution of pure dextrin made with dis-
tilled water 10 ,,

Pour this solution over the plates two or three days before they are

ADDENDA. 405

used ; run off the excess ; allow the plates to dry slowly in a hori-
zontal position and protected from dust.

Arrange the series of sections in rows on the plates. Place the
plates in a paraffin-oven, which is kept at a temperature slightly
above the melting-point of the paraffin employed, and leave it there
for a few minutes, when the embedded tissues stick fast to the
prepared surface.

Remove the surplus paraffin with xylol or naphtha, and then
wash with methylated spirit or absolute alcohol.

The spirit is run off, and the plates are covered with the following
celloidin solution : —

Phytoxylin 6 grm.

Absolute alcohol ..... loo cc.
Pure ether loo ,,

The plates are placed horizontally. After the thin sheet of celloidin
solidifies, run a knife along between the rows of sections, and allow
further evaporation to take place.

When the sections are required plunge the plate into water ; the
ribbons float off as the sugar is dissolved. The ribbons may be
stained with any reagents except those which dissolve or overstain
celloidin. Stain with very dilute Ehrlich's acid-hsematoxylm (p.
69), and then wash in dilute eosin. Dehydrate the sections, and
clarify in a mixture of xylol 3 parts and carbolic acid crystals i part,
and mount in balsam. We have tried this method, as recom-
mended by Gulland, and find that it works very well. Moreover,
we found that the sections, after being floated off, can be kept in
80 per cent, alcohol until they are required.

APPENDIX.

A.— SOME WORKS OF REFERENCE.

A. — Systematic Histology.

Schwann, Mikrosk. Untersuch., 1838 (translated by the Sydenham Society
1847).— E. Virchow, Die Cellular Pathologic (translated by Chance), 1860. —
Henle, Handbuch der systernatischen Anatomie des Menschen, 3rd ed.,
1866-83. — W- Krause, Allgemeine und mikroskopisclie Anatomie, Han-
nover, 1876.— F. Leydig, Lehrbuch der Histologie des Menschen und
der Thiere, Hamm, 1857 ; his Untersuchungen, 1883 ; and his Zellen.
Gewebe, Bonn, 1885. — L. Ranvier, Traite technique d'histologie, Paris, 2nd
ed., 1889. — G. Schwalbe, Lehrbuch der Neurologie, Erlangen, 1881 ; Lehrb.
d. Anat. d. Sinnesorgane. — S. Strieker, Handbook of Histology (trans-
lated by the New Sydenham Society), 1871-73. — L. Beale, The Struc-
ture of the Elementary Tissues, London, 1881. — A. Kblliker, A Manual of
Human Microscopic Anatomy, London, 1860 ; and his I cones Histolog.,
Leip., 1864; vol. i. of his Handbuch d. Gewebelehre, Leipzig, 1889. —
Bindfleisch, A Manual of Pathological Anatomy (translated by R. Baxter),
London, 1873.— C. Toldt, Lehrbuch der Gewebelehre, Stuttgart, 311! ed., 1888.
— E. Klein and E. Noble-Smith, Atlas of Histology, London, 1872.—
H. Frey, Handbuch der Histologie und Histochemie des Menschen, Leipzig,
1876, Grundziige, 1885.— Cadiat, Traite d'anat. gen., Paris, 1879.— Brass,
Kurzes Lehrbuch d. Histologie, Leipzig, 1888. — Heitzmann, Mikrosk. Mor-
phology, 1882. — Purser, Man. of Hist., Dublin. — E. Klein, Elements of
Hist., London, 1883. — W. Flemming, Zellsubst. u. Zelltheilg, Leipzig, 1882. —
Bizzozero, Hand. d. klin. Mikroskop., Erlang., 2nd ed., 1888. — Carnoy, Gilson,
andDenys, Biol.Oellul., Louvain, 1884-88. — Renaut, Traite d'Histologie, Paris,
1885. — Frommann,Unters.ii. thier. u. pflanz. Zellen, Jena, 1884. — Wiedersheim,
Lehrb. d. vergl. Anat., Jena, 1888. — S. L. Schenk, Grundriss der Histologie
d. Menschen, Vienna, 1885.— Orth, Cursus d. norm. Histol., 4th ed., 1886. —
S. Mayer, Histolog. Taschenbuch, Prag., 1887.— Stbhr, Lehrb. d. Histol., 5th
ed., Jena, 1892.— Lee and Henneguy, Traite de meth. d'Anat, Paris, 1888. —
Schafer, Essentials of Histology, 3rd ed., 1892. — Owsjannikow, Text-Book
of Histology (Russian), iS88. — Ftisari and Monti, Compendio di Istologia

APPENDIX. 407

generate, Torino, 1891. — Ellenberger, Vergleich. Histol. der Hausthiere,
vol. i., Berlin, 1887, vol. ii., 1892.— Altmann, Die Elementarorganismen u.
ihre Beziehung z. d. Zellen, Leipzig, 1890 (with 21 plates). An abstract of
the methods by which Altmann prepares his " granula " in cells will be found
in Zeit. f. mik. Anat., vii. p. 199, 1890.— Quain's Anatomy, loth ed., edited
by Schafer and Thane, 1892-93.— Obersteiner, Anleitung b. Studium d.
Banes d. nerv. Centralorg., 2nd Germ, ed., 1892. English ed. translated by
Hill from ist Germ. ed. — Edinger, Zwb'lf Vorlesung. v. d. Bau d. Central
Nerv. System, 2nd ed., and English trans, by Vittum and Biggs, 1890.

B. — The Microscope, Microscopical Technique, and Manuals of Practical

Histology.

Some of the works already mentioned contain descriptions of microscopical
methods, e.g., those of Ranvier, Stohr, Orth.

Dippel, Das Mikroskop und seine Anwendung.^Beale, How to Work
with the Microscope, London, 1880. — H. Frey, Das Mikroskop. , 8th ed., 1886. —
Carpenter's The Microscope and its Revelations, edited by Dallinger, yth ed.,
1892. — J. Hogg, The Microscope, i2th ed., 1887. — Naegeli and Schwen-
dener, The Microscope in Theory and Practice (translated from 2nd Germ,
ed.), 1892. — Gage, The Microscope and Microscopical Methods, Philadelphia,
1892. — Amstrom, Anleit. z. Benntz. d. Polaris- Mikroskop., Leip., 1892. —
Rutherford, Outlines of Histology, 1876.— Schafer, Pract. Histol., London,
J877.—W. Stirling, Text-Book of Pract. Histol., London, 1881.— Foster and
Langley, Pract. Phys., 5th ed., 1884.— Friedlander and Martinotti, Tec-
nica microscopica, Turin, 1885. — Garbini, Manuale per la tecnica mod. del
Mikroscopio, 3rd ed., 1891. — Fol, Lehr. d. vergleich. mikros. Anatomic, Leip.,
Pt. i., 1885. — Behrens, Tabellen z. Gebrauchb. mik. Arbeiten, Braunschweig,
2nd ed., 1892.— Fearnley, Pract. Histol., 1887.— W. Stirling, Histological
Memoranda, Aberdeen, 1880. — Lee and Henneguy, Traite demeth. de 1'Anat.,
Paris, 1888.— Friedlander and Eberth, Mik. Technik., 4th ed. , Berlin, 1888.
— Gierke, Farberei z. mik. Zwecken, Braun., 1887. — Renaut, Traite d'Histol.
pratique, Paris, 1889. — Behrens, Kossel, and Schiefferdecker, Das Mikro-
skop., Pt. i., Braunschweig, 1889, Pt. ii., 1891.— Ramon yCayal, Manual de
Histologia normal, Valencia, 1889.— Bbhm and Oppel, Taschenb. d. mik.
Technik., Miinchen, 1890. — B. Rawitz, Leitfaden f. hist. Untersuch., Jena,
1889.— Neelsen, Grundriss d. Path. -Anat. Technik, Stuttgart, 1892.— Squire,
Methods and Formulae used in the Preparation of Animal and Vegetable
Tissues for Micros. Exam., London, 1892.— Strassburger, Das botan. Practi-
cum, and English trans. — Zimmermann, Die botan. Mikrotechnik, Tubingen,
1892.

C. — Journals.

Archiv f. mik. Anatomic, Bonn, formerly edited byM. Schultze and now
by Hertwig, La Valette, St George, and Waldeyer.— Archiv fur Anat. u.

408

APPENDIX.

Physiol., edited by Du Bois-Reymond. — Virchow's Archiv.— Quarterly Micro-
scopical Journal, London. — Journal of the Royal Microscopical Soc., London. —
Jour, of Anat. and Physiol., edited by Humphry, Turner, and M'Kendrick. —
La Cellule, Louvain. — Journal de Micrographie, Paris, edited by Pelletan. —
Zeits. f. wiss. Alikrosk., edited by Behrens.— Zoolog. Anzeiger, edited by
V. Cams. — Internats. Monats. f. Anat. u. Phys., edited byKrause. — Journal
of Physiology, edited by Foster. — Archiv ital. de Biologie, edited byMosso. —
Proceedings and Transactions of the Royal Society. — Comptes rendusde 1'Acad.
des Sciences. — Sitzb. d. k. Akad. d. Wissenschaft, Wien. — Archiv f. d.
gesammte Physiologic, edited by Pfliiger. — Journal of Morphology, edited
by Whitman, in which will be found elaborate papers by Minot on the Uterus
and Placenta, and on the Ear by H. Ayers.

B.— TABLES OF MAGNIFYING POWER OF OBJECTIVES
AND OCULARS.

Objective.

Magnifying
Power of Ob-

Eyepiece No. i (A)
magnifies 5
times ; combined

Eyepiece No. 2 (B)
magnifies 73
times ; combined

Eyepiece No. 4 (D)
magnifies 20
times ; combined

jective alone.

with Objective
magnifies

with Objective
magnifies

with Objective
magnifies

i inch

10

50

75

200

A

25

125

I87

500

i

40

2OO

275

800

^

50

250

300

1000

•^

60

300

450

I2OO

i

80

400

600

l6oo

TV

. 100

500

750

2000

T*

120

6co

900

2400

This table is calculated for a xo-inch tube, and gives approximately the
magnifying power ; but if accuracy be required, each combination of lenses
and objectives must be measured by the method already described at page 19.
(After Gfibbes.)

HARTNACK'S DRY LENSES.

Ocular.

Objective

No. 1.

No. 2.

No. 3.

No. 4.

4

40

50

65

IOO

8

150
250

22O
360

300
400

450
600

9

360

430

520

850

APPENDIX. 409

MAGNIFYING POWER OF ZEISS'S OBJECTIVES AND OCULARS.

Length of Tube 155 mm.
Ocular.

Objective.

No. 1.

No. 2-

No. 3-

No. 4.

A

7

II

15

22

A*

4-12

7-17

10-24

A, AA

38

52

71

97

B, BB

70

95

130

175

C, CO

1 20

US

195

270

D, DD

175

230

320

435

E

270

355

490

670

F

405

540

745

IOIO

The lens A* is a particularly useful low-power lens, as by merely rotating
a collar, a great variety of magnifying power is obtained without changing
the leus.

MAGNIFYING POWER OF LEITZ'S OBJECTIVES AND OCULARS.

Number

Magnifying
Power of

Length of Tube 160 mm.
Ocular.

of
Objective.

Objective
without

Ocular.

0 = 5.6.

I. =6. 9.

H. =8.5.

III. =12. 7.

IV. = 16.3.

V. = i9.i.

I

3-2

16

21

25

39

5°

60

2

5

26

34

40

63

81

95

3

9

47

62

72

114

146

171

4

ii

58

75

88

139

179

210

5

26

137

179

208

330

423

496

6

34

1 80

234

272

43i

554

649

7

47

250

325

380

600

770

900

8

60

3<8

414

480

762

978

1146

C.— LIST OF MAKERS OF MICROSCOPES, <fcc.

MICROSCOPES.
American Makers and Agents.

Bausch & Lomb Optical Co., Rochester, N. Y.

J. W. Queen & Co., 1010 Chestnut Street, Philadelphia.

Williams, Brown & Earle, N. E. cor. Tenth and Chestnut Sts., Philadelphia

Eimer & Amend, 205-211 Third Avenue, New York.

Joseph Zentmayer, 209 S. Eleventh Street, Philadelphia.

36

41O APPENDIX.

Foreign Makers.

Powell & Lealand, 170 Euston Road, N. W., London.

R. & J. Beck, 68 Cornhill, E. C., London.

Swift & Son, 8 1 Tottenham Court Road, London.

C. Baker, 244 High Holborn, London.

H. Crouch, 66 Barbican, London.

Carl Zeiss, Jena.

E. Hartnack, Waisenstrasse, 39, Potsdam.

W. & H. Seibert (successors to Gundlach), Wetzlar.
C. Reichert, Bennogasse, 26, Vienna.
Ernest Leitz, Wetzlar.

F. W. Shieck, Hallesche Strasse, 14, Berlin, S.W.
Nachet et Fils, Rue St. Severin, 17, Paris.

C. Verick, Rue des Ecoles, Paris.

MICROTOMES.

To be had from most of the above Firms, and also from —

Zimmermann, Albert Strasse, Leipzig (Maker of Minot's Microtome).

R. Jung, Heidelberg (Maker of Thoma's Microtome).

Schanze, Pathologisches Institut, Liebig Strasse, Leipzig.

W. Hume, Lothian Street, Edinburgh.

A. Fraser, Lothian Street, Edinburgh (Maker of Cathcart's Microtome).

J. Gardner, Teviot Place, Edinburgh (Maker of Rutherford's Microtome).

Cambridge Scientific Instrument Co.

Kanthack, Golden Square, off Regent Street, London.

CHEMICALS AND HISTOLOGICAL REAGENTS.

Hopkin & Williams, 16 Cross Street, Hatton Garden, London, E.G.

R. & J. Beck, London.

Baker, 244 High Holborn, London.

Southall Brothers & Barclay, Dalton Street, Birmingham.

Dr. Georg Grubler, Bayersche Strasse, 12, Leipzig.

D. — WEIGHTS AND MEASURES, EQUIVALENTS.

Centigram ..... = .154 English grains.

Decigram = 1.543 „

Gram =15.432 ,,

Kilogram = 2.2 Ibs. (avoird.).

I fluid ounce .... =28 cubic centimetres.

I fluid drachm . . . . = 3.9 ,, ,,

I inch. .....= 2.539 centimetres.

I foot ......= 3.047 decimetres.

ic oo micros (ju) .... = I millimetre.

10 millimetres (mm.) . . . = I centimetre.

100 centimetres (cm. or ctm.) . = I metre (unit of length).

i/x = 0.000039 inch . . . =-5T$Tn7th inch (approximately).

I cm = 0.3937 inch.

I metre =39.3704 inches.

INDEX.

ABBE'S condenser, 10.
Absolute alcohol, 28.
Absorption of fat, 283.
Acid-alcohol, 65.
Acid-fuchsin, 74, 154.
Acids, 30.
Adenoid reticuhim, 236.

tissue, 172, 398.

Adipose tissue, 168, 398, 401.
Agminated follicles, 274.
Air-bubbles, 101.
Albo-carbo light, 23.
Albuminous glands, 258.
Alcohol, 27.

• • dilute, 25.

Alkalies, 93.
Alum carmine, 65.
Ammoniacal carmine, 63.
Ammonium bichromate, 25, 29.

chromate, 25, 29.

Amoeboid movement, in, 117.
Angle of aperture, 13.
Aniline-blue, 73.

blue-black, 76.

dyes, 72.

oil, 73.

Anodon's muscle, 201.

Aorta, 226.

Apochromatic lenses, 14.

Aponeurosis, 397.

Aqueous humour, 24.

Arachnoid, 328.

A reolar tissue, 159, 161, 397, 400.

Arrector pili, 322.

Arsenic acid, 37.

Arteriole, 229.

Artery, 223.

• • elastic fibres in, 233.

endothelium of, 228.

Articular cartilage, 1 50.
A trophic fat-cells, 171.

Auerbach's plexus, 277.
Axis-cylinder, 206, 211.

BACTERIA, 104.
Balsam, 82, 85, 87.
Baryta-water, 26.
Basement membrane, 311.
Bayerl's fluid, 36.
Benzo-azurin, 131.
Bile-ducts, 290.

auto-injection of, 291.

Bismarck brown, 75.
Bladder, 313.

cells of, 190, 313.

frog's, 134, 190.

crayfish's, 115.

Blood, colourless corpuscles of, no,
117, 122, 402.

circulation of, 231.

division of, 113.

• effects of reagents on, 112, 114.

feeding of, 113.

colourless corpuscles of, glycogen

in, US- ,

migration of, 113.

Blood-corpuscles, 400.

— amphibians, 106.
— • bird, no.

— cover-glass preparations, 114.
— • crenation of, 1 19.

— crystals from, 120, 402.
— • double staining, 114.
— • enumeration of, 121.

— fish, no.

— frog, 1 06.

— human, 115.

— leukaemio, 121.

— pseudo-membrane, 114.

— tablets, 1 06, 123.

Blood, effect on, of acetic acid, 107,

112.

412

INDEX.

Blood aniline dyes, 121.

• of boracic acid, 109.

of hydrochloric acid, 108.

• of magenta, 109.

of osmic acid, 1 10.

of syrup, 108.

• of tannic acid, lop.

of water, 108.

Blood-plates, 106, 123.
Blood-serum, 24.
Blood-vessels, 223.

injection of, 230.

development of, 228.

Bone, 174.

blood-vessels of, 179, 181.

cancellated, 1 80.

corpuscles, 177.

decalcified, 177, 181.

development of, 182.

marrow of, 186.

perforating fibres, 178.

polarised light, 181.

Borax-carmine, 64.
Boveri's fluid, 213.
Bowman's glands, 375.
Bronchus, 297.
Brownian movement, 100.
Brunner's glands, 278.

CABINET, 2.
Calcified cartilage, 183.
! Caliciform cells, 273.
Camera lucida, 17.

Abbe's, 17,

Chevalier's, 18.

Malassez's, 18.

Camera, Zeiss's, 17.
Camera obscura shade, 23.
Canada balsam, 85.
Capillaries, 224, 231.
Capillary attraction method, 240.
Carbolic acid and xylol, 83.
Cardiac glands, 267.

muscle, 199.

Carmine, 63, 64.

acid -chloral, 282.

and Dahlia fluid, 67.

Frey's, 641.

Carter's injection, 89.
Cartilage, 146.

articular, 150.

cellular, 146.

costal, 148.

cuttlefish, 151.

encrusting, 184.

• epiphysial, 184.

Cartilage, fibrous, 151.

— hyaline, 147, 397.
parenchymatous, 146.

— • transition, 155.
Caustic potash, 25.
Cayal's methods, 222.
Cedar-wood oil, 83.
Cell, animal, 141.
Celloidin, 45.
Cell-spaces, 162, 168.

of cornea, 361.

Cellular cartilage, 146.
Cement of tooth, 251.
Central nervous system, staining of,
338.

— freezing method, 339.
Golgi's methods, 344.

— Pal's method, 343.

— Vessale's method, 344.

Weigert's method, 338.

Weigert-Pal method, 340.

Central tendon, 129, 237.
Centrifuge, 94.
Cerebellum, 349.

blood-vessels of, 351.

Golgi's method, 355.

Cerebrum, 351.

— blood-vessels of, 355.

Golgi's method, 357.

Weigert's method, 357.

Ceruminous glands, 371.
Chalice-cells, 139, 273.
Choroid, 362.

pigment-cells of, 363.

Chromic acid, 25, 28.

and nitric acid, 36.

Chromo-acetic acid, 31.

aceto-osmic acid. 32.

-formic acid, 31.

-osmic acid, 37.

Ciliary muscle, 363.

motion, 135.

effects of reagents on, 1 36.

— processes, 363.
Ciliated epithelium, 135.

isolated cells, 138.

Circulation of blood, 231.

in frog's tongue, 234.

Circumvallate papillae, 247.
Clarifying reagents, 82.
Clarke's column, 330.
Clasmatocytes, 162.
Cloves, oil of, 83.
Coarsely-granular cells, 162.
Cochineal, 67.
Cochlea, 371.

INDEX.

413

Cockroach, salivary glands of, 265.
Coh u helm's areas, 200.
Collateral fibres, 346.
Collodion, 211.
Colostrum, 395.
Columnar epithelium, 131.
Condenser. 10.
Cones, 368.

Connective tissues, 156.
Convoluted tubules, 305.
Copper acetate, 338.
Cornea, 358.

cell-spaces of, 361.

— corpuscles, 359, 396.

fibrils of, 369.

lymph-spaces, 396.

nerves of, 359.

Corrosive sublimate, 33.
Cortex cerebri, 352.
Cortical arteries, 355.
Corti's organ, 373.
Costal cartilage, 148.
Cotton fibres, 102.
Cover-glasses, I.

to clean, 98.

Cover-glass tester, 23.

Crab's muscle, 196.

Crayfish blood, 115.

Crenation, 119.

Crista acustica, 374,

Creosote, 83.

Crusta petrosa, 251.

Cutting sections in series, 53, 60.

Cuttlefish cartilage, 151.

DAHLIA, 73.
Dammar lac, 86.
Decalcifying fluids, 36.
Decussation of pyramids, 347.
Demilunes, 258, 260, 265.
Dentine, 251.

Descemet's membrane, 359.
Diaphragm, 129, 237, 401.
Diaphragms, 7.
Digestion methods, 26.
Dilute alcohol, 25.
Dissecting case, 2.

microscopes, 22.

Dissociating fluids, 24.

Dogiel's method, 167.

Drawing materials, 3.

Dry cover-glass preparations, 1 14.

Duck's bill, 378.

Ductule, 257.

Dura mater, 328.

EAR, 371.

cartilage, 154.

Ebner's fluid, 37.

Ehrlich-Biondi fluid, 81, 140.

Ehrlich's hsematoxylin, 69.

Eimer, organ of, 381.

Elastic fibres, 157, 397.

Herxheimer's method, 161.

— - Martinotti's reaction, 161.
Embedding, 40.

boxes, 45.

in celloidirr, 45.

in gum, 41.

— in paraffin, 41, 44.

• interstitial, 41.

Enamel, 251.
End-bulbs, 378.
Endocardium, 223.
Endothelium, 129,

of arteries,

End-plates, 219.
Eosin, 72.

Eosin-hsematoxylin, 70.
Eosinophilous cells, 122.
Epidermis of man, 317, 325.

• of newt, 126.

Epididymis, 387.
Epiglottis, 153.
Epiphysis, 184.

Epithelial cells, fibrillation of, 327.
Epithelium, 124, 398.
— • ciliated, 135.

— columnar, 131.

— germinating, 239.
— -glandular, 133.

— secretory, 133.

— squamous, 124.

— transitional, 133.
Erlicki's fluid, 29.
Eternod's rings, 77.
Eye, 358.

blood-vessels of, 369.

— triton's, 370.
Eyelid, 369.

FALLOPIAN tube, 390.
Farrant's solution, 85.
Fat-cell, 169.

absorption of, 283.

action of reagents on, 169.

atrophic, 171.

development of, 171.

Fenestrated membranes, 158, 160,

227.

Fibres of Tomes, 253.
Fibrin, 119, 123.

414

INDEX.

Fibro-cartilages, 151.

Filiform papillae, 246.

Fixatives, 60.

Fixing fluids, 27.

Flemnung's fluid, 32.

Fol's solution, 32.

Formatio reticularis, 348.

Free nerve-endings, 376.

Freezing fluid, 50.

Fresh tissues, examination of, 93, 396.

Frog's bladder, 1 34.

heart, nerve-cells of, 233.

tongue, 140.

Frommann's lines, 207, 211.
Fuchsin, 74.
Fundus glands, 268.
Fungiform papillae, 247.

GAMBOGE, 100.
Ganglion, spinal, 214.

sympathetic, 216.

Gasserian ganglion, 215.
Gastric gland-cells, 268.
Gaule's method, 61.
Genital corpuscles, 378.
Gentian violet, 73.
Germinal epithelium, 389.
Germinating epithelium, 239.
Gland-ducts, 265.
Glandular epithelium, 133.
Glia-cells, 343.
Glisson's capsule, 285.
Glycerine, 85.

jelly, 85.

Glycogen, 293.
Goblet-cells, 138, 140, 273.
Gold chloride, 78.
Golgi's methods, 78, 220, 344.

slow method, 344.

• sublimate method, 345.

• rapid method, 345.

• for retina, 369.

Coil's column, 330.
Graafian follicles, 389.
Gram's method, 105.
Grandry's corpuscles, 377.
Granular cells, 156, 293, 401.
Guanin cells, 173.
Gustatory cells, 250.

HAM-ALTTM, 71.
Hamatein, 71.
Hsematoxylin, 68, 71.

acid, 69.

Bohmer's, 68.

Delafield's, 69.

Hsematoxylin, Ehrlich's, 69.

eosin, 70.

. glycerine, 70.

• Hamilton's, 69.

Heidenhain's, 70.

• Kleinenberg's, 69.

— - Kultschitzky's, 340.

nucleus-staining, 69.

— Weigert's, 338.
Hiemin, 126.
Haemoglobin, 120.
Hsemolymph, 115.
Hair, human, 323.

blood-pigment in, 327.

development of, 327.

elements of, 324.

rabbit's, 324.

Hair-follicles, 321.

coverings of, 321.

development of, 327.

double-staining of, 327.

Hairs, tactile, 327.
Half-drying method, 159.
Hamilton's hsematoxylin, 69.
Hardening fluids, 27, 33.
Hard palate, 126.
Hartnack's dry lenses, 408.
Hassall's corpuscles, 242.
Hay em's fluid, 122.
Heart, 223.

valves, 225.

Heidenhain's method, 70, 259, 282.
Henle's tubule, 305.
Herbst's corpuscles, 379.
Hermann's fluid, 384.
Herxheimer's method, 161, 326.
Horny epidermis, 127.
Hot stage, 117.
Howship's lacunae, 184.
Hyaline cartilage, 147.
Hypodermic syringe, 161.
Hypophysis cerebri, 357.

ILLUMINATION, artificial, 21.

direct and oblique, 7.

Immersion lenses, 12.
Impregnacion doble, 222, 346.
Incremental lines, 252.
Indigo-carmine, 67.
Indirect cell-division, 141.
Inflammation, 233.
Injection mass, 89.

methods of, 91.

Intercellular bridges, 127.

— channels, 127.
Intercostal nerve, 207.

INDEX.

Interglolnilar spaces, 252.
Iiiterlobular artery, 310.
Intervertebral disc, 151.
Intestinal glands, 283.
Intestine, large, 280.

small, 272.

Iodine green, 74.
Iodised serum, 25.
Iris, 363.

Irregular tubules, 305.
Irrigation, 107.

KARYOKINESIS, 141, 145.
Kidney, 303.

blood-vessels of, 306.

convoluted tubules, 308.

fresh, 311.

glomerulus, 307.

injected, 309.

irregular tubules, 308.

isolated tubules, 311.

• medullary ray, 309.

Kleinenberg'a lisernatoxylin, 69.

— fluid, 30.
Klein's fluid, 29.
Kochs-Wolz lamp, 22.
Kronecker's fluid, 24.
Kiihne's method, 211.
Kultschitzky's haematoxylin, 340.
method, 344.

LABELS, 4.

Lachrymal gland, 371.
Lacteal, 273, 282.
Landois' fluid, 26.
Large intestine, 280.
Leitz's lenses, 409.
Lei.-s, crystalline, 364.

epithelium of, 369.

Lenses, apochromatic, 15.

dry, II.

immersion, II.

Leucocytes, 122, 402, 403.

Leukaemia, 121.

Lieberkiihn's glands, 273, 280.

mitosis in, 283.

Ligamentuhn nuchae, 157.
Linen fibres, 102.
Lithium carmine, 65.
Liver, 285.

bile capillaries of, 291, 292

bile ducts, 290.

blood-vessels of, 285, 289.

cells, 133.

connective tissue of, 292.

frog's, 288.

Liver, glycogen in, 293.

— granular cells of, 293.
human, 288.

— iron in, 293.
. methods for, 286.

• pigment in, 293.

pig's, 286.

rabbit's, 288.

Loffler's blue, 93.
Logwood, 71.
Lugol's solution, 93.
Lungs, 294.

blood-vessels of, 294.

— — dried, 300.

• elastic fibres in, 300, 302.

foetal, 300.

fresh, 300.

frog's, 302.

newt's, 302.

Lymph, 112.

channels, 237.

- gland, 234, 235, 245.
Lymphatics, 234.

MACROPHAOES, 285.
Macula lutea, 370.
Magenta, 74.
Magnifying power, 19.

— powers of objectives, 408.
Malpighian capsule, 304.

pyramid, 303.

Mammary gland, 393.

— active, 395.
Marchi's method, 212, 347.
Margarine crystals, 170.
Marrow, 186, 401.
Martinotti's methods, 161.
Mastzellen, 67, 156, 162, 401.
May's methods, 219.
Mayer's embedding bath, 37.
Measures, 410.

Medulla oblongata, 347.

— olivary bodies, 348.
Medullary ray, 303.
Medullated nerve-fibres, 202, 399.
Meissner's corpuscles, 380.

plexus, 277.

Membrana tympani, 371.
Mercuric chloride, 33.

method for nervous system, 345.

Merkel's corpuscles, 377.
Methylated spirit, 28.
Methylene-blue, 73, 131, 192, 222,

370, 284.
Methyl -green, 74.
Methyl-mixture, 26.

416

INDEX.

Methyl-violet, 73.
Micrococci, 104.
Micrometer, 20.
Micro-organisms, 103.
Microphages, 285.
Microscope, 5.

choice of, 15.

dissecting, 23.

illumination of, 7.

lamp, 21.

mngnifying power of, 19.

makers of, 409.

parts of, 15.

Microtomes, 49.

Cambridge, 53

• Cathcart's, 53.

Jung's, 56.

makers of, 410.

Malassez's, 56.

• • Minot's, 55.

Ranvier's, 59.

Roy's, 53.

Rutherford's, 50.

Swift's, 59.

Thoma's, 56.

Williams', 58.

Migratory cells, 157.
Milk, 98.
Mitosis, 141, 145.
Mounting block, 89.

— fluids and methods, 85.
Mole's nose, 381.
Mucigen, 139, 260.
Muco-salivary glands, 257, 262, 264.
Mucous cells, 265.

glands, 256, 258.

— tissue, 171, 174.
Miiller's fluid, 29.

— . and spirit, 29.
Multipolar nerve-cells^ 217, 343.
Muscle, 189, 398.

striped, 189, 398.

Myelin, 205.

drops, 205.

Myeloplaxes, 187,
Myocardium, 223.

NAIL, 326.

. double-stained, 327.

Needles, 2.
Nerve-cells, 213, 399.

cover-glass preparations of, 218.

crayfish, 223.

in frog's heart, 22 1 , 233.

— multipolar, 217, 343.
pyriform, 221.

Nerve-cells, spinal cord, 217.

sympathetic, 217, 222.

transverse markings on, 207.

Nerve-endings, 376.
Nerve-plexuses, 276.
Nerve-fibres, 202.

axis-cylinder of, 205, 208, 211.

• degeneration of, 213.

in osmic acid, 206, 211.

intercostal, 207.

living, 212.

medullated, 202, 399.

non-medullated, 203, 209.

size of, 212.

spinal cord, 213.

— - to muscle, 219.

transverse markings on, 207.

Nerve-plexus in intestine, 277.
Nerve-trunks, 203.
Neuroglia, 341, 343.
Neurokeratin, 210.
Newt's cartilage, 145.
Nitric acid, 31.
Nodes of Ranvier, 202.
Non-medullated nerve- fibres, 203.
Non-striped muscle, 189.

cement of, 191.

grooving on, 193.

plexus in, 192.

Normal fluids, 24.

saline, 24.

Nose, 374, 376.
Nuclear stains, 63.

OBJECTIVES, 9.
Obreggia's method, 404.
Oculars, 5, 10.
Odontoblasts, 253.
(Esophagus, 255.

and stomach, 271.

Oikoid, 109.
Olfactory bulb, 376.

cells, 375.

Omen turn, 129.
Onion, 103.

Opaque injections, 289.
Optic nerve, 368.

entrance of, 370.

Origanum oil, 83.
Osmic acid, 25, 32.
Osmico-bichromale mixture, 345.
Ossification, 182.
Osteoblasts, 178.
Osteoclasts, 183.
Ovary, 388.
Ovum, 390.

IXDEX.

417

PACINIAN corpuscle, 378.
Palate, hard, 126.

glands of, 251.

soft, 250.

Palm of hand, 317.
Pal's method, 343.
Pancreas, 262, 404.

cells of, 265.

nerves of, 266.

Papillae foliatse, 249.

of skin, 315.

of tongue, 246.

Paraffin, 41.
Penicillium, 103.
Penis, 313.
Perenyi's fluid, 31.
Perforating fibres, 178.
Pericardium, 223.
Pericesophageal membrane, 163.
Periodontai membrane, 252.
Periosteum, 177.
Peyer's patch, 272, 274, 284.
Phagocytosis, 284.
Phenylene-brown, 75.
Phloroglucin, 37.
Photophore, 27.
Pia mater, 228.
Picric acid, 30, 36.
Picrin-glycerine, 192.
Picro-carmine, 66, 80.
Picro-glycerine mixture, 192.
Picro-lithium carmine, 66.
Picro-nitric acid, 31.
Picro-sulphuric acid, 30.
Pigment-cells, 173, 397.

— choroid, 363.
Pipettes, 4.
Pituitary body, 357.
Placenta, 395.

— injected, 396.
Plasma-cells, 156.
Polariscope, 200.
Potassic bichromate, 25, 29.
Potato starch, 100.
Preparation of tissues, 38, 396.
Prickle cells, 127, 318.

Pulp cavity, 253.
Purkinje's cells, 351.

— fibres, 225.
Pyloric glands, 270.
Pyloro-duodenal region, 271.
Pyriform nerve-cells, 221.

RABL'S fluid, 31.

Ran vier's crosses, 207, 211.

fluid, 25.

37

Ranvier's nodes, 206.
Razor, 3.

Rectified spirit, 28.
Red marrow, 187.
Respiratory organs, 295.
Retina, 365, 370, 399.
cones of, 367.

frog's, 368.

Golgi's methods for, 369.

macula lutea, 370.

Retro-lingual membrane, 201.
Rice-starch, 100.
Ripart arid Petit's fluid, 24.
Rosaniliu, 74.

SAFRANIN, 75.
Salivary corpuscles, 125.

glands, 256 404.

of cockroach, 265.

Sarcolemma, 193.
Sarcostyles, 198.
Scalp, 323.

Schiefferdecker's fluid, 26.
Schizomycetes, 105.
Schreger's lines, 252.
Sebaceous glands, 323.
Section cutting, 48.

flatteners, 59.

in series, 60.

lifter, 3.

to place on slide, 86.

Semicircular canals, 374.
Semidesiccation method, 159.
Seminiferous tubules, 382.
Sensory nerve-terminations, 376.
Septum cisternse, 238.
Serial sections, 60.
Serous fluids, 113.

glands, 257, 261.

nerves in, 265.

Serum, action of, 124.

and osmic acid, 78.

Sharpey's fibres, 178.
Silver lines, 129.

nitrate, 76.

Skin, 315.

— blood-vessels of, 325.
cutis vera, 315.

— elastic fibres in, 326.
epidermis of, 325.

— finger, 317.

fcetal, 320.

injected, 325.

— negro's, 320.

— nerves of, 377.

sebaceous glands of, 322.

2D

4i8

INDEX.

Skin, sweat-glands of, 319, 327.

touch-corpuscles of, 320.

to clean. 98.

Slides, i.

Small intestine, 272.

absorption of fat, 283.

• blood-vessels of, 275.

nerve-plexuses of, 276.

nerves of, 284.

Soft palate, 250,
Solitary follicles, 275, 280.
Spermatogenesis, 383, 385.
Spermatozoa, 386.

cover-glass preparations, 387.

frog's, 386.

human, 387.

newt's, 386.

Spiller's purple, 74.
Spinal cord, 328.

cells of, 217, 329,

collateral fibres, 346.

columns of, 330.

commissures of, 329.

cornua, 329.

degeneration of, 332, 347.

dry preparation of, 342.

fissures of, 328.

ganglia, 213.

grey matter of, 334.

human, 337.

longitudinal section, 338.

nerve-cells, 343.

nerve-fibres of, 341.

neuroglia of, 341.

staining of, 341.

substantia gelatinosa, 329.

tracts in, 542.

white matter of, 335.

Spleen, 242.

injected, 245.

Squames, 125.
Staining in bulk, 44.

general remarks, 81,

multiple, 80.

reagents, 63.

Starch, potato, loo.

— rice, 100.
Stomach, 266.

blood-vessels of, 271.

cardiac end, 267.

double staining of> 271.

methods for, 267.

• nerves of, 284.

pyloric end, 270.

Stomata, 239.

Stratified epithelium, 126.

Striped muscle, 193, 398.

Anodon's, 201.

cardiac, 199.

• • crab's, 196, 200.

discs, 195.

fibrillse of, 195.

frozen, 200.

injected, 198.

isolated fibres, 194.

living, 200.

red, 199.

sarcolemma, 193.

tendon of, 196.

Sublingual gland, 259.
Submaxillary glands, 259, 262, 264.
Suprarenal capsule, 314.

nerves of, 315.

Sweat-glands, 319, 327.
Sympathetic nerve-fibres, 209.

ganglia, 216.

nerve-cells, 222.

Cayal's method, 222.

Syringes, 91.

TACTILE cells, 377, 381.

disc, 377.

hairs, 327.

Taste-buds, 249.
Teasing, 26.
Tendon, 163, 397.

nerves in, 221.

Terminal corpuscles, 377.
Testis, 387.
Thymus gland, 241.
Thyroid gland, 301.
Tizzoni's reaction, 293.
Tongue, 246.

circulation in, 234.

frog's, 140.

gland of, 251.

injected, 248.

nerves of, 251.

papillae of, 246.

Tonsils, 239.
Tooth, 251.

development of, 253.

— softened, 252.
Touch-corpuscle, 380.
Trachea, 294.

Transitional epithelium, 133.
Tubules isolated of kidney, 311.

UMBILICAL cord, 395.
Unna's method, 327.
Ureter, 312.
Uterus, 392.

INDEX.

410

VALENTIN'S knife, 49.

Valves of heart, 225.

Vas deferens, 388.

"Vegetable cells, 103.

Veins, 224, 230.

Vermiform appendix, 281, 284.

Vessale's method, 344.

Vesuvin, 76.

Villus of intestine, 272, 280, 282.

injected, 275.

Volkmann's canals, 176.
Von Ebner's fluid, 37.

WAGNER'S touch-corpuscles, 380.
Warm stages, 117.
Weigert's hsematoxylin, 338.
method for staining central ner-
vous system, 338.

Weigert-Pal method, 340.
Weights, 410.
Welsbach light, 22.
Westphal's fluid, 67.
Wharton's jelly, 171.
White nbro-cartilage, 152.

— zinc cement, 89.
Wool, 102, 324.
Works of reference, 406.

XYLOL, 83.
balsam, 86.

YEAST, 104.

Z KISS'S lenses, 409.
Zinc cement, 89.
Zooid, 109.

Catalogue No. 8. December, 1895.

CLASSIFIED SUBJECT
CATALOGUE

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Materia Medica 12

Medical Jurisprudence 13

Microscopy 13

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CLEVELAND. Pocket Medical Dictionary, ^d Edition. Very
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MAXWELL. Terminologia Medica Polyglotta. By Dr.
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TREVES AND LANG. German-English Medical Dictionary.

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EAR (see also Throat and Nose).

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GOUT AND RHEUMATISM.

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HYGIENE AND WATER ANALYSIS.

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JOURNALS, ETC.

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12 SUBJECT CATALOGUE.

KIDNEY DISEASES.

RALFE. Diseases of the Kidney and Urinary Derange-
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THORNTON. The Surgery of the Kidney. 19 Illus. Clo., $1.50
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POWELL. Diseases of the Lungs and Pleurae, including
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MASSAGE.

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MURRELL. Massotherapeutics. Massage as a Mode of Treat-
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OSTROM. Massage and the Original Swedish Move-
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MATERIA MEDICA AND THERA-
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ALLEN, HARLAN, HARTE, VAN HARLINGEN. A
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BRACKEN. Outlines of Materia Medica and Pharmacology. By
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DAVIS. Materia Medica and Prescription Writing. $1.50

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MAYS. Therapeutic Forces ; or, The Action of Medicine in
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MAYS. Theine in the Treatment of Neuralgia. ^ bound, .50

MEDICAL BOOKS. 13

NAPHEYS. Modern Therapeutics, gth Revised Edition, En-
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I SMITH, M.D., and J. AUBREY DAVIS, M.D.

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POTTER. Hand-Book of Materia Medica, Pharmacy, and
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POTTER. Compend of Materia Medica, Therapeutics, and
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SAYRE. Organic Materia Medica and Pharmacognosy. An
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WARING. Practical Therapeutics. 4th Edition, Revised and
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WHITE AND WILCOX. Materia Medica, Pharmacy, Phar-
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MEDICAL JURISPRUDENCE AND
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REESE. Medical Jurisprudence and Toxicology. A Text-Book
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MANN. Forensic Medicine and Toxicology. Illus. $6.50

MURRELL. What to Do in Cases of Poisoning. 7th

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TANNER. Memoranda of Poisons. Their Antidotes and Tests.

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MICROSCOPY.

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BEALE. How to Work with the Microscope. A Complete
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them colored. $6-5°

CARPENTER. The Microscope and Its Revelations. ?th
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14 SUBJECT CATALOGUE.

LEE. The Microtomist's Vade Mecum. A Hand-Book of
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M ACDONALD. Microscopical Examinations of Water and Air.
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REEVES. Medical Microscopy, including Chapters on Bacteri-
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WETHERED. Medical Microscopy. A Guide to the Use of the
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MISCELLANEOUS.

BLACK. Micro-Organisms. The Formation of Poisons. A
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BURNETT. Foods and Dietaries. A Manual of Clinical Diet-
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DAVIS. Biology. Illustrated. $3.00
GOWERS. The Dynamics of Life. .75
HAIG. Causation of Disease by Uric Acid. A Contribution to
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GOWERS. Manual of Diseases of the Nervous System. A
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Vol. II. Diseases of the Brain and Cranial Nerves ; General and
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GOWERS. Syphilis and the Nervous System. $1.00

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FLOWER. Diagram of the Nerves of the Human Body.
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MEDICAL BOOKS. 15

HORSLEY. The Brain and Spinal Cord. The Structure and
Functions of. Numerous Illustrations. $2.50

OBERSTEINER. The Anatomy of the Central Nervous Or-
gans. A Guide to the Study of their Structure in Health and Dis-
ease. 198 Illustrations. $5-5°

ORMEROD. Diseases of the Nervous System. 75 Wood En-
gravings. $1.00

OSLER. Cerebral Palsies of Children. A Clinical Study. $2.00

OSLER. Chorea and Choreiform Affections. $2.00

PAGE. Injuries of the Spine and Spinal Cord. In their Surgical
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PAGE. Railroad Injuries. With Special Reference to Those of the
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THORBURN. Surgery of the Spinal Cord. Illustrated. £4.00

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WOOD. Brain Work and Overwork. .40

NURSING.

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CANFIELD. Hygiene of the Sick-Room. A Book for Nurses and
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Subjects for the Use of Nurses and Other Intelligent Women. $1.25

CULLINGWORTH. A Manual of Nursing, Medical and Sur-
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CULLINGWORTH. A Manual for Monthly Nurses. 3dEd. .40

DOMVILLE. Manual for Nurses and Others Engaged in At-
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FULLERTON, Obstetric Nursing. 40 Ills. 4th Ed. $1.00

FULLERTON. Nursing in Abdominal Surgery and Diseases
of Women. Comprising the Regular Course of Instruction at the
Training-School of the Women's Hospital, Philadelphia. 2d Edition.
70 Illustrations. t^-S0

HUMPHREY. A Manual for Nurses. Including General
Anatomy and Physiology, Management of the Sick-Room, etc. i3th
Edition. Illustrated. $1.00

SHAWE. Notes for Visiting Nurses, and all those Interested
in the Working and Organization of District, Visiting, or
Parochial Nurse Societies. With an Appendix Explaining the
Organization and Working of Various Visiting and District Nurse So-
cieties, by HELEN C. JENKS, of Philadelphia. $1.00

STARR. The Hygiene of the Nursery. Including the General
Regimen and Feeding of Infants and Children, and the Domestic Man-
agement of the Ordinary Emergencies of Early Life, Massage,- etc. 4th
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TEMPERATURE CHARTS. For Recording Temperature, Res-
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VOSWINKEL. Surgical Nursing, in Illustrations. Ji.oo

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16 SUBJECT CATALOGUE.

OBSTETRICS.

BAR. Antiseptic Midwifery. The Principles of Antiseptic Meth-
ods Applied to Obstetric Practice. Authorized Translation by
HENRY D. FRY, M.D.. with an Appendix by the Author. $1.00

CAZEAUX AND TARNIER. Midwifery. With Appendix by
MUNDE. The Theory and Practice of Obstetrics, including the Dis-
eases of Pregnancy and Parturition, Obstetrical Operations, etc.
8th Edition. Illustrated by Chromo- Lithographs, Lithographs, and
other full-page Plates, seven of which are beautifully colored, and
numerous Wood Engravings. Cloth, $4.50 ; Full Leather, $5.50

DAVIS. A Manual of Obstetrics. Being a Complete Manual for
Physicians and Students, zd Edition. 16 Colored and other Plates
and 134 other Illustrations. $2.00

LANDIS. Compend of Obstetrics. $th Edition, Revised by WM.
H. WELLS, Assistant Demonstrator of Clinical Obstetrics, Jefferson
Medical College. With many Illustrations, 80; Interleaved, $1.25.

SCHULTZE. Obstetrical Diagrams. Being a series of 20 Col-
ored Lithograph Charts, Imperial Map Size, of Pregnancy and Mid-
wifery, with accompanying explanatory (German) text illustrated
by Wood Cuts. 2d Revised Edition.

Price in Sheets, $26.00 ; Mounted on Rollers, Muslin Backs, $36.00

STRAHAN. Extra-Uterine Pregnancy. The Diagnosis and
Treatment of Extra-Uterine Pregnancy. .75

WINCKEL. Text-Book of Obstetrics, Including the Pathol-
ogy and Therapeutics of the Puerperal State. Authorized
Translation by J. CLIFTON EDGAR, A.M., M.D. With nearly 200 Illus-
trations. Cloth, $5.00; Leather, $6.00

FULLERTON. Obstetric Nursing. 4th Ed. Illustrated. $1.00

SHIBATA. Obstetrical Pocket-Phantom with Movable Child
and Pelvis. Letter Press and Illustrations. $i.co

PATHOLOGY AND HISTOLOGY.

BLACKBURN. Autopsies. A Manual of Autopsies Designed for
the Use of Hospitals for the Insane and other Public Institutions.
Ten full-page Plates and other Illustrations. $1.25

BLODGETT. Dental Pathology. By ALBERT N. BLODGETT,
M.D., late Professor of Pathology, and Therapeutics, Boston Dental
College. 33 Illustrations. $1.25

GILLIAM. Pathology. A Hand-Book for Students. 47 Illus. .75

HALL. Compend of General Pathology and Morbid Anatomy.
91 very fine Illustrations. .80; Interleaved, $1.25

STIRLING. Outlines of Practical Histology. 368 Illustrations.
2d Edition, Revised and Enlarged. With new Illustrations. $2.00

VIRCHOW. Post-Mortem Examinations. A Description and
Explanation of the Method of Performing Them in the Dead House
of the Berlin Charity Hospital, with Special Reference to Medico-
Legal Practice. 3d Edition, with Additions. .75

PHARMACY.

Special Catalogue of Books on Pharmacy sent free upon application.

COBLENTZ. Manual of Pharmacy. A New and Complete
Text-Book by the Professor in the New York College of Pharmacy.
2d Edition, Revised and Enlarged. 437 Illustrations. $3-5°

MEDICAL BOOKS. 17

BEASLEY. Book of 3100 Prescriptions. Collected from the
Practice of the Most Eminent Physicians and Surgeons — English,
French, and American. A Compendious History of the Materia
Medica, Lists of the Doses of all the Officinal and Established Pre-
parations, an Index of Diseases and their Remedies. 7th Ed. $2.00

BEASLEY. Druggists' General Receipt Book. Comprising
a Copious Veterinary Formulary, Recipes in Patent and Proprietary
Medicines, Druggists' Nostrums, etc. ; Perfumery and Cosmetics,
Beverages, Dietetic Articles and Condiments, Trade Chemicals,
Scientific Processes, and an Appendix of Useful Tables, zoth Edi-
tion, Revised. $2.00

BEASLEY. Pocket Formulary. A Synopsis of the British and
Foreign Pharmacopoeias. Comprising Standard and Approved
Formulae for the Preparations and Compounds Employed in Medical
Practice, nth Edition. $2.00

PROCTOR. Practical Pharmacy. Lectures on Practical Phar-
macy. With Wood Engravings and 32 Lithographic Fac-simile
Prescriptions. 3d Edition, Revised, and with Elaborate Tables of
Chemical Solubilities, etc. $3.00

ROBINSON. Latin Grammar of Pharmacy and Medicine.
2d Edition. With elaborate Vocabularies. $i .75

SAYRE. Organic Materia Medica and Pharmacognosy. An
Introduction to the Study of the Vegetable Kingdom and the Vege-
table and Animal Drugs. Comprising the Botanical and Physical
Characteristics, Source, Constituents, and Pharmacopeial Prepar-
ations. With Chapters on Synthetic Organic Remedies, Insects
Injurious to Drugs, and Pharmacal Botany. A Glossary and 543
Illustrations, many of which are original. $4.00

SCOVILLE. The Art of Compounding. A Text-Book for the
Student and a Reference Book for the Pharmacist. $2.50

STEWART. Compend of Pharmacy. Based upon " Reming-
ton's Text-Book of Pharmacy." 5th Edition, Revised in Accord-
ance with the U. S. Pharmacopoeia, 1890. Complete Tables of
Metric and English Weights and Measures. .80; Interleaved, $1.25

UNITED STATES PHARMACOPO2IA. 1890. 7th Decennial
Revision. Cloth, $2.50 (postpaid, $2.77); Sheep, $3.00 (postpaid,
$3.27); Interleaved, $4. oo (postpaid, $4.50); Printed on one side of
page only, unbound, $3.50 (postpaid, $3.90).

Select Tables from the U. S. P. (1890). Being Nine of the Most
Important and Useful Tables, Printed on Separate Sheets. Care-
fully put up in patent envelope. .25

WHITE AND WILCOX. Materia Medica, Pharmacy, Phar-
macology, and Therapeutics. 2d American Edition. Revised
by REYNOLD W. WILCOX, M.D., LL.D. Cloth, $2 75 ; Leather, $3.25

POTTER. Hand-Book of Materia Medica, Pharmacy, and
Therapeutics. 600 Prescriptions and Formulae, sth Edition.

Cloth, $4.00 ; Sheep, $5.00

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PHYSICAL DIAGNOSIS.

TYSON. Hand-Book of Physical Diagnosis. For Students and
Physicians. By the Professor of Clinical Medicine in the University
of Pennsylvania. Illus. 2d Ed., Improved and Enlarged. $1.25

MEMMINGER. Diagnosis by the Urine. 23 Illus. $1.00

2

18 SUBJECT CATALOGUE.

PHYSIOLOGY.

BRUBAKER. Compend of Physiology, yth Edition, Revised
and Illustrated. .80; Interleaved, $1.25

KIRKE. Physiology, (i3th Authorized Edition. Dark Red Cloth.)
A Hand-Book of Physiology. i3th London Edition, Revised and
Enlarged. 516 Illustrations, some of which are printed in colors.

Cloth, $3.25; Leather, $4.00

LANDOIS. A Text-Book of Human Physiology, Including
Histology and Microscopical Anatomy, with Special Reference to
the Requirements of Practical Medicine, sth American, translated
from the last German Edition, with Additions by WM. STIRLING, M.D.,
D.SC. 845 lllus., many of which are printed in colors. In Press.

STARLING. Elements of Human Physiology. loollls. £1.00

STIRLING. Outlines of Practical Physiology. Including
Chemical and Experimental Physiology, with Special Reference to
Practical Medicine. 3d Edition. 289 Illustrations. $2.00

TYSON. Cell Doctrine. Its History and Present State. 2d
Edition. Ji.5o

YEO. Manual of Physiology. A Text-Book for Students of
Medicine. By GERALD F. YEO, M.D., F.R.C.S. 6th Edition. 254
Illustrations and a Glossary. Cloth, $2.50 ; Leather, $3.00

PRACTICE.

BEALE. On Slight Ailments; their Nature and Treatment.
2d Edition, Enlarged and Illustrated. $1.25

CHARTERIS. Practice of Medicine. 6th Edition. Thera-
peutical Index and Illustrations. $2.00

FAGGE. The Practice of Medicine. Cloth, $7.00 ; Leather, $9.00

FOWLER. Dictionary of Practical Medicine. By various
writers. An Encyclopaedia of Medicine. Clo.,$3.oo; Half Mor. $4.00

HUGHES. Compend of the Practice of Medicine, sth Edition,
Revised and Enlarged.

Part I. Continued, Eruptive, and Periodical Fevers, Diseases of the
Stomach, Intestines, Peritoneum, Biliary Passages, Liver, Kid-
neys, etc., and General Diseases, etc.

Part II. Diseases of the Respiratory System, Circulatory System,
and Nervous System; Diseases of the Blood, etc.

Price of each part, .80; Interleaved, $1.25

Physician's Edition. In one volume, including the above two

parts, a Section on Skin Diseases, and an Index, sth Revised,

Enlarged Edition. 568 pp. Full Morocco, Gilt Edge, $2.25

ROBERTS. The Theory and Practice of Medicine. The

Sections on Treatment are especially exhaustive, gth Edition,

with Illustrations. Cloth, $4.50; Leather, $5. 50

TAYLOR. Practice of Medicine. Cloth, $2.00; Sheep, $2.50

PRESCRIPTION BOOKS.

BEASLEY. Book of 3100 Prescriptions. Collected from the
Practice of the Most Eminent Physicians and Surgeons — English,
French, and American. A Compendious History of the Materia,
Medica, Lists of the Doses of all Officinal and Established Prepara-
tions, and an Index of Diseases and their Remedies. 7th Ed. $2.00

MEDICAL BOOKS. 19

BEASLEY. Druggists' General Receipt Book. Comprising
a Copious Veterinary Formulary, Recipes in Patent and Proprie-
tary Medicines, Druggists' Nostrums, etc. ; Perfumery and Cos-
metics, Beverages, Dietetic Articles and Condiments, Trade Chem-
icals, Scientific Processes, and an Appendix of Useful Tables,
loth Edition, Revised. $-2.00

BEASLEY. Pocket Formulary. A Synopsis of the British and
Foreign Pharmacopoeias. Comprising Standard and Approved
Formulae for the Preparations and Compounds Employed in Medical
Practice, nth Edition. Cloth, $2.00

DAVIS. Essentials of Materia Medica and Prescription
Writing. $1.50

PEREIRA. Prescription Book. Containing Lists of Terms,
Phrases, Contractions, and Abbreviations Used in Prescriptions, Ex-
planatory Notes, Grammatical Construction of Prescriptions, etc.
i6th Edition. Cloth, .75 ; Tucks, $1.00

WYTHE. Dose and Symptom Book. The Physician's Pocket
Dose and Symptom Book. Containing the Doses and Uses of all
the Principal Articles of the Materia Medica and Officinal Prepara-
tions, i jth Ed. Cloth, .75; Leather, with Tucks and Pocket, $1.00

SKIN.

ANDERSON. A Treatise on Skin Diseases. With Special
Reference to Diagnosis and '1 reatment, and Including an Analysis
of n,ooo Consecutive Cases. Illus. Cloth, $3.00; Leather, $4.00

BULKLEY. The Skin in Health and Disease. Illustrated. .40

CROCKER. Diseases of the Skin. Their Description, Pathol-
ogy, Diagnosis, and Treatment, with Special Reference to the. Skin
Eruptions of Children. 92 Illus. zd Edition. Enlarged. $4.50

VAN HARLINGEN. On Skin Diseases. A Practical Manual
of Diagnosis and Treatment, with special reference to Differential
Diagnosis. 3d Edition, Revised and Enlarged. With Formulae
and 60 Illustrations, some of which are printed in colors. $2.75

SURGERY AND SURGICAL DIS-
EASES.

CAIRO ANDCATHCART. Surgical Hand-Book. 5th Edition,
Revised. 188 Illustrations. Full Red Morocco, $2.50

DULLES. What to Do First in Accidents and Poisoning.
4th Edition. New Illustrations. $1.00

HACKER. Antiseptic Treatment of Wounds, Introduction to
the, According to the Method in Use at Professor Billroth's Clinic,
Vienna. With a Photo-engraving of Billroth in his Clinic. .50

HEATH. Minor Surgery and Bandaging. loth Ed Revised
and Enlarged. 158 Illustrations, 62 Formulae, Diet List, etc $1.25

HEATH. Injuries and Diseases of the Jaws. 4th Edition.
187 Illustrations. ' $4 50

HEATH. Lectures on Certain Diseases of the Jaws. 64 Illus-
trations. Boards, .50

HORWITZ. Compend of Surgery and Bandaging, including
Minor Surgery, Amputations, Fractures, Dislocations, Surgical Dis-

, eases, and the Latest Antiseptic Rules, etc., with Differential Diagno-
sis and Treatment. $th Edition, very much Enlarged and Rear-
ranged. 167 Illustrations, 98 Formulae. Clo., .80 ; Interleaved, $1.25

SUBJECT CATALOGUE.

JACOBSON. Operations of Surgery. Over 200 Illustrations.

Cloth, $3.00 ; Leather, $4.00

JACOBSON. Diseases of the Male Organs of Generation.
88 Illustrations. $6.00

MACREADY. A Treatise on Ruptures. 24 Full-page Litho-
graphed Plates and Numerous Wood Engravings. Cloth, $6.00

MOULLIN. Text-Book of Surgery. With Special Reference to
Treatment. 3d American Edition. Revised and edited by JOHN B.
HAMILTON, M.D., LL.D., Professor of the Principles of Surgery and
Clinical Surgery, Rush Medical College, Chicago. 623 Illustrations,
over 200 of which are original, and many of which are printed in
colors. Just Ready. Handsome Cloth, $6.00; Leather, $7.00

" The aim to make this valuable treatise practical by giving special

attention to questions of treatment has been admirably carried out.

Many a reader will consult the work with a feeling of satisfaction that

his wants have been understood, and that they have been intelligently

met." — The American Journal of Medical Science.

PORTER. Surgeon's Pocket-Book. 3d Ed. Lea. Cover, £2.00.

SMITH. Abdominal Surgery. Being a Systematic Description of
all the Principal Operations. 80 Illus. sth Edition. In Press.

VOSWINKEL. Surgical Nursing, in Illustrations. #1.00

WALSHAM. Manual of Practical Surgery. 5th Ed., Re-
vised and Enlarged. With 380 Kngravings. Clo., $2.75 ; Lea. ,$3.25

WATSON. On Amputations of the Extremities and Their
Complications. 250 Illustrations. $5.50

THROAT AND NOSE (see also Ear).

COHEN. The Throat and Voice. Illustrated. .40

HALL. Diseases of the Nose and Throat. Two Colored
Plates and 59 Illustrations. $2.50

HALL. Compend of Diseases of the Ear and Nose. Illus-
trated. .80; Interleaved, $1.25

HUTCHINSON. The Nose and Throat. Including the Nose,
Naso-Pharynx, Pharynx, and Larynx. Illustrated by Lithograph
Plates and 40 other Illustrations. 2d Edition. In Press.

MACKENZIE. The Pharmacopoeia of the London Hospital
for Diseases of the Throat, sth Edition, Revised by Dr. F.
G. HARVEY. $1.00

McBRIDE. Diseases of the Throat, Nose, and Ear. A Clinical
Manual. With colored Illus. from original drawings. 2d Ed. $6.00

MURRELL. Chronic Bronchitis and its Treatment. (Author-
ized Edition.) A Clinical Study. $1.50

POTTER. Speech and its Defects. Considered Physiologically,
Pathologically, and Remedially. $1.00

WOAKES. Post-Nasal Catarrh and Diseases of the Nose
Causing Deafness. 26 Illustrations. $1.00

URINE AND URINARY ORGANS.

ACTON. The Functions and Disorders of the Reproductive
Organs in Childhood, Youth, Adult Age, and Advanced Life,
Considered in their Physiological, Social, and Moral Relations.
Sth Edition. ^.75

ALLEN. Albuminous and Diabetic Urine. Illus. $2.25

MEDICAL BOOKS. 21

BEALE. One Hundred Urinary Deposits. On eight sheets,
for the Hospital, Laboratory, or Surgery. Paper, $2.00

HOLLAND. The Urine, the Gastric Contents, the Common
Poisons, and the Milk. Memoranda, Chemical and Microscopi-
cal, for Laboratory Use. Illustrated and Interleaved. 5th Ed. $1.00
LEGG. On the Urine. 7th Edition, Enlarged. Illus. $1.00

MEMMINGER. Diagnosis by the Urine. 23 Illus. $1.00

MOULLIN. Enlargement of the Prostate. Its Treatment and
Radical Cure. Illustrated. $1.50

THOMPSON. Diseases of the Urinary Organs. 8th Ed. $3.00
THOMPSON. Calculous Diseases. The Preventive Treatment
of, and the Use of Solvent Remedies. 3d Edition. .75

TYSON. Guide to Examination of the Urine. For the Use of
Physicians and Students. With Colored Plate and Numerous Illus-
trations engraved on wood, gth Edition, Revised. $1.25
VAN NUYS. Chemical Analysis of Healthy and Diseased
Urine, Qualitative and Quantitative. 39 Illustrations. $1.00

VENEREAL DISEASES.

COOPER. Syphilis, ad Edition, Enlarged and Illustrated with
20 full-page Plates. $5.00

GOWERS. Syphilis and the Nervous System. $1.00

HILL AND COOPER. Venereal Diseases. Being a Concise De-
scription of Those Affections and Their Treatment. 4th Ed. .75

JACOBSON. Diseases of the Male Organs of Generation. 88
Illustrations. $6.00

VETERINARY.

ARMATAGE. The Veterinarian's Pocket Remembrancer.
Being Concise Directions for the Treatment of Urgent or Rare Cases,
Embracing Semeiology, Diagnosis, Prognosis, Surgery, Treatment,
etc. 2d Edition. Boards, $1.00

BALLOU. Veterinary Anatomy and Physiology. 29 Graphic
Illustrations. .80; Interleaved, $1.25

TUSON. Veterinary Pharmacopoeia. Including the Outlines of
Materia Medica and Therapeutics, sth Edition. $2.25

WOMEN, DISEASES OF.

BYFORD (H. T.). Manual of Gynecology. With 234 Illustra-
tions, many of which are from original drawings and photographs.
Just Ready. $2.50

BYFORD (W. H.). Diseases of Women. 4th Edition. 306
Illustrations. Cloth, $2.00; Leather, $2.50

DUHRSSEN. A Manual of Gynecological Practice. 105
Illustrations. Just Ready. tl-5°

LEWERS. Diseases of Women. 146 Illus. 3d Edition. $2.00

WELLS. Compend of Gynecology. Illus. .80; Interleaved, $i. 25

WINCKEL. Diseases of Women. Translated by special authority
of Author, under the Supervision of, and with an Introduction by,
THHOPHILUS PARVIN, M.D. 152 Engravings on Wood. 3d Edition,
Revised. In Preparation.

FULLERTON. Nursing in Abdominal Surgery and Diseases
of Women. 2d Edition. 70 Illustrations. $i-5°

22 SUBJECT CATALOGUE.

COMPENDS.

From The Southern Clinic.

" We know of no series of books issued by any house that so fully
meets our approval as these ?Quiz-Compends?. They are well ar-
ranged, full, and concise, and are really the best line of text-books that
could be found for either student or practitioner."

BLAKISTON'S ?QUIZ-COMPENDS?

The Best Series of Manuals for the Use of Students.
Price of each, Cloth, .80. Interleaved, for taking Notes, $1.25.

4®" These Compends are based on the most popular text-books
and the lectures of prominent professors, and are kept constantly re-
vised, so that they may thoroughly represent the present state of the
subjects upon which they treat.

JKg* The authors have had large experience as Quiz-Masters and
attaches of colleges, and are well acquainted with the wants of students.

J8®~ They are arranged in the most approved .form, thorough and
concise, containing over 600 fine illustrations, inserted wherever they
could be used to advantage.

4®" Can be used by students of any college.

4Sf They contain information nowhere else collected in such a
condensed, practical shape. Illustrated Circular free.

No. I. POTTER. HUMAN ANATOMY. Fifth Revised and
Enlarged Edition. Including Visceral Anatomy. Can be used
with either Morris's or Gray's Anatomy. 117 Illustrations and 16
Lithographic Plates of Nerves and Arteries, with Explanatory
Tables, etc. By SAMUEL O. L. POTTER, M.D., Professor of the
Practice of Medicine, Cooper Medical College, San Francisco ; late
A. A. Surgeon, U. S. Army.

No. 2. HUGHES. PRACTICE OF MEDICINE. Part I. Fifth
Edition, Enlarged and Improved. By DANIEL E. HUGHES, M.D.,
Physician-in-Chief, Philadelphia Hospital, late Demonstrator of
Clinical Medicine, Jefferson Medical College, Phila.

No. 3. HUGHES. PRACTICE OF MEDICINE. Part II.
Fifth Edition, Revised and Improved. Same author as No. 2.

No. 4. BRUBAKER. PHYSIOLOGY. Seventh Edition, with
new Illustrations and a table of Physiological Constants. Enlarged
and Revised. By A. P. BRUBAKER, M.D., Professor of Physiology
and General Pathology in the Pennsylvania College of Dental
Surgery ; Demonstrator of Physiology, Jefferson Medical College,
Philadelphia.

No. 5. LANDIS. OBSTETRICS. Fifth Edition. By HENRY G.
LANDIS, M.D. Revised and Edited by WM. H. WKLLS, M.D.,
Assistant Demonstrator of Obstetrics, Jefferson Medical College,
Philadelphia. Enlarged. 47 Illustrations.

No. 6. POTTER. MATERIA MEDICA, THERAPEUTICS,
AND PRESCRIPTION WRITING. Sixth Revised Edition
(U. S. P. 1890). By SAMUEL O. L. POTTER, M.D., Professor of
Practice, Cooper Medical College, San Francisco ; late A. A. Sur-
geon, U. S. Army.

MEDICAL BOOKS. 23

PQUIZ-COMPENDS ?— Continued.

No. 7. WELLS. GYNECOLOGY. A New Book. By WM.

H. WELLS, M.D., Assistant Demonstrator of Obstetrics, Jefferson
College, Philadelphia. Illustrated.

No. 8. FOX AND GOULD. DISEASES OF THE EYE AND
REFRACTION. Second Edition. Including Treatment and
Surgery. By L. WEBSTER Fox, M.D , and GEORGE M. GOULD,
M.D. With 39 Formulae and 71 Illustrations.

No. 9. HORWITZ. SURGERY, Minor Surgery, and Bandag-
ing. Fifth Edition, Enlarged and Improved. By ORVILLE
HORWITZ, B. s., M.D., Clinical Professor of Genito-Urinary Surgery
and Venereal Diseases in Jefferson Medical College ; Surgeon to
Philadelphia Hospital, etc. With 98 Formulae and 71 Illustrations.

No. 10. LEFFMANN. MEDICAL CHEMISTRY. Fourth

Edition. Including Urinalysis, Animal Chemistry, Chemistry of
Milk, Blood, Tissues, the Secretions, etc. By HENRY LEFFMANN,
M.D., Professor of Chemistry in Pennsylvania College of Dental
Surgery and in the Woman's Medical College, Philadelphia.

No. ii. STEWART. PHARMACY. Fifth Edition. Based upon
Prof. Remington's Text-Book of Pharmacy. By F. E. STEWART,
M.D., PH.G., late Quiz-Master in Pharmacy and Chemistry, Phila-
delphia College of Pharmacy ; Lecturer at Jefferson Medical
College. Carefully revised in accordance with the new U. S. P.

No. 12. BALLOU. VETERINARY ANATOMY AND PHY-
SIOLOGY. Illustrated. By WM. R. BALLOU, M.D., Professor
of Equine Anatomy at New York College of Veterinary Surgeons;
Physician to Bellevue Dispensary, etc. 29 graphic Illustrations.

No. 13. WARREN. DENTAL PATHOLOGY AND DEN-
TAL MEDICINE. Second Edition, Illustrated. Containing
all the most noteworthy points of interest to the Dental Student
and a Section on Emergencies. By GEO. W. WARREN, D.D.S.,
Chief of Clinical Staff, Pennsylvania College of Dental Surgery,
Philadelphia.

No. 14. HATFIELD. DISEASES OF CHILDREN. Second
Edition. Colored Plate. By MARCUS P. HATFIELD, Profes-
sor of Diseases of Children, Chicago Medical College.

No. 15. HALL. GENERAL PATHOLOGY AND MORBID
ANATOMY. 91 Illustrations. By H. NEWBERRY HALL, PH.G.,
M.D., Professor of Pathology and Med. Chem., Chicago Post-
Graduate Medical School; Mem. Surgical Staff, Illinois Charit-
able Eye and Ear Infirmary ; Chief of Ear Clinic, Chicago Med.
College.

No. 16. DISEASES OF NOSE AND EAR. Illustrated. Same
Author as No. 15.

Price, each, Cloth, .80. Interleaved, for taking Notes, $1.25.

Handsome Illustrated Circular sent free upon application.

In preparing, revising, and improving BLAKISTON'S ? Quiz-Co M-
PHNDS f the particular wants of the student have always been kept in
mind.

Careful attention has been given to the construction of each sentence,
and while the books will be found to contain an immense amount of
knowledge in small space, they will likewise be found easy reading ;
there is no stilted repetition of words ; the style is clear, lucid, and dis-
tinct. The arrangement of subjects is systematic and thorough ; there
is a reason for every word. They contain over 600 illustrations.

Moullin's
Surgery. Thlr<] Editlon,

Just Ready.

EDITED BY

JOHN B. HAMILTON, M.D.,

Professor of the Principles of Surgery and Clinical Surgery,
Rush Medical College, Chicago, etc.

This is not only the latest, but the most uniform
and complete one-volume Text-Book of Surgery.
The relative value of each subject has
been carefully considered, the constant aim
of author and editor having been to make it
practical and useful. It is systematically
arranged and pays special attention to treat-
ment.

Royal 8vo. 1250 Pages. 600 Illustrations.
Cloth, net, $6.00. Sheep, net, $7.00.

Illustrated circular free upon application.

DATE DUE SLIP

UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL LIBRARY

THIS BOOK IS DUE ON THE LAST DATE
STAMPED BELOW

fMAY
SEP

; 1935

lwi-4,'29