THE THE GIFT OF ROSWELL P FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE. Cornell University Library RB 37.M25 1897 Pathological technique; a practical manua 3 1924 000 227 961 Cornell University Library The original of tiiis bool< is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000227961 Pathological TECHNiQUE'^^^^^ii«^ A PRACTICAL MANUAL FOR THE PATHO- LOGICAL LABORATORY BY FRANK BURR MALLORY, A.M., M.D. Assistant Professor of Pathology, Harvard University Medical School; Assistant Pathologist to the Boston City Hospital ; Pathologist to the Children's Hospital and to the Carney Hospital JAMES HOMER WRIGHT, A.M., M.D. Director of the Laboratory of the Massachusetts General Hospital ; Instructor in Pathology, Harvard University Medical School W/Tff 105 ILLUSTRATIONS PHILADELPHIA W. B. SAUNDERS 925 Walnut Street 1897 W-Ca^u^-. 1 .^<-a>-u Copyright, 1897, By W. B. SAUNDERS. Mb. 74^-^ /{6 mi ELECTROTYPED BY WESTCOTT & THOMSON. PHILADA "^^^ °^ w. B. SAUNDERS, PHfLADA. TO HENRY F. SEARS, A.M., M. D., WHO BY HIS LIBERALITY FIRST RENDERED POSSIBLE PATHOLOG- ICAL RESEARCH IN BOSTON, AND BY HIS PERSONAL WORK ADVANCED AND STIMULATED IT, THIS BOOK IS RESPECTFULLY DEDICATED BY THE AUTHORS PREFACE. This book is designed especially for practical use in pathological laboratories, both as a guide to beginners and as a source of reference for the advanced. We believe that the book will also meet the wants of practitioners who have more or less opportunity to do general pathological work. Every autopsy presents for solution a problem which may be simple or complex. The known quantities are certain clinical symptoms and physical signs ; the unknown quan- tities are not only the gross and microscopic lesions that may or may not have given rise to the symptoms and signs, but also the etiology of the lesions and the order of their sequence. The solution of the problem often requires the highest skill in post-mortem, bacteriological, and histo- logical technique, but in its solution lies the fascination of pathological work. It has seemed advisable to us to present, so far as possible, a consecutive statement of the methods employed in solving the various problems that arise, so as to avoid the repetitions that necessarily occur when the three usual divisions of the subject are separately considered by different writers. It is hoped that this method of presenting the subject will bring the student to the realization that the mechanical per- formance of a post-mortem examination and the inspection of the gross lesions constitute usually only the beginning of the solution of the problem, which should be investigated 11 12 PREFACE. bacteriologically, histologically, and chemically as far as our present knowledge will permit. We should particularly advise the routine bacteriological and histological examination of the more important organs in all suitable cases. Naturally, the autopsies in which the lesions are due to a single etiological factor are the most valuable and instructive for a clear understanding of the pathological processes present. Besides the methods of post-mortem examinations and of bacteriological and histological investigations connected with autopsies, we have added the special methods em- ployed in clinical bacteriology and pathology. In the parts devoted to Bacteriology and to Pathological Histology we have not endeavored to make an exhaustive collection of methods and formulae, but rather to select those which have been found of the greatest service in practical work. To Dr. A. H. Wentworth, Assistant in Children's Dis- eases in the Harvard University Medical School, we are indebted for the sections on the Blood and on Malaria, and for the notes in regard to Lumbar Puncture. Boston, August, 1897. CONTENTS, PART I. POST=MORTEM EXAMINATIONS. Introduction, 17. — Instruments, 18. — General Rules, 21. — Suggestions to Beginners, 23. — Private Autopsies, 24. I. EXTERNAL EXAMINATION OF THE BODY. Inspection of the Body as a Whole, 26. — Special Inspection of the Different Parts of the Body, 27. II. INTERNAL EXAMINATION OF THE BODY. Opening of the Abdominal Cavity, 27. — Inspection of the Abdominal Cavity, 29. — Opening of the Thorax, 30. — Inspection of the Pleural Cavities, 31. — Opening of the Pericardium, 32. — External Inspection of the Heart, 32. — Opening of the Heart, 33. — Removal of the Lungs, 37. — Organs of the Neck, 39. — The Abdominal Cavity, 40. — The Spleen, 41. — The Gastro-intestinal Tract, 41. — The Liver, 44; The Kidneys and Adrenals, 45. — The Pelvic Organs, 47. — Removal of the Brain, 50. — External Examination of the Brain, 55. — Section of the Brain, 56. — Virchow's Method, 58; Pitre's Method, 59. — Removal of the Spinal Cord, 60. — The Eye, 62. — The Ear, 63. — The Naso- pharynx, 64. — Examination of New-born and Very Young Children, 65. — Restitution of the Body, 67. PART II. BACTERIOLOGICAL EXAMINATIONS. I. BACTERIOLOGICAL APPARATUS. Steam Sterilizer, 70. — Hot-air Sterilizer, 70. — Blood-serum Coagulator, 70. — Gas-stove, 71. — Test-tubes, 71. — Hypodermic Syringes, 71. — Cornet Cover- glass Forceps, 71. — Dropping-bottles, 71. — The " Platinum Wire " or " Loop," 72. — Bacterial Filtering Apparatus, 72.^ — Wire Baskets, 72. — Tin Cups, 73.^ Thermo-regulator, 73. 13 14 CONTENTS. II. CULTURE=MEDIA. The Preparation of Test-tubes, 74.— Preparation of Culture-media : Bouillon, 75; Glucose Bouillon, 76; Agar-agar (Plain), 77; Glucose Agar- agar, 79.— Glycerin Agar-agar, 80; Gelatin (Plain), 80; Glucose Gelatin, 81; Blood-serum (Loffler's Mixture), 81 ; Litmus-mill^, 85 ; Potato-cultures accord- ing to Bolton, 85 ; Dunham's Pepton Solution, 86. — The Filling of Test-tubes, 86. — Sterilization of Culture-media, 87. III. BACTERIOLOQICAL EXAMINATIONS AT AUTOPSIES. Cover-glass Preparations, 89. — Staining Methods for Cover-glass Preparations: Gram's Method of Staining, 91 ; Stain for the Bacillus Tuberculosis (Gabbet's Method), 92; Welch's Method of Staining the Capsule of the Pneumococcus, 93 ; Curry's Method of Staining the Capsule of the Pneumococcus, 93. — Ex- amination by Cultures, 94. — Method of Preparing Cultures on Blood'serum, 94; The Inoculation of Animals, 96. IV. THE METHODS OF STUDYING BACTERIA IN CULTURES. I. Cover-glass Preparations from Cultures, 98. — The Staining of Spores, 100.- -The Staining of Flagella, 100 : Loifler's Method, 102; Pitfield's Method, 103 ; Bunge's Method, 103 ; Van Ermengem's Method, 104. — 2. Methods of Obtaining Pure Cultures, 105. — Method of Isolation of a Bacterium in Pure Culture from a Mixed Growth, 107 : The Plate Method of Petri, no; Esmarch's Method of Roll-cultures, no.— The Determination of the Motility of Bacteria, iii. — 3. The Inoculation of Animals, 112. — Guinea-pigs, 112. — Rabbits, 113. — Mice, 115. — The Care of Animals, 117. — 4. Cultivation without Oxygen (Anaerobic Cultures) : Method of Liborius, 117; Method of Buchner, 119; Esmavch's Method, 119; Bouillon Cultures under Hydrogen, 119. V. BACTERIOLOQICAL DIAGNOSIS. Staphylococcus Pyogenes Aureus, 121. — Staphylococcus Pyogenes Albus and Citreus, 124. — Staphylococcus Epidermidis Albus, 124. — Staphylococcus Cereus Albus and Flavus, 124. — Streptococcus Pyogenes, 124. — Pneumococcus, 128. — Gonococcus, 130: Special Culture-media, 131; Serum Agai'-agar, 131 ; Urine- Serum-Agar-agar, 132. — Micrococcus Tetragenus, 133. — Diplococcus Intracel- lularis Meningitidis, 135. — Bacillus Diphtherise, 137. — Bacillus of Typhoid Fever, 141 : Differential Diagnosis between the Bacillus of Typhoid Fever and the Bacillus Coli Communis, 143. — Bacillus Coli Communis, 145. — Bacillus Tuberculosis, 148. — Spirillum of Asiatic Cholera (Comma Bacillus), 152. — Bacillus of Anthrax, 156. — Bacillus Pyocyaneus (Bacillus of Green Pus), 160. — Bacillus of Influenza, 162. — Bacillus of Glanders (Bacillus Mallei), 164. — Bacillus Proteus (Proteus Vulgaris), 167. — Bacillus Mucosus Capsulatus, 168. — Bacillus of Tetanus, 171. — Bacillus Aerogenes Capsulatus, 173. — Bacillus of Malignant Edema, 175. — Actinomyces, 176. CONTENTS. 1 5 VI. CLINICAL BACTERIOLOQY. General Considerations, 178; Cover-glass Examinations, 180; Preparation of Cultures, 180; Animal Inoculations, 181. — Suppurative Processes, 181. — Erysipelas, 181. — Peritonitis and Appendicitis^ 181. — Pleural, Pericardial, and Joint Exudates, 182. — Anthrax, or Malignant Pustule, 182. — Diphtheria, 182: Special Methods of Staining the Bacillus Diphtherise, 184. — Influenza, 185. — Examination of Sputum for Tubercle Bacilli, 186. — Tubercle Bacilli in Urine, 188. — Surgical Tuberculosis, 189. — Cultures from the Blood during Life, 190.— Gonorrhea, 190: Method of Staining for Gonococci, 191. — Gonorrheal Con- junctivitis, 192. — Pyosalpinx, 192. — Cerebro-spinal Meningitis, 192. — Glan- ders, 193. — Tetanus, 193 : Method of Isolation, 193. — Asiatic Cholera, 195. — Typhoid Fever, 198: Isolation of the Typhoid Bacillus from the Feces, 198; Capaldi's Culture-medium, 200 ; The Blood-serum Reaction in Typhoid Fever, 201. — Rabies (Hydrophobia), 201. — Leprosy, 203. — Actinomycosis, 203. PART III. HISTOLOGICAL METHODS. Introduction, 204. — Laboratory Outfit : Microscopes, 204. — Freezing Micro- tome, 206. — Celloidin Microtome, 209. — Paraffin Microtome, 210. — Paraffin Bath, 210. — Vulcanized Fiber, 211. — Knives, 212. — Running Water, 213. — Slides, 213. — Cover-slips, 214. — Staining Dishes, 214. — Metal Instruments, 215. — Bottles, 2t6.^Exainination of Fresh Material, 216. — Indifferent Fluids, 217. — Macerating Fluids, 218. — Examination of Fluids, 218. — Injections, 218. — Cold Injection-masses, 219. — Warm Injection-masses, 219. — Fixing Re- agents, 220. — Alcohol, 222. — Zenker's Fluid, 223. — Orth's Fluid, 224. — Flemming's Solution, 224. — Hermann's Solution, 225. — Pianese's Solution, 225. — Rabl's Chromo-formic Acid Solution, 225. — Corrosive Sublimate, 225. — Formaldehyde, 226. — Boiling, 227. — Miiller's Fluid, 227. — Marchi's Fluid, 228. — Erlicki's Fluid, 228. — Decalcification, 228.- — Directions for Using Nitric Acid, 229. — Phloroglucin and Nitric Acid, 229. — Picric Acid, 230. — Trichlor- acetic Acid, 230. — Imbedding Processes, 230. — Celloidin, 231. — Imbedding in Celloidin, 231. — Imbedding in Paraffin, 234. — Serial Sections by the Celloidin Method, 237. — Serial Sections by the Paraffin Method, 239. — Staining Solu- tions : Hematoxylin and Hematein Stains, 239. — Carmine Stains, 244. — Aniline Dyes, 245. — Diffuse Stains, 249. — Combination Stains, 250. — Pianese's Staining Solutions and Staining Methods, 250. — Orcein, 253. — lodin, 253. — Lugol's Solu- tion, 254. — Acid Alcohol, 254. — Aniline Water, 254.- — Carbolic-acid Water, 255. — -Mayer's Glycerin-albumin Mixture, 255. — Clearing Reagents, 255. — Mount- ing Reagents, 258. — Metallic Stains or Impregnations, 258. — Silver, 258. Gold, 260. — Osmic Acid, 261. — Staining Methods, 262. — Nuclear Stains, 265. — Alum-hematoxylin Stains, 266. — Aqueous Alum-hematoxylin ; Delafield's Hematoxylin; Ehrlich's Acid Hematoxylin, 267. — Mayer's Hemalum, 268. — • Hcidenhain's Hematoxylin Stain, 268. — Carmine Stains, 268. — -Aniline Dyes as 1 6 CONTENTS. Nuclear Stains, 269. — Diffuse or Contrast-stains, 271. — Combination Stains, 272. — Staining in Mass, 273. — Mitosis, 273. — ^Directions for Staining Karyomi- totic Figures with Safranin, 274. — The Staining of Bacteria in Tissues, 275. ^Pathogenic Bacteria which do not Stain by Gram, 277. — Gonococcus, 278. — Typhoid Bacillus, 278. — Influenza Bacillus, 279. — Glanders Bacillus, 279, — Friedlander's Capsule-bacillus, 280. — Pathogenic Bacteria which Stain by Gram, 280. — Bacillus of Rhinoscleroma, 282. — Actinomyces, 282. — Bacteria that Stain by the Tubercle-bacillus Method, 283. — Tubercle Bacillus, 283. — • Bacillus of Leprosy, 285. — Syphilis Bacillus, 286. — Smegma Bacillus, 286. — Methods of Examination of Animal Parasites, 287. — Protozoa, 287. — Malarial Organisms, 287. — Ameba Coli, 292. — Sporozoa, 294. — Round-worms, 294. — Trichinas, 295. — Tape-worms, 296. — Taenia Solium, 296. — Taenia Medio- canellata s. Saginata, 298. — Taenia Echinococcus, 298. — Bothriocephalus Latus, 298. — Special Stains for Certain Tissue-elements other than Nuclei, 298. — Mastzellen, 298. — Plasma-cells, 300. — Connective-tissue Fibrillse, 300. — Elastic Fibers, 301. — The Central Nervous System, 303; General Stains, 305; Stains for Ganglion-cells, 307; Stains for the Myelin-sheath, 316; Stains for the Neuroglia-fibers, 321. — Degenerations of the Nervous System, 325. — Examination of the Blood, 326. — Apparatus Used in the Examination of the Blood, 327. — Preparation of Apparatus, 330. — Obtaining the Blood, 331. — The Hemoglobin Test, 332. — Estimation of Number of Red Corpuscles, 333. — Cover-glass Preparations, 336. — The Elements of the Blood, 338. — Methods of Staining, 342. — Methods of Fixing and Examining Special Organs and Tissues, 344. — Acute Inflammatory Exudations ; Granulation-tissue, 345. — Lung, Spleen, Bone-marrow, Kidney, 346. — Gastro-intestinal Tract, 347. — Liver, 347. — Bone and Cartilage, 348. — Museum Preparations, 350. — Patho- logical Products, 352. — Cloudy Swelling; Albuminous Degeneration, 352. — Fatty Degeneration, 352; Necrosis, 354. — Caseation, 355. — Demonstration of Fibrin, 355. — Mucin, 356. — Pseudo-mucin, 358. — Colloid and Hyaline, 358. — Glycogen Infiltration, 361. — Amyloid Infiltration, 362. — Pigmentation, 365. — Petrifaction, 368. — Clinical Pathology, 368. — Examination of Tissues from Clinical Cases for Diagnosis, 369. — Uterine Scrapings, 369.— Examination of Fluids obtained by Puncture, 370. — Lumbar Puncture, 371. — Ovarian and Parovarian Cysts, 374. — Pancreatic Cyst or Fistula, 374.^ — Dropsy of the Gall- bladder, 375. — Hydronephrosis and Renal Cysts, 375. — Echinococcus Cysts, 375. — Examination of the Sputum, 376: Macroscopic Examination, 376; Micro- scopic Examination, 378. — Examination of the Gastric Contents, 380. — Exami- nation of the Feces, 382. — Examination of the Urine, 382. PATHOLOGICAL TECHNIQUE. PART I. POST-MORTEM EXAMINATIONS. Introduction. — The method of making post-mortem examinations as originally taught by Virchow has been variously modified in its details by his pupils and followers. We have endeavored, while following in general his plan, to select those modifications that have proved simplest and of greatest value. In certain instances we have not hesitated to adopt, or at least to call attention to, useful methods of procedure originating in the Rokitansky school of pathology, as now best exemplified by Chiari. The problem offered by an autopsy is often solved in part or wholly by the macroscopic post-mortem examination. More frequently, however, the complete and final solution is reached only after careful bacteriological and histological study. The post-mortem examination may, therefore, be looked upon as the beginning of the solution of the prob- lem. Its particular function is to demonstrate in the indi- vidual case all congenital or acquired abnormalities, all macroscopic lesions, and to explain all gross mechanical questions. It furnishes the material for bacteriological and histological study. Perfectly to accomplish its purpose a post-mortem examination must be made in a careful, sys- tematic manner. While a general method of procedure is advisable, it will often be found advantageous, or even necessary, to depart 2 17 1 8 PATHOLOGICAL TECHNIQUE. from it. According to Orth, " the chief reqiiisite of every exact post-mortem examination is this, that no part shall be displaced from its position until its relations to the surround- ing parts are established, and that no part shall be taken out by whose removal the further examination of other parts is affected." The order and method of procedure in making a post- mortem examination, including the various incisions, may be said to have been planned for the routine examination of normal or diffusely diseased organs. As soon as a notice- able focal lesion is present the order of procedure and the customary method of removal and of incision must be so altered as best to display the lesion. Instruments. — The following instruments will be found extremely useful in the autopsy-room, although not all of them are necessary : The autopsy-table should be large, so as to accommodate on it the instruments and several dishes in addition to the body. It should have a slightly raised edge, and should slope gently toward an opening in the center for the escape of fluids. The table is best made of zinc, and along one edge should have a centimeter scale. The water for use on the table is best supplied by a rubber tube from an overhead pipe reaching to within 6o to loo cm. of the table. The scales for weighing the various organs should have a large pan and gram and kilogram weights. A band-saw will be found very useful for sawing bones for the inspection of the marrow, and for calcified and osseous tumors. The best autopsy-knife is a stout, broad-bladed knife with bellied edge and heavy handle. The blade should measure about 1 2 cm. in length and 3 cm. in width ; the handle should be 12 cm. in length. Many operators prefer a somewhat smaller knife than this. Amputating-knives of different sizes are useful for long, deep cuts into organs and tumors. A myelotome is a short, thin, narrow knife -blade, 1.4 cm. long and 4 mm. wide, set obHquely on a slender steel stalk POST-MORTEM EXAMINATIONS. 19 ending in a wooden handle (Fig. 2). It is used only for cutting the cord squarely across in removing the brain. Fig. I. — Instruments for use in the autopsy-room : a, saw ; b, holder for the head ; c, steel hammer with wedge end and blunt hook on the handle ; d, costo- tome ; e, bone-cutter ; /, hatchet-chisel ; g, autopsy-knife. Cartilage-knives and scalpels of different sizes are used for a variety of purposes. Scissors, both straight and curved, should be of various 20 PATHOLOGICAL TECHNIQUE. sizes. A medium-sized and a fine pair should each have one probe-pointed blade. An enterotome is a long, straight pair of scissors, of which Fig. 2, — Myelotome. one blade is longer than the other and blunt at the extremity (Fig. 3). A hook at the end is not advisable. The instru- ment is used in opening the heart and the intestines. Fig. 3. — Enterotome. A saw with movable back and rounded end will be found the most generally useful for opening the skull and the Fig. 4. — Luer's double rachiotome. spinal canal. An ordinary meat-saw is preferred by some, but cannot be used on the vertebrae. Luer's double rachiotome, or adjustable double saw (Fig. 4), is very useful in removing the cord, and is the safest instru- ment to put into the hands of beginners. POST-MORTEM EXAMINATIONS. 21 Forceps : several sizes, large and small, mouse-toothed. Costotome : heavy bone-shears for cutting the ribs. A powerful bone-cutter, with short blades, 5 cm. long, set at an angle of about 45° to the handles, which are 36 cm. in length, is employed for dividing the arches of the cervical vertebrae and for other purposes where ordinary bone-cutters will not do. A chisel with 2 cm. cutting edge, for exposing the marrow of the long bones, removing portions of the base of the skull, etc. A hatchet-chisel of steel for starting the calvaria and spinous processes after sawing the skull and the vertebral column. Soft-iron hammer with wooden handle. Steel hammer with wedge end, and blunt hook on the handle. Holder for the head while sawing the skull. Autopsy-needles, long and a little curved. Probes of flexible metal ; also fine glass probes for small blood-vessels or ducts. Grooved director. Pans for holding water, organs, etc. Boards, square or oblong, 30X30 or 30X50 cm., on which to lay instruments or cut organs. Sponges. Catheters. Strong hemp twine is the best for sewing up the body. Glass graduates for measuring fluids. A block of wood with shallow depression for the neck ; for use while opening the head. Vise. Small cup or dish for removing fluid from cavities. General Rules. — The room for an autopsy should be well lighted, otherwise the finer changes in the tissues cannot be recognized. Artificial illumination is not good, because the colors of the tissues are entirely changed by the yellow- ness of the light. Before beginning an autopsy the necessary instruments 22 PATHOLOGICAL TECHNIQUE. should be arranged on a short board on the autopsy-table in the order in which they are most likely to be used. The operator stands on the right side of the body. This position he rarely leaves except for some definite purpose ; for example, in opening the skull he stands at the head. Order and cleanliness are the first points to be insisted upon at every autopsy. Clean water should always be at hand for washing the instruments and for keeping the hands free from blood and pus. The cut surface of an organ should not be washed with water except to remove blood ; gently scrape the surface with the knife held obliquely. In cutting, the knife should be drawn, not pressed or shoved into the tissues. According to Virchow, a broad, clean cut into an organ, even if incorrectly made, is much better than several short cuts which leave a ragged surface. The autopsy-knife should be grasped in the hand as if to cut bread. In using this knife the main movement should be from the shoulder, not from the wrist as in dissecting. It goes without saying that the sharper the knife the better. In cutting the brain and cord, especially if their consistency is lessened, moisten the knife to prevent the tissue from stick- ing and tearing. Before beginning an autopsy it is important to know the main points in the clinical history of the case, as they may greatly lighten the work of investigation by calling attention to those organs that require special examination. The record of an autopsy should be dictated by the ope- rator as he proceeds with the examination of the case, and should be as nearly as possible an objective description of the appearances found. Only the anatomical diagnosis should express the opinion of the operator. If it is not convenient to dictate the autopsy during its performance, the description of the lesions certainly ought to be made with the organs in sight, and not from memory after the lapse of hours or even days, when many of the details may be forgotten. Later, the results of the bacteriological and histological examinations should be added to the autopsy report, so as to make the case complete. POST-MORTEM EXAMINATIONS. 23 Rubber gloves are sometimes worn to protect the hands while making a post-mortem examination ; but they greatly dull the sense of touch, and cannot be recommended for routine work except while opening the stomach and intes- tines. Rubber cots for the fingers are often useful. For cuts on the fingers use celloidin dissolved in equal parts of alcohol and ether, instead of flexible collodion, because the latter will not stick. A cut received during an autopsy should immediately be washed thoroughly, and then sucked so that the blood will flow freely. For protection during the rest of the autopsy use a rubber glove or cover ' the cut with celloidin. It is always well to wash the hands after an autopsy with an antiseptic solution, such as corrosive sublimate (i : 2000). For removing odors from the hands turpentine will often be found serviceable, or a saturated solution of permanganate of potassium followed by oxalic acid. Orth highly recom- mends a dilute solution of formaldehyde for this purpose. Suggestions to Beginners. — In a case of general miliary tuberculosis the older focus from which the organ- isms have spread must always be found. Look especially for tubercular thrombi in the pulmonary veins as the frequent source of the general infection. In a case of embolism hunt for the thrombus, bearing in mind, however, that the whole of a thrombus may become free and form an embolus. An arterial embolus may be due to a venous thrombus, in which case it must have passed through an open foramen ovale, except in the case of thrombi of the pulmonary veins. In acute peritonitis always seek for a source of infection (appendix, female genitals, gastro-intestinal tract, etc.). It cannot always be found. In hemorrhage from the stomach associated with cirrhosis of the liver look for rupture of dilated esophageal veins. In cases of more or less sudden death, especially if pre- ceded by signs of asphyxia, always examine the pulmonary artery in situ for possible emboli. In cases of instantaneous death examine the coronary arteries. 24 PATHOLOGICAL TECHNIQUE. Private autopsies must often be made under many dis- advantages, and, when out of town, not infrequently in a short space of time. It is always important to warn the attending physician not to allow the undertaker to inject the body before the autopsy, because the color and consistency of the organs are so changed by most injecting fluids that it is difficult to recognize the pathological processes. If there is danger of post-mortem changes, have the body packed in ice. A regular autopsy-bag will be found very convenient for carrying to private autopsies. It is made of black leather lined with rubber, and measures about 40 X 1 8 X 20 cm. Loose within it is carried a rubber bag 40 X 24 X 20 cm., shaped like a short envelope with a flap (22 cm. long) on one side, for bringing away any organs that demand further examina- tion. The case of instruments should contain one or two autopsy-knives, two scalpels, a pair of forceps, one or two pairs of scissors, an enterotome, a steel hammer with wedge- end and with a blunt hook on the handle, a small chisel, a saw with detachable handle and back, an autopsy-needle, and a probe ; free within the bag should be carried a spool of strong twine, a costotome, a long slender knife for use in removing the brain, a hammer with soft iron head, and a sponge. In rare cases additional instruments may be re- quired. A white duck apron for personal use will always be found convenient. It is also well to carry along several blood-serum tubes and a platinum needle for making cultures at the autopsy. When there is a lesion of the nervous sys- tem it is advisable to bring a jar of a 4 per cent, solution of formaldehyde and to place the tissue in the fluid at the autopsy, as otherwise it is not easily gotten to the laboratory in good condition. At the house can always be obtained a slop-pail, a wash- bowl, a pitcher of water, several newspapers, and an old sheet. The body is usually on an undertaker's frame, but it may be in an ice-box or on the bed. The examination of the chest and abdomen can be made in any of these posi- tions. If, however, the body is in an ice-box, it must be POST-MORTEM EXAMINATIONS. 25 raised to the level of the top. of the box in case it is neces- sary to open the head. The clothing on the body can be removed, or, if only a shirt or a night-dress, is best slit down the middle and turned out over the arms. Tear the sheet into four equal pieces. Fold and tuck in one piece on each side of the trunk and neck, allowing the outer portion to fall over the arms. Fold and lay the third piece on the lower extremities, tucking the upper end beneath the clothing below the pubes. The fourth piece can be placed beneath the head if it is to be opened. This procedure leaves the front of the thorax and abdomen free for operation and protects the rest of the body and the clothing. On the thighs place one or two folded newspapers, and on these the necessary instruments. On the legs place the bowl containing only a dampened sponge. If the undertaker has not put a rubber sheet on the floor beneath the body and on the side where the operator is to stand, newspapers should be spread to protect the carpet. Place the slop-pail on the rubber sheet within convenient reach. Having thus made all arrangements, even to the threading of his needle, the operator is ready to begin. If the cord and brain have to be examined as well as the body, it is best to do the cord first, so as to avoid the leak- age that might otherwise occur from the trunk-cavities if they had been opened first. To support the head while opening it, use a stick of wood, a brick, or, in case of neces- sity, the instrument-box wrapped in a newspaper. At a private autopsy cleanliness is extremely important. If there is no undertaker or nurse present, the operator himself must see that everything is cleaned and put in order before leaving, that all the blood-stains are removed from the dishes, and that all papers and soiled cloths are burned or rolled up and left in a neat bundle for the undertaker to dis- pose of Ground coffee thrown on a shovelful of burning coals will be found an excellent deodorizer for the room after the autopsy. 26 PATHOLOGICAL TECHNIQUE. EXTERNAL EXAMINATION OF THE BODY. External examination is often of great importance, espe- cially in medico-legal autopsies, and should never be ne- glected, as it may throw great light on lesions found within the body. It should be systematic and careful, and is best taken up in the following order: I. Inspection of the Body as a Whole. 1. Sex. 2. Age. 3. The body-length should be measured on the table beside the body, between points opposite the vertex of the head and the sole of the foot beneath the ankle. 4. The development of the skeleton has reference to the bony framework, which may be powerful, slender, or de- formed. 5. The general nutrition is shown by the amount of mus- cular development and of subcutaneous fat-tissue. The latter is judged by pinching up a fold of skin. 6. The general condition of the skin includes amount of elasticity, bronzing, jaundice, edema, and decubitus. 7. Post-mortem discolorations may be divided into three varieties : {a) Hypostasis of blood, or the settling of blood into the lowest lying blood-vessels ; this form of discoloration dis- appears on pressure. ((5) Diffusion of blood-coloring matter out of the vessels into the surrounding tissues (due to blood-pigment being set free by post-mortem decomposition) ; does not disappear on pressure. (i:) The greenish discoloration, usually seen earliest over the abdomen, is due to sulphate of iron formed through decomposition of the tissues. This discoloration is import- ant, as it may modify the interpretation of appearances observed in the internal organs. 8. Post-mortem rigidity, degree and extent. It begins in the maxillary muscles, and spreads gradually from above downward, disappearing later in the same order. It is most marked and lasts longest in muscular individuals who have POST-MORTEM EXAMINATIONS. 2/ been ill but a short time. Cholera furnishes the most marked cases. The rigor disappears quickest in cachectic diseases. When once it has been forcibly overcome, it does not recur. The time of beginning after death varies widely — from ten minutes to seven hours. II. Special Inspection of the DiflFerent Parts of the Body. The examination should begin with the head. Any lesion or abnormality found should be carefully noted. Particular attention should be paid to the condition of the pupils and to the color of the sclera. Then follow in order the neck, the thorax (size and shape), the abdomen (distended or retracted), the genitals, and the extremities. INTERNAL EXAMINATION OF THE BODY. The opening of the body-cavities is described first, be- cause the brain is relatively much less frequently the seat of disease, and because in this country it is often impossible to obtain permission to open the head. Moreover, the lesions in the body often throw much light on those to be expected in the brain. The advantage of examining the brain first, particularly in those cases in which the important lesions are cerebral, is said to be that the amount of blood in the cerebral vessels can be more accurately determined. After the heart has been removed some of the blood in the brain may escape through the severed vessels below. In routine examinations, however, the body is usually examined first, then the brain, and finally the cord. It is not a bad practice to remove the calvaria, to examine the meninges over the upper surface of the cerebrum, and then to make the examination of the body before removing the brain. In this way any change in the blood-supply of the cerebral vessels would be observed. Opening of the Abdominal Cavity, — In the exam- ination of the body the peritoneal cavity is opened first, the two pleural cavities next, and the pericardial cavity last. The cavities and their contents are to be inspected in the order and at the time that each is opened, but the organs are to 28 PATHOLOGICAL TECHNIQUE. be removed from the cavities for further examination in the reverse order, beginning with the heart. The primary or long anterior incision to bare the thorax and to open the abdomen (Fig. 5) should extend from the larynx to the pubes, passing to the left of the umbilicus, so as not to cut the- round ligament. In cutting, the handle of the knife is depressed so as to use the belly of the blade rather Fig. 5. — Primary incision in the body (Nauwerck). than the point. An incision beginning as high as the chin is, unfortunately, rarely allowable. Over the sternum the cut should extend down to the bone; over the abdomen, however, only into the muscles, or in fat people through the muscles into the subperitoneal fat-tissue. To open the ab- dominal cavity, nick carefully through the peritoneum just below the sternum, introduce the first and second fingers of POST-MORTEM EXAMINATIONS. 29 the left hand, and while making strong upward and outward traction on the right abdominal flap extend the incision to the pubes. Some operators prefer to separate the fingers and to cut between them. The abdominal flaps are rendered much less tense by cut- ting the pyramidales and recti muscles from below just above the pubis. Care must be taken not to injure the overlying skin. The abdominal cavity can now be examined, but more room will be obtained if the skin and the underlying muscles be first stripped back from the thorax to about 5 cm. outside of the costochondral line. The operation is most easily and neatly done by lifting the skin directly away from the chest-wall or turning it forcibly out with the left hand, and then cutting the tense tissue close to the cartilages and ribs with long sweeps of the knife held almost flat. The operation begins over the lower border of the ribs and extends upward. In dissecting off the skin and muscles from the left side the right hand works underneath the left. The mammae can easily be incised from the under side of the flap, and if necessary the axillary glands can be reached by dissecting the skin farther out, especially over the clavicle. Before beginning 'the inspection of the peri- toneal cavity it is important to examine first the surface of the incision into the abdomen, noting the thickness and color of the fat-tissue and the condition of the muscles. Inspectionof the Abdominal Cavity. — The character of any fluid present should be determined and its amount measured or estimated. The simplest way to remove it is to dip it up with a small cup or dish and pour it into a glass graduate for inspection and measurement. If the presence of gas within the peritoneal cavity is suspected, a small pouch should be formed in the first incision as soon as it has been made and water poured in. The first opening into the abdominal cavity should then be made with the point of a scalpel at the bottom of the water, through which the gas, if present, will escape in bubbles. The various abdominal organs and their relations to each other are to be investigated in situ by sight and by touch. 30 PATHOLOGICAL TECHNIQUE. As a rule, examine first the gastro-intestinal tract, including the appendix and the mesenteric lymph-glands. Ulcerations of the intestine can often readily be made out through the walls. The examination of the spleen, liver, kidneys, and pelvic organs follows. The pancreas is easily reached by tearing through the omentum between the stomach and the colon, so as to open the lesser peritoneal cavity. After the inspection of the abdominal organs the position of the diaphragm is to be ascertained on both sides in the costochondral line by measuring with the right hand passed palm upward underneath the ribs, and the left hand outside at the corresponding height to mark the position of ribs or intercostal spaces. On the right side the hand is to be passed up on the outside of the falciform ligament. Nor- mally, the diaphragm stands at the fifth rib on the left side, and at the fourth rib or fourth interspace on the right. Opening of the Thorax. — To open the thorax, cut through the cartilages close to the ribs from the second down (about 5 mm. distant) with a scalpel held nearly hori- zontal, so that as one cartilage is cut through the handle of the scalpel will strike the next below and prevent the blade from penetrating too far and injuring the lung. In young people the cartilages can be cut easily by one long stroke on each side, but care must be taken not to go too deep. If the intercostal muscles are not divided by the same opera- tion, the sternum can be depressed by the left hand and the muscles severed by one pass of the knife on each side. The lower end of the sternum can now be elevated and freed from below upward from the diaphragm and pericardium until the first rib is reached. The cartilage of this rib is to be cut about i cm. farther out than the others, and from below upward toward the clavicle, with the handle of the knife beneath the elevated sternum and with the point and edge of the knife directed upward and a little outward. The sternum is then to be still further freed from the anterior mediastinal tissue until its upper end is reached. The sterno- clavicular joint on the left side can now be easily opened from below by entering a scalpel just above the cartilage of POST-MORTEM EXAMINATIONS. 3 1 the first rib, and following the irregular line of the joint around the end of the clavicle, while at the same time draw- ing the sternum over to the right side of the body. The right sterno-clavicular articulation is to be opened by con- tinuing the incision of the scalpel over the upper end of the sternum and into the second joint. The advantage of this method is that there is much less danger of wounding the large vessels at the base of the neck, and thus of mingling blood with any exudation which may happen to be present in the pleural cavities. If preferred, however, the articulations can be opened and the cartilages of the first ribs cut from above before freeing the sternum from the diaphragm. In this case enter a short, sharp, narrow-bladed scalpel held vertically, but loosely, into the left joint on its upper side, starting the incision just outside of the attachment of the sternal end of the sterno-mastoid muscle, and cut around the end of the clavicle by a series of short up-and-down strokes, allowing the blade to follow the irregular line of the joint. After cut- ting through the joint continue the incision outward and cut through the cartilage of the first rib. If the cartilages are calcified, use the costotome and cut through the ribs, as more room can be gained in this way, and they are more easily cut than calcified cartilages. When for any reason it is not permitted to open the thorax, the organs within it can be obtained through the opening into the abdominal cavity by freeing the diaphragm from the ribs, and removing first the heart and then the lungs. The sternum should be inspected at the time of its removal. It is perhaps best to examine next, especially in children, the epiphyses of the ribs at the costochondral line for any evi- dence of thickening. Inspection of the Pleural Cavities. — In the pleural cavities, as in the peritoneal cavity, the character and amount of any abnormal contents must be determined. If, from the cHnical history or from any other reason, the presence of air in a pleural cavity is suspected, a pouch should be formed over the ribs by aid of the skin-flap and filled with water. The pleural cavity is then to be pierced with a scalpel 32 PATHOLOGICAL TECHNIQUE. through the bottom of the pouch. Air, if present, will bubble up through the water. Slight adhesions are best torn through or cut. If the lungs are firmly attached, it is best to strip off the costal layer of the pleura with the lung. This is most easily done by starting the anterior edge of the costal pleura with the handle of the scalpel, and working in first a finger and then the whole hand until the pleura is entirely free. In passing the hand into the pleural cavities protect the back of it, especially if the ribs have been cut through, by folding the skin-flap in over the edge of the ribs. If desired, the lungs can be drawn forward, examined over their whole extent, even incised, and then replaced until the heart has been removed. In the connective tissue of the anterior mediastinum there is almost always a certain amount of emphysema due to the removal of the sternum. Emphys- ema due to laceration of lung-tissue is more marked in the upper half of the mediastinum, and usually extends up into the neck. The thymus gland attains its full development at the end of the second year, after which time it usually gradually disappears. Opening of the Pericardium. — To open the pericar- diurri, seize the sac near the middle with fingers or forceps, snip through the wall with knife or scissors, and with either instrument cut upward to where the pericardium is reflected over the large vessels, downward to the lower right border, and lastly to the apex. By gently raising the apex of the heart the amount of fluid in the pericardial cavity can be seen. The normal amount is about a teaspoonful, but it may be increased to loo c.c. in cases where the death-agony is prolonged. Pericardial adhesions should be broken through with the fingers. If this is impossible, the heart must be incised through the pericardium. External Inspection of the Heart. — Determine first the position, size, and shape of the heart, and the degree of distention of the different parts. The right ventricle and both auricles are usually distended with blood, which may be fluid as in death from suffocation or more or less coagu- POST-MORTEM EXAMINATIONS. 33 lated. The left ventricle is contracted and empty unless the individual has died from paralysis of this part of the heart, when it will be found distended with blood (condition of greatest diastole). Opening of the Heart. — The heart may be opened in situ or after removal from the body. Except in certain cases, to be spoken of later, it usually will be found advisable to remove the heart before making any incision into it, for the reason that it can be more perfectly opened after removal, especially by beginners, and the danger of contaminating any bacterial lesions of the valves is lessened. To remove the heart, grasp it gently near the apex with the left hand, supporting it further, if necessary, by one or two fingers placed above the coronal suture, and lift the whole heart vertically upward. Then cut its vessels from below upward with the knife held transverse and oblique. Divide in turn the inferior vena cava, the pulmonary veins on both sides, the superior vena cava, the pulmonary artery, and the aorta. Go deep enough to remove the auricles entire, but avoid injury to the underlying esophagus. For making the incisions to open the heart either a long, slender-bladed knife or long, straight scissors may be used. The heart should be placed on a board with its anterior sur- face up. The right auricle is opened by cutting from the orifice of the inferior vena cava into that of the superior, and from the latter into the auricular appendage. The first incision to open the right ventricle is made through the tri- cuspid valve and the wall of the ventricle along the under surface of the right border of the heart. It should be carried to the end of the ventricle, which does not reach quite to the apex of the heart. The second incision begins about the middle of the first, just above the insertion of the anterior papillary muscle (which should not be cut), and is carried through the pulmonary valve well over on the left side along the left border of a narrow, projecting ridge of fat-tissue usually present, so as to pass between the left ante- rior and the posterior segments of the valve. The left auricle is opened in a manner similar to the right 3 34 PATHOLOGICAL TECHNIQUE. by incisions joining the four orifices of the pulmonary veins and extending into the auricular appendage. The first incision into the left ventricle is through the mitral valve along the left border of the heart (i. e. the middle of the external wall of the left ventricle), between the two bundles of papillary muscles, to the apex of the heart. The second incision begins at the termination of the first at the apex, and is carried up close to the interven- tricular septum, parallel to the descending branch of the Fig. 6. — Heart, showing incisions. anterior coronary artery and about i cm. from it. The upper portion of the incision should pass midway between the pulmonary valve and the left auricular appendage. Ordinarily, one of the aortic cusps is divided, but this may be avoided, if desired, by dissecting away to some extent the pulmonary artery from the aorta and carrying the incision well over to the right between the right posterior and ante- rior valve-segments. As each auricle is opened the blood and clots it contains should be carefully removed and the auriculo-ventricular valves carefully inspected from above. In certain cases — as, for instance, extreme stenosis — it may be preferable not to cut through the valve, but to begin the POST-MORTEM EXAMINATIONS. 35 incision in the ventricular wall below the valve. The ven- tricular cavities should in like manner be freed from clots and the valves closely inspected. The coronary arteries should always be opened by means of small, narrow-bladed, probe-pointed scissors as far as they can be followed. The examination of the descending branch of the anterior artery is especially important. The posterior coronary is best opened by placing the tip of the left fore finger in the aorta over the orifice of the artery, and cutting from without in toward the finger-tip until the vessel is reached, when it can easily be slit up. In this way injury to the aorta is avoided. In cases of more or less sudden death with symptoms of asphyxia the pulmonary artery should always be opened in situ before removal of the heart, in order to examine for pos- sible emboli, because they often lodge just at the point where the vessels are severed in removing the heart and lungs, and easily may slip out unobserved. The simplest operation is to thrust a sharp-pointed scalpel through the artery just above the valve on the left side in the line of incision already described, and to cut upward until the branches to the right and left lungs are reached. If desired, this incision may be extended down through the pulmonary valve and the ven- tricular wall along the line given for the second incision in the right ventricle. The water-test for the competence of the valves of the heart is not very reliable, especially for the auriculo-ven- tricular valves, and is not so much used as formerly. Inspection and measurement of the valve after the heart has been opened will usually enable one to judge fairly accurately concerning the degree of competence. Before applying the test to the aortic valve the first incision into the left ventricle must be made and the cavity freed from clots, so that no obstruction will exist below the valve. Then the heart is to be held so that the aortic valve is per- fectly horizontal, and water poured in from above to float the cusps out. If competent, they should keep the water from flowing through. If, however, in holding the heart the nor- mal relations of the valve and the surrounding parts are not 36 PATHOLOGICAL TECHNIQUE. maintained, the valve may leak. A second source of error is that the water may escape through the coronary arteries, branches of which have been cut in opening the ventricle. In testing the mitral valve the left auricle is first opened and the clots removed, so as to expose the upper surface of the valve. Then the nozzle of a syringe is introduced through the aortic valve and water forced in so as to float the mitral curtains up. The test, however, is very unreliable, because the parts cannot be placed under natural conditions. The pulmonary and tricuspid valves can, of course, be tested by methods similar to those already described. Increase or diminution in the size of the heart is best determined by weighing the organ after the removal of the clots. In certain cases, however, and in special investigations measurements of different parts of the heart are desirable. Roughly, the heart is the size of the individual's fist. The following weights and measurements are taken from Nauwerck' s Sectionstechnik : Weight of the heart averages in men, 300 gr- \ /n ji " " " " women, 250 " ' Krause gives the average weight of the heart as 292 gr. Relative weight of heart to body in men, 1-169 "1 „, " " " " women, 1-162 i Length of heart in men, 8.5-9 cm. 1 „ . " " women, 8.0-8.5 " i Circumference of heart at base of ventricles, 28.8 cm. {Sappey). Thickness of wall of left ventricle, 1.1-1.4 cm.") „ ^ )■ Krause. " " right " 0.5-0.7 " J Thickness of wall of left ventricle (without trabeculee), 7-10 mm. 1 ^ i " " right " " " 2-3 " J Circumference of mitral orifice, 10.4 (W.), 10.9 (M.) ") " " tricuspid " 12.0 (W.), 12.7 (M.) j " aortic " 7.7 (W.), 8.0 (M.) j" ^''"^"^^■ " " pulmonary orifice, 8.9 (W.), 9.2 (M.) J " " ascending aorta, 7.4 cm. " " pulmonary artery, 8.0 cm. {Bukl). The directions given for the removal and opening of the heart apply only when the organ is normal or contains lesions within itself which are not in continuity with any of the vessels entering into it. In aneurysm of the ascending POST-MORTEM EXAMINATIONS. 37 aorta, in thrombosis of a vena cava, and in a number of dif- ferent lesions connected with the heart or with the vessels given off from it, it is important to examine these vessels and to open them while they are still in continuity with the heart. For this purpose it is often necessary or advantageous to remove the thoracic organs in one piece, so as to be able to examine the central circulatory apparatus in continuity from the front and back before disturbing any of its relations. This is done by cutting across the trachea and adjoining tissues as high in the neck as necessary or possible, and dis- secting them free from the cervical vertebrae and the first ribs. Then by drawing the trachea and surrounding tissues forcibly forward the aorta and overlying organs can be easily stripped from the vertebral column as low as the diaphragm. The left hand is now placed around the lower end of the pericardial sac, the aorta, and the esophagus just above the diaphragm, and the vessels are severed by cutting between the hand and the diaphragm. More space for the examination in situ of the vessels at the base of the neck can be obtained by freeing the clavicles from all attachments above and to the first ribs and drawing them forcibly outward ; this operation will be found especially useful in following up the subclavian vessels. Removal of the I^ungs. — Pleural adhesions have al- ready been spoken of If the base of the lung is adherent to the diaphragm, it is usually advisable to remove the latter with the lung by cutting through its insertion into the ribs. According to Orth, there is less danger of wounding the ab- dominal organs if scissors be used for the performance of the operation. After the lung is free it is drawn forward out of the pleural cavity, and the root of it is grasped from above downward between the separated fingers (first and second or second and third) of the left hand. The lung, thus resting in the palm of the left hand, is first drawn downward toward the pubes until the primary bronchus is divided by a nearly vertical incision above and behind the left hand. Then the lung is lifted vertically upward, and the rest of its attachments cut in the same direction from above 38 PATHOLOGICAL TECHNIQUE. downward by the knife held transverse and flat, so as to avoid injuring the esophagus and aorta. The procedure is the same for both lungs. Once in a great while the apex of a lung will be found so firmly adhe- rent by dense scar-tissue that it can be freed only by using the knife. The primary or main incision into a lung is a long, deep Fig. 7. — Method of incising the lung (Nauwerck). cut from the apex to the base and from the convex surface to the root, slitting the primary bronchus, and thus not cut- ting it off from its branches to the upper and lower lobes (Fig. 7). To incise the left lung, place it with its inner or median surface and root downward on a board and with its base toward the operator. The left thumb steadies the lower lobe ; the first finger reaches between the two lobes almost to the primary bronchus ; and the rest of the fingers should hold the upper lobe. POST-MORTEM EXAMINATIONS. 39 The right lung is most easily incised by placing it in the same position, but with the apex toward the operator; in other words, always place the anterior edge of a lung beneath the palm of the hand. Some prefer to place each lung on its lower or diaphragmatic surface for incision. The right middle lobe is incised separately by a cut extending trans- versely in its greatest diameter. The bronchi and blood-vessels should be opened up for some distance with small probe-pointed scissors — as a rule from the surface of the section — cutting through the over- lying lung-tissue. In some cases, however, it is best to open up both the blood-vessels and the bronchi from the outside of the lung before incising it. The order to follow is vein first, then artery, and finally the bronchus. Secondary cuts into the lung are to be made parallel to the main incision. The bronchial lymph-glands should be incised from the outside of the lung. Organs of the Neck. — The operation of the removal of the organs of the neck is greatly facilitated if it is pos- sible to continue the primary skin-incision up to the chin. In other cases dissect the skin from the larynx and muscles of the neck as far up as possible. In like manner free the muscles, esophagus, and trachea from their attachments later- ally and posteriorly. Then allow the head to drop well back over the end of the table, and pass a long, slender-bladed knife up between the skin and the larynx, just behind the symphysis of the lower jaw, until the point of the knife appears beneath the tip of the tongue. From this point the knife is carried with a sawing motion down first one ramus of the jaw and then the other, dividing laterally the glossal muscles as far back as the posterior pharynx. The knife is next carried up behind the esophagus, and the posterior wall of the pharynx divided as high as possible. Pass the left hand up inside of the neck and draw down the tongue. Then cut the attachments of the soft to the hard palate, carrying the knife well out so as not to injure the tonsils. Any remaining attachments are usually easily severed by 40 PATHOLOGICAL TECHNIQUE. pressing the tongue first to one side and then to the other, and cutting close to the roof of the pharynx. Each lobe of the thyroid gland is to be incised in its greatest diameter. Next cut through the middle of the uvula and examine all of the pharynx removed. Incise the tonsils vertically. The esophagus is to be slit in the median line posteriorly, and the larynx and trachea anteriorly. The Abdominal Cavity. — The order of removal of the abdominal organs varies with diiferent operators, and under varying circumstances with the same operator. The gastro-intestinal tract, including the liver and pancreas, may be removed before or after the genito-urinary tract. The spleen as an organ by itself is often the first to be removed. The early removal of the liver is occasionally advantageous for the sake of the additional space obtained for the exami- nation of the other organs. It is well to practise the different methods of procedure, so that in a difficult case the best may be selected, because the examination of the abdominal cavity, especially in cases of extensive disease with numer- ous adhesions, is often one of the hardest tasks in post- mortem technique. As a rule, it is best to follow the usual order as long as possible, gradually removing the more or less normal or uninvolved organs. Occasionally it may be advisable to remove the organs en bloc, so as to be able to approach the problem from all sides. In all cases of acute peritonitis it is best before removing any organ to search for the source of the infection, paying particular attention to the vermiform appendix, to the gastro- intestinal tract, and, in females, to the pelvic organs. The order of removal of the abdominal organs adopted in this book for the majority of cases is that which seems the simplest and most natural — namely, to remove first the spleen as an organ essentially by itself; secondly, the gastro- intestinal tract, including the pancreas and liver, which forms the upper layer ; thirdly, the genito-urinary tract or middle layer, leaving the circulatory tract, the lowest layer, to be opened and inspected in situ. If, however, it proves neces- POST-MORTEM EXAMINATIONS. 41 sary to open a part of the gastro-intestinal tract in situ, it will be neater perhaps to remove the kidneys and spleen first. Occasionally at private autopsies it may be unneces- sary to examine the intestinal tract; under these circum- stances it is important to be able to get at the different organs without taking out the intestines. The Spleen. — As a rule, the spleen can easily be drawn forward from its bed behind the fundus of the stomach, be- neath the diaphragm, and lifted on to the lower edge of the ribs on the left side without cutting its vessels. The organ is then to be incised in its greatest diameter while thus firmly fixed between the left hand and the ribs ; or the vessels may be cut close to the hilus and the spleen incised after being placed on a board. In cases of adhesion to the diaphragm the spleen must be handled carefully while the fibrous attachments are torn or cut through, for the capsule is easily ruptured. Occasion- ally it is advisable to cut out with the spleen the portion of diaphragm attached to it. The important anatomical structures to be noted in the macroscopic examination are the capsule, trabeculae, blood- vessels, follicles, and pulp. The weight of the spleen, ac- cording to Orth, varies from 150 to 250 grams. The aver- age weight is put at 171 grams. The spleen measures 12 X 7.5 X 3 cm. The Gastro-intestinal Tract. — The first step is to examine externally, more or less carefully according to the clinical symptoms, the whole tract from the stomach to the rectum, if it has not already been done at the primary in- spection of the peritoneal cavity. The main points to notice are distention or contraction of the intestines, injection of the blood-vessels, thickening of the wall, especially in the lower part of the ileum, adhesions, exudations, etc. Inspect the mesentery, its length, the amount of fat, and the size of the lymph-glands ; incise the latter to determine color and con- sistency. Examine the mesenteric vessels if any evidence of infarction of the intestine is noticed. The portal vein and its branches should be opened up in situ, in all cases of ab- 42 PATHOLOGICAL TECHNIQUE. scess of the liver or of secondary deposits in it of malignant growths, before the gastro-intestinal tract is removed. As a rule, it is not necessary to open any part of the gastro-intes- tinal tract in situ. The operation can be performed much more neatly at the sink. The duodenum is often opened for the sake of investigating the flow of bile from the gall-duct, but except in cases of jaundice the operation must be looked upon largely as a physiological experiment. Free the omentum from the transverse colon by putting it on the stretch and dividing it with the knife close to the colon. Then begin the removal of the large intestine by drawing the sigmoid flexure forcibly forward and cutting the mesocolon close to the gut, first down to the rectum, then upward to the transverse colon. Free the latter by dividing the two folds of the lesser omentum, if not already cut through, which unite it to the stomach. The ascending colon is to be freed in the same manner as the descending portion. Care should be taken not to injure the appendix. If the lower part of the sigmoid flexure be now stripped up- ward a short distance with the fingers, so as to force the in- testinal contents out of the way, the gut can be divided just above the rectum without fear of the feces escaping. Place the freed intestine in a pan or pail, and as the small intestine is divided from its mesentery deposit it in the same receptacle. To remove the small intestine, begin at the ce- cum, and, while lifting the ileum with the left hand strongly enough to keep the mesentery constantly tense, cut the latter close to the intestine by playing the knife easily backward and forward across it with a fiddle-bow movement. Con- tinue the operation until the duodenum is reached. The mesentery can now be dissected from the duodenum and re- moved, or the mesentery, duodenum, pancreas, and stomach can be removed in continuity with the intestine by carefully dissecting them off the underlying structures. The opera- tion is perhaps more easily accomplished by freeing the organs from below upward. First cut down through the diaphragm and free it around the esophagus. Then separate the stomach from the liver by means of the thumb and fingers POST-MORTEM EXAMINATIONS. 43 of the left hand in such a way as to put on the stretch the vessels of the hepato-duodenal ligament. These vessels (hepatic artery, common gall-duct, and portal vein) are then carefully divided in the order named. As each vessel is cut the character of its contents should be observed to see if anything abnormal is present. The mesentery, if still present, the duodenum, the pan- creas, and the stomach, are now to be dissected carefully away from the underlying vessels from below upward until the esophagus is reached. This may be constricted by the fingers at any point desired, and cut across without danger of the gastric contents escaping and without the necessity of tying. In certain cases of hemorrhage from the stomach associated with cirrhosis of the liver it is important to re- move the esophagus in continuity with the stomach, because in these cases the hemorrhage usually takes place from dilated esophageal veins. The stomach and intestines are now to be opened at the sink by means of the enterotome, the colon along one of its longitudinal muscular bands, the small intestine along its mesenteric attachment, because the most important lesions usually occur opposite this line in the follicles and Peyer's patches. The stomach is opened by many along the greater curvature ; others, however, prefer to cut along a line 3 cm. from the lesser curvature, on the ground that better museum preparations are thus obtained. In case any tumor or focal lesion is perceived from the outside, it is advisable to cut the stomach, if possible, in such a way as to leave the patho- logical part uninjured. Whenever jaundice is present the duodenum must be opened in situ in order to examine the bile apparatus in con- tinuity, so as to determine whether the coloring is due to obstruction of the hepatic or common gall- ducts, or is of so- called hematogenous origin. To open the duodenum make a transverse fold in the ante- rior wall and incise with the scissors. Continue the longi- tudinal slit thus made up as far as the pylorus and down to where the duodenum passes beneath the mesentery. Notice 44 PATHOLOGICAL TECHNIQUE. the contents of the duodenum and their color both above and below the opening of the gall-duct. The ductus cho- ledochus usually opens in common with the ductus pan- creaticus on the posterior wall of the duodenum a little below the middle of the head of the pancreas, at a point marked by a small papilla which can easily be recognized by putting the mucous membrane on the stretch transversely. Press first on the common duct gently and in the direction of the papilla, watching the opening to see if any obstructing material is forced out. Pressure is then to be made on the gall-bladder to see if its contents also will flow. If neces- sary, the common duct and its branches are to be opened in situ. In certain cases the ductus pancreaticus is likewise to be opened up. Several cross-sections of the pancreas are usually better than one in the greatest diameter, because the duct is left in a better condition for slitting up if necessary. The weight of the pancreas varies from 90 to 120 grams (Orth). It measures 23 X 4.5 X 2.8 cm. The Liver.^— The liver is usually the last organ of the gastro-intestinal tract to be removed. This is ordinarily done by lifting up the right lobe and freeing it from all attachments as far as the vertebral column : the right lobe is then lifted and placed on the edge of the ribs on the right side, while the left lobe is elevated and freed. If the dia- phragm is firmly adherent, remove it with the liver. The incision to display the liver is a long deep cut passing through the right and left lobes in the greatest diameter of the organ. In a good many cases it is very convenient to remove the liver at the beginning of the special examination of the ab; dominal cavity, because more room can be obtained for the investigation of the other organs. This latter fault can to some extent be obviated by cutting the diaphragm on the right side and allowing the liver to slide forward somewhat into the right thoracic cavity. There can be no objection to the removal of the liver when jaundice is not present or when the liver is not con- nected by continuity with the lesion of some other organ POST-MORTEM EXAMINATIONS. 45 (pylephlebitis, malignant growth extending through portal vein or along gall-ducts, etc.). The operation is performed as follows : Pass the left hand in between the diaphragm and the right lobe and push the liver forward out of the right hypochondrium. Incise it deeply in its greatest diameter through the left and right lobes. Next free the gall-bladder from its bed by means of the fingers, and cut it off near the ductus hepaticus after compressing its lower end. It can then be opened length- wise and washed without danger of discoloring the liver or other organs. The liver is now to be grasped by placing the thumb on the under surface of the liver and the fingers in the incision. Elevate the organ, and, while carefully watching, cut through the ligamentum hepato-duodenal, which includes the blood-vessels and the ductus hepaticus. The ligamentum hepato-gastrium, the inferior vena cava, the suspensory ligament, the ligamentum coronarium, and the tissue between the inferior surface of the liver and the upper end of the kidney follow next : the adrenal is to be left on the kidney, and the diaphragm ought not to be injured. Even in the ordinary way of removing the liver the organ will be found much easier to handle if the usual incision is made in situ, so as to furnish a hold for the left hand. Other cuts into the liver are best made parallel to the primary one. Orth gives the weight of the liver for adults as varying from 1000 to 2000 grams. The average weight is usually put at 1500 to 1800 grams. The liver measurements are as follows : Length from right to left 25-32 cm. Width of right lobe 18-20 Width of left lobe 8-10 " Vertical diameter of right lobe 20-22 " Vertical diameter of left lobe 15-16 " Greatest thickness 6- 9.5 " The Kidneys and Adrenals. — If the adrenals are to be removed with the kidneys, it is necessary to cut first to the inside, and secondly above the adrenal, and then to make 46 PATHOLOGICAL TECHNIQUE. from the outer end of the second cut a curved incision along the outer convex border of the kidney through the perito- neum and the perinephritic fat-tissue. The left hand is to be inserted into the cut, the mass of tissue drawn forcibly- forward, and the vessels divided as close to the aorta as pos- sible, so that the renal vessels may be slit up and examined in connection with the renal lesions. The adrenal should be incised crosswise. The kidney is to be held firmly in the left hand between the thumb and fingers while a longitudinal incision is made from the convex border to the hilus. As a rule, it is better to shell it out of its investing fat-tissue before incising it. It will often be found convenient to make simply the curved incision above given, to shell the kidney out of its fat-cap- sule, and then to divide its vessels, leaving the adrenal behind to be incised in situ or removed separately. As a rule the left kidney is removed first. In all cases in which the bladder is involved in patho- logical changes in common with the kidneys the whole urinary tract should be removed intact, so that the lesions may be examined in continuity. For this reason it is a good plan to open up the pelvis of the kidney and the ureter from the primary incision, in order to see if any lesion is present before dividing the ureter. If it is desired to remove the kidneys before the intestines, the latter must to some extent be freed from their normal attachments. The splenic flexure of the colon is first to be drawn for- cibly forward and its attachments divided where they hide the left kidney. If the ureter is to be taken out also, it is best to free the whole of the descending colon from its mesocolon. Then the colon and the coils of small intestine are drawn over to the right side of the body, so as to leave the left kid- ney and adrenal exposed. They are then removed in exactly the same manner as already described. To remove the right kidney the hepatic flexure must be freed from over it. If the ureter is to be taken out, the de- scending colon and the cecum are dissected from over it. POST-MORTEM EXAMINATIONS. A.'J The right adrenal is firmly attached to the under surface of the liver, and must be carefully dissected from it by turning the latter upward. If the urinary tract is to be removed in continuity, each ureter is dissected down to the brim of the pelvis, and then left with its kidney attached until the pelvic organs have been taken out. After the kidney has been incised the capsule is to be stripped off, at least in part, so that the appearance of the surface of the kidney and the presence or absence of adhe- sions between the capsule and the renal tissue can be deter- mined. The points to be noted in the macroscopic examination of the kidney are size, consistency, and, on section, color, rela- tive proportion of cortex to pyramids, and thickness of each ; finally, the normal markings of the kidney, including blood- vessels, glomeruli, convoluted and straight tubules of cortex, collecting tubules of pyramids. The average weight of the kidney is 150 grams. The left kidney is always 5 to 7 grams heavier than the right (Orth). A kidney measures 11-12X5-6X3-4.5 cm. The cortex measures in thickness 4-6 mm. The relation of the cortex to the medulla is i to 3. The Pelvic Organs. — The pelvic organs are most easily and neatly removed by stripping the peritoneum from the pelvic wall with the fingers. Begin over the bladder and extend down the sides of the pelvis until the fingers meet beneath the rectum. Brace the backs of the hands laterally on the brim of the pelvis and lift the fingers forcibly upward ; this movement will free the pelvic organs cleanly from the sacrum, and leave them attached only anteriorly at the rectal and genital openings, and posteriorly by the peritoneum and the vessels at the brim of the pelvis. Anteriorly, the attachments may now be divided with the knife at whatever point seems advisable, ordinarily close to the pubes just anterior to the prostate (or through the ure- thra and vagina in females) and through the lower end of the rectum. Posteriorly, cut through the tissues at the brim 48 PATHOLOGICAL TECHNIQUE. of the pelvis, taking care not to cut the ureters if the kid- neys are still attached to them. The rectum is to be opened with the enterotome along the posterior wall, and the inner surface thoroughly washed off so as to avoid soiling the other organs. To open the bladder in males, especially if the penis has been removed in continuity with it, incise with the scissors a transverse fold in the anterior wall of the fundus, and carry the incision through the urethra and along the dorsum of the penis. To accomplish the latter act perfectly the penis must be firmly stretched by having an assistant pull at the frenum while the bladder is held fixed by the operator. In females it is usual to enter the scissors into the bladder through the urethra and to cut through the middle of the anterior wall of the fundus. In males the rectum should be dissected from the bladder, so as to lay bare the vesiculse seminales and the prostate, which are examined by means of several transverse incisions. In females, if the bladder is normal, the vagina is incised in the anterior wall through the middle of the bladder. Or the vagina may be incised laterally until the cervix is reached, and then the cut be carried up to the median line. The uterus is incised in its anterior wall from the cervix to the fundus. From the upper part of this incision second- ary incisions are carried out on each side to the oriiices of the Fallopian tubes. The ovaries are incised in their greatest diameter, from the convex border to the hilus. Weight of ovaries, 7 grams. The testicles can readily be examined without external injury to the scrotum by cutting underneath the skin over the pubes down to the scrotum on either side of the penis, and shoving the testicles up through the incision. Cut care- fully through the overlying tissues until the cavity of the tunica vaginalis is opened. Remove the testicle by severing the cord. The incision to display a testicle should be in the long diameter, beginning on the side opposite the epididymis and extending through into it. Weight of testicles, 15-24.5 grams. In cases of tuberculosis of the testis and epididymis POST-MORTEM EXAMINATIONS. 49 it is advisable not to cut through the cord, but to remove the testicles and cords with the bladder, so that the whole genital tract may be examined in continuity and the asso- ciated lesions in the vesiculae seminales demonstrated, if present. The penis, or at least the larger portion of it, can be re- moved in connection with the bladder by continuing the pri- mary body-incision out to about the middle of the dorsum of the penis, which is then to be freed from the investing skin and divided just posterior to the corona. It is next dissected back to the pubic arch, and freed from it partly by cutting from without, partly from within, the pelvis, until the penis can be passed underneath the arch into the pelvis. Other methods are to cut through the symphysis, which can then readily be sprung apart by swinging one of the legs out into a horizontal plane, or even to saw out a small sec- tion of bone including the symphysis, so as to have more room for freeing the attachment of the penis and for re- moving it. The structures now remaining in the abdominal and thoracic cavities which require examination are the large blood-vessels, the thoracic duct, the celiac ganglion, and the retroperitoneal lymph-glands. The inferior vena cava and its branches are first examined (especially in all cases of pulmonary embolism) by slitting them with scissors along the anterior wall. If it is necessary to follow the iliac ves- sels into the thigh, it will be found easier in sewing up if the primary abdominal incision is continued off to the side in question, thus giving a single though curved incision. It is sometimes advisable to open up the inferior vena cava and its branches before removing the pelvic organs, so that thrombi extending into the pelvic vessels may be examined before they are disturbed. The semilunar ganglia lie on the aorta, around the celiac axis, above the pancreas. The thoracic duct lies behind and to the right of the aorta. In the thorax it is most easily found by dissecting on the right side between the aorta and the azygos vein. The re- 4 50 PATHOLOGICAL TECHNIQUE. ceptaculum chyli lies to the right and behind the aorta upon the second or third lumbar vertebra. Examination of the thoracic duct is of especial importance in cases of tubercu- losis of the intestine and mesenteric lymph-glands with secondary miliary tuberculosis. The aorta is to be opened in situ along the anterior wall throughout its whole extent, and the iliacs as far as the femoral ring. Besides the brain, the spinal cord, and the thoracic and abdominal organs, it is often necessary to examine or remove for study other portions of the body that are affected by dis- ease. A little ingenuity will enable one in appropriate cases to get at almost any part desired. A view of the marrow in a long bone is most easily ob- tained in the femur by extending the body-incision down over one of the thighs, dissecting the muscles way, and then chiselHng off a portion of the upper part of the shaft. In tuberculosis of the spine it is quite easy to remove any part, or even the whole, of the vertebral column, including the pelvis and portions of the femurs, without other incisions than the one from the neck to the pubes, with extension down the thighs in case parts of the femurs are to be taken out. Divide the ribs a few centimeters from the vertebral column on each side of the portion that is to be removed, cut through intervertebral disks both above and below it, and then carefully dissect it free, taking great care not to button-hole the skin. Removal of the Brain. — The incision into the scalp should begin from one to two centimeters behind the right ear, near its lower border, at the edge of the hair, and ex- tend over the vertex of the skull to a corresponding point behind the left ear. The cut is most easily made by thrust- ing a small narrow-bladed scalpel, with its back toward the calvaria and its point toward the vertex, through the skin behind the ear and shoving it along in the desired direction. By making the incision in this manner the hair is not cut, but simply parted. The anterior flap should be stripped from the calvaria and the temporal muscles by putting it on POST-MORTEM EXAMINATIONS. 5 I the stretch and dividing the loose connective tissue hold- ing it by sweeping strokes of the scalpel nearly as far for- ward as the orbits. After a part of the flap has been freed it is often possible to strip the rest without using the scalpel. For the posterior flap, which should be removed back as far as the occipital protuberance, the scalpel nearly always has to be used. If the hair is long, the anterior portion can be rolled into the anterior flap over the face and thus protected. The posterior portion is gathered at the nape of the neck, and then a towel is wrapped tightly around the head and neck, extending from the line where the flaps are reflected down to the shoulders, and is pinned over the lower part of the forehead. In this manner the hair is perfectly protected from being soiled and ample room is left for work. Of the two methods of opening the skull, the circular and the wedge-shaped, the former makes the better museum preparation, but the latter is in greater use in this country, and has the advantage of rendering the calvaria less likely to slip out of place after the head has been sewed up. The wedge-shaped incision consists of three cuts, which should be outlined on the periosteum of the skull with a scalpel. The first cut begins just above and behind the left ear, and is carried over the forehead just back of the edge of the hair or over the frontal eminences to a corresponding point above and behind the right ear. The two other cuts begin at each end of the first incision, forming there an obtuse angle, and are carried back to meet in the median line behind at an angle of about 160° a little in front of the occipital protuberance. The temporal muscle on each side is now to be scraped back from the line of incision out of the way of the saw, but is not to be cut off". The holder, if one is used, is attached with a foot in each obtuse angle in the temporal region. If a holder is not employed, the head is best steadied by hands on the calvaria and face. Use towels or cloth to prevent slipping. Start the incision with the saw over the forehead and extend it back along the line marked out. It is best not to 52 PATHOLOGICAL TECHNIQUE. carry the incision clear through the inner table of the bone, for two reasons : first, on account of the danger of injuring the brain-substance ; secondly, because if the inner table or a part of it is cracked through with a chisel and hammer, it can be done without injuring the underlying tissue, and the irregular overlapping fragments of bone thereby formed serve afterward for holding the calvaria firmly and steadily in place. After sawing along the lines marked out, insert a chisel in the frontal region, and with a quick, sharp blow crack through the rest of the inner table'. In like manner insert the chisel in the middle of the other incisions and free the calvaria posteriorly. To remove the calvaria insert the chisel end of the hammer in the incision in the frontal region, and press down with the left hand while swinging the handle around in a horizontal plane. By means of the powerful purchase obtained the calvaria is easily started. Then catch the hook of the hammer over the calvaria and strip it off. If the dura is adherent to the calvaria, it may be freed by using the point of the closed enterotome to pry it off. In young children, and sometimes in old people, it is necessary to remove the dura with the calvaria. To do this, cut through the dura with the point of a scalpel along the lines of incision in the skull ; then cut the falx cerebri in the median line, both anteriorly and posteriorly. An infant's skull is best opened by cutting with a pair of scissors through the dura along the sutures (in the longi- tudinal suture on each side of the falx) well down to the floor of the skull. This gives five bone-flaps which may be turned out like the petals of a flower, leaving the brain unin- jured. It is often necessary to cut half of the base of each flap in a horizontal line to aid its being turned out. The falx cerebri must of course be divided anteriorly and drawn back before the brain is removed. In sewing up, the bone- flaps are turned in over a bag of sand or sawdust filHng the cranial cavity, and are kept perfectly in place by the skin. In a case of fracture of the skull no cracking with hammer POST-MORTEM EXAMINATIONS. 53 and chisel is allowable ; the calvaria must be freed entirely by sawing. The calvaria should be examined at the time of removal. The next step is to inspect the dura. Under normal con- ditions it is not tense in the frontal region, but can be picked up with the forceps or fingers. If the dura is not thickened, the convolutions normally should be visible through it. The longitudinal sinus is opened with knife or scissors and its contents examined. Pacchionian granulations are not infre- quently found projecting into it. To remove the dura, cut through it with scissors or knife along the same lines in which the calvaria was sawn. Turn back each half of the dura and examine the surface of the convolutions and the inner surface of the dura. The con- volutions should be distinct and rounded, not flattened, with obliteration of the gyri, as occurs when there is internal pressure. The Pacchionian granulations are situated along the longi- tudinal fissure and may grow through the dura and form depressions in the calvaria. There may be apparent adhe- sions between the dura and pia due to veins passing from one to the other. The dura is still further freed by seizing the two halves anteriorly and lifting them up until the falx is tense at its insertion into the crista galli. Pass a knife in parallel to the falx, on the left side, with the edge forward, as far as the cribriform plate ; turn it to the right and cut until the falx yields. Withdraw the knife in the same manner in which it was inserted. Next draw the dura back. It is usu- ally more or less attached along the longitudinal fissure by Pacchionian granulations and by blood-vessels. These may be cut or torn through. Do not cut the dura posteriorly, but let it hang down. To remove the brain, insert the two fore fingers, or the first and second fingers of the left hand, anteriorly between the dura and the frontal lobes, one on each side of the falx cerebri, and draw the brain gently back until the optic nerves are visible. Ordinarily, the olfactory nerves come away from the cribriform plate without trouble, but sometimes have to 54 PATHOLOGICAL TECHNIQUE. be freed with the point of the knife. With a long, slender- bladed knife divide the optic nerves as far forward as possible while holding the brain back with the left hand. Continue to draw the brain carefully back and divide the cranial nerves N. opt. N. oculom.\^ /d N. trochl. N. abdvc. Hypoph. ^arotis ijit. N. trigem. N. facial. N. acust. N. glossophar..^ a N. vagus^ V N. recurr. Vi^^J^ N. hypogiy^ Tentoriuv S. cavernos. S. petr. inf. S.petr.sup, Cut edge of tento- rium. Fig. 8. — Base of skull (Nauwerck). and the carotids. Then draw forward first the left, then the right temporal lobe, and cut the tentorium close to its attach- ment to the petrous portion of the temporal bone with a sawing motion, using the tip of the knife. Insert the knife at the side close to the squamous bone, and cut from there POST-MORTEM EXAMINATIONS. 55 in toward the foramen magnum. Then cut the nerves given off from the medulla oblongata while supporting the con- vexity of the brain in the left hand. Lastly, carry the knife as far as possible into the spinal canal, and divide the cervical cord by an oblique incision from each side, severing the vertebral arteries with the same stroke. Better than a knife is the myelotome, because it gives a cross-section of the cord and allows more of it to be removed. The brain is now to be removed by passing the first and second fingers of the right hand in on either side of the cord, and everting the brain while still supporting it posteriorly with the left hand. Before proceeding to open the brain it is best to examine the base of the skull, particularly the dura, of which the sinuses should be incised, and the hypophysis cerebri. If there is a suspicion of a fracture at the base, strip off the dura, so as to give a better opportunity for examination of the bone. The brain should be weighed before it is dissected. The average weight in an adult male is 1358 grams; in an adult woman, 1235 grams. IKxtemal ISxamination of the Brain. — Place the brain with the base uppermost and with the cerebellum to- ward the operator. Examine first the pia and the cranial nerves, then the arteries, especially the middle cerebral and its branches on each side in the fissure of Sylvius, for it is here that emboli most frequently lodge. The pia bridging the fissure of Sylvius can sometimes be torn through, but usually has to be cut. It is important, particularly in cases of obscure cerebral symptoms, to feel gently with the finger-tips all over the sur- face of the brain for any areas of increased density, because patches of sclerosis may in that way be found which might otherwise be overlooked. By stripping off the pia — a procedure not often advisable — adhesions over pathological areas can sometimes be found pointing to the lesions beneath, but the pia should not be 56 PATHOLOGICAL TECHNIQUE. stripped from those portions which are to be examined mi- croscopically. To remove the pia an incision is made on the median surface of each hemisphere just above the corpus callosum from . one extremity to the other, and the pia stripped back first from the median and then from the con- vex surface. The stripping is done by means of the fingers, with occasional aid from the forceps. Section of the Brain. — There are several methods of cutting up the brain, no one of which is particularly suitable to all occasions. That method must be chosen which is most fitted to the individual case and to the use to which the tissue is to be put. The ideal method from a neuro-pathological standpoint would undoubtedly be to harden the brain entire, and then to make serial frontal sections thin enough for microscopical purposes through the whole organ. The nearest approach to this ideal method is to harden the brain entire in formal- dehyde, a process occupying ten days to two weeks (see page 303), to make thin serial sections, to mordant the sec- tions, divided if necessary into smaller pieces, in a chrome salt (preferably by Weigert's quick method), and then to carry through a number of series from the important parts for microscopical examination. By this means the relations of the various cerebral structures and of the pathological lesions can be perfectly preserved and studied. This method can be particularly recommended for tracing degenerations in the motor tract. If there is a noticeable focal lesion, such as a tumor or hemorrhage, it should be so incised, generally frontally or horizontally, as best to show its relations to the important cerebral tracts and ganglia. In these cases also the best re- sults are obtained by hardening the brain entire in formal- dehyde, and later making serial sections for macroscopic study or for carrying through for histological purposes. In many cases, however, it is necessary or advisable to examine the lesions in the fresh state. For instance, if it be desired to study the neuroglia-fibers, it is positively necessary to cut out thin slices of fresh tissue and to fix them immediately in POST-MORTEM EXAMINATIONS. 57 the proper solution. Often, too, the lesion cannot be or is not found except on fresh examination, or the clinician whose case it is desires to see at once the cause of certain symp- toms. Under such circumstances the more ideal method must be sacrificed, and as much made out of the case as is possible in the condition in which it is left after the exami- nation. For the routine examination of the brain, to demonstrate G.forn. G. front. I. G fi out II G front III G. centr, u. S Rol. G. centr. p. Fig. C. call. -First cut in the brain (Nauwerck). its topography and to bring to light suspected or unsus- pected lesions, probably no method is more generally used than Virchow's. The objection most often made against it is that the cerebral cortex is too much cut up. In case, however, it is desired to preserve the cortex or parts of it for microscopic purposes, the longitudinal incisions after the first may be omitted, and the cortical portion, after being sepa- rated from the stem, may be cut in any way that seems ad- visable. In like manner, the brain-stem or any other part 58 PATHOLOGICAL TECHNIQUE. may be left uncut, and hardened entire in formaldehyde for histological purposes. Virchow's Method. — The brain is to be placed on its base in the same position as one's own. Press the hemispheres apart a little so as to expose the corpus callosum. Hold the left half of the cerebrum in the left hand with the fingers on the lateral aspect and the thumb in the longitudinal fissure. Then make an almost vertical incision with a long, slender knife through the roof of the left ventricle in its middle third, 2 to 3 mm. from the median raphe of the corpus callosum. The roof of the ventricle is to be slightly raised vertically by the thumb, so that the incision, which must not be too deep, may not injure the basal ganglia. The incision is to be continued into the anterior and posterior cornua. Then make a long incision from one end of the above cut to the other, passing just outside of the basal ganglia at an angle of about 45°. Repeat the process on the right side, turning the brain half around. Next seize what remains of the corpus callosum and fornix in the middle, lift them, and cut through from be- low up, passing the knife through the foramen of Munroe. The parts are then turned back, exposing the velum inter- positum and the choroid plexuses. By drawing back the velum interpositum the third ventricle is uncovered. The corpora quadrigemina are exposed by cutting trans- versely the right posterior pillar of the fornix and adjoining brain-substance and carrying them over to the left. Each ventricle as it is opened is to be carefully inspected and any abnormal condition of its ependyma noted. The cortex is further divided on one side, and then on the other, by hold- ing it in the left hand and making vertical straight sections from the upper angle of the previous cut into the convex cortex, allowing the sections to fall apart, so as to avoid touching and soiling the surface with knife or fingers. Each portion thus cut represents a prism. The incisions should go well into the cortex, but not so far as to separate the different pieces. The basal ganglia are examined by means of a number of frontal sections. For this purpose the left hand is placed palm upward underneath the brain, so that as POST-MORTEM EXAMINATIONS. 59 each section is made over the tips of the fingers by one long stroke of the knife it falls forward, exposing a clean surface of which the two halves can be compared. An incision is next carried through the middle of the pineal gland, the corpora quadrigemina, and the vermiform process of the cerebellum, opening the aqueduct of Sylvius and the fourth ventricle. Each half of the cerebellum is divided by a median hori- Corp. call Cotp call O font dtSL Cr forn. desc. Fig. io. — Section of the brain (Nauwerck). zontal section into halves, and these portions are still further subdivided by a series of cuts radiating from the peduncles. In order to make sections of the pons and medulla the brain is folded together and turned over. Several cross- sections are then made with the left hand placed beneath as in sectioning the basal ganglia. Before making the sections it is well to remove the basilar and vertebral arteries, especially if they are calcified. In Pitre's method of dissecting the brain the lateral ven- tricles are opened as in Virchow's method. Then the pedun- culi cerebri are cut squarely across, so as to remove the pons 6o PATHOLOGICAL TECHNIQUE. and cerebellum, and a longitudinal incision is carried down through the third ventricle, halving the cerebrum. Through each half of the cerebrum a series of six sections is then made parallel to the fissure of Rolando. The names of the sections and the important parts which they show are as follows : 1. The pre-frontal section through the frontal lobe, 5 cm. anterior to the fissure of Rolando, shows the gray and white substance of the frontal convolutions. 2. The pediculo-frontal section through the posterior por- tions of the three frontal convolutions shows the anterior extremity of the island of Reil, the lenticular and caudate nuclei, and the internal capsule. 3. The frontal section through the ascending frontal con- volution, parallel to the fissure of Rolando, shows the optic thalamus, the lenticular and caudate nuclei, the claustrum, the external and internal capsules, the anterior portion of the descending horn of the lateral ventricle, and the island of Reil. 4. The parietal section through the ascending parietal con- volution shows portions of the same structures as the pre- ceding, and a transverse view of the hippocampus. 5. The pediculo-parietal section through the parietal lobe, 3 cm. posterior to the fissure of Rolando, shows the tail of the caudate nucleus in two places and the posterior portion of the optic thalamus. 6. The occipital section through the occipital lobe, i cm. in front of the parieto-occipital sulcus, shows simply the white and gray matter of the occipital lobe. The cere- bellum, pons, and medulla are incised in the manner already described. Removal of the Spinal Cord. — The body is to be placed face downward, with the head over the end of the table and a block under the chest. The incision is made over the spinous processes from the occiput to the sacrum. Dissect the skin and muscles back on each side, so as to leave the vertebral laminae as bare as possible. The laminae may be cut through by means of several instruments, of POST-MORTEM EXAMINATIONS. 6 1 which the double-bladed saw (Luer's rhachiotome) is perhaps the safest, at least for beginners. The single-bladed saw with rounded end is also very useful and can be thoroughly- recommended. The operation can be done most quickly by biting off the spinous processes with the heavy bone-forceps and cutting through the laminae with chisel and hammer, but there is greater danger of injuring the cord. The numerous artifacts in the cord, reported as neuromata and heteroplasia even within very recent times by competent pathologists, would seem to indicate that the need of careful and dehcate technique in the removal of the spinal cord is not yet fully appreciated. The laminae should be sawn nearly or entirely through in a line with the roots of the transverse processes from the third or fourth lumbar vertebra to the cervical region. The arches of the cervical vertebrae are best divided with a heavy bone-cutter, because they cannot be easily sawn, and there is sufficient room here for the points of the bone-cutter with- out danger of their pressing on the cord. It is important to strike the outside limits of the spinal canal, so as to get as much room as possible for the removal of the cord. Test if the sawing be deep enough by the mobility of the spinous processes. If necessary, they can be freed by means of the hatchet-chisel and a hammer in the same way that the calvaria is loosened. As the cord reaches only to the second lumbar vertebra, cut through between the third and fourth, free with the heavy bone-cutter the lower end of the row of the spinous processes, which are held together by their ligaments, and strip them up to the neck; then cut through the cervical arches with the bone-cutter, taking care that the point within the canal does not come in contact with the cord. The nerve-roots are to be divided with a sharp scalpel by means of a long cut on each side of the cord. Then cut across the dura and the nerve-roots at the lower end of the exposed canal, and, while holding the dura with forceps, carefully free the cord from below up with scissors or scalpel, taking care all the time not to pull or bend the cord, be- 62 PATHOLOGICAL TECHNIQUE. cause in either way artifacts may be produced. Cut the cord squarely across as high in the cervical canal as possible, so that the remaining portion may be easily removed with the brain. Lay the the cord after removal on a flat surface and incise the dura longitudinally, first posteriorly and then in front. A series of cross-sections, usually i to 2 cm. apart, is made through the cord while supported on the fingers during the Fig. II.- -Base of skull, showing lines of incision for removing internal eye, etc. (Nauwerck). cutting, so that the cut surfaces shall fall apart. The dif- ferent segments should ordinarily be left attached to the dura, so that their position in the cord can easily be deter- mined. A diagnosis from the fresh, macroscopic appearances of the cord is often very difficult to make, according to the best authorities. The :Eye. — The contents of the orbit, including the poste- rior part of the eye, can be readily examined by chiselling POST-MORTEM EXAMINATIONS. 63 off the roof of the orbit. The posterior half of the eye can be removed by cutting around the eyeball with sharp scissors without changing the hold of the forceps on the sclera. If done quickly, the retina remains quite well spread out. The anterior half of the eyeball is to be propped in place by a plug of cotton dipped in ink or in a solution of perman- ganate of potassium. The ^ar, — The middle ear can be exposed by chipping off with a chisel its roof, which lies in the middle of the petrous portion of the temporal bone. The roof can also be very easily bitten off with the heavy bone-cutters. If, how- ever, it be desired to examine the ear more carefully by means of a section through the external meatus and the middle ear, it will be necessary to remove the whole of the petrous bone. For this purpose the incision behind the ear must be carried back along the anterior edge of the trapezius muscle halfway down the neck. Then the skin-flaps, in- cluding the external ear and the underlying tissues, must be dissected back for some distance on each side of the incis- ion. Two converging incisions are then to be sawn, the anterior passing through the root of the zygomatic arch, the posterior just back of the sigmoid sinus, so as to come together at the apex of the pyramid of the petrous bone, or, better still, to meet in the foramen magnum. An ordinary chisel and a hammer or mallet will be found very convenient for freeing the petrous bone after the incisions have been sawn. In the examination of the petrous bone after it has been removed the first step is to chisel off the tegmen tympani so as to get a view of the middle ear. Next remove the lower wall of the external meatus, so as to expose the outer sur- face of the membrana tympani. Finally divide the petrous bone with a fine hair-saw by an incision starting in at the styloid process and coming out at the carotid canal, parallel to the crest of the pyramid of the petrous bone. This incision divides the cavum tympani into halves. In the lateral half can be seen the membrana tympani with the hammer and the anterior half of the mastoid cells. In the 64 PATHOLOGICAL TECHNIQUE. median half are the labyrinthine wall of the cavum tympani with the stapes and the posterior half of the mastoid cells. It is best to remove the anvil before sawing through the bone. The Eustachian tube can be easily exposed by start- ing from its termination in the middle ear. The Naso-pharynx. — Although a fair view of the nares and pharynx can be obtained by chiselling off the portion of the base of the skull lying over them, the method does not begin to offer the satisfactory view that can be obtained by the method of Harke/ a method which is not so difficult as might at first sight seem, and which consists in halving the base of the skull by a longitudinal incision. To do this the original incision in the scalp must be extended on each side over the mastoid processes and along the anterior edge of the trapezius muscle to a point below the middle of the neck. Then the posterior flap and the underlying muscles must be freed from the occipital bone and the upper portion of the occipital vertebrae. In like manner, the anterior flap must be dissected from over the root of the nose and the upper edge of the orbits, and be drawn down over the face. Then flex the head strongly forward and saw through the occipital bone and the base of the skull, dividing the occipital and frontal bones, the sella turcica, the cribriform plate, and the basilar process into equal halves. Anteriorly, it is well to go a little to the left or right, so as not to injure the nasal septum. The next step is to cut the pachymeninx and the appara- tus ligamentosis between the anterior edge of the foramen occipitale magnum and the processus odontoideus, as well as the inner side of the atlanto-occipital joint from within. Then the two halves of the skull are to be drawn forcibly apart. The nasal bones, the hard palate, and the alveolar process of the upper jaw break, and the two halves of the base of the skull open like a book, revolving around an axis which passes through the joint of the lower jaw and the atlanto-occipital joint. If the foramen occipitale magnum offer too much resist- 1 Berliner klin. Wochenschrift, 1892, No. 30. POST-MORTEM EXAMINATIONS. 65 ance, break through it with a chisel, and also if necessaiy through the anterior and posterior arches of the atlas. It is now easily possible to inspect the sinus sphenoidales, the nasal septum, the frontal sinuses, and the nasal passages. The antrum of Highmore is easily opened with forceps and a pair of bone-shears. After the operation the two halves of the base of the skull are brought together, and wired if necessary. When the skin-flaps have been replaced all evidence of the operation is covered up. Bxamination of New-bom and Very Young Chil- dren. — I. The head is preferably opened by the method given on page 52. 2. According to Nauwerck, the spinal canal can be opened by dividing the vertebral arches with strong scissors. 3. The umbilical cord, if present, and the umbilical arte- ries demand close attention in children who have lived a few days or weeks, for the purpose of determining if infection has taken place at that point. Nauwerck advises a modification of the primary long incision. A little above the umbilicus it should divide into two diverging incisions running to the pubes. In this way a triangular flap is left containing the umbilical arteries, while from the upper end is given off the umbilical vein. The vessels may be ligated or opened at any point that seems advisable. 4. Anomalies of circulation should be looked for in all " blue babies." The closure or non-closure of the ductus Botalli (arteriosus) is best determined in situ by dissecting off the thymus and opening up the pulmonary vein in the middle of its anterior surface. The cut may be extended downward, if desired, through the pulmonary valve and the wall of the right ventricle. The duct lies in the median line of the pulmonary artery, a little above its division into its two main branches. A small probe can be passed through it into the aorta. The condition of the foramen ovale be- tween the auricles is easily examined. For other anomalies of the circulation it will usually be found most satisfactory to remove the thoracic organs in 5 66 PATHOLOGICAL TECHNIQUE. mass, so as to be able to open up the heart and the vessels given off from it before any of the vessels have been severed from their connections. 5. In medico-legal cases especially it is important to de- termine whether or not a child has breathed. The main steps of the process are as follows : {a) Position of the diaphragm before the chest is opened. When the lungs are fully distended it is at the fifth or sixth rib on the right and at the sixth rib on the left. When the lungs contain no air or are but partially distended the diaphragm reaches to the fourth rib. (^) Ligate the trachea above the sternum before opening the thorax. ( I %, Fig. 38. — Di]>lococcus intracellularis meningitidis; from a cnlture (Jaeger). siderable variation in size in cultures (Fig. 38). The larger forms are regarded as involution or degenerate forms. In cover-glass preparations from the meningeal exudate the diplococcus frequently is situated inside leucocytes, and sometimes within the nucleus (Fig. 39). The appearances are veiy much like those of gonorrheal pus. It is decolor- ized by Gram's method. 1 Weichselbaum : Fovtschriltt dey Medidne, Bd. 5, 1S87; Jaeger: Zeit- schrift fiir Hygiene und Infeetionskrmikheiten, Bd. 19, 1S95 ; Councilman: Transaetions of the Assoeiation of American Physiciatts, 1897. 136 FA TIIOL GICA L TE CHNIQ UE. Blood-scriini. — The colonies appear after about twenty-four hours, and after forty-eight hours may attain a diameter of 2 or 3 mm. They are round, colorless, shining, slightly convex or flat, moist, and viscid-looking. They may be- come confluent. Agar-agar. — Round, flat, grayish, translucent, moist, shin- ing colonies, attaining a diameter of 2 or 3 mm. after tvventy- FlG. 39. — Diplococcus mtracellularis meningitidis in polynuclear leucocytes of meningeal exudate (Jaeger). four hours in the incubator. They may become confluent, and in a "slant" culture the growth appears as a grayish, translucent, moist, shining streak about 3 mm. in width, with smooth margins. Under a low magnifying power the colo- nies are homogeneous, semi-translucent, and not granular. Siigar-agar Stab-cidtnrc. — Feeble growth not extending all along the line of inoculation. Bouillon. — Medium slightly to moderately clouded. At the bottom of the tube a whitish sediment, which may rise as a viscid string" when the tube is shaken. BACTERIOLOGICAL EXAMINATIONS. I 37 Potato. — Very feeble or doubtful growth, giving the surface of the potato a moist appearance at the most. Litmus-milk. — Growth without visible change in the medium. Gelatin. — Feeble growth. No liquefaction. Vitality. — The organism quickly dies out under cultivation. It seems to survive somewhat better on blood-serum than on agar-agar, but cultures on the former only seventy-two hours old may be found no longer capable of growth after transplantation. Pathogenesis. — Intraperitoneal inoculation of guinea-pigs and rabbits gives very uncertain results. Mice are said to succumb to subcutaneous inoculation, with some invasion of the blood by the organism. Exceptionally, we have found that the intraperitoneal in- jection of a bouillon suspension of a twenty-four-hour blood-serum culture in the quantity of about i c.c. would kill guinea-pigs within forty-eight hours. At the autopsy there is an accumulation of a cloudy or blood-stained fluid in the peritoneal cavity, some little en- largement of the spleen, and some injection of the perito- neum. Microscopical examination of the exudate shows numerous leucocytes crowded with the diplococci. The culture-test gives no evidence of general infection of the blood. Occurrence. — Found in the meningeal exudate of certain cases of epidemic cerebro-spinal meningitis. It is situated mainly inside the pus-cells, some of which may contain many diplococci. In some cases the presence of the organ- ism in the exudate may be difficult or impossible to demon- strate, and it is probable that it rapidly dies out. It has been observed in the lung in the broncho-pneumonia of one case, and in the nasal secretions of a number of cases. A general invasion of the circulation by the organism does not occur. Bacillus Diphtheriae.' — Blood-serum. — Round, elevated, smooth colonies of the color of the medium. They may 'Loffler: Mitth. a. d. Kais. Gesundheitsamte, Bd. 2, 1884; Roux and Yer- sin: Annales de V Institut Pasteur, T. 2-4, 1888-90. 138 PATHOLOGICAL TECHNIQUE. attain a diameter of 2 mm. after forty-eight hours in the incubator. Morpliology. — Bacilli varying markedly in size and shape, of irregular outline, and showing great variability in the staining of different parts of their protoplasm (Fig. 40). The presence in a palely tinted rod of deeply stained granules and points, frequently situated at the extremities, and the occurrence of irregular forms, often club-like in shape with a constriction in the middle, are appearances which are very characteristic of the bacillus when grown upon blood- Fig. 40. — Bacillus diphtherice, from a culture upon blood-serum ; x looo (Frankel and Pfeiffer). serum and stained with Loffler's methylene-blue solution. Its morphology and staining peculiarities are so characteristic when cultivated upon blood-serum that the microscopical examination is in most cases sufficient for its identification. When grown upon other culture-media than blood-serum, however, its morphology and staining peculiarities are not so characteristic, and they may vary markedly in different media. Stained by Gram's method. Not motile. Bouillon. — Grows usually in the form of fine grains at the bottom of the tube and adherent to the sides, the bouillon BA CTERIOL OGICA L EXAMIiVA TIONS. 139 remaining clear or becoming sliglitly clouded. The reac- tion of the media rapidly becomes acid, but changes to alka- line after a variable length of time. Potato. — Growth not visible to the naked eye. The bacil- lus grows, however, to a certain extent, and usually assumes veiy atypical and irregular forms (involution forms). Agar-agar and Gelatin. — The growth on these media is slower and more feeble than upon blood-serum (Fig. 41). It presents nothing remarkable. Patliogencsis. — Subcutaneous inocu- lations of guinea-pigs are fatal in thirty- six to seventy-two hours in the case of virulent cultures. The lesions pro- duced consist usually of edema, hem- orrhage, and fibrino-purulent exuda- tion about the point of inoculation in the subcutaneous tissue, hemorrhagic enlargement of the lymphatic glands, congestion and edema of the lungs, hemorrhages into the suprarenal cap- sules, and less frequently necrosis of the liver and pleural effusions. His- tological examination of the lymph- glands shows marked "fragmentation " of the nuclei of the cells, giving rise to numerous deeply staining globules of chromatin scattered throughout them. The bacilli are ordinarily found only about the point of inocu- lation, but cultures from the various organs will sometimes show the presence of the bacilli in some of them. Toxin-production. — The effects produced by infection with the bacillus diphtheria; are due to the action of a so-called toxalbumin or " toxin " which the organism manufactures in its growth. The poisonous substance is produced in cultures. Its presence may be demonstrated by inoculating an animal with a small quantity of the filtrate, obtained by passing a bouillon culture some weeks old Fig. 41. — Bacillus di]:)h- theria^ ; agar-agar culture (photograph by Dr. Henry Koplik). I40 PATHOLOGICAL TECHNIQUE. through an unglazed porcelain filtering apparatus, by which all the bacteria are removed from the fluid. The "toxin" is contained in solution in the filtrate. If this be fairly rich in "toxin," the injection of -^ c.c. subcutane- ously into a guinea-pig should lead to the death of the animal in three or four days with the various lesions above described. The local reaction, however, is not so marked as in the case of inocu- * lation with the bacilli. With the ordinary bouillon the produc- tion of a great amount of "toxin " by the growth of the diph- theria bacilli in it is very uncertain. Theobald Smith has recently shown that this uncertainty is due to the presence of variable amounts of muscle-sugar from the meat used in the preparation of the bouillon, and that this substance prevents the accumulation of toxin. He has found that that bouillon yields the most toxin which has the least muscle-sugar in it. According to Smith, the best means of obtaining a fluid rich in toxin is as follows : The meat destined for use in the preparation of the bouillon is to be allowed to decompose during three or four days, in which pro- cess most of the muscle-sugar disappears from it. With this partially decomposed meat a bouillon is made as described else- where, but of such a reaction that lo c.c. of a normal solu- tion of sodium hydrate ^ per liter of bouillon is necessary to give a faint pink coloration with phenophthalein. This bouillon is placed in two 500 c.c. Erlenmeyer flasks, 250 c.c. in each flask. In these cultures are made and kept for at least eight days in the incubator. After this time a fair amount of toxin may be assumed to have developed, and the contents of the flask are then filtered through a porcelain cylinder. A filtrate is to be regarded as containing a reasonable amount of toxin if yij- c.c, injected subcutaneously, kills a medium-sized guinea-pig in three days. The filtrate containing the "toxin" can be preserved by the addition of 0.5 per cent, pure carbolic acid. Occurrence. — The bacillus diphtheriae occurs in the local lesions in all cases of true diphtheria, in rhinitis fibrinosa, and in many cases of the milder forms of acute inflammation of the air-passages. It may persist in the mucous mem- brane of the throat and nose long after convalescence has been established. In fatal cases of diphtheria the organism is nearly always present in the lungs, and it may be often found by culture- methods more or less generally distributed in compara- tively small numbers throughout the internal organs. In the majority of diphtheria autopsies an invasibn of the blood- 1 40 grams of sodium hydrate c. p. (by alcohol) dissolved in 1000 c.c. of distilled water. BACTE RIO LOGICAL EXAMINATIONS. 141 stream by the streptococcus pyogenes, and sometimes b)' other bacteria, may be demonstrated by cuhures. The ba- cillus may also be found in company with other bacteria in ulcerated or excoriated surfaces on the skin, as well as in other suppurative processes, in individuals affected with diphtheria, and on the soiled linen of the patient. The in- fection of wounds with the bacillus diphtherias has also been observed without coincident diphtheria. Bacillus of Typhoid Fever.' — Synonyms: Bacillus typhi abdominalis ; Bacillus typhosus ; Typhoid bacillus (see also Clinical Bacteriology). Blood-scrum. — Round, grayish, \-iscid-looking colonies. Fig, 42. — Bacillus of typhoid fever, from a twenty-four-hour agar-agar culture ; X 650 (Heim). which may attain a diameter of 2 mm. after fortj'-eight hours in the incubator. Fig. 43. — Diagrammatic representation of retraction of protoplasm, with produc- tion of pale points, in the bacillus of typhoid fever (Abbott). morphology. — Medium-sized bacilli with rounded ends, ' Eberth: r?nLiow's Arch. f. Path. Anal., Bd. Si, lS8o; Bd. 83, 18S1 ; Gaffky : Alitlh. a. d. Kais. GesnnJheilsatnti, Bd. 2, 18S4. 142 PATHOLOGICAL TECHNIQUE. generally short (Fig. 42), but sometimes long or thread-like, and frequently showing faintly-stained, sharply-defined areas in their protoplasm (Fig. 43). Gelatin Slant. — Broad translucent streak with wavy, irregular margins. The gelatin is not liquefied. Growth is slower than that of the bacillus coli communis in the same medium. An isolated colony, slightly magnified, on gelatin, is shown in Fig. 44. Glucose-gelatin Stab. — Growth all along the line of inocu- lation in the form of confluent spherical grayish colonies, and Fig. 44. — Bacillus of typhoid fever; superficial colony two days old, as seen upon the surface of a gelatin plate ; X 20 (Helm). on the surface about the point of entrance of the platinum wire in the form of a circular translucent grayish layer. No production of gas-bubbles. No liquefaction. Glucose Agar-agar. — Growth similar to that in the pre- ceding. No gas-formation. Lituius-niilk. — No visible change. Potato. — Growth occurs, but it is usually invisible. Dunham's Pepton Solution. — No indol-production — i. e. no red color appearing in the twenty-four- to forty-eight- hour cultures after the addition of 5 drops of concentrated sulphuric acid, c, p., and i cubic centimeter of a solution of sodium nitrite, i : 10,000. Motility. — Very marked. BACTERIOL O GICAL EX A MINA TIONS. 143 Flagella (F"ig. 45) may be demonstrated by the special methods of staining described elsewhere. Decolorized by Gram's method. Does not form spores. Bouillon. — Clouded, with the formation of some sediment. The clouding of the medium is not so marked as in the case of the bacillus coli communis. In general, the growth of the typhoid organism is not so vigorous on culture-media as is the growth of the bacillus coli communis. When to a bouillon culture a small quantity of the blood- FlG. 45. — Bacillus of typhoid fever, from an agar-agar culture six hours old, showing the flagella stained by Loffler's method ; X looo (Friinkel and Pfeiffer). serum of a typhoid-fever patient is added, the bacilli lose their motility and aggregate in clumps ("serum reaction"). Pathogenesis. — The inoculation of animals is usually with- out results if moderate quantities of the organism are used. Sometimes, however, death occurs apparently from the effects of the toxic material injected. Occurrence. — Found in the spleen in large numbers at autopsies in typhoid fever. Its presence may also be demon- strated in the intestinal lesions, mesenteric lymph-glands, liver, bile, kidneys, and blood of the heart. As a rule, the number of bacilli found in the liver, kidneys, and blood of 144 PATHOLOGICAL TECHNIQUE. the heart is small. In the bile they may be numerous and may persist in it for a long period of time after the disease has subsided. The typhoid bacillus may also occur in the suppurative sequelae of typhoid fever, especially those involving bones. In these conditions, however, it may be accompanied by the pyogenic cocci. Occurs in contaminated water. Diflferential Diagnosis between the Bacillus of Typhoid Fever and the Bacillus Coli Coniinunis. — The most im- portant points of difference between these two organisms are as follows, and to distinguish with certainty between them it is necessary that attention be paid to all of them : Motility. — The typhoid bacillus is actively motile, the bacillus coli communis not motile or exceptionally motile. Potato Cultures. — The typhoid bacillus usually grows in- visibly, the bacillus coli communis as a dirty, slimy layer. Gas-production in Media containing Glucose. — The bacillus coli communis produces gas, the typhoid bacillus does not. Litmus-milk Cultures. — The bacillus coli communis changes the blue color of the medium to a pink color and usually coagulates the milk. The typhoid bacillus produces no visible change. Indol-pr eduction. — The bacillus coli communis produces indol, the typhoid bacillus does not. Serum or Clump Reaction. — The typhoid bacillus shows the clump reaction, while the bacillus coli communis does not. As it is not always possible to have a typhoid serum at hand by which to determine whether this reaction is present, a stock of dried blood from a typhoid case, con- tained in filter-paper, may be kept ready for use. That this is quite practicable has been recently shown by Dr. Mark W. Richardson. The blood may be obtained from the heart at the autopsy of a typhoid-fever case by soaking a piece of filter-paper with it. This is allowed to dry, and then is cut into pieces about i cm. square. When it is desired to make the test, one of these pieces is extracted with ten or fifteen drops of water, and a drop of this extract is mixed with a drop of an eighteen- to twenty-four-hour bouillon culture BACTERIOLOGICAL EXAMINATIONS. 1 45 on a slide, covered with a cover-glass, and examined with the high-power dry lens. Dr. Richardson has found that the blood under these conditions retains for months its " clumping " power with reference to the typhoid bacillus. Other differences are — the production of a red color in litmus-lactose agar-agar by the bacillus coli communis, and no change in color of this medium by the typhoid bacillus, and the slower and less vigorous growth of the typhoid bacillus in culture-media. Bacillus Coli Communis.' — Synonyms : Bacterium coli commune ; Colon bacillus. Blood-scnini. — Rounded, grayish-white, slightly elevated, .J* Fig. 46. — Bacillus coli communis, from an agar-agar culture; X 1000 ( Itzerott and Niemann). viscid-looking colonies, which may attain a diameter of 3 mm. after twenty-four hours in the incubator. Morphology. — A medium-sized bacillus with rounded ends, often short or even coccus-like, but may grow in long forms (Fig. 46). Faintly staining, sharply defined areas are present in the protoplasm. Gelatin Slant. — Grayish translucent strip with wavy mar- gins. The gelatin is not liquefied. Growth is more rapid than in the case of the typhoid bacillus. ' Escherich: Fortschr. d. Mediciti, 1SS5, Nos. 16, 17. 10 146 PATHOLOGICAL TECHNIQUE. A single colony on a gelatin plate is shown in Fig. 47. Glucose-gelatin Stab. — Growth along the line of stab in the form of confluent spherical colonies, and on the surface about the point of entrance of the needle as a thin gray circular layer. Gas-bubbles are produced in the gelatin from fermentation of the glucose. The gelatin is not liquefied. Glucosc-agar-agar Stab. — Growth essentially the same as in the preceding, except that the gas-formation is more marked. Litiims-viilk. — Turned pink and usually coagulated. Fig. 47. — Bacillus coli communis : suj^tcrficial colony two days old upon a gela- tin plate ; X 21 (Heim). Potato. — Dirty grayish or brownish, viscid-looking layer. Dunham's Pepton Solution. — Marked indol-production. This is shown by the appearance of a red color in the cul- ture after the addition of 5 drops of concentrated sulphuric acid, c. p., and i c.c. of a i : 10,000 solution of sodium nitrite. The culture in the pepton solution should have been at least twenty-four hours — or, better, forty-eight hours — in the incubator before the test is made. Jllotilitj'. — Usually not motile, but some varieties show sluggish independent movement. Flagclla. — May be demonstrated by the special methods of staining. They are less numerous than in the case of the typhoid bacillus. BACTERIOLOGICAL EXAMINATIONS. \a;j Decolorized by Gram's method. Bouillon. — Markedly clouded with formation of a sedi- ment. The clouding is more marked than in the case of the typhoid bacillus. Lactose-litmus-agar-agar Slant. — Growth has a pink color, and the blue color of the medium is changed to red. Pathogenesis. — " Its virulence as tested upon animals is variable, but is generally manifest only after inoculation of large doses, which kill by intoxication rather than infection " (Welch). The lesions produced are not sufficiently characteristic to be detailed here. Occurrence. — Occurs constantly in the intestinal canal, and is widely distributed in the external world. " The colon bacillus is a frequent invader of the internal organs in all sorts of diseases, especially when there are in- testinal lesions. It manifests no evident pathogenic action in most of these cases, and is then without clinical signif- icance. It occurs frequently associated with other bacteria in infected wounds and other inflammations of exposed sur- faces. Here also it does not usually appear to cause serious disturbance. The fact that the colon bacillus is so common and widely distributed, and found so often as a harmless in- vader, should lead to much caution in interpreting the sig- nificance of its presence when it occurs in definite lesions. There is no doubt, however, that it may be pathogenic for man. It plays an important role in inflammations of the urinary tract and biliary passages ; also, but usually with less independence, in peritonitis and appendicitis. " The list of diseases in which it may be found is a very long one and includes inflammations in all organs and parts of the body. In general its pathogenic properties are of a mild character. One of its leading roles is to invade terri- tory already occupied by other bacteria or previously damaged. It may be concerned in the production of gall- stones, in the interior of which it has been found by the writer with great frequency " (Welch, Dennis's System of Surgery, vol. i.). 148 PATHOLOGICAL TECHNIQUE. The bacillus above described is to be regarded as a type of a group of bacilli constituting the so-called "colon group" of bacilli. These present certain quantitative differences among themselves which are not quite sufficient to characterize them as distinct species. Welch regards as belonging to this group the Bacillus pyogenes fmtidus (Fig. 48), distinguished by the stinking odor of its cul- tures, and the Bacillus lactis aerogenes, which is characterized »• •* * *» -I i.» " *, » «• • -/ . c "" FlG. 48. — Bacillus pyogenes foetidus, from agar-agar; X looo (Itzerott and Niemann). chiefly by its plumper form, its more energetic gas-production, its rapid coagulation of milk, and its denser growth in cultures. Bacillus Tuberculosis.' — Synonyms : Tubercle bacillus ; Bacillus of Koch. (See also Clinical Bacteriology.) Blood-serum. — After three or four weeks in the incubator the colonies appear as dry, cream-colored, granular, slightly elevated patches with irregular margins, i to 2 mm. in diam- eter. They may become confluent, to form a dense dry, granular mass with irregular surface and of a creamy-white color. The growth is very friable, but coherent, and may be picked up in clumps on the platinum wire. The first generation from tissues is very slow in developing, but suc- ceeding generations grow more rapidly, and may form a ' Koch : Berlin, klin. Wochensckr., 1882, No. 15 ; Mitth. a. d. Kais. Ge- sundheitsamte, Bd. 2, 1884. BACTEKWI.OGICAL EXAMINA TIONS. 149 wrinkled, dry, cream-colored membranous layer on the surface of the medium. Morphology. — Slender rods, usually shorter than when observed in sputum, and in fresh cultures staining homo- geneously ; in older cultures presenting a segmented or irregularly stained appearance. They frequently occur in pairs of short rods and in closely adhering clumps and strands (Fig. 49). When once stained with fuchsin or gen- tian-violet they are not decolorized by treatment with Gab- FlG. 49. — Bacillus tuberculosis : cover-glass preparation from a fourteen-day-old blood-serum culture ; X loo (Frankel and Pfeiffer). bet's solution or with a 20 per cent, solution of any of the mineral acids, followed by alcohol. Stained by Gram's method. Not motile. Does not form spores. Glycerin Agar-agar Slant. — Growth siinilar to that on blood-serum, but not so vigorous. By continued inoculation of this medium through a number of generations, however, the organism may eventually grow luxuriantly upon it. Glycerin Bouillon. — Growth on the surface in the form of floating patches or as a membrane similar in appearance to the growth on blood-serum. The growth sinks to the bottom from time to time. The glycerin-bouillon culture is I50 PATHOLOGICAL TECHNIQUE. best contained in Ehrlenmeyer flasks, filled to such a depth as to give a wide surface to the fluid and thus permit the access of plenty of oxygen to the growth. Potato. — The growth is not remarkable. Agar-agar or bouillon not containing glycerin is not suit- able for the cultivation of this bacillus. Pathogenesis. — The inoculation of guinea-pigs or rabbits by any method is followed by the development of general mili- ary tuberculosis. Guinea-pigs are most susceptible. These animals usually survive about two or three months, with marked emaciation. The lesions in the spleen and liver in the guinea-pig are characterized by extensive areas of ne- crosis not confined to the tubercular tissue, large parts of these organs being transformed into a firm yellow, opaque, friable material. Isolation of the Bacillus Tuberculosis from Tubercular Le- sions. — The best method of procedure is to inoculate a guinea- pig subcutaneously in the abdominal wall with tubercular material, and after four to six weeks, when the inguinal lym- phatic glands have become enlarged, to kill the animal. The object of killing the animal, rather than allowing it to die spontaneously, is to secure fresh tissue and to avoid the chance of an invasion of the lesions by other bacteria. Cultures on blood-serum are then to be made from the tuberculous lymphatic glands of the inguinal and retro- peritoneal regions of the animal. A number of tubes are to be inoculated, say three or four, from each of the two or three glands, a large quantity of material being spread upon the surface of each tube. Great care is to be exer- cised to avoid contamination with other bacteria in preparing these cultures. The culture-tubes used should contain freshly prepared moist medium, and immediately after inoculation should be sealed air-tight to prevent evaporation. This may conveniently be done by first cutting off the projecting por- tion of the cotton stopper and inserting a cork into the mouth of the tube in such a way as to push the cotton stopper before it. In order to prevent the invasion of fungi from the cotton. BACTERIOLOGICAL EXAMINATIONS. 151 the neck of the tube should be heated in the Bunsen flame until the cotton begins to brown before inserting the cork, which should also be charred in the Bunsen flame before in- sertion. The tubes may also be sealed with wax or paraffin or covered with small rubber caps. Cultures may also be made from other organs containing tubercular lesions, but the lymphatic glands seem to offer the best examples of large tubercular foci, and therefore are preferred for the purpose in view. The tubercular lesions in human tissues are not ordinarily favorable for the isolation of the bacillus, on account of the frequent presence of other bacteria in them and because of the small number of tubercle bacilli usually present in tissue otherwise suitable. We have found the firmly coagulated, opaque blood-serum medium above mentioned to be quite adequate for the cul- tivation of the bacillus from its lesions, both in man and guinea-pigs. A special culture-medium for the cultivation of the bacillus tu- berculosis is that recommended by Lubinski. Its composition and mode of preparation are as follows — to make one liter of medium : Take i kilo of potato in small pieces, and, after thoroughly washing, cook in the steam sterilizer for three or four hours with 1500 c.c. of water. Of the mass thus formed take 1000 c.c, and add to it 500 grams of finely chopped beef, and allow it to stand in the cold for twenty-four hours, then boil the mixture for a few minutes and filter. Add to the filtrate i to 1.5 per cent, agar-agar, and boil until the agar-agar is thoroughly dissolved. This will require say half an hour's boiling. Next add i per cent, pepton and 0.5 per cent, sodium chlorid. When these are thor- oughly in solution, and after about five minutes' continued boiling, the mixture is to be neutralized with sodium hydrate solution, boiling meanwhile. When brought to the proper reaction it is to be filtered, and to the filtrate add 4 per cent, glycerin. The medium is then to be run into test-tubes and sterilized as in the case of agar-agar. The loss by evaporation in boiling is to be made up with water. A bouillon may be also made by leaving out the agar-agar. Another special culture-medium is that recommended by Ca-. paldi. This consists in mixing with fluid agar-agar in tubes, at a temp'erature of 45° C, three or four large loopsful of the yelk of a fresh egg to each tube. When the portion of the yelk taken is thoroughly mixed with the fluid agar, the tube is placed on its 152 PATHOLOGICAL TECHNIQUE. side, and its contents allowed to become solid by cooling so as to form a " slant. ' ' In securing the yelk aseptic precautions must be used. The egg must be quite fresh. By suitable manipulations the yelk is to be freed from the "white " and placed in a small, clean dish. Then the yelk-membrane is to be seared with a hot knife and an opening made in it. Through this opening the yelk is obtained with the loop. Before using the culture-tubes so pre- pared, they should be tested as to their sterility by placing in the incubator for twenty-four hours. Occurrence. — In tubercular lesions generally and in the sputum of pulmonary phthisis, in the urine in many cases of genito-urinary tuberculosis, and in the feces in intestinal tuberculosis. The tuberculosis of cattle is generally con- sidered to be due to this organism, while the tuberculosis of birds is probably due to a different variety. Does not multiply outside of the body except in cultures. May occur on the surface of objects contaminated with the excreta of tuberculous individuals or in the dust of places inhabited by such individuals. Spirillum of Asiatic Cholera (Comma Bacillus)' (see also Clinical Bacteriology). — Morphology (Figs. 50, 51). — In fresh cultures the organism appears usually as a slightly curved rod somewhat shorter than the tubercle bacillus, but much thicker. The curving of the rod varies, being very marked in some individuals and absent in others. Sometimes two rods are joined end to end with their convexity pointing in opposite directions, or moderately long, undulating threads may be found. It seems probable that the curved rods rep- • resent the segments of a spirillum, and hence the name of the organism. In cultures some days old degenerated and atypical forms are found (involution forms). The organism is motile, and a single flagellum is attached to the end of the rod. It is not stained by Gram's method. Colonies on Gelatin Plates (Fig. 52). — After twenty-four to forty-eight hours at a temperature of 20° to 22° C. the largest colonies will appear as masses of indefinite granular material lying in circular areas of hquefied gelatin in which 'Koch: Deutsche vied. Wochenschr., 1884 and 1885. BACTERIOLOGICAL LiXAM/XATIOXS. I 53 granular shreds are scattered. Within the next twenty-four hours the areas of liquefaction increase, and the colonies ap- '^r o . ^- .V '^/ ,<^7-'t ^' , ^' r A -•> ( 1^. , ■-'■'v '# »• \ Lz\ ■"■>■ ■ ' V' ,:^''* \ .t>.»»^ V-^ V '*>.■-:" fe.V^N * v'^ 7 - ' \ J . i»» *? ' ' - * » »»ff. Fig. 56. — Bacillus of anthrax, stained to sliow the spores; X looo (Frankel and Pfeiffer). and show square or slightly concave ends. The capsule is not present in cultures. Stained by Gram's method. Not motile. Forms oval spores in the middle of the short segments or rods. The spores may be seen in blood-serum cultures after forty-eight hours in the incubator (Fig. 56). Gelatin Stab. — Growth along the line of stab, with radiat- ing filaments extending laterally into the gelatin, which is slowly liquefied in funnel form (Fig. 57). Bouillon. — Growth in the form of cotton-like flakes and filamentous masses. No clouding of the medium. Asiar-as'ar. — Matted network of translucent filaments. BA CTERIOL GICAL EXAMLYA TIOiVS. 159 Under a lower magnifying power the growth is seen to be made up of twisted and contorted masses of filaments, giv- ing the appearance of curled hair (Fig. 58). Fig. 57. — Bacillus of anthrax : sjelrttin stah-ciilture seven days old (Giinthcr). Potato. — Grayish-white, rather thick, dry layer, having the appearance of frosted glass. Occiiyycnce. — In malignant pustule, wool-sorter's disease, ^ r^^ Fig. 58. — Colony of bacillus of anthrax, slighth' magnified (Fliigge). and intestinal anthrax. Found in the blood of animals dead of anthrax. In man the infection is usually localized at l6o PATHOLOGICAL TECIINLQUE. first at the point of inoculation, cither on the skin or on the mucous membrane of the air-passages or intestinal tract. Later, a general invasion of the blood may occur and a fatal septicemia result. The organism or its spores may be pres- ent in wool or hides, and infection may take place from these. Bacillus Pyocyaneus (Bacillus of Green Pus).' — Colonies on blood-scrum grow rapidly, are not especially characteristic in form, and liquefy the medium, imparting to it a dark greenish color. Morpliology. — Small bacilli with rounded ends (Fig. 59). r V Fig. 59. — Bacillus pyocyaneus, from an agar-agar culture ; X looo ( Itzerott and Niemann). Decolorized by Gram's method (Welch). Motile, and is provided with a flagellum at one end. Does not form spores. Gf/at/;/ Stall. — Liquefaction in funnel form, with green fluorescence of the upper portions of the medium. The liquefied gelatin is densely clouded, and there may be a viscid pellicle on the surface. Agar-agar Stab. — A green fluorescence in the upper layers of the medium, which later becomes a dark blue-green. Potato. — Slightl}' elevated, brownish, \'iscid la)-er. The potato in some cases assumes a green color, in others a brown color. In some cultures the potato when touched 'Gessard: Annates de l' Lnstiliit Pasteur, T. 5, 1S91. BA CTERIOL O GICAL EX A MINA TIONS. i6i with the platinum wire takes on a green color at the point touched. This is the so-called " chameleon phenomenon," and it is best observed in cultures several days old. Bouillon. — The growth is in the form of flocculi and a delicate surface pellicle. The fluid acquires a green color. Litums-viilk. — Acid reaction with coagulation. Diuiliam's Pepton Solution. — Indol is produced. Colonies on Gelatin Plates (Fig. 60). — Development is rapid. "Young colonies are provided with a fringe of delicate fila- J Fig. 60. — Bacillus p> :)l) mpus l jlunies upon gelitin ( \1 bott), ments about their periphery. . . . As growth progresses and liquefaction becomes more advanced, the central mass of the colony sinks into the liquefied depression, while at the same time there is an extension of the colony laterally. . . At this stage the colony, when slightly magnified, may present vari- ous appearances, the most common being that shown in Fig. 60. The gelatin between the growing colonies takes on a bright yellowish-green color, but, as growth is comparatively rapid, it is quickly entirely liquefied, and one often sees the colonies floating about in the pale-green fluid." ^ Pathogenesis. — Subcutaneous inoculation of guinea-pigs and rabbits with i c.c. of a virulent bouillon culture may produce purulent infiltration and inflammatory edema of the tissue about the point of inoculation, and death may follow in eighteen to thirty-six hours. Intraperitoneal inoculation ^ .Vbbott : PrtJicfp/t's of Bacteriology. 11 l62 PATHOLOGICAL TECHNIQUE. may result in a sero-fibrinous or purulent peritonitis with fatal result. In fatal inoculations the bacillus is found in the various viscera, but not in great numbers. Animals inocu- lated with small amounts may survive with merely local lesions, and an immunity may be produced. Several varieties of this bacillus have been described, but their differences do not seem to be of sufficient importance to justify their separation into distinct species. Occurrence. — " Is widely distributed, occurring often on the human skin, in the feces, and outside of the body. In wounds, stains the dressings bluish-green and produces a somewhat characteristic offensive odor. " Increases suppuration of wounds, usually with little con- stitutional disturbance. Is found not infrequently in perfora- tive peritonitis and appendicitis, sometimes in phlegmons, otitis media, broncho-pneumonia, and inflammation of serous membranes, associated usually with other bacteria. " It was found by H. C. Ernst in tuberculous pericarditis. Often found in diarrheal and dysenteric discharges. May cause general infection in human beings. With or without general infection it may cause hemorrhagic and necrotic en- teritis, a form of pyocyaneous infection in human beings which we have repeatedly observed at autopsy. Instances of in- vasion of the body from wounds by the bacillus pyocyaneus have not been observed " (Welch). Bacillus of Influenza ^ (see also Clinical Bacteriology). — Morphology. — Very small bacilli, with rounded ends and of variable length, sometimes growing into long forms, more or less bent or curved. Stains more deeply at the ends than in the middle, and in the long forms shows irregularity of staining. The faintly stained areas are very sharply defined, as in the case of the typhoid bacillus. Cultivation. — Does not grow in the ordinary culture-media, but maybe cultivated on agar-agar "slants," the surfaces of which have been smeared with a few drops of sterile ipfeiffer: Zeitschrift f. Hygiene u. Infectionskrankheiten, Bd. 13, 1893. BACTERIOLOGICAL EXAMINA TIONS. 163 blood. The blood of man, rabbits, guinea-pigs, pigeons, or frogs will serve for this purpose, the best growth being obtained with pigeon's blood. The blood may be ob- tained from a needle-prick, and spread over the surface of the agar-agar by means of the platinum loop. The skin should be previously thoroughly washed with alcohol and ether, and the first drops of blood should not be used. Human blood is best obtained from the lobe of the ear Fig. 61, — Bacillus of influenza: colonies on blood agar-agar; low magnifving power (Pfeiffer). or from the finger. Tubes thus prepared are only rarely contaminated. Colonics. — After twenty-four hours in the incubator the colonies appear as minute colorless, glassy, transparent points resembling small drops of dew. They never attain any size, and do not become confluent. They are barely visible to the unpractised eye, and require a low magnifying power to be seen clearly. Under the low magnifying power they are translucent, homogeneous, not granular, and cir- cular in outline (Fig. 61). 164 PATHOLOGICAL TECHNIQUE. Decolorized by Gram's method. Not motile. Will not grow without oxygen. Pathogenesis. — The ordinary laboratoiy animals are not susceptible to infection with this organism. Occurrence.— YounA in the exudate of the respiratory tract in influenza, frequently inside of leucocytes (Fig. 62). It may be present in the small bronchi and in the exudate of broncho-pneumonia in this disease, sometimes unaccompanied Fig. 62. — Bacillus of influenza; cover-glass preparation of sputum from a case of influenza, showing the bacilli in leucocytes; highly magnified (Pfeiffer). by other organisms. It has been observed in purulent meningitis secondaiy to influenza. Bacillus of Glanders (Bacillus Mallei).' — Blood- scritni. — Rounded, elevated, colorless, viscid-looking colonies, growing slowly and becoming well de\'eloped after thirty-six hours in the incubator. They may attain a diameter of 2 or 3 mm., and after a time they assume a brownish tint. RIorpliology. — Bacilli of medium size, variable in length, having rounded ends, and frequently showing faintly stained areas in their protoplasm (Fig. 63). The larger forms of the ' Loftier: Arbciten a. d. A'ais. Gcsundhcitsamte, Bd. I, i8S6. BACTERIOLOGICAL EXAMIAATIONS. 165 bacillus are usually slightly bent or wavy in outline, and may be slightly swollen at their ends. Potato. — After thirty-six hours in the incubator a rather thick, colorless, viscid-looking layer appears, which soon assumes a brownish tint and resembles honey in appearance. Later the brown color changes to a dark reddish-brown, and the growth becomes thicker and more opaque, while the potato takes on a dark-gray color. Pathogenesis. — When inoculated subcutaneously into K -- Fig. 63. — BacciUus mallei, from a culture upon gl)'cerin agar-agar; x 1000 (Frankeland Pfciffer). guinea-pigs the characteristic result is swelling and inflam- mation of the scrotum, appearing after a variable number of days, often about a week. The animals usually survive several weeks, with ulceration at the point of inoculation. The lesions produced consist in suppurative processes or abscess-formations in or about the testes, in the lymph-glands, in the anterior nares, about the joints, and in other situations, besides small grayish nod- ules or areas in the viscera — the so-called " glanders tuber- cles." The suprarenal capsules usually show red areas, and they may be enlarged. On microscopic examination, the small nodules as well as the extensive suppurative areas l66 PATHOLOGICAL TECHNIQUE. will be found to be composed of necrotic material containing leucocytes and fragments of chromatin. The distribution and extent of the lesions vary with each animal, but the involve- ment of the testis or its membranes is practically constant and pathognomonic of the bacillus of glanders. This in- volvement of the testis may consist, in early cases, in the presence of yellow foci in or about the tunica vaginalis, or in later cases the organ may show large yellow areas with purulent softening. Intraperitoneal inoculation with virulent cultures may be followed by death within forty-eight hours, with fibrinous exudate on the peritoneum in which minute grayish nodules are seen. The nodules are made up of a material which is apparently mainly dead or degenerated leucocytes and des- quamated peritoneal endothelium, together with many chro- matin fragments. In these acute cases also microscopical examination of the spleen and liver may show the presence of small nodules identical in structure with those seen in the more chronic cases. For the purpose of producing with cultures the characteristic lesions of the testis or its coverings it is better to inoculate the animal subcutaneously, for in the rapidly fatal intraperitoneal inoculations with virulent cultures these may not show any marked changes. The bacilli may be cultivated from the lesions, but not from the blood of the heart, in the chronic cases. They may be present in the blood of the heart, however, in small numbers in rapidly fatal infections following intraperitoneal inoculation. Field-mice may die from subcutaneous inoculation in about seventy-two hours. The most conspicuous lesion produced is enlargement of the spleen, with the presence in it of minute grayish nodules. White mice are immune. Rabbits are not so susceptible as guinea-pigs to the infection. Decolorized by Gram's method. Not motile. Spore- formation not probable. Rate of growth is slow. Bouillon. — Diffusely clouded, with the formation of a vis- cid sediment. BACTERIOLOGICAL EXAMINATIONS. 1 6/ Litmus-milk. — Gradually turned red and coagulated. Agar-agar and Gelatin. — Growth not especially charac- teristic. Occurrence. — Found in the lesions of glanders, and may invade the blood-stream in small numbers in acute cases of infection. Grows in the tissues in clumps or groups as well as scattered. In lesions on exposed surfaces it may be ac- companied by the pyogenic cocci. Bacillus Proteus (Proteus Vulgaris). — Morphology. — Bacilli of very variable length, sometimes appearing like cocci or as filaments. Motile, being provided with terminal flagella. Does not stain by Gram's method. Colonies in Gelatin. — Rapid growth with liquefaction of the gelatin. In a medium containing 5 per cent., instead of 10 per cent., of gelatin prolongations from the margins of the colonies may be formed. These may become sep- arated from the mother colonies and form daughter colonies. Motions may be observed in these prolonga- tions. Gelatin Stab. — Rapid liquefaction along the line of inocu- lation with cloudiness of the liquefied gelatin and a floccu- lent deposit. Agar-agar Slant. — Widely spreading, thin, moist, grayish- white layer. Potato. — Dirty white, moist layer. Litmus-milk. — Turned pink and slowly coagulated. Odor. — The cultures generally have a putrefactive odor. Pathogenesis. — Intravenous, intraperitoneal, or intramus- cular inoculations of rabbits may produce death in twenty- four to thirty-six hours after moderately large doses. Liq- uefied gelatin-cultures are said to be more virulent than bouillon cultures. Guinea-pigs seem to be less susceptible than rabbits to infection with this organism. Occurrence. — This bacillus and its varieties are among the most common and widely distributed putrefactive bacteria. It occurs in the intestinal contents. In pathological exam- inations it may be found in peritonitis and in abscesses, i6S FA THOL GICA L TE CHXIQ I 'E. usually associated with other bacteria. It may also invade the circulating blood. The so-called " proteus group" includes several varieties of similar organisms — viz. the proteus vuli^aris, \h• .J Fig. 69. — Bacillus of tetanus : five-days-old colony ui glucose-gelatin ; x 1000 (Frankel and Pfeiffer), extended backward. At the autopsy the bacillus is to be found only at the point of inoculation, and may be difficult or impossible to demonstrate there. Glucose-gelatin Stab. — Growth along the line of inocula- tion, beginning 2 or 3 cm. below the surface, with delicate filaments radiating laterally into the gelatin (Fig. 68). Liq- uefaction and gas-production occur. In the vegetative forms the organism is sluggishly motile. Stained by Gram's method. BACTERIOLOGICAL EXAMINATIONS. 173 In anaerobic cultures in glucose-agar-agar and in glucose- bouillon growth is good, but is not remarkable. Occurrence. — Found in the soil, and often in the feces of herbivorous animals. In cases of tetanus the bacillus is to be found only in the wound or at the point of inoculation. It does not invade the blood-current. The bacillus of tetanus acts by the production of a " toxin " or " toxalbuminr This is also produced in cultures. It may be demonstrated in the bacteria-free filtrate of bouillon cul- tures some days or weeks old. A very few drops of this fluid will give rise to fatal tetanus in a mouse. Bacillus Aerogenes Capsulatus.'— Will not grow in the presence of oxygen. Morphology. — Bacilli of about the thickness of the anthrax bacillus, variable in length, but usually 3 to 6// long. Ends rounded or square cut. Occurs singly, in pairs, in clumps, and sometimes in short chains, less frequently in threads and long chains. May show unstained spots or deeply staining granules in the protoplasm. Capsules may be frequently demonstrated in the specimens from the tissues, and sometimes in agar- agar cultures. Colonies in anaerobic cultures are grayish to brownish- white, with a central darker spot by transmitted light. In time they may attain a diameter of 2 to 3 mm. or more. Colonies in the depths are spherical or oval, sometimes pre- senting knob-like or feathery projections. Effects on Animal Tissues. — Not pathogenic for rabbits. If a rabbit that has received 0.5 to i c.c. of a bouillon culture injected into the ear-vein be killed immediately after- ward and the body kept for twenty-four hours at a tempera- ture of 18° to 20° C, or for four to six hours at a tempera- ture of 30° to 35" C, the vessels and organs will be found to contain a great quantity of gas and large numbers of the bacilli. The organism multiplies post-mortem in the blood of the animal and produces the gas. This effect upon the tissues of the dead animal is characteristic of the bacillus. ' Welch and Flexner : Journal of Experini. Medicine, vol. i. No. i, 1896. 174 PATHOLOGICAL TECHNIQUE. Gas-prodiictioji is marked in agar-agar and gelatin cultures containing glucose. The gas produced burns with a blue flame and is odorless. Gelatin is liquefied slowly and to a limited extent. Glucose Bouillon.— V>\^ws&\y clouded at first, later becom- ing clearer, with an abundant whitish, more or less viscid sediment. il/z7/f'.— Coagulated, the clot being firm, retracted, and fur- rowed with the marks of gas-bubbles. Fig. 70. — Bacillus of malignant edema from the edema fluid of a guinea-pig inoculated with garden-earth ; X 1000 (Friinkel and Pfeiffer). Potato. — Growth thin, moist, and grayish-white, or it may not be visible. The bacillus is stained by Gram's method. It does not form spores. The vitality of the organism depends upon the character of the culture-medium and the mode of cultivation. It sur- vives longer when cultivated by Buchner's method (see page 119) than when cultivated under hydrogen. Cul- tures on glucose media are shorter lived than those on plain media. Occurrence. — Occurs at autopsies in which gas-bubbles are present in the larger vessels, accompanied by the formation BACTERIOLOGICAL EXAMINATIONS. I/S of numerous small cavities irf the liver containing gas. It has been found also in emphysematous phlegmons, in puer- peral sepsis, in peritonitis, and in other conditions. Bacillus of Malignant Edema/ — This bacillus will not grow in the presence of oxygen. Morphology. — Rather large bacilli, sometimes growing into threads (Fig. 70), but occurring frequently in pairs, in which the proximal ends are square while the distal ends are rounded. Forms oval spores in the middle of the rod, which may give the rod a spindle or oval shape. The colonies in anaerobic glu- cose-gelatin cultures appear as spheres of cloudy liquefied gela- tin marked by delicate radiating f • streaks. Gas-bubbles are formed in the medium (Fig. 71). Pathogenesis. — Subcutaneous in- oculation of mice, guinea-pigs, and rabbits is followed by death in from sixteen to forty-eight hours, de- pending upon the animal, mice being most susceptible. The typical lesions are extensive sub- cutaneous edema containing gas- bubbles and more or less blood, and enlargement of the spleen. The bacilli are found in the edema, in the viscera, and on the serous surfaces of the organs, but not in the blood of the heart if the examination be made imme- diately after death, except sometimes in mice. The organism is not capable of multiplying in the living blood, owing to the presence of oxygen. In inoculating subcutaneously a deep pocket should be made in the skin, and the material 1 Liborius : Zdlschrift f. Hypenc ii. Infectionskrankheileii^ Ed. I, lS86. Fig. 71. — Bacillus of malig- nant edema; colonies growing in glucose-gelatin (Frankel and PfeiiTer). 176 PATHOLOGICAL TECHNIQUE. for inoculation introduced info the tissue as far away from the opening as possible. This is to prevent the access of too much oxygen to the organism. Slightly motile. Flagella may be demonstrated by special staining methods. Decolorized by Gram's method. Growth in anaerobic agar-agar and bouillon culture is good, but not characteristic. Occurrence. — Widely distributed in the soil and in putre- fying substances. Only a very few cases are on record of infection in man by this bacillus. Actinomyces (Fig. 72). — The position of this organism among the other bacteria is not yet definitely settled, owing to its peculiar morphology, described below. The general tendency of late has been to classify it as a cladothrix. A number of varieties of actinomycetes is claimed, but only the probably identical variety affecting cattle and man is considered here. Morphology. — In tissues the organism appears as discrete and very characteristic colonies, less often as loose or com- pact tangles of branching filaments or as bacilli and cocci. The colonies are often large enough to be visible to the naked eye. In form they are spherical, oval, or slightly curved ; in color, gray and gelatinous, opaque white, yellow, or even brown. Occasionally they have a greenish tint. Cover-slip preparations from actively growing colonies in tissues show cocci, long and short bacilli, and single and branching filaments, of which some stain uniformly, while others consist of longer or shorter joints, each of which corresponds to a coccus or to a bacillus. Transparent club- shaped bodies, in which bacilli and cocci similar to those in the threads can often be found, are also present. Sections of colonies in the tissues show that they are solid or hollow, depending somewhat on their size. Around the outside is a layer of transparent bodies, the so-called " clubs " (Fig. 72). Inside of this layer is a thick tangle of branching filaments composed of bacilli and cocci. In the centers of the large colonies usually only a few threads are found. BACTERIOLOGICAL EXAMLNATLONS. I77 Besides the colonies, careful examination of the tissue often shows numerous bacilli and filaments lying in or between the cells. The filaments may form closely-woven tangles sug- gesting colonies, or may spread out loosely in every direction. In cattle the organism appears most frequently as discrete colonies with marked development of the clubs, and as bacilli in and between the cells. In man this polymorphous bacterium seems to grow more luxuriantly ; discrete colonies with typical formation of clubs are often hard to find, while bacilli and loosely or closely-woven tangles of filaments are very abundant. The transparent clubs, which lie in the peripheries of the i-rf Fig. 72. — Actinomyces (von Jaksch). colonies and form the characteristic peculiarity of the organ- ism, are due to a hyaline degeneration or swelling of the membrane or sheath of the bacilli and filaments under con- ditions which oppose their free growth. I/! cultures the organism appears as straight and curved rods of varying length and breadth, and on special media as straight, wavy, or spiral filaments which are often dichot- omously divided. No clubs are ever formed in artificial culture-media. Cultures. — The organism is anaerobic, but not strictly so, and grows slowly on agar-agar or blood-serum and in raw or boiled hen's or pigeon's eggs. On agar-agar and blood- serum it appears in tlie form of dry, discrete colonies which 12 178 PATHOLOGICAL TECHNIQUE. send roots into the culture-medium. The older colonies are often yellowish in color. Microscopically, the colonies con- sist mainly of bacilli. In eggs a tangle of threads is the commonest form. Pathogenesis. — The organism is extremely disappointing from the experimental standpoint, because of its very slight pathogenesis when cultures are inoculated into animals. Only a few observers, of whom Wolff and Israel are the most prominent, claim to have obtained positive results. By inoculating rabbits and guinea-pigs intraperitoneally with anaerobic cultures they succeeded in producing typical colonies with formation of clubs.' For staining cover-slip preparations Gram's method is the most satisfactory. Partial decolorization in alcohol leaves the clubs lightly stained, while the filaments, etc. are stained intensely. The methods of staining sections of colonies in tissues are described elsewhere. VI. CLINICAL BACTERIOLOGY. General Considerations. — The end in view in the bac- teriological examination of pathological material obtained from the individual during life is the determination of the species of bacteria which may be present in it. The exam- ination is effected by various methods of cover-glass prepara- tion, of culture, and of animal inoculation. In this work it is of obvious importance that the material be protected from the invasion of bacteria from without, and that in its collec- tion every object with which it comes in contact be free from living bacteria. To fulfil these requirements the material may be conve- niently collected in any of the following ways : I. It may be obtained directly from the individual by 1 Bostroem : " Untersuchungen iiber die Aktinomykose des Menschen,'' Ziegler's Beitrage, z. path. Anat., vol. ix. pp. 1-240; M. Wolff and J. Israel: Virchow's Archiv f. path. Anat., vol. cxxvi. pp. 11-58. BACTERIOLOGICAL EXAMINATIONS. 179 means of the sterilized platinum wire, and cover-glass prep- arations, cultures, and, if necessary, animal inoculations, made at once. 2. Since a veiy small quantity of the material usually suf- fices for the purposes of examination, it may often be very conveniently collected and brought to the laboratory on the so-called " swabs," where it can be subjected to the vari- ous manipulations at leisure. The " swab " consists of a piece of rather stiff wire about six inches long, on one end of which ;s firmly twisted a pledget of absorbent cotton, so that ^he end of the wire is well covered. This is placed, cotton end' first, in a test-tube, which is then provided with a cotton stopper (Fig. 73), and the whole sterilized in a hot-air sterilizer by heating to 150° to 180° C. during about half an hour. A large number of " swabs " in test-tubes may be kept on hand sterilized and ready for use. When it is desired to secure material for bacteriological examination on a " swab," the cotton stopper is removed, the swab taken out, and the cotton end brought in contact with the pus or exudate in such a manner that some adheres to the cotton. The swab is then immediately replaced in the test- tube, the cotton stopper returned to its place, and the whole then carried to the laboratory. In these manipulations care should be taken to avoid touching with the swab anything but the material which it is desired to examine, otherwise the material may be con- taminated with other bacteria than those originally present in it. Fig. 73. — Sterilized test- tube and swab for collect- ing pus and fluids for bac- teriological examinations (Warren). l8o PATHOLOGICAL TECHNIQUE. By means of swabs material for examination from pus or exudates may be secured and brought to the laboratory in most instances. They are especially useful in surgical work, in which it is often desirable to determine the character of the organism present in a pus-formation or exudation with- out waiting to summon a bacteriologist or to collect the necessary cover-glasses, culture-tubes, platinum needle, etc. The swabs and their test-tubes may be kept on hand in a sterile condition, so that they may be handled by the ope- rator or an assistant. 3. Fluid material may be collected by aspiration or other- wise. In the case of fluids care should be taken that every- thing with which the fluid comes in contact be clean and sterilized by heat if possible. The use of antiseptics, such as carbolic acid or corrosive subhmate, is to be avoided. If a hypodermic syringe is used in obtaining material, it should be of a construction which will admit of sterilization by heat, and it should be so sterilized before using. Cover-glass Examinations. — For methods of preparation and staining see page 89. The cover-glass examination is of great importance, for it may give valuable information, not only as to the species, but also as to the number of bac- teria present. Preparation of Cultures. — The best culture-medium for general purposes is, we think, the coagulated blood-serum described elsewhere. In certain instances other special media are to be used, as will be pointed out in the following pages. In the preparation of cultures the same general di- rections given on page 94 are to be followed. If the material is on a " swab," the surface of the blood- serum or other media may be conveniently inoculated directly by gently rubbing the swab over it. In this case it is usually best to make a dilution or two by means of the platinum wire, as described on page 95, especially if there be a large amount of material on the swab or if the cover-glass examination has shown that a large number of bacteria are present. In any case it is important that the infected material be spread BACTERIOLOGICAL EXAMINATIONS. l8l over all of the surface of the medium, and not in the form of one or two narrow streaks. Animal Inoculations. — The general methods of animal inoculation have already been described. The inoculation of animals directly with pathological material is often of im- portant diagnostic value, and the special methods of doing so when it is necessary for diagnosis will be treated under the special headings of this section. Suppurative Processes. — The bacteria commonly found associated with these conditions are the pyogenic cocci. In general it may be said that the staphylococci will be found in the localized suppurations with abscess-formation, while the streptococcus will be found in the spreading inflamma- tions producing little pus and accompanied by lymphangitis. The streptococcus pyogenes may often be identified by the cover-glass examination alone through its characteristic chain-formation, but this may not be apparent and the result of cultures must then be awaited. The staphylococcus pyogenes aureus cannot usually be identified with any certainty by the cover-glass examination alone. Cultures are necessary in order to differentiate from the other staphylococci and from the streptococcus. For practical purposes the identification of the pyogenic cocci may be made by the appearances of their colonies on blood-serum and by their morphology; no secondary cul- tures are usually necessary. Erysipelas. — The streptococcus is most readily found in the extreme margin of the affected area where the process is newest. The skin should be cleansed with soap and water, and with alcohol. Then with a sterile knife-point or a large needle a small wound should be made, and some of the blood and exudate pressed out from the tissue beneath. From this, cultures and cover-glasses may be prepared. Peritonitis and Appendicitis.— The exudate or puru- lent material is best obtained at the operation by means of swabs. A great variety of bacteria may be met with in these situations, the most frequent among which is the bacillus coli communis. l82 PATHOLOGICAL TECHNIQUE. Pleural, pericardial, and joint exudates may con- tain various organisms. In purulent exudates of the pleura and pericardial cavities the pneumococcus is frequently pres- ent, as are also the pyogenic cocci. Other bacteria also occur here. The pneumococcus may be identified from the cover-glass examination alone by the demonstration of its capsule with one of the special staining methods (see p. 92), and by its peculiar morphology. In sero-purulent exudates from the joints the gonococcus may be present. This may be identified with some degree of certainty by the special staining method for the gono- coccus (see p. 191). Cultures on the special media (see p. 131) should be made if it is desired to remove all doubt as to the identity of the organism. The other pyogenic cocci also occur in purulent joint-inflammations. If the exudate from these situations is not purulent, and if tuberculosis is suspected, the results of cover-glass exam- ination for the presence of tubercle bacilli will usually be negative. The main reliance in the determination of the tu- berculous nature of such an exudate is in the results of the inoculating of guinea-pigs (see p. 1 89). The animal is best inoculated with a sterile hypodermic syringe in the tissues of the abdominal wall, about i c.c. of the fluid being injected. Anthrax, or Malignant Pustule. — The bacilli may be found by the cover-glass examination of the contents of the small blebs and vesicles. The bacillus of anthrax may be identified by its morphology (see p. 156), its special charac- teristics being its large size and its square or concave ex- tremities. The inoculation of a mouse at the root of the tail with some of the material from the pustule, and the production of the characteristic fatal septicemia, will render the identifi- cation certain. Diphtheria. — The bacteriological diagnosis of infection with the bacillus diphtheriae depends upon the characteristic BACTERIOLOGICAL EXAMINATIONS. 183 morphology and peculiarities of staining, as well as rapidity of growth, which this organism presents when cultivated upon coagulated blood-serum. The identification by direct cover-glass examination of the exudate is very uncertain. The method is as follows : A blood-serum culture-tube is inoculated with a small amount of the material from the mucous membrane affected, and is placed in the incubator twelve to eighteen hours. After this length of time the re- sulting growth is examined by cover-glass preparations stained either with Loffler's methylene-blue solution or by one of the special methods given below. The bacillus diphtheriae, if present, may then be recog- nized and differentiated from other bacteria present in the preparation by its characteristic morphology and peculiarity of staining, described on page 138. The gross appearances of the culture present little that is characteristic, as a rule, and the main reliance is to be placed on the microscopic ex- amination. Early in the infection the greater part of the growth may be made up of the specific bacilli, but toward convalescence they fall into the minority. The ordinary ■forms of agar-agar culture are not suitable for use in the bacteriological diagnosis of diphtheria, owing to the com- parative feebleness of the growth of the organism on these media, and because of the fact that its microscopic appear- ances when cultivated on such media are not sufficiently characteristic. The material for culture is very conveniently obtained by means of sterilized cotton swabs. In collecting this material the swab is removed from its test-tube and touched to the affected areas of the mucous membrane of either the nose or throat. It is then to be gently rubbed over the surface of a blood-serum culture-tube, or it may be replaced in the test- tube and the inoculation of the culture-tube made later in the laboratory. In the latter case the inoculation should be made within an hour or two after the material has been col- lected, the infected swab meanwhile being prevented from drying by firmly replacing the cotton plug. In cases with membrane-formation the greatest number 1 84 PATHOLOGICAL TECHNIQUE. of bacilli are on the surface or in the upper layer of the membrane^ and the swab should therefore be touched to these portions rather than to the tissue beneath. In addition to the coagulated blood-serum here recom- mended, another practical medium is the following : Special Culture-medium of Kanthack and Stephens. — This con- sists of a mixture of ascitic, pleuritic, or other similar fluid with agar-agar. The method of preparation is as follows : To every loo c.c. of the serous fluid add 2 c.c. of a 10 per cent, solution of potassium hydrate, by which the serum-albumin of the fluid is transformed in the after-heating into alkali-albumin. To this add 1.5 to 2 per cent, agar-agar which has been softened by soaking in weakly acidulated (HCl) water, and heat the mix- ture in the steam sterilizer until the agar-agar is dissolved. The mixture is then filtered through filter-paper and 4 to 5 per cent, of glycerin added. It is then to be run into test-tubes and sterilized as in the case of agar-agar (see p. 88). In the diagnosis of diphtheria it is used in the form of ' ' slants ' ' in the same manner as the coagulated blood-serum. In order to obtain this medium clear, it is necessary that the serous fluid should be worked into the medium as soon as possible after it has been withdrawn from the body. The advantages of this medium are — 1 . It can easily be prepared. 2. It is clear and transparent. 3. Its basis is a body fluid which may be readily obtained in a hospital. 4. The bacillus diphtheriae and certain other pathogenic bac- teria grow upon it rapidly and vigorously, while many common organisms grow upon it slowly and feebly. 5. It can be used in the plate method of Petri. Note. — Before adding the potassium hydrate to the serous fluid, the latter should be tested in a test-tube by heating to the boiling temperature to determine the amount of albumin present. If a great amount of coagulation results, add to the serous fluid twice its volume of distilled water and then proceed as above indicated. If this dilution is not made, the mass will be gelatinous and there- fore useless. The serous fluid after treatment with the alkali forms also a good fluid culture-medium. Special Methods of Staining the Bacillus Diphtheriae. — Owing to the fact that the bacillus diphtheriae may be recognized by its peculiar morphology and characteristic staining in cover- glass preparations from its growth upon certain culture-media, as already pointed out, various special staining methods have been devised for accentuating and rendering more striking to the eye the peculiar deeply-stained points and granules in the bodies of BACTERIOLOGICAL EXAMINATIONS. 185 the individual bacilli, which have been referred to as of great im- portance in the identification of the organism. These special methods of staining are said to be of great advan- tage in cases where only a few specific bacilli may be suspected to be present among a large number of other bacteria. Hunt's Method. — i. Stain in saturated aqueous solution of methylene-blue one minute without heating. 2. Wash in water. 3. Cover with aqueous solution of tannic acid, lo per cent., for ten seconds. 4. Wash in water. 5. Stain in saturated aqueous solution of methylene-orange one minute, without heating. 6. Wash in water. 7. Dry, and mount in balsam. By this method the granules, etc. are dark blue or almost black, and stand out very sharply against the light-green coloring of the body of the bacillus. The solution of methyl-orange should be freshly prepared, for it deteriorates in a few days. Crouch's Method. — Stain the cover-glass preparation in the fol- lowing mixture for a few seconds, then wash in water and mount : I per cent, aqueous solution of methyl green, 5 parts; I per cent, aqueous solution of dahlia, i part ; Water, 4 parts. By this method the deeply stained portions of the bacillus take on a reddish color, while the remainder of the organism is colored pale green. Method used at the Boston City Hospital. — i. Stain with Loffler's methylene-blue solution, heating the preparation for a few seconds. 2. Wash in a |- per cent, solution of acetic acid for a few seconds. 3. Wash in water and mount. The partial decolorization with the acetic acid brings out the deeply-blue stained granules more strongly. Another method may be mentioned here. Whether it is of any use in the routine diagnosis of diphtheria we are unable to say. We are indebted to Dr. A. C; Abbott of Philadelphia for it. The method consists in staining the preparation by Gram's method and then staining with Bismarck-brown. The granules will be found to be black or blue-black and the body of the bacillus of a brown color. Influenza (see also p. 162). — Microscopic examination of cover-glass preparations of the bronchial sputum shows very small, short, round-ended bacilli, often in very large 1 86 PATHOLOGICAL TRCHNIQUE. numbers and frequently in the pus-cells. These bacilli fre- quently occur in pairs, and resemble pairs of cocci. Their ends may be more deeply stained than the central portions. For the staining of cover-glass preparations of the sputum Pfeiffer recommends that a very dilute carbol-fuchsin solution be applied for five to ten minutes. The cover-glass prepara- tion is to be made from a distinctly purulent portion of the sputum. Staining with Loffler's methylene-blue solution also gives good results. The bacillus of influenza may be cultivated from the sputum by breaking up a small portion of a distinctly purulent cha- racter in I or 2 c.c. of bouillon, and then spreading a platinum loopful of the suspension over the surface of a blood-agar-agar slant, which is then placed in the incubator. After eighteen to twenty-four hours the characteristic col- onies may be visible with the aid of a hand-lens. These should not grow in ordinary media unless blood or hemo- globin be present, and should have the morphology of the bacillus of influenza. :Bxamination of Sputum for Tubercle Bacilli. — The morning sputum should be taken for examination. Se- lect one of the dense, grayish-white particles, and with the aid of small pointed forceps or the platinum wire rub it over the surface of a cover-glass, breaking it up as much as pos- sible. The material should be spread in a very thin layer. The preparation is next to be " fixed " in the ordinary way described for cover-glass preparations (see p. 89), and is then to be treated as follows : 1. Stain in carbol-fuchsin solution, steaming for one minute over the Bunsen flame, with the staining solution thoroughly covering all the surface of the cover-glass. None of the surface of the cover-glass should be allowed to become dry by evaporation, as this causes a precipitate to form, but more of the staining fluid should be added from time to time to keep it completely covered as evapora- tion occurs. 2. Wash in water. 3. Cover with Gabbet's solution for twenty seconds. The BACTERIOLOGICAL EXAMINATIONS. 1 87 solution should also be applied to the uncharged slide of the cover-glass to remove any dried stain which may have col- lected thereon. 4. Wash thoroughly in water. 5. Mount in water or balsam. Water is to be preferred, for the reason that the apparent size of the bacilli is larger when examined in water. • - -*,-^jgs«.ai«r 1 ir^ ^ ^ ( f - ^ . .... - -. - i%, ^ = -^ 1 S" ],i: ~ Fig. 74. — Tubercle bacilli in sputum (carbol-fuchsin and methylene-blue). Another method is as follows : 1 . Stain in carbol-fuchsin solution or aniline-fuchsin solu- tion in the manner above indicated. 2. Wash in water. 3. Wash in 95 per cent, alcohol for a few seconds. 4. Decolorize in a solution composed of — Water, 150 c.c. ; Alcohol, 50 " Concentrated sulphuric acid, 30 drops, until the preparation has a faint pink color. l88 PATHOLOGICAL TECHNIQUE. 5. Wash thoroughly in water. 6. Stain for thirty seconds with Loffler's methylene-blue solution without heating. 7. Wash in water and mount. By both of these methods the tubercle bacilli are stained red, while other bacteria and the nuclei of cells are stained blue (Fig. 74). There are other methods for staining tuber- cle bacilli, but these fulfil all the requirements of practical work. Tubercle bacilli, when present in sputum in very small numbers, may sometimes be demonstrated by methods of sedimentation. A good means of sedimentation consists in heating the sputum in a test-tube either with boiling water or in the steam sterilizer for fifteen minutes. The heat coagulates the cells and albuminous constituents, which sink to the bottom, carrying with them the bacilli. The supernatant liquid may then be poured off and the sediment examined as above. Another method of treating the sputum when only a small number of bacilli may be present is as follows : Place 10-15 c-c. of the sputum in a wide-mouthed bottle of 100 c.c. capacity. Add 10 c.c. of water and 6 c.c. of liquefied carbolic-acid crystals. Close the flask with a rubber cork and shake for one minute. After shaking, fill the bottle with water and shake again. Then pour the contents of the bottle into a sedimenting glass, let stand for twelve to twenty-four hours, and examine the sediment. The cover-glass preparations of the sediment before staining are to be washed in ether or chloroform, and then in alcohol, or they may be washed in a mixture of alcohol and ether, equal parts. Tubercle Bacilli in Urine. — The sediment of the urine should be examined. This may be rapidly thrown down by the centrifuge. In urine smegma bacilli may be mistaken for tubercle baciUi, especially in the urine of females. The smegma bacillus resembles the tubercle bacillus very closely in form, and, like the latter, it retains its stain in the presence of acids. It differs from the tubercle bacillus, however, in that it is de- colorized by alcohol. Therefore in staining for tubercle bacilH in the urine alcohol should be used in decolorizing. The procedure is as follows : I. Make a cover-glass preparation from the sediment. BACTERIOLOGICAL EXAMINATIONS. 189 2. Stain with carbol-fuchsin solution for one minute, as described in the case of sputum. 3. Wash in water. 4. Decolorize in 20 per cent, sulphuric acid until the prep- aration has a pink color. 5. Wash thoroughly in water. 6. Wash in alcohol (95 per cent.) thirty seconds. 7. Wash in water. 8. Stain with Loffler's methylene-blue solution twenty seconds, without heat. 9. Wash in water and mount. Surgical Tuberculosis. — The demonstration of the tu- berculous nature of material removed at operations may be made by the histological examination, by the demonstration of the tubercle bacilli on cover-glass preparations, as in sputum, and by the results of the inoculation of guinea-pigs with the material. Cultures are not ordinarily practicable. The examination of cover-glass preparations is commonly of little value, owing to the small number of bacilli usually present. The histological examination may be often made very sat- isfactorily by frozen sections of the tissues. If possible, how- ever, regularly hardened and imbedded tissues, cut in fairly thin sections and stained with hematoxylin and eosin, are preferable. The inoculation of guinea-pigs is to be made subcutane- ously in the abdominal wall, either with a hypodermic syr- inge if the material is fluid, or, if it be in the form of tissue, by inserting a small piece beneath the skin. Material obtained on a swab may also be used for inoculation by introducing the infected swab beneath the skin and moving it back and forth a few times. If tubercle bacilli are present in the ma- terial, the animal will show enlargement of the inguinal lymphatic glands in about three weeks and will usually die of miliary tuberculosis in the course of six to ten weeks. If necessary, the glands in the inguinal region may be examined histologically after three weeks for the presence of tubercu- lar lesions, or examined by cover-glass preparations for tu- I go PATHOLOGICAL TECHNIQUE. bercle bacilli. The discharges from sinuses, etc. may also be tested for the presence of tubercle bacilli as above indi- cated, the material being obtained on a " swab." Cultures from the Blood during I/ife. — With a steril- ized hypodermic syringe secure a cubic centimeter of blood from one of the large veins at the flexure of the elbow. Mix this immediately with one or two tubes of fluid agar-agar which has been kept at hand at a temperature of about 40° C. and form a " slant," or make a Petri plate of the mixture. (For keeping the agar-agar tubes at-the desired temperature an ordinary cup or similar vessel filled with water of the proper temperature will suffice.) When the " slant " or " plate " has become solid it is to be placed in the incubator for development. It may be mentioned that this mixture of blood and agar- agar is a good medium for the growth of the gonococcus, and that this organism has been cultivated from the blood in gonorrheal endocarditis by this means. Before puncturing the vein the skin over it should be thoroughly cleansed with alcohol and ether, but no antiseptics, such as carbohc acid or corrosive sublimate, should be used. The syringe should be thoroughly sterilized by steam for half an hour, and should be brought to the patient in a sterilized test-tube, from which it should be removed only when it is to be used. Intraperitoneal inoculation of mice may also be made with J to I c.c. of the blood. This may give rise to strepto- coccus or pneumococcus septicemia. Gonorrhea. — Cover-glass examination of the pus shows cocci in the form of paired hemispheres, mainly inside pus- cells (Fig. 7S). For the identification of these cocci as gono- cocci their morphology and position inside the leucocytes are not alone sufficient, for essentially the same appearances may be sometimes seen in pus infected with staphylococci or streptococci. A positive diagnosis of infection with the gonococcus is not justified until it has been determined that the cocci are decolorized by Gram's method, for the ordinary pyogenic cocci are stained by this method. BACTERIOLOGICAL EXAMINATIONS. 191 Prepare a cover-glass with the pus, spreading it thinly with the platinum wire. The practice of spreading a small drop of pus between two cover-glasses and drawing them apart is objectionable. After " fixing " (see p. 89) stain the preparation by the following method: Fig. 75. — Gonococci in leucocytes : cover-glass preparation of gonorrheal pus. Method of Staining for Gonococci. — i. Stain with aniline- gentian-violet solution for thirty seconds, without heating. 2. Wash in water. 3. Cover the preparation with Gram's iodin solution for thirty seconds. 4. Wash in water. 5. Wash with alcohol (95 per cent.) until the color ceases to come out of the preparation. 6. Wash in water. 7. Stain in saturated aqueous solution of Bismarck brown for thirty seconds. 8. Wash in water and mount. This method is nothing but Gram's method and after- staining with Bismarck brown. With it the gonococci are stained brown, and other pyogenic cocci stained blue-black. If it is desired to obtain cultures of the gonococcus from the pus of gonorrheal urethritis, the case should not be more than of a few days' duration, because cases of longer dura- 192 PATHOLOGICAL TECHNIQUE. tion will usually show the presence of other bacteria whose colonies overgrow the feebly growing colonies of the gono- coccus. An organism which may be mistaken for the gono- coccus is a coccus growing in large milk-white colonies on all media, but staining by Gram's method. This coccus is frequently found in gonorrheal pus after the discharge has lasted several days. Other cocci also occur. The pus for culture purposes may be collected on a "swab," and the special culture-medium (see page 131) di- rectly infected with this. The gonococcus retains its vitality in the pus on the swab for a number of hours, but care should be taken to avoid drying. Gonorrheal Conjunctivitis. — Prepare cover-glass prep- arations of the pus and stain by the special method for gonococci given above. If gonococci are present, they must be stained brown — i. e. they must be shown to be decolor- ized by Gram's method before the diagnosis of gonorrheal infection is justified. If it is desired to obtain cultures of gonococci from this condition, the special medium must be used as above men- tioned. Pyosalpinx. — In a certain proportion of cases of puru- lent inflammation of the Fallopian tubes gonococci may be found and cultivated, as above indicated. The majority of cases, however, will have sterile pus, while in a small per- centage of cases the ordinary pyogenic cocci will be present. Cerebro-spinal Meningitis (see also the section on Lumbar Puncture). — It is claimed that the nasal secretion in the epidemic form of this disease contains the diplococcus intracellularis meningitidis (see Bacteriological Diagnosis). There is no doubt that diplococci, decolorizing by Gram's method and often situated in polynuclear leucocytes, are to be found in the nasal secretion of certain cases of this dis- ease, but whether their presence is pathognomonic of cere- bro-spinal meningitis is not yet determined. The material for examination is best obtained with the platinum loop from the superior portions of the nasal cav- ities. From it cover-glass preparations are made and stained BACTERIOLOGICAL EXAMINATIONS. 1 93 by the method described above for gonococci. The diplo- cocci, if present, will be stained brown. The microscopical appearances presented by the prepara- tion may be very like those of preparations of gonorrheal pus. Cultures on blood-serum may be also made from the nasal secretion, but the growth of the diplococci on them is uncertain. Glanders. — In a case of suspected glanders the dis- charges from sinuses or ulcerated surfaces, or the contents of pustules, are to be examined for the presence of the bacillus of glanders by the usual methods. The material for examination may be collected on " swabs." With this a guinea-pig is to be inoculated and cultures and cover-glass preparations are made. If the material be from sinuses or ulcerated surfaces, the isolation of the bacillus by cultures will be difficult, owing to the presence of other or- ganisms. The guinea-pig is to be inoculated in the perito- neal cavity by introducing the infected swab into it through an incision in the abdominal wall, or by injecting about i c.c. of a suspension in bouillon of the suspected material into the peritoneal cavity with a hypodermic syringe. If the bacillus of glanders is present, the scrotum will usually show the characteristic swelling and inflammation in the course of three or four days, and death will occur after some weeks. In some cases the animal may die in thirty- six or forty-eight hours. In any case the characteristic lesions of glanders will be found as described elsewhere, and the bacillus may be isolated from them by cultures. The spleen will practically always yield glanders bacilli in pure culture even if no macroscopic lesion can be made out. In cultures the organism should show those characteristics of morphology, of culture, and of pathogenesis which have been described in the section on Bacteriological Diagnosis. Tetanus. — The isolation of the tetanus bacillus is a very difficult task, and attempts to cultivate it from cases of tetanus are often without success. Method of Isolation. — Inoculate several mice subcutane- ously with the secretion of the wound. If tetanus results 13 194 PATHOLOGICAL TECHNIQUE. in the mice, there will usually be some suppuration about the point of inoculation owing to the presence of other organisms. Smear some of this inflammatory exudation from the point of inoculation on the surface of several blood-serum tubes, and place in the incubator. After twenty- four hours the microscopical examination of the growth may reveal the presence of a few of the characteristic drumstick-shaped, spore-bearing bacilli (p. 170). If these are present, the culture is then replaced in the incubator for twenty-four hours, and then heated in a water-bath at 80° C. for about three-quarters of an hour. Then make from the growth several anaerobic cultures by any of the methods (see section on. Methods of Cultivation without Oxygen). In the anaerobic cultures the colonies which develop should not only be composed of the bacilli of the charac- teristic morphology, but they should show the characteristic pathogenesis for mice (see section on Bacteriological Diag- nosis). If gelatin is used as a culture-medium, the colonies appear after about a week. Langdon Frothingham^ has used the following method with success in the isolation and identification of the bacil- lus from cases of tetanus in horses : 1. Inoculate alkaline bouillon with the pus from the wound, or, pus being absent, with small bits of tissue from the inside of the wound, or, in the case of experiment ani- mals inoculated with pure cultures, with bits of tissue from the region of the point of inoculation. 2. Place the tubes in an atmosphere of hydrogen and in the incubator for forty-eight hours. 3. Examine microscopically, and if tetanus bacilli are present — 4. Heat from three-quarters of an hour to an hour in water-bath kept at a temperature of 80° C. 5. From the heated tubes inoculate fresh bouillon cultures (under hydrogen) and place in the incubator for forty-eight hours. ^ Am. Journ. Med. Sci., May, 1894. BACTERIOLOGICAL EXAMINATIONS. 1 95 6. Test the purity by microscopic examination and by cultures in glucose-gelatin, glucose-agar, etc. 7. Test the virulence on mice, white or gray. Asiatic Cholera. — Bacteriological Diagnosis.^ — Because of the manifold channels which are open for the dissemination of this disease it is of the utmost importance that its true nature should be recognized as quickly as possible, for with every moment of delay in its recognition opportunities for its spread are multiplying. It is essential, therefore, when employing bac- teriological means in making the diagnosis to bear in mind those biological and morphological features of the organism that appear most quickly under artificial methods of cultivation, and which at the same time may be considered as characteristic of it — viz. its peculiar morphology and grouping ; the much greater rapidity of its growth over that of other bacteria with which it may be associated ; the characteristic appearance of its colonies on gela- tin plates and of its growth in stab-cultures in gelatin ; its property of producing indol and coincidently nitrites in from six to eight hours in pepton solution at 37° to 38° C. ; and its power of causing the death of guinea-pigs in from sixteen to twenty-four hours when introduced into the peritoneal cavity, death being preceded by symptoms of extreme toxemia, characterized by prostration and gradual and continuous falling in temperature of the animal's body. In a publication recently made by Koch ''■ he called attention to a plan of procedure that is employed in this work in the Institute for Infectious Diseases in Berlin. In this scheme the points that have been enumerated are taken into account, and by its employ- ment the diagnosis can be established in the majority of cases of Asiatic cholera in from eighteen to twenty-two hours. In general, the steps to be taken and the points to be borne in mind are as follows. The material should be examined as early as possible after it has been passed : I. Microscopic examination. From one of the small slimy particles that will be seen in the semifluid evacuation prepare a cover-slip preparation in the ordinary way and stain it. If, upon microscopic examination, only curved rods or curved rods greatly in excess of all other forms are present, the diagnosis of Asiatic cholera is more than likely correct ; and particularly is this true if these organisms are arranged in irregular linear groups, with the long axis of all the rods pointing in nearly the same direction ; that is to say, somewhat as minnows arrange themselves when swimming in schools up stream (Koch). In 1886, Weisser and Frank' expressed their opinion upon the value of microscopic examination in these cases in the following terms : ' Abbott : Principles of Bacteriology. ' Zeitschrift f. Hygiene u. Infectionskrankheiten, Bd. 14, 1893. ' Ibid., Bd. I, p. 397. 196 PATHOLOGICAL TECHNIQUE. (a) In the majority of cases microscopic examination is suf- ficient for the detection of the presence of the comma bacillus in the intestinal evacuations of cholera patients. (Ji) Even in the most acute cases, running a very rapid course, the comma bacillus can always be found in the evacuations. {c) In general the number of cholera spirilla present is greater the earlier death occurs ; when death is postponed and the disease continues for a longer period, their number is diminished. (d) Should the patient not die of cholera, but from some other disease, such as typhoid fever, that may be engrafted upon it, the comma bacilli may disappear entirely from the intestines. 2. With another slimy flake prepare a set of gelatin plates. Place them at a temperature of from 20° to 22° C, and at six- teen, twenty-two, and thirty-six hours observe the appearance of the colonies. Usually at about twenty -two hours the colonies of this organism can easily be identified by one familiar with them. 3. With another slimy flake start a culture in a tube of pepton solution — either the solution of Dunham or, as Koch proposes, a solution of double strength of that of Dunham (Witte's pepton is to be used, as it gives the best and most constant results). Place this at 37° to 38° C, and at the end of from six to eight hours prepare cover-slips from the upper layers (without shaking) and examine them microscopically. If comma bacilli are present and capable of multiplication, they will be found in this locality in almost pure culture. After doing this prepare a second pepton culture from the upper layer, also a set of gelatin plates, and with what remains make the test for indol by the addition of 10 drops of concentrated sulphuric acid for each 10 c.cm. of fluid con- tained in the tube. If comma bacilli are growing in the tube, the rose color characteristic of the presence of indol should appear. By following this plan "a bacteriologist who is familiar with the morphological and biological peculiarities of this organism should make a more than probable diagnosis at once by micro- scopic examination alone, and a positive diagnosis in from twenty to, at the most, twenty-four hours after beginning the examination' ' (Koch). There are certain doubtful cases in which the organisms are present in the intestinal canal in very small numbers, and micro- scopic examination is not, therefore, of so much assistance. In these cases plates of agar- agar, of gelatin, and cultures in the pep- ton solution should be made. The plates of agar-agar should not be prepared in the usual way, but the agar-agar should be poured into Petri dishes and al- lowed to solidify, after which one of the slimy particles may be smeared over its surface. The comma bacillus being markedly aerobic, develops very much more readily when its colonies are located upon the surface than when they are in the depths of the medium. A point to which Koch calls attention in connection BACTERIOLOGICAL EXAMINATIONS. I97 with this step in the manipulation is the necessity for having the surface of the agar-agar free from the water that is squeezed from it when it solidifies, as the presence of the water interferes with the development of the colonies as isolated points and causes them to become confluent. To obviate this, he recommends that the agar-agar be poured into the plates and the water allowed to separate from the surface at the temperature of the incubator before they are used. It is wise, therefore, when one is liable to be called on for such work as this to keep a number of sterilized plates of agar-agar in the incubator ready for use, just as sterilized tubes of media are always ready and at hand. The advantage of using the agar plates is the higher temperature at which they can be kept, and consequently a more favorable condition for the development of the colonies. As soon as iso- lated colonies appear they should be examined microscopically for the presence of organisms having the morphology of the one for which we are seeking, and as soon as such is detected gelatin plates and cultures in pepton solution (for the indol reaction) should be made. The pepton cultures started from the original material should be examined microscopically from hour to hour after the sixth hour that they have been in the incubator. The material taken for examination should always come from near the surface of the fluid, and care should be taken not to shake the tube. As soon as comma bacilli are detected in anything like considerable numbers in the upper layers of the fluid, agar-agar plates and fresh pepton cultures should be made from them. The colo- nies will develop on the agar-agar plates at 37° C. in from ten to twelve hours to a size sufficient for recognition by microscopic examination, and from this examination an opinion can usually be given. This opinion should always be controlled b)'' cultures in the pepton solution made from each of several single colonies, and finally the test for the presence or absence of indol in these cultures. In all doubtful cases in which only a few curved bacilli are pres- ent or in which irregularities in either the rate or mode of their development occurs, pure cultures should be obtained by the agar-plate method and the method of cultivation in pepton solu- tion as soon as possible, and their virulence tested upon animals. For this purpose cultures upon agar-agar from single colonies must be made. From the surface of one of such cultures a good- sized wire-loopful should be scraped, and this broken up in about I c.c. of bouillon, and the suspension thus made injected by means of a hypodermic syringe directly into the peritoneal cavity of a guinea-pig of about 350 to 400 grams weight. For larger animals more material should be used. If the material injected is from a fresh culture of the cholera organism, toxic symptoms at once begin to Appear ; these have their most pronounced expression in the lowering of temperature, and if one follows this decline in temperature from time to time with the thermometer, it will be iqS pathological technique. seen to be gradual and continuous from the time of injection to the death of the animal, which occurs in from eighteen to twenty- four hours after the operation.' In general, this is the procedure employed at Berlin in the In- stitute for Infectious Disease under Koch's direction. Typhoid Fever. — The bacteriological diagnosis of this disease may be made by two methods. One of these coa- sists in the isolation of the typhoid bacillus from the feces by means of special culture-media; the other depends upon a pecuhar effect which the blood-serum of a typhoid patient exerts upon a suspension of typhoid bacilH when a mixture of the two is made. Isolation of the Typhoid Bacillus from the Feces. — This may be accomplished in a certain proportion of cases by making Petri plate cultures from the feces with either of the following special culture-media. The material for cul- tures may be very conveniently secured by means of a "swab," and brought to the laboratory in the sterile test- tube. A few grams of fecal material should be obtained, as this quantity will not readily dry up. We are indebted to Dr. Mark W. Richardson of Boston for the following directions for preparing the special culture- medium of Eisner, and for the description of the method of applying it to the isolation of the typhoid bacillus from the stools, as practised by him : 1. Pare carefully and cut up into small pieces J kilogram of potatoes. The potatoes should be old rather than new, and not too large. The best size it that of an egg or slightly larger. New or large potatoes are too acid in reaction, hence are not to be used. 2. Add I liter of water and boil for one and a half hours. 3. Mash potatoes thoroughly. 4. Measure and make up to i liter the loss of water by evaporation. 5. Strain through a cloth. 6. Make up amount once more to i liter. 7. Boil with 1 5 per cent, gelatin for five or ten minutes. ^ Pfeiffer : loc. cit. BACTERIOLOGICAL EXAMINATIONS. I99 8. Cool down to 60° C, and add a well-beaten egg to clear. Boil. ' 9. Filter through cotton. 10. Filter through paper. 11. Test acidity with decinormal (4 grams to 1000 c.c.) sodium hydrate solution. The acidity should be such, ac- cording to Eisner, that 10 c.c. of gelatin are neutralized by 2.5 to 3 c.c. of the solution of alkali. The acidity may be somewhat lower, however (1.7 c.c), with no harmful results. An acidity above 3 is to be corrected with normal sodium hydrate solution (40 grams to looo c.c). 12. Add potassium iodid i per cent. This may be added — {a) Before tubing the medium, in which case add to i liter of media 10 c.c. of a solution in which i c.c. is equivalent to I gram of potassium iodid, and mix thoroughly ; or, {B) Just before using the medium, in which case we add to 10 c.c. of medium ^ c.c. of the above solution. 13. Tube, and sterilize three times. In the examination of a suspected typhoid stool the first dilution is made with one " loop " of stool into bouillon (to economize on the medium) or into the medium itself Three or four Petri plates are generally sufficient, the dilu- tions being made as follows : Plate II. = I loop from plate I. or from the bouillon tube. Plate III. = I loop from plate II. Plate IV. = 6 loops from plate III. The plates are now kept at room-temperature (as near 20°-22° C. as possible) for forty-eight hours. The colonies of the bacillus coli communis appear in twenty-four hours, but it is rare to see the typhoid colonies before the end of forty-eight hours. Naked-eye appearances have little or no value. The plates are best examined under the microscope with a Zeiss AA or Leitz No. 3 lens. Seen under these cir- cumstances, at the end of forty-eight hours the colon col- onies appear as larger, rounder, more well defined, coarsely granular, distinctly brown-colored colonies, while the typhoid colonies are much smaller, paler, less sharply defined, very finely granular, and are more often oval than round in shape. 200 PATHOLOGICAL TECHNIQUE. It is, however, very important that the plates should not be too crowded, otherwise the small colonies of the bacillus coli communis may give rise to considerable confusion. The suspicious colonies are now picked up (always under the microscope) and transferred to bouillon and glucose agar-- agar tubes. If, after twenty-four hours in the incubator, there has been no formation of gas on the glucose agar-agar, and the bouillon culture shows, in the hanging drop, an organism resembling in morphology and motility the typhoid bacillus, then the other tests for the typhoid bacillus (see Bacterio- logical Diagnosis) are indicated ; for the appearances alone of the colony upon the potato-gelatin is certainly no more proof of the identity of the bacillus than any other one of the differential tests, unless it be the serum-reaction of Pfeiffer. Capaldi's Culture-medium. Distilled water, loooc.c. ; Pepton (Witte), 20 grams ; Gelatin, 10 Glucose, 10 " Sodium chlorid, 5 " Potassium chlorid, 5 " Make a solution of this composition and filter. To the fil- trate add 20 grams of agar-agar and 10 c.cm. of a normal solution of sodium hydrate (40 grams to lOOO c.c). With this medium Petri plate-cultures are to be made from the stools by first pouring the medium into the plates, allowing it to become solid, and then inoculating the sur- face by gently rubbing the infected platinum loop over it. The loop may be infected either by plunging it into the stool or into a bouillon suspension of the stool. In consequence of this manner of inoculating the plates the colonies all grow on the surface, and thus show more clearly anything charac- teristic in their appearance than if they developed in the depths of the medium. The colonies of the typhoid bacil- lus, when grown upon this medium, are shining, transparent. BACTERIOLOGICAL EXAMINATIONS. 20I almost colorless, and smaller than the opaque grayish-white colonies of the bacillus coli communis. Of course suspected colonies must be shown to be com- posed of bacilli which conform to the various tests described elsewhere before they can be identified with any degree of certainty as those of the typhoid bacillus. The Blood-serum Reaction in Typhoid Fever. — A few drops of the blood of a suspected case of typhoid fever are collected in a small test-tube, either from the finger or the ear. After clotting has taken place, transfer a drop of the serum by means of a medicine-dropper to 0.5 or i.o c.c. of a twenty-four-hour bouillon culture of the typhoid bacillus. After mixing, place a drop of the mixture on a slide, cover it with a cover-glass, and examine it with either an oil-im- mersion lens or a high-power dry lens. If desired, the mix- ture may also be examined as a hanging-drop preparation. If the patient has typhoid fever, the majority of the bacilli on the preparation will be seen to lose their motility and to " agglomerate " into clumps within a very few minutes. This constitutes the serum-reaction. It is only demonstrable, as a rule, after the first week of the disease. The reaction may also be obtained from the dried blood. A few drops of the blood may be collected on a glass slide or a piece of paper and allowed to dry. It may then be brought to the laboratory, where the dried blood is extracted with 15 or 20 drops of water, and the test made with a drop of the solution thus obtained, by mixing it with a drop of an eighteen- to twenty-four-hour bouillon culture of the typhoid bacillus, and observing the mixture with the high- power dry lens after covering with a cover-glass. Rabies (Hydrophobia). — The diagnosis of this disease from a pathological standpoint is usually made by the pro- duction of experimental rabies in a rabbit by intradural in- oculation with material from the nervous system of the ani- mal suspected to have died of it. The poison of the dis- ease is found in the brain, spinal cord, salivary glands, and panci'eas. For purposes of inoculation a piece (i or 2 c.c.) of the medulla or brain, preferably the former, is rubbed up 202 PATHOLOGICAL ILCHXIQCE. in a sterilized mortar with about lo c.c. of sterilized dis- tilled water. The resulting fluid is filtered through absorb- ent cotton, and then through filter-paper, to remove tissue- shreds. Of the clear fluid thus obtained 4 or 5 drops are injected beneath the dura of a rabbit by means of a hypo- dermic syringe, the skull being trephined with a small trephine about 4 mm. in diameter. The most favorable place for opening the skull is at a point in the median line just pos- terior to a line drawn through the middle of each eye. The symptoms of experimental rabies in the rabbit first Fig. 76. — Bacillus of leprosy: section tlirousjh a subcutaneous node, showing the bacilli in tissue-cells; X 500 (Frankel and Pfeiffer). manifest themselves after two weeks, never earlier, but they may not appear until later, not even until two months have passed. The first symptom is a weakness of the hind legs, followed by paralysis. The paretic condition soon extends to the fore legs, dyspnea appears, and death usually occurs in about three days after the onset of the symptoms. Paralytic symptoms developing before two weeks are not due to infec- tion with rabies, but to some other cause ; for instance, in- BACTERIOLOGICAL EXAMINATIONS. 203 fection with the pneumococcus or other bacteria which may be present in the material inoculated. During the course of the disease the animal never appears stupid, with dull eyes, as in other infections, but remains " conscious," so to speak, until the last. I/Cprosy. — The bacillus of this disease shows essentially the same staining reactions as the bacillus tuberculosis. In sections of the lesions the bacilli are found in large numbers, mostly inside the tissue-cells (see Fig. 76). Actinomycosis. — The microscopical diagnosis has been discussed in the section on Bacteriological Diagnosis. PART III. HISTOLOGICAL METHODS. Introduction. — The ideal function of the technique of pathological histology is so to fix tissues for microscopic examination that every tissue-element or pathological prod- uct is perfectly preserved with all its morphological and chemical properties intact, and so to stain tissues that every tissue-element or pathological product can be readily differ- entiated from any other tissue-element or pathological prod- uct that resembles it. In certain respects only has this ideal yet been reached, but the number of differential stains is increasing yearly. In the following pages the various steps in the prepara- tion and staining of tissues have been arranged, so far as possible, in logical sequence. LABORATORY OUTFIT. Microscopes. — The most important laboratory instru- ment is the microscope. It should be, so far as means will permit, the best that skill can produce. The two foreign makes of microscopes most in favor in this country are the Zeiss and the Leitz. Excellent microscopes are also made by Reichart, Hartnack, and Seibert. American microscopes have greatly improved during the past few years, but do not yet reach the standard set by the best foreign makes. Un- doubtedly the best microscopes in every particular and the most expensive are those manufactured by Zeiss. It is important for a beginner in microscopy, before buy- ing a microscope of any make, to have it carefully examined and its lenses tested at a pathological or other laboratory by 204 HISTOLOGICAL METHODS. 20S some one skilled in its use. The continental form of stand of medium size is to be preferred to all others. The large stand is undesirable, because it is too heavy and too high for comfortable use. It should be furnished with rack and pin- ion, and with micrometer screw for coarse and fine adjust- ment, with a triple nose-piece, and with an Abbe condenser and iris diaphragm. The necessary objectives are a low and a high dry, and a ^ oil-immersion. Two eye-pieces, a low and a high, will be found sufficient for all ordinary purposes. The stands, oculars, and objectives generally used are the following : Zeiss : Stands, \\a, IVa, V<[b. Oculars, 2 and 4. Objectives, AA, D, and ^ oil-immersion. Leitz : Stands, la, Id, II, lie. Oculars, i and 3. Objectives, 3, 7, and -^^ oil-immersion. Even if all these different parts cannot be purchased at the same time, it is important to buy a stand to which they afterward may be added, for the list includes only what every medical practitioner should have at his service for the proper examination of urine, sputum, blood, etc. The apochromatic lenses and compensation oculars are too expensive to come into general use. Fortunately, they are more important for photomicrography than for general microscopic work. The oil-immersion lens should always be cleaned after using by wiping off the oil with an old linen or silk hand- kerchief or with the fine tissue-paper now manufactured for that purpose. If the lens is sticky, moisten the cloth with benzol or xylol ; Leitz recommends alcohol. The same process can be used if necessary for the dry lenses, but it must be done quickly, so as not to soften the balsam in which the lenses are imbedded. Ordinarily a dry cloth is sufficient. In using the Abbe illuminating apparatus it is important to bear in mind that the best results are obtained, according to Zeiss, by employing the plain mirror, for the condenser is 206 PATHOLOGICAL TECHNIQUE. designed for parallel rays of light. The concave mirror is to be used only when some near object, such as the window- frame, is reflected into the field of vision or when artificial light is employed. A 'niechanical stage is now made which can be instantly attached to any microscope. It is exceedingly useful for blood-counting or for searching carefully the whole surface of a stained cover-slip. For ordinary work it is undesirable. For microscopic work the best illumination is that obtained from a white cloud. When artificial light is necessary, the Welsbach burner, or, better still, the Edison electric lamp with ground-glass globe, will be found very satisfactory. The slight yellowish tint of the light can be corrected, if neces- sary, by means of a piece of blue glass inserted over the mirror or just below the object to be examined. For drawing, the Abbe camera lucida will be found ex- tremely useful and convenient. Leitz has recently con- structed two new drawing oculars, of which the one to be used with the microscope inclined at an angle of 45 ° would seem to be very practicable. With the other the drawing surface has to be inclined at an angle of 12" to avoid dis- tortion of the image. Freeijing Microtome.— Freezing by means of the evaporation of ether, more rarely of rhigolene, is the method in general use. The process is both expensive and slow. A much cheaper and more rapid method of freezing was originated several years ago in the Sears Pathological Labo- ratory by Dr. S. J. Mixter, and has since been in constant use both here and in the hospitals in Boston. This method consists in the employment of compressed carbon-dioxid, which is found in commerce in iron cylinders containing each about twenty pounds of liquefied gas. It is commonly used for charging beer and soda-water. As a rule, the cylinders are loaned, so that it is necessary to pay for the contents only. The cylinder must be securely fastened in an upright posi- tion near the microtome, with its valve end below and with its escape-tube on a level with the entrance-tube into the HISTOLOGICAL METHODS. 207 freezing-box. The cap covering the escape-tube of the cylinder should have a small hole bored through it, and into this hole a small brass tube about 5 cm. long, with a fine bore, should be tightly driven. This permits the use of a smaller stream of gas than the escape -pipe of the cylmder Fig, 77, — Freezing microtome. would otherwise furnish. The same cap can be kept to use on all future cylinders. The c}'linder is connected with the microtome by means of a short piece of thick, strong rubber tubing with small bore, so as to fit snugly over the escape-tube of the cylinder and the entrance-tube into the freezing-box. Sometimes it is advisable to tie with string each end of the rubber tubing around the tube it encloses. The rubber tubine acts as a 208 PATHOLOGICAL TECHNIQUE. safety-valve in case the pressure of gas should by any acci- dent become too great. In order to obtain better leverage and more perfect con- trol over the escape of the gas than is needed for the pur- poses for which the cylinders are ordinarily used, it is neces- sary to lengthen to about 25 cm., in whatever way seems best, the handle of the key which opens the escape-valve. The first time the cylinder is used for freezing a little water may escape, causing considerable sputtering. It is important that the water should all be gotten rid of In freezing, the valve should be turned carefully, so that the gas may escape slowly and evenly. Pieces of tissue can easily be frozen in a few seconds, but it is much better to freeze the tissue neither too rapidly nor too hard, otherwise the sections will show on microscopic examination multiple parallel lines of fracture Fig. 78. — Knife for freezing microtome, made from the blade of a carpenter's plane. due to their being bent by the knife while still brittle. Ac- cordingly, when the tissue is frozen too hard, it is best to wait until it thaws a little, or else to soften the surface each time just before cutting a section by rubbing over it the tip of a finger moistened in water or salt solution. Pieces of tissue for freezing should not be over 2 to 4 mm. thick. Al- cohol specimens must first be thoroughly freed from spirit by soaking in running water for some time, usually several hours. The freezing-box of the microtome should be stronger than when intended for the use of ether. An excellent microtome designed for the use of carbon-dioxid has recently been put on the market by Bausch & Lomb. For cutting frozen sections the blade of a carpenter's plane mounted in a wooden handle (Fig. 78) will be found very serviceable and easy to sharpen. HISTOLOGICAL METHODS. 209 Celloidin Microtome. — There are two types of celloid- in microtomes — one in which the object is raised by a screw, a second in which the object is raised by being moved up an inchned plane. The first type of machine is the better, for two reasons ; the screw affords greater accuracy in the even elevation of the object than is possible with an inclined plane, and the object remains at all times in the same rela- tive position with regard to the knife, so that an equally long sweep of the blade can be obtained for every section. An excellent instrument of this type is made by Bausch & Lomb (Fig. 79). For practical work it is much to be preferred to Fir.. 79. — Large laboratory microtome. the elaborate Schiefferdecker-Becker microtome, designed for cutting sections under alcohol. A new and wholly original microtome, in which the knife remains fixed and is clamped at both ends, while the object- holder, which is raised by a screw, moves back and forth be- neath the knife, has just been designed by Dr. C. S. Minot and is bemg manufactured by Bausch & Lomb (Fig. 80). It is intended both for celloidin and for paraffin work. When but one instrument can be afforded, it is believed that this model will be found the most serviceable for both kinds of work. A drop-hottlc on an elevated stand, with screw arrange- 14 210 PATHOLOGICAL TECHNIQUE. merit for regulating the amount of alcohol, is the most con- venient method for keeping the object and the knife wet while cutting ; 8o per cent, alcohol should be used. Fig. 8o, — Minof s latest microtome. Paraffin Microtome. — Although paraffin sections can be cut on a celloidin microtome, it is preferable to have an instrument designed for the purpose. The Minot ribbon microtome (Fig. 8i), especially the latest and heaviest model, can be thoroughly recommended. It is manufactured in this country by the Franklin Educational Supply Co., which also furnishes with it an excellent heavy, biconcave knife. Paraffin Bath. — The best bath for keeping paraffin at a constant temperature is a thermostat of suitable size with hot-water jacket, such as is used for growing cultures of bacteria. The paraffin is kept in it on shelves in glass dishes of various sizes. The advantages of this method over the old way of using copper cups set into the top of a water- bath are that the paraffin is kept absolutely free from dust, each worker can have his own set of dishes, and the smallest bits of tissue can be readily found in them, because they are transparent. A paraffin melting at 50^ C. will be found convenient for HISTOLOGICAL METHODS. 211 use throughout the year if the laboratory is kept fairly warm in winter. Many workers, however, prefer one paraffin melt- ing at 45° C. for winter, and another melting at 48° C. for summer. A preliminary bath of soft paraffin is wholly unneces- sary, and only prolongs the objectionable stage of heating. The regulator should register only one or two degrees above the melting-point of the paraffin. Paraffin should be melted and decanted or filtered before use, as it often contains foreign material. When hot it runs Fig. 81. — Minol's paraffin microtome. through an ordinary filter without trouble. A hot-water jacket to the funnel is not at all necessary. Vulcanized Fiber. — For mounting celloidin prepara- tions nothing is so poor as cork, although it has been in use for years. The chief objections to it are that it does not fur- nish a rigid support to the imbedded object ; that, unless weighted, it floats in alcohol with the specimen downward ; and that it j-ields a coloring material which stains both the 212 PATHOLOGICAL TECHNIQUE. alcohol and the specimen. Wood is not much better, although, of course, much firmer. Glass blocks have been proposed, and might do fairly well if there did not exist an ideal substance — viz. vulcanized fiber. This can be obtained in boards or strips, preferably ^ or -| inch in thickness and sawn to any desired dimensions. It is perfectly rigid, is Fig. 82. — Blocks of vulcanized fiber. heavy enough to sink specimens to the bottom of the jar in an upright position, is unaffected by alcohol or water, except that the surface swells and softens very slightly, is light red in color, so that it is easily written on with a lead pencil, gives off no coloring material, and is practically indestructible. Two or three parallel cuts, i to 2 mm. in depth, should Fig. 83. — Diagram of the direction of the movements in honing. be sawn into the upper surface of each block, so as to give the celloidin a firm hold. Knives. — The knives for both the celloidin and the paraffin microtomes should be heavy and not too long, so as to afford as great rigidity as possible ; they should be bicon- cave, so that they may be easily sharpened. It is important that every one who does much work in a pathological labor- HISTOLOGICAL METHODS. 213 atory should learn to sharpen his own knives. The requisite skill is not difficult to acquire, and the time spent in learning is fully compensated for by the ability always to have a sharp knife when it is wanted. For honing a knife either a fine water-stone or a glass plate with diamantine and Vienna chalk may be used. In honing, the edge of the knife is for- ward and the motion is from heel to toe. The knife should always be turned on its back, and the pressure on it should be at all times rather light. In stropping, the movement is reversed. The back of the knife necessarily precedes the edge, and the motion is from Fig. 84. — Diagram of the direction of the movements in stropping. toe to heel. The direction of the movements in honing and stropping is best illustrated by the diagrams (Figs. 83, 84). Running water for washing out specimens which have been fixed in Flemming and other solutions is most easily supplied by having a water-pipe, furnished with numerous cocks 5-10 cm. apart, run horizontally over a slightly slop- ing shelf adjoining the sink. Attached to each cock is a rubber tube, with a glass tube in the end of it long enough to reach to the bottom of the jar (Fig. 85). By this arrange- ment the amount of water supplied to each specimen can be easily regulated. Slides should be of colorless glass with ground edges. The English form, measuring 1X3 inches (76 X 26 mm.), is 214 PATHOLOGICAL TECHNIQUE. to be preferred for ordinary use. Occasionally broader slides are needed. Cover-slips should be square or oblong according to the shape of the specimen. Most dry lenses are adjusted for cover-glasses measuring i6 or 17/^ in thickness, so that if possible no cover-slips ranging outside of 15 to 18// should be used. With an oil-immersion the exact thickness is not quite so important. Slides and cover-slips are cleaned by dipping in alcohol Fig. 85. — Large laboratory sink, showing adjoining slielf and arrangement for running water. and wiping dry with a soft crash towel or old linen handker- chief To clean old slides place them in waste alcohol until the cover-slips can be easily removed. The slides and cover- slips are then treated separately with nitric acid. A 10 per cent, solution is usually sufficient, but occasionally the strong acid will be found necessary. A thorough washing in run- ning water, followed by alcohol, completes the process. Alkalies are not so good for cleaning purposes, because they attack the glass. Staining Dishes. — Watch-glasses are not satisfactory, HISTOLOGICAL METHODS. 215 on account of their instability. Concave dishes with flat bottoms are much better for ordinary use, and can be ob- tained of several patterns. They should be large enough to hold 25 c.c. of fluid. The Syracuse solid zvatch-glasscs are very good dishes of this shape. Stender dislies (Fig. 86) of various sizes will be found use- ful for many purposes. Sectii:)nal view. Fig. -Slender dislies. Oblong rectangular Petri dishes are very convenient for staining preparations mounted on the slide. Steiuach's sieve-dish is valuable where a number of sections are to be stained in the same manner. Large concave dishes holding 100 c.c. will be found the most convenient for holding frozen sections of fresh tissue, because a slide can be dipped into them and under the sections. Metal Instruments. — Spatulas of different sizes are "•muiI^ Fig, 87.— Spatula. needed. The}' should be thin, smooth, and large enough, so that a section will not curl over the edge (Fig. 87). The best instrument for transferinc sections under all cir- 2l6 PATHOLOGICAL TECHNIQUE. cumstances is a piece of platinum wire mounted in an ordi- nary screw needle-holder. It is pliable and can be bent to any shape, will not break like a glass needle when dropped, and is not affected by acids. Ladies' hat-pins form a cheap but serviceable substitute. They are readily bent to any desired shape by heating. Forceps, scissors, scalpels, and many other instruments required in microscopical work do not need any special mention. Bottles. — For cover-slip work and for staining on the slide dropping-bottles will be found extremely convenient. The patent T. K. pattern of 50 c.c. capacity is probably the best form and size. EXAMINATION OF FRESH MATERIAL. Fresh tissues may be examined either in teased prepara- tions or in sections. Teased preparations are made by cutting out a very small bit of the tissue in question and dividing it as finely as pos- sible, by means of two sharp, clean needles, on a slide in a drop or two of some indifferent fluid, such as the normal salt solution. Teased preparations are often made, for in- stance, of the heart-muscle when fatty degeneration is sus- pected. If the tissue is soft, the cells are easily obtained by simply scraping the cut surface with the edge of the knife. Sections of fresh tissues can be made with a razor or with a double knife, but much the better way, at least for general diagnostic purposes, is to use frozen sections, which can be very quickly and perfectly made with the freezing microtome. The fresh sections are put into salt solution in a glass dish large enough to permit of a slide being dipped into it, so that a section can be floated and spread out evenly on its surface. The slide is then carefully raised, the excess of fluid wiped off, and a cover-slip put on. If it is desired to stain the section, a few drops of Loffler's methylene-blue solution are poured over it after it is spread evenly on the sHde. In a few seconds the coloring fluid is HISTOLOGICAL METHODS. 21/ thoroughly washed off with salt solution, a cover-slip put on, and the section examined in the salt solution. If sections of fresh tissues are put directly into a staining fluid in the ordi- nary manner, they pucker up and do not stain evenly. Fresh preparations are often treated with chemicals for various purposes. Of these chemicals, acetic acid is the most generally useful in pathological work. It shrinks the nuclei and renders their outlines more distinct. It swells connective tissue, making it more transparent, so that the elastic fibers which are unaffected Stand out distinctly. It precipitates mucin and dissolves or renders invisible the al- buminous granules so abundantly present in the protoplasm in the cloudy swelling of various organs in disease. Its main use as a reagent for fresh tissues is to demonstrate fat and to differentiate that substance from albuminous gran- ules. Acetic acid is ordinarily used in a i to 2 per cent, aqueous solution, a few drops of which are placed at one edge of the cover-slip, and then drawn beneath it by placing a piece of filter-paper on the opposite side. If in a hurry, however, stronger solutions, or even glacial acetic acid, may be used. Other reagents are of less importance, but are occasionally used. Osmic acid is sometimes employed in a i per cent, aque- ous solution to demonstrate fat, which it stains brown to black. Hydrochloric acid in a 3 to 5 per cent, solution is used to demonstrate calcification. Phosphate of lime is simply dis- solved, while from carbonate of lime bubbles of carbon- dioxid (CO2) are set free. Indiflferent Fluids. — Fresh tissues are usually examined in norrrial salt solution, a -^-^ per cent, solution of common salt in water. It has the advantage over water that tissues do not swell up so much in it, blood-globules are unaffected, and the finer structures are better preserved. A very few drops of Lugol's solution added to the stock-bottle of salt solution will be found useful in preventing the growth of mould. 2l8 PATHOLOGICAL TECHNIQUE. Serous fluids, such as hydrocele fluid, are occasionally used. Artificial serum is made by adding i part of egg- albumin to 9 parts of normal salt solution. Macerating fluids are little used in pathology. Occa- sionally, however, when tissues are tough, so that they cannot be readily teased apart, they are macerated in certain fluids which dissolve the substances that hold the different elements together. The reagents most commonly used are the fol- lowing : 1. Ranvier's one-third alcohol is made by taking i part of 96 per cent, alcohol and 2 parts of water ; twenty-four hours are usually enough. 2. Very dilute solutions of chromic acid are recommended — TW to ^ of I per cent. 3. ^3 per cent. Caustic Potash. — Tissues are macerated in a few minutes to one hour : they must be examined in the same fluid, because the cells are destroyed if the solution is weakened. iExaminatiou of Fluids. — Small fragments of tissue should be picked out with forceps. If much blood is ad- herent, wash the tissue well in salt solution. When the cell- ular elements are few in number they are obtained with a pipette, just as in urine- work, after allowing them to settle at the bottom of the glass. A centrifugal machine will be found of great service when the sediment is slight. INJECTIONS. Injections are not much used in pathology. The process is an art that requires much patience and considerable ex- perience. The purpose of an injection is to render vessels and vessel-walls more visible than under ordinary circum- stances. Transparent, deeply-colored fluid mixtures are used which will become hard in the vessels. Some injec- tion-masses are employed cold, others warm. The warm injection-masses contain gelatin, and are much more trouble- some to use, but give much the more perfect results. For HISTOLOGICAL METHODS. 219 coloring the mass carmine is the best material, because it is a permanent color. The instruments required are cannulas of various sizes and a syringe, or, better still, a constant-pressure apparatus. When a warm injection-mass is used the bottle containing the mass must be placed in a water-bath and kept at a tem- perature of about 45° C. The organ or aninial to be in- jected must hkewise be placed in a water-bath of the same temperature. It is very important that in connecting the end of the tube carrying the injection-mass with the cannula inserted in the vessel no air-bubbles shall enter. When blood-vessels are to be injected it is advisable to wash them out first with normal salt solution. Cold Injection-masses. — i. Blue Coloring Mass. — Soluble Berlin blue, I ; Distilled water, 20. 2. Carmine Injection-mass (Kollmann). — Dissolve i gram of carmine in i c.c. of strong ammonia plus a little water ; dilute with 20 c.c. of glycerin. To this solution add i gram of common salt (NaCl) dissolved in 30 c.c. of glycerin. To the whole solution add an equal quantity of water. Warm Injection-masses. — i. Berlin Blue. — Warm the solution of Berlin blue given above, and add it, with con- tinual stirHng, to an equal quantity of a warm, concentrated solution of gelatin prepared as follows : Allow clean sheets of the best French gelatin to swell up for one to two hours at room-temperature in double the quantity of water. Then dissolve them by warming gently over a water-bath. Filter the combined solution through flannel. 2. Carmine-gelatin Mass. — This is by all means the best injection-mass to use, because it is permanent, but it is very difficult to prepare. Dissolve 2 to 2.5 grams of best carmine in about 15 c.c. of water, to which just enough ammonia is added, drop by drop, to effect the solution. Filter the fluid obtained, and 220 PATHOLOGICAL TECHNIQUE. " add it, with continual stirring, to a filtered warm, concen- trated solution of gelatin (prepared as above) over the water- bath. Then add acetic acid slowly until the color changes to a bright-red shade. The exact amount desired is when the solution loses its ammoniacal odor and has a peculiar sweetish aroma free from acid. Examined under the micro- scope, no granular precipitate of carmine should appear. If too much acetic acid has been added, so that the carmine is precipitated, the mass must be thrown away and a new lot prepared. Organs which have been injected with a cold mass are placed directly in 80 per cent, alcohol. After a few hours they are to be cut up into pieces that are not too small. After a warm injection-mass the organ or animal is placed first in cold water to hasten the solidification of the gelatin, and then transferred to 80 per cent, alcohol. Masses already prepared for injecting cold or warm can be obtained from Gruebler. FIXING REAGENTS. The various reagents used for the preservation of fresh tissues possess the properties of penetrating, killing, fixing, hardening, and preserving in different degrees. Of these properties " fixing " is the most important, and to a certain extent implies or includes the others. The term " fixative " has been used more particularly, perhaps, for reagents which preserve faithfully the various changes of the nucleus in karyomitosis. In a broader sense, however, it refers to the faithful preservation of any tissue-element or pathological product, and of the chemical properties peculiar to that ele- ment or product. A good fixative is a reagent that pene- trates and kills tissues quickly, preserves the tissue-elements, and particularly the nuclei, faithfully in the condition in which they are at the moment when the reagent acts on them, and hardens or so affects them that they will, not be altered by the various after-steps of staining, clearing, and HISTOLOGICAL METHODS. 221 mounting. Most fixatives are mixtures of different reagents so combined that all the desirable properties may be present in as large a degree as possible. The choice of the proper fixing reagent for a given tissue is often difficult, and must depend largely on the nature of the pathological lesions present or suspected, and on the purposes for which the tissue is preserved. For diag- nosis, for general bacteriological study of tissues, and for many valuable and important chemical reactions alcohol is to be preferred to any other reagent. Although by no means an ideal fixative, it will be found exceedingly useful. For finer histological study it is important to preserve val- uable tissue in some more perfect fixative than alcohol. Zenker's and to some extent Orth's fluid will be found the most generally useful. Flemming's solution is especially to be recommended for the study bf renal lesions with fatty degeneration. For general pathological work, aside from the nervous system, these four fixatives will be found the most valuable. It is strongly advised that pieces of tissue in all important cases be hardened both in alcohol and in Zenker's fluid — in alcohol for bacteria and for chemical reactions ; in the other for bacteria, nuclear figures, blood, and general histo- logical study. For special investigations other fixatives are sometimes desirable. Tissues fixed in alcqhol or in a solution of formaldehyde may remain as long as desirable in those fluids. Tissues hardened in most of the other fixatives must be transferred, after thorough washing in water, to alcohol for preservation. It is usually recommended to pass the specimens through graded alcohols, either through 30, 60, 90, and 96 per cent, or through 50, 70, and 96 per cent, allowing them to remain from a few hours to a day in each strength. For most purposes it will be found sufficient to transfer the specimens directly from water to alcohol of 70 to 80 per cent., in which they may remain until it is desired to imbed them. 222 PATHOLOGICAL TECHNIQUE. Alcohol extracts chrome salts from tissues hardened in solutions of them. As these salts are precipitated in the alcohol under the action of light, it is desirable, although by no means necessary, to keep all such specimens in the dark. Alcohol. — The strength of alcohol ordinarily used in laboratories is 95-96 per cent. Absolute alcohol is much more expensive. Tissues hardened in either of these strengths shrink a great deal. The exposed surface becomes ex- tremely hard, and the outer layers of the cells of tissues like a rabbit's kidney, for example, are as shrunken and flattened as though dried in the air. It is only inside of this hard casing, where the alcohol has penetrated more slowly and has been somewhat diluted by the fluid of the tissue, that the cells are better preserved. Moreover, this extreme hardening of the surface hinders the penetration of the alco- hol into the deeper parts. Tissue which is to be hardened in absolute or 95 per cent, alcohol should be cut into thin pieces, preferably not over ^ cm. thick. The volume of alcohol used for hardening should be fifteen to twenty times as great as the specimen, and should be changed after three or four hours. The tissue should be kept in the upper part of the alcohol by means of absorbent cotton, or the jar may be frequently inverted and the alcohol thus kept of even strength. The advantages of strong alcohol, 95 per cent, and abso- lute, are that the tissue is more qyickly fixed than with weaker strength, and that at the same time it is made quite hard — a quality more necessary formerly than now when tis- sues are so generally imbedded. Tissues hardened in strong alcohol should later be transferred to 80 per cent, alcohol for preservation or the staining properties will gradually become impaired. For general purposes it will be found better to place tissues at first into 80 per cent, alcohol, which should be replaced in two to four hours by 95 per cent, alcohol. In this way less shrinkage is caused and the surface of the tissues is not made so hard. HISTOLOGICAL METHODS. 223 Zenker's Fluid. — Bichromate of potassium, 2.5 grams ; Sulphate of sodium, i Corrosive sublimate, 5 Glacial acetic acid, 5 c.c. ; Water, ad 100 The solution is practically Miiller's fluid saturated with corrosive sublimate, plus 5 per cent, of glacial acetic acid. It is advisable not to add the acetic acid to the stock solu- tion, but only in the proper proportion to the part taken for hardening pieces of tissue, because the acid evaporates so readily. Directions for Use. — i. Fix tissues in the solution one to twenty-four hours, rarely forty-eight hours, according to thickness. 2. Wash in running water twelve to twenty-four hours. 3. Preserve in 80 per cent, alcohol until used. Tissues float at first in this solution, which will be found a most admirable general fixative. It penetrates quickly, so that pieces of tissue do not need to be so thin as with most other fixatives, but it is advisable not to let them exceed J cm. in thickness. Nuclear figures, red blood-globules, and protoplasm are all perfectly preserved. The greatest draw- back to the fluid is the precipitation of mercury which takes place to a varying degree in the tissues. This precipitation may be removed by adding a little tincture of iodin (up to \ per cent.) to the alcohol in which the specimens are pre- served. As soon as the color of the iodin disappears, on account of its forming a colorless, soluble compound with mercury, more iodin must be added until the alcohol re- mains stained faintly yellow. It will sometimes be found difficult to get rid of all the deposit even after fairly pro- longed treatment (two weeks) with iodin in this manner, so that it is sometimes necessary to treat sections, after cutting, with Lugol's solution for a short time. Sometimes, indeed, it is preferable to reserve the treatment with iodin for the sections only. 224 PATHOLOGICAL TECHNIQUE. Zenker preparations stain slowly but beautifully in alum- hematoxylin. Excellent results can also be obtained with eosin, followed by Unna's alkaline methylene-blue solution. Fuchsin and safranin stains are sometimes useful. Ortli's Fluid. — Recently Orth has highly recommended as a general fixative a solution consisting of the well-known Miiller's fluid plus 4 per cent, of formaldehyde : Bichromate of potassium, 2 to 2.5 ; Sulphate of sodium, i ; Water, lOO; Formaldehyde (40 per cent, solution), 10. The formaldehyde should be added only at the time of using, for in two days the solution becomes darker, and by the fourth day a crystalline deposit begins to take place. As fixation is ordinarily complete in three to four days, this deposit does not matter. The tissue should not be over i cm. in thickness. Small pieces \^o \ cm. in .thickness can be readily hardened in the incubator in three hours. The specimens should be washed thoroughly in running water six to twenty-four hours before placing in 80 per cent, alcohol. The method is particularly recommended for mitosis, red blood-globules, bone, and colloid material (in cystomata, etc.), as it gives a very good consistency to the tissues. Flemming's Solution. — Osmic acid, 2 per cent, aqueous solution, 4; Chromic acid, i per cent, aqueous solution, 1 5 ; Glacial acetic acid, i. I. Fix in the solution one to three days. 2. Wash in running water six to twenty-four hours. 3. Alcohol, 80 per cent. It is best to keep the osmic acid in a 2 per cent, solution, and the chromic acid in a i per cent, solution. The mixture can then be quickly made up fresh at the time it is needed. The best stains after hardening in Flemming are Babes' saf- ranin, aniline-gentian-violet, and carbol-fuchsin. Pieces of tissue for hardening in Flemming's solution should not be over 2 mm. in thickness, because it has very slight penetrating properties. HISTOLOGICAL METHODS. 22$ Hermann's Solution. — Osmic acid, 2 per cent, aqueous solution, 4 ; Platinic chlorid, i per cent, aqueous solution, 15 ; Glacial acetic acid. i. — -— — - — — — 7 - f- Glacial acetic acid, This modification of Flemming's solution is, perhaps an even better fixative than the model on which it is based, but is more expensive. It should be employed in the same manner. Pianese's Solution. — Chlorid of platinum and sodium, i per cent. aqueous solution (platinic), 15 c.c. Chromic acid, \ per cent, aqueous solution, 5 " Osmic acid, 2 per cent, aqueous solution, , 5 " Formic acid, C. P. i drop. Fix small pieces of tissue, not over 2 mm. thick, in the solu- tion for thirty-six hours. Wash in running water for twelve hours, then 80 per cent, alcohol. Stain paraffin sections by Pianese's special methods (see p. 250). This fixative and the special staining methods are particu- larly recommended for the study of karyomitosis and of the so-called cancer bodies. Rabl's Clironio-fomiic Acid Solution. — Chromic acid, 0.33 per cent, aqueous solution, 200 ; Formic acid, 4 to 5 drops, to be added just before the solution is used. Directions for Use. — i. Harden in the fixing solution twelve to twenty-four hours ; 2. Wash in running water twelve to twenty-four hours ; 3. Dehydrate in 80 per cent, alcohol. Rabl used after this fixative a very faint stain with hema- toxylin, followed by safranin. Corrosive Sublimate. — Use a saturated solution (made by heat) in normal salt solution. The addition of 5 per cent, of glacial acetic acid is sometimes advisable, i. Harden thin pieces of tissue (2 to 5 mm.) for one to six hours ; 2. Wash in running water twenty-four hours ; 3. Preserve in 80 per cent, alcohol. 15 226 PATHOLOGICAL TECHNIQUE. An even better method is, perhaps, to transfer the tissue directly from the corrosive-sublimate solution to 70 per cent, alcohol, to which enough tincture of ipdin is added to give it a light-red color. When the color due to the iodin dis- appears more of the tincture must be added until a Ught color of iodin becomes permanent. Tissues hardened in corrosive stain quickly and brilliantly in nearly all staining solutions. It is the only fixative after which the Heidenhain-Biondi triple stain gives good results. Formaldehyde. — The gas formaldehyde (HCOH) is soluble in water to the extent of 40 per cent. Solutions of this strength are manufactured by different commercial houses under the names of formaline, formol, and formalose. The best strength of formaldehyde to use for fixing tissues is a 4 per cent, solution ; that is, 10 parts of the aqueous 40 per cent, solution, no matter what name is given to it, to 90 parts of water — or, better still, perhaps, of normal salt solution. This new fixing reagent penetrates very quickly. Its hardening action is not understood. It does not precipitate albuminous bodies, but makes them quite firm. It also hardens nerve-sheaths, acting toward them and red globules like the chrome salts. Formaldehyde is very useful for pre- serving gross specimens, because it gives them a rather tough, elastic consistency, and preserves the normal colors better than other hardening fluids, and also the transparency of many parts, such as the cornea. In histological work it has been found most useful, so far, for the preservation of nervous tissue. Sections of ordinary tissues not over i cm. thick are hardened in twenty-four hours. They may then be trans- ferred to alcohol, or may remain indefinitely in the formal- dehyde. Large pieces require more time. Formaldehyde is much used for hardening tissues quickly, so that frozen sections can be cut and permanent preparations stained and mounted in the course of a few hours. Thin pieces are suf- ficiently hardened in one to three hours. They may be frozen directly in the formaldehyde solution or in water. HISTOLOGICAL METHODS. 22/ Almost any stain is applicable after the sections have been washed in water and treated for a short time with alcohol. Although formaldehyde is an excellent fixative from the point of view of killing quickly, it does not seem to pre- serve tissue-elements so that they will not later undergo changes when treated with various reagents. For this reason it is best combined with some more purely hardening re- agent, as, for example, bichromate of potassium in Orth's solution. Tissues such as muscle or the contents of a mul- tilocular cystoma which have been made very hard by the fixing reagent used, can be softened, even when mounted in celloidin, so that they will cut perfectly, by placing in a 4 per cent, solution of formaldehyde for twenty-four hours. Boiling. — Boiling precipitates the soluble albumin in tissues as a granular material which can be readily recog- nized. The method is used particularly for the demonstra- tion of albumin in renal diseases and in edema of the lungs. By means of boiling the quickest permanent mounts of tissues can be obtained. The method is not advocated, on account of the shrinkage caused by the heat, but will some- times be found useful. Small pieces of tissue not over 1.5 cm. in diameter should be dropped into the boiling water for one half to two minutes ; cool quickly in cold water and make frozen sec- tions, or put into 80 per cent, alcohol. Any stain can be used; methylene-blue will be found excellent. Miiller's Fluid.— Bichromate of potassium, 2 to 2.5 grams ; Sulphate of sodium, i " Water, 1 00 c.c. Harden tissues six to eight weeks. Change the fluid daily during the first week ; once a week thereafter. Ordinary tissues are then washed in running water over night before being placed in alcohol. Nervous tissue is transferred directly from the fluid to the alcohol. This famous hardening solution seems destined before long to give way entirely to better fixatives. It hardens tissues slowly, evenly, and with little or no shrinkage, but it 228 PATHOLOGICAL TECHNIQUE. is a poor nuclear fixative, and does not encourage any great variety of stains. For ordinary tissues it will undoubtedly be replaced by Zenker's or Orth's fluid, both of which fix very quickly, besides having all its good qualities. For nervous tissues formaldehyde followed by other solutions of the chrome salts is a great deal quicker and better. MarcM's Fluid. — Muller's fluid, 2 parts ; Osmic acid, i per cent, aqueous solution, i part. Place small pieces of tissue in the mixture for five to eight days, wash thoroughly in running water, and harden in alcohol. For its application to degenerated nerve-fibers see page 325. Brlicki's Fluid.— Bichromate of potassium, 2.5 grams ; Sulphate of copper, i. Water, 100 c.c. Hardening is quicker than with Muller's fluid, requiring eight to ten days, otherwise the treatment is the same. DECALCIFICATION. Tissues which are to be decalcified should be sawn with a fine hair-saw into thin slices, so that they will decalcify quickly. It is usually desirable to saw the tissue into pieces of proper size for imbedding in celloidin. Very dense bone ought not to be over 2 or 3 mm. thick ; softer tissues do not need to be thinner than 4 to 6 mm. In cutting sections after decalcifying and imbedding it is necessary to throw away the first half-dozen sections or so, because the tissue is so lace- rated to a slight depth by the movement of small fragments of bone in the process of sawing as to be useless for micro- scopic purposes. The extent of the decalcification can be tested at any time by thrusting a needle into the tissue. The following steps in the decalcification of tissues must be carefully borne in mind : HISTOLOGICAL METHODS. 229 1. The tissues must first be thoroughly hardened. The three most usefiil reagents for this purpore are alcohol, Zenker's and Orth's fluids. After the two latter reagents the tissues must have been washed thoroughly in water and placed in alcohol for at least twenty-four hours. They will then be ready for decalcification. 2. The decalcifying fluid must be used in large amounts, and if necessary be frequently changed. 3. After decalcification the tissues must be thoroughly washed in running water for twenty-four hours to get rid of every trace of the acid. 4. The tissues finally must be hardened again in alcohol. Of the various agents used for decalcifying bone, ni- •tric, hydrochloric, chromic, picric, trichloracetic acids, etc., the most important is nitric acid. It acts quickly, without swelling the tissues or attacking injuriously the tissue-ele- ments, and does not interfere to any marked degree with any subsequent staining process. Red blood-globules will be found uninjured in tissues hardened in Zenker's fluid even after remaining four days in 5 per cent, nitric acid. This acid is used in dilute solution alone or in combination with phloroglucin. Directions for Using Nitric Acid. — i. Decalcify in large quantities of a 5 per cent, aqueous solution of nitric acid, changing the solution every day for one to four days. 2. Wash twenty-four hours in running water to remove every trace of acid. 3. Harden in 80 per cent., and then 95 per cent., alcohol. Imbed in celloidin. Phloroglucin and Nitric Acid. — Phloroglucin is not a decalcifying agent, but is added to nitric acid to protect the tissues while allowing a stronger solution of the acid to be used than would otherwise be possible. The solution is prepared by dissolving i gram of phloroglucin in 10 c.c. of nitric acid. Solution takes place quickly, with generation of considerable heat. The fluid is reddish brown at first, but becomes light yellow in the course of twenty-four hours. Dilute with 100 c.c. of a 10 per cent, solution of nitric acid. This gives nearly a 20 per cent, solution of nitric acid. The 230 PATHOLOGICAL TECHNIQUE. process of decalcification in this fluid is extremely rapid ; a few hours only, as a rule, are required. It is not advisable to dilute the solution by the simple addition of water, but by the use of less acid, because the phloroglucin must be present to the amount of i per cent, or it will not protect the tissues so well. The following slower-acting solution may be found useful : Phloroglucin, I ; Nitric acid, 5 J Alcohol, 70 ; Water, 30. A rather deep single stain with alum-hematoxylin (either aqueous solution or Delafield's) will usually be found to give the best results with tissues decalcified with nitric acid. It is very important to leave the sections after staining in a large dish of water over-night, otherwise the stain will not be so sharp and clear. Picric Acid. — A saturated aqueous solution containing an excess of crystals is sometimes used for decalcifying. It has no injurious action on tissues, but is extremely slow, fre- quently requiring months. Fresh tissues may be placed directly in the solution, which hardens and decalcifies at the same time. Instead of being washed out in water, in which they would macerate, the pieces of tissue are placed directly in 70 per cent, alcohol to remove the acid. Trichloracetic Acid. — A 5 per cent, solution of this acid has lately been recommended for the decalcification of bone and teeth. It acts more slowly than nitric acid, and seems to possess no advantages over it. Tissues must be washed out in running water, as after nitric acid. IMBEDDINQ PROCESSES. Sections of hardened tissues can be cut with a razor by hand, or with a microtome knife after fastening the specimen in the microtome clamp either directly or between pieces of amyloid liver. Fair sections of firm tissues can often be ob- HISTOLOGICAL METHODS. 23 1 tained in this way. Thinner sections can be gotten by means of the freezing microtome, but these methods are all open to the objection that unless the tissue is very cohesive por- tions of it are likely to fall out of the sections. The best results would, therefore, naturally be expected from some imbedding process, employing a substance to in-r filtrate the tissues thoroughly and to hold the different parts in proper relative position even in the thinnest sections. The two substances in common use for this purpose are celloidin and paraffin. Each has its advantages and disad- vantages. Neither can be employed in pathological histology to the exclusion of the other. Paraffin affords the thinnest sections, but they must be small if the best results are de- sired, and cannot be properly handled except when fastened to the slide. Hard tissues like muscle, and tissues of vary- ing consistency like skin, are cut with great difficulty by the paraffin method. Staining is rather simpler than after im- bedding in celloidin. On the other hand, tissues of almost any consistency or size can be cut by the celloidin method, which is also capable of furnishing very thin sections. Both methods of imbedding should be learned and used. Celloidin sections are especially good for general work, for studying the extent and relations of pathological processes, and for much of the finer histological work. Paraffin sec- tions are better for the finest details of processes — for special work on special tissues. Celloidin. — Schering's celloidin is the best preparation of gun-cotton (pyroxylin) to use. It is sold now in a con- venient granular form in small bottles. It keeps well, dis- solves somewhat slowly, and gives a fairly transparent im- bedding mass which is firm and tough, so that very thin sections can be cut. Other forms of gun-cotton are not so reliable ; they often contain impurities and do not yield so firm an imbedding mass. Imbedding in Celloidin.— The process consists in soaking the tissues for twenty-four hours to a number of days in two different solutions of celloidin. The two solu- 232 PATHOLOGICAL TECHNIQUE. tions are spoken of as thin and thick celloidin. To make thick celloidin 30 grams of the dry celloidin are dissolved in 500 c.c. of a mixture of equal parts of ether and absolute or 95 per cent, alcohol. This gives a 6 per cent, solution. Diluted with an equal amount of the ether-and-alcohol mix- ture, it forms thin celloidin. The steps of the- imbedding process are as follows : Pieces of tissue which have been properly fixed and finally pre- served in 80 per cent, alcohol are first to be cut up with in- telligence. They should rarely be over 4 to 8 mm. thick ; for most purposes 2 mm. will be found sufficient. Pieces of this thickness will furnish several hundred sections, will im- bed more quickly than larger masses, and will be more rigid when mounted on a block. They should never be broader or longer than is necessary to show the whole of the process under study. Very thin celloidin sections cannot usually be obtained with tissues over i^ to 2 cm. square, and smaller dimensions are preferable. Beginners usually imbed larger pieces than are necessary. The trimmed pieces of tissue are first hardened and de- hydrated for twenty-four hours in 95 per cent, alcohol ; then soaked in equal parts of alcohol and ether for the same length of time to prepare them for the thin celloidin. In the latter they remain at least twenty-four hours, preferably for a number of days if at all thick, for in this solution occurs most of the infiltration with celloidin. Finally, the pieces are soaked twenty-four hours or more in the thick celloidin. They are then mounted on blocks of vulcanized fiber, ex- posed to the air for two or three minutes till the surface hardens a little, and placed in 80 per cent, alcohol for six to twenty-four hours to allow the celloidin to harden. Briefly summed up, the steps of imbedding in celloidin are as follows : 1. 95 per cent, or absolute alcohol, twenty-four hours. 2. Ether and 95 per cent, or absolute alcohol, aa. twenty- four hours. 3. Thin celloidin, twenty-four hours to one or more weeks. 4. Thick celloidin, twenty-four hours to one or more weeks. HISTOLOGICAL METHODS. 233 5. Mount on blocks of vulcanized fiber: dry a minute or two in the air. 6. Harden celloidin in 80 per cent, alcohol, six to twenty- four hours. Although absolute alcohol is perhaps preferable for dehy- dration and for mixing with the ether in the above mixtures, 95 per cent, alcohol will be found to answer all requirements. The second step may be omitted, especially after dehydration in absolute alcohol, if time is pressing. Instead of mounting directly from the thick celloidin, it is often advisable to allow the celloidin to evaporate until a firm mass is obtained. This is particularly true when very loose tissues are to be imbedded. The simplest method is to place the pieces of tissue, which have been soaking in thick celloidin, in proper position in a glass dish and pour thick celloidin over them. The dish is then covered, but not too tightly, and the ether is allowed to evaporate for one or more days until the proper consistency of celloidin is reached, so that it can be cut out in blocks enclosing the specimens. If the ether evaporates too rapidly, place a large dish or a bell-jar over the covered dish. Mount the blocks, after they have been cut out and trimmed, by dipping the bases in thick celloidin and then pressing them on to blocks of vulcanized fiber. In two or three minutes they can be placed in 80 per cent, alcohol. After the cel- loidin mounts have been in 80 per cent, alcohol for six to twenty-four hours the celloidin is of the proper consistency for cutting. It is best to take a sharp knife or an old razor and trim the top of the celloidin down to where the first good section of the specimen can be cut ; this will save con- siderable wear on the microtome knife. In cutting, the microtome knife should be fastened very obliquely, so that as much of the edge of the knife as pos- sible shall be used in making each section. The surface of the knife should be kept well wet with 80 per cent, alcohol, preferably from an overhanging drop-bottle. If the sections curl, as often happens when they are thin, they are best flattened by unrolling them on to the surface 234 PATHOLOGICAL TECHNIQUE. of the knife with a camel's-hair brush just before the last edge of celloidin is cut through, as this serves to keep them fixed in place during the process. This method can be used when the simple transferring of sections from alcohol to water is not sufficient to uncurl them. Celloidin sections can be stained by nearly all methods, without the necessity of removing the celloidin. When necessary, however, the celloidin is readily removed by pla- cing the sections from absolute alcohol in oil of cloves or in the alcohol-and-ether mixture for five or ten minutes, and then passing them back through absolute into ordinary alcohol. To Attach Celloidin Sections to the Slide. — A celloidin sec- tion can be fairly well attached to a slide by transferring it from water to a slide freshly washed in alcohol and dried with a doth. The section is then to be firmly blotted with filter-paper so as to apply it closely to the slide and to re- move all wrinkles. It should not be allowed to dry. A section treated in this way will ordinarily stand considerable manipulation without becoming loose. Celloidin sections can be more securely attached by trans- ferring them from 95 per cent, alcohol to clean slides and pouring over them ether-vapor from a bottle half full of ether. With a little practice sections can be fastened in a few seconds. Follow slowly along the edge of the celloidin, and the frills in it will soften down. Then wash the speci- men with 80 per cent, alcohol to harden the celloidin. Imbedding in Paraffin. — Paraffin imbedding is particu- larly useful when very thin sections are desired. To obtain the best results the pieces of tissue should be small, soft, and of uniform consistency. In pathological work it is much better to cut the sections and to stain them after they are fastened to the slide than to stain in the mass beforehand, because then a variety of stains may be used. A complete or perfect series is not so important as in embryology, but with a little care can be obtained. The first step in the preparation of hardened tissues for the paraffin bath is to cut them into small, thin square or HISTOLOGICAL METHODS. 235 rectangular pieces, not over i cm. square, perhaps, for the best results, and not over 2 to 3 mm. thick. It should be stated, however, that with proper skill, a heavy, sharp knife, and a rigid microtome very thin paraffin sections can be ob- tained with tissues measuring 4X3 cm. The pieces of tissue are then thoroughly dehydrated by soaking first in 95 per cent, and then in absolute alcohol. From alcohol they are put in some substance, such as chloroform or oil of cedar, which has the property of mixing with alcohol and of dissolving paraffin. From the chloroform they are trans- ferred to a saturated solution of paraiifin in chloroform, and then passed through two or three separate baths of the melted paraffin to get rid of every trace of the chloroform. If oil of cedar is used, the specimens are transferred directly from it into the melted paraffin. One advantage of the chloroform method is that the dura- tion in the hot paraffin, the objectionable feature of the paraffin method, is shortened, because the tissues are already somewhat infiltrated with paraffin. Another advantage is that the paraffin bath purifies itself, because the chloroform rapidly evaporates. When oil of cedar is used the paraffin must be renewed frequently. The methods of imbedding in paraffin are briefly stated as follows : Method No. i. 1. 95 per cent, alcohol, 2. Absolute alcohol, 3. Chloroform, 4. Chloroform saturated with paraffin, 5. Paraffin bath, three changes, 6. Imbed and cool quickly in cold water. Method No. 2. 1. 95 per cent, alcohol, 2. Absolute alcohol, 3. Oil of cedar, two changes, 4. Paraffin, three changes, till no odor of oil of cedar. 5. Imbed and cool quickly in cold water. 6-24 hours. 6-24 " 6-24 " 6-24 I- 6 " 6-24 hours. 6-24 " 6-24 •' 236 PATHOLOGICAL TECHNIQUE. In the second method other substances than oil of cedar can be used, such as xylol, equal parts of oil of cloves and turpentine, or oil of cloves and xylol. For imbedding paraffin specimens metallic boxes can be used, or forms made round or square from strips of sheet lead or tin. Many prefer paper boxes, which can be made easily of any size desired from stiff writing-paper. Melted paraffin is poured into the paper box to the depth of about I cm. The pieces of tissue are then placed in the box with that side down from which sections are preferred. When all of the pieces are arranged in order with about half a centimeter or more between them, the box is placed on the surface of a large dish of cold water, on which it floats, so that the paraffin may cool quickly without crystallizing. After the paraffin has hardened the paper is removed and the paraffin is divided up according to the pieces in it. One of the blocks is then fastened to the object-holder by heating the latter in a flame until it will just melt the paraffin when the block is held in proper position against it. The holder is then quickly cooled in cold water. The upper surface of the paraffin should now be shaved down to the specimen. The four sides are to be carefully trimmed as close as possible to the specimen, but care must be taken to preserve always a perfect rectangular parallelo- gram. The holder is finally carefully adjusted in the paraffin microtome. To get good sections which will adhere to each other and form a ribbon the temperature of the room must be regulated to suit the degree of hardness of the paraffin used. An open window will often make all the difference needed to obtain good results. The harder the paraffin the warmer the room must be. The temperature can be raised by burning a Bun- sen flame near the niicrotome or lowered by the presence of a lump of ice. The ribbons of sections as cut, usually a slideful, are laid on the surface of a large dish of warm water at about 44° C, and if necessary gently stretched so as to remove all wrinkles. Paint the surface of a slide with a thin layer HISTOLOGICAL METHODS. 237 of Mayer's glycerin-albumin mixture, wipe off all excess with a towel so that only a faint layer is left, dip the slide under the sections, arrange them in order, lift the slide, and drain off the water. The slide is then placed in a slanting position until dry, when it is put in the incubator for two to twelve hours at a temperature of 54° to 60° C. The heat coagu- lates the albumin, which thus attaches the sections firmly to the slide. To get rid of the paraffin in the sections they are treated with two or three changes of xylol, and then with absolute followed by 95 per cent, alcohol. If for any reason the celloidin-and-oil-of-cloves mixture is used, the paraffin is removed by means of xylol, followed by origanum or bergamot oil, and finally by 95 per cent, alco- hol, because absolute alcohol will dissolve the celloidin. Serial Sections by the Celloidin Method. — i. For Tissues in General. — With a little care perfect serial sections can be made by the following method, and each slide of sec- tions can be stained in whatever way seems best. The specimen is imbedded, mounted on vulcanized fiber, and hardened in 80 per cent, alcohol in the usual way. In cut- ting moisten the microtome knife with 95 per cent, alcohol. As the sections are cut they are drawn up on the surface of the knife and arranged in regular order by means of a camel's- hair brush until a slideful is ready. They are then drawn on a clean and numbered slide held against the back of the knife. After being carefully arranged the sections are fast- ened to the slide by means of ether-vapor (see p. 234) poured over them from a half-full bottle. Care must be taken that every edge of the celloidin is fully softened down. The slides are then placed in a jar of 80 per cent, alcohol to be stained at leisure. 2. Another method, often convenient where the stain is of little importance, is as follows : The tissue is stained, in bulk, in alum-cochineal or some other staining fluid that will pene- trate, and then imbedded in celloidin in the usual way. After being mounted on vulcanized fiber the specimen is hardened in chloroform instead of in 80 per cent, alcohol. From the 238 PATHOLOGICAL TECHNIQUE. chloroform the specimen is transferred to oil of thyme. After it is thoroughly penetrated by the latter it is ready to be cut. The knife is to be moistened with oil of thyme. The sections as cut are arranged on the knife, and then trans- ferred to slides placed against the back of the knife. The slides covered with sections can be placed under a bell-jar as fast as they are ready until all are cut, because the oil of thyme evaporates slowly. Balsam and cover-slips can be added after the cutting is finished. 3. Darkschewitsch has recently proposed a comparatively simple method for preparing a series of celloidin sections. A glass cylinder without a neck, of about the diameter of the specimen to be cut, is filled with alcohol. Then a series of circles of filter-paper is cut of a size just to fit the bottle, numbered in order, and wet with alcohol. Each section is removed from the microtome knife by pressing one of the paper circles upon it and drawing it off The paper is then inverted so that the section is uppermost, and deposited in proper order in the bottle, where the series forms a column, each section resting upon a numbered paper. The sections can be kept indefinitely. When ready to stain the alcohol is poured off, the sections washed with water if necessary, and then the staining solution poured into the bottle. Other reagents are used in the same manner, or sections can be treated with the reagents in flat plates, as they do not readily slip off the papers. 4. Weigert's method for a series of celloidin sections was designed especially for the nervous system and is rather complicated. The process depends on transferring the sec- tions as cut to narrow strips of tissue-paper. To do this each section as cut is arranged in proper position close" to the edge of the knife. Then a strip of tissue-paper twice as wide as the section is gently placed upon it. The section adheres, and is removed by drawing the paper to the left and in an upward direction over the edge of the knife. The success of the process depends on having but little alcohol on the knife, otherwise the specimen will not stick. Each specimen is placed on the paper to the right of the last one. HISTOLOGICAL METHODS. 239 The strips of paper when full are kept moist by being placed with the specimens uppermost on a moist surface composed of a layer of blotting-paper wet with alcohol, covered with a sheet of tissue-paper, and lying in a shallow dish. When all the sections have been cut, each strip of them is taken in turn and coated on both sides with a thin film of celloidin in the following way : A strip of sections with the specimens below is first pressed gently down upon the sur- face of a slide covered with a thin layer of celloidin. This fastens the sections and the paper can be removed. Then a thin coat of celloidin is poured over the sections and the slide is placed on its edge to drain. When the surface of the celloidin is dry, the strips can be marked by a fine brush dipped in methylene-blue. As soon as the slides are placed in the staining solution the celloidin peels off, taking the specimens with it. Later, the strips of specimens can be divided as desired. On account of their thickness they should be cleared, after dehydrating in 95 per cent, alco- hol, in a mixture of xylol 3, carbolic-acid crystals i. To obtain serial sections by the paraffin method it is only necessary to avoid losing any of the sections from the ribbon as ordinarily cut. Perhaps the easiest and safest way is to cut long ribbons, a yard or more in length, and to place them on sheets of paper in proper order. They can then readily be divided by means of needles into short series of any desired number of sections, and fastened to numbered slides by means of albumin fixative. STAINING SOLUTIONS. Hematoxylin and Hematein Stains. — The active coloring agent in most hematoxylin stains is hematein, which is gradually formed in the ordinary solutions from the hematoxylin by oxidation, a process occupying a number of days or weeks and spoken of as " ripening." The selec- tive staining power of alum-hematoxylin solutions is due to 240 PATHOLOGICAL TECHNIQUE. the combination of this hematein with alumina. The result- ing blue-colored solution is precipitated in the tissues (chiefly in the nuclei) by certain organic and inorganic salts there present, as, for instance, phosphates. Mayer and Unna have shown that it is possible to oxidize and to ripen in an instant a solution of alum and hema- toxylin by adding to it a little peroxid of hydrogen neutral- ized by a crystal of soda. By employing hematein or its ammonium salt, instead of hematoxylin, Mayer has been able to obtain immediately ripened solutions which compare fairly favorably with old and well-known solutions prepared from hematoxylin by the slow process of ripening. They do not stain any better, however, and it is doubtful if, for the present at least, they become generally accepted. Most solutions of alum and hematoxylin are not stable. A continuous chemical change is the formation from hema- toxylin, by oxidation, of hematein, which, uniting with the alum, gives a bluish or purplish solution. The degree of blueness depends largely on the freshness of the alum. As the solution becomes older free sulphuric acid is gradually formed from the alum, causing the solution to lose its bluish or purplish tint and to become reddish. A third chemical change is the continuous formation of a precipitate due to the further oxidation of the hematein, in consequence of which it is always necessary to filter alum-hematoxylin solu- tions just before they are used. More alum than is needed to combine chemically with the hematoxylin is always added to the solution, for the reason that it acts as a differential decolorizer, limiting^he stain largely to the nuclei of the cells. As alum-hematoxylin solutions be- come older they stain more quickly, but also more diffusely. This diffuseness of staining can be counteracted by adding enough alum-water to make the stain precise again. A good alum-hematoxylin solution ought not to stain the celloidin in which the section is imbedded. If the celloidin stains more or less deeply, it shows that the solution requires more alum. HISTOLOGICAL METHODS. 24 1 Aqueous Alum-hematoxylin Solution. — Hematoxylin crystals, i ; Saturated aqueous solution of ammonia alum, 100; Water, 300 ; Thymol, a crystal. The hematoxylin crystals are dissolved in a little hot water. The combined solution is then exposed to the light in a bottle lightly stoppered with a plug of cotton. The solution will be ripened sufficiently for use in about ten days, after which time it should be kept in a tightly-stoppered bottle. The solution is very easily prepared, gives beautiful results, and will keep at its best for two to three months. The pro- portions of alum and of hematoxylin are the same as in Del- afield's solution. Delafleld's Hematoxylin. — Hematoxylin crystals, 4 grams ; Alcohol, 95 per cent, 25 c.c. ; Saturated aqueous solution of ammonia alum, 400 " Add the hematoxylin dissolved in the alcohol to the alum solution, and expose the mixture in an unstoppered bottle to the light and air for three to four days. Filter, and add — Glycerin, 100 c.c. ; Alcohol, 95 per cent., ido " Allow the solution to stand in the light until the color is sufficiently dark, then filter and keep in a tightly-sto'ppered bottle. The solution keeps well and is extremely powerful. So long as it is good the solution has a purplish tinge. It would seem advisable, both in this solution and in Ehr- lich's, to combine the alum, hematoxylin, and the water, and to ripen the solution for two or three weeks before adding the other ingredients which have a tendency to prevent oxi- dation. A fully-ripened solution would then be obtained more quickly and surely. 16 242 PATHOLOGICAL TECHNIQUE. Ehrlich's Acid Hematoxylin. — Hematoxylin crystals, 2 grams ; Absolute alcohol, 60 c.c. ; ,./ ' ^ ,, \ saturated with Water, 60 " > . , _, . ' - ■ , „ I ammonia alum. Glacial acetic acid, The solution is to be exposed to the hght for a long time until it acquires a deep-red color. If it then be kept carefully stoppered, its staining powers will remain constant for years. The acetic acid is added to prevent the formation of insoluble compounds of hematein and as a decolorizer to limit the stain to nuclei. Mayer's Hemalum. — Hematein, or its ammonia salt, i gram ; 90 per cent, alcohol, 50 c.c. ; Alum, 50 grams ; Water, looo c.c. ; Thymol, a crystal. Dissolve the hematein or its ammonia salt in the alcohol by the aid of heat, and add it to the alum dissolved in the water. The solution can be diluted with 20 parts of water or of weak alum solution. Mayer's acid hemalum is prepared by adding 2 per cent, of glacial acetic acid to the above solution. The acid stain is more precise than the alkaline. Mayer's Glycerin-alum-hematein Solution. — According to Mayer's latest investigations, glycerin is the only reliable preservative of hematein solutions. Unfortunately, it slows the staining power to a considerable extent and makes the stain less precise. He recommends the following solution for its keeping properties : Hematein, 0.4 grams (dissolve by rubbing up in a few drops of glycerin) ; Alum, 5 grams ; Glycerin, 30 c.c. ; Water, 70 " HISTOLOGICAL METHODS. 243 Mayer's Muchematein. — Hematein, 0.2 grams; Chlorid of aluminum, 0.1 " Glycerin, 40 c.c. ; Water, 60 " Rub up the hematein with a few drops of glycerin, add the chlorid of aluminum, and dissolve the mixture in the glycerin and water. Weigert's Aloohol Hematoxylin. — Hematoxylin crystals, 10 grams; Alcohol (absolute or 95 per cent), 90 c.c. The solution ripens in a week or two to a brown color, and keeps perfectly for a long time. It is used only in the Weigert stain for myelin sheaths, for which purpose it is diluted at the time of using with water and combined with carbonate of lithium (see page 318). Phosphomolybdic Acid Hematoxylin {Mallory). — Hematoxylin crystals, 1.75 grams; \ per cent, aqueous solution of phos- phomolybdic acid, 200 c.c. The hematoxylin will dissolve- almost immediately if powdered, or it may be dissolved in water by the aid of heat. The solution must be exposed to the light in a bottle plugged with cotton for five to six weeks before it is fully ripened. It will keep for several months, and can be used over and over. It is employed for staining the nervous system and connective tissue (see page 301). The solution seems to keep better without the addition of any antiseptic, as for- merly advised. Phosphotungstio Acid Hematoxylin {Mallory). — Hematoxylin, .1 gram; I per cent, aqueous solution of phospho- tungstic acid, 1 00 c.c. Dissolve the hematoxylin in a little water by the aid of heat, and add it after it is cool to the dilute acid. The solution 244 PATHOLOGICAL TECHNIQUE. has a greenish-brown, then brownish color of slight intensity. It is ready to use at once, keeps well, and requires no anti- septic. It is used mainly for staining neuroglia-fibers, but gives interesting results with elastic fibers, striated muscle-fibers, fibrin, and cartilage. Carmine Stains. — The active staining principle in car- mine solutions is carminic acid. In cochineal carminic acid is combined with an alkaline base. Carmine itself is a com- mercial compound containing carminic acid combined with aluminum and calcium. Carminic acid itself does not stain, but it forms compounds with certain metals, mainly with the aluminum contained in alum, which have selective staining properties. All of the alkaline and acid solutions made with carmine owe their staining properties to carminic acid combined with the aluminum, and perhaps also to the calcium contained in the carmine. Alum Carmine. — Carmine, 2 grams ; Alum, 5 Water, lOO c.c. Boil twenty minutes, adding enough water to make up for that lost by evaporation. When cool, filter and add a crystal of thymol to prevent the growth of mould. Alum Cochineal. — Powdered cochineal, 6 grams ; Ammonia alum, 6 " Water, lOO c.c. Boil for half an hour ; add water to make up for that lost by evaporation. Filter and add a crystal of thymol. Orth's Lithium Carmine.— >- Carmine, 2.5 to 5 grams ; Saturated aqueous solution of car- bonate of lithium, lOO c.c. ; Thymol, a crystal. The carmine dissolves at once in the cold solution. When HISTOLOGICAL METHODS. 24$ used as a counter-stain for bacteria in the Gram-Weigert method this solution should be carefully filtered, because organisms occasionally grow in it and may give rise to con- fusion in the stained preparations. Neutral Carmine. — Dissolve, without heating, i gram of best carmine in 50 c.c. of distilled water plus 5 c.c. of strong aqua ammoniae. Expose the fluid in an open dish until it no longer smells ammoniacal (about three days) ; then filter and put away in a bottle for future use. The odor of the solution will soon become bad, but the staining properties will remain unaffected. Aniline Dyes. — It is extremely important that all aniline dyes used in histology should be obtained, with possibly a few exceptions, from Griibler, either directly or from his authorized agents. In no other way is it possible to obtain with certainty the results expected. In this country Eimer & Amend of New York City are the chief agents for Griibler. Aniline dyes come in the form of a powder or as crystals, and most of them keep well in that condition. Methylene- blue for one, however, seems to be an exception. After the original package has been opened for a short while the dye loses in intensity of staining power. It is well to keep on hand saturated alcoholic solutions of certain of the dyes, because they keep well in that form, and are ready for use when a saturated alcoholic solution is wanted. This is par- ticularly true of methylene-blue, fuchsin, and gentian-violet. Aniline dyes are derived from either aniline or toluidin, or from both together. They may be regarded as salts having basic or acid properties. The basic colors stain cell-nuclei, including bacteria, for which they show a marked affinity. The acid colors stain diffusely. The basic dyes most com- monly employed in pathological histology are methylene- blue, fuchsin, gentian-violet, and safranin. Of the acid colors, eosin, picric acid, and acid fuchsin are most in use. As a rule, every aniline dye has one or more standard solutions which are used largely to the exclusion of others, for the reason that, being required for certain purposes, they are kept in stock. As they are thus always at hand, they 246 PATHOLOGICAL TECHNIQUE. are used where simple solutions might be used. For in- stance, Loffler's methylene-blue solution is often used, be- cause ready and convenient, when a simple aqueous solution would do as well. In the following pages we have arranged under each dye the solutions of it most in use : Methylene-blue. 1. Saturated solution in 95 per cent, or absolute alcohol. A stock solution to be used in making other solutions. It can be used as a stain by adding i part to 9 parts of water. 2. Aqueous solutions of various strengths are often used, and can be made up as needed. 3. Loffler's Methylene-blue Solution. — Saturated alcoholic solution of methylene-blue, 30 c.c. ; Solution of caustic potash in water, i : 10,000, 100 " This is one of the most useful of the aniline staining solu- tions, and will keep for a long time without losing much in staining power. 4. Kuhne's Methylene-blue Solution. — Saturated alcoholic solution of methylene-blue, 10; 5 per cent, carbolic-acid water, 90. This is a stronger staining solution than Loffler's, but the resulting stain does not seem so sharp and clear. 5. Gabbefs Methylene-blue Solution. — Methylene-blue, 2 ; Sulphuric acid, 25 ; Water, 75. It is used as a decolorizer and contrast-stain for tubercle bacilli. 6. Unna's Alkaline Methylene-blue Solution. — The strongly alkaline solution of methylene-blue recommended by Unna for staining plasma-cells has been found extremely valuable as a general stain in connection with eosin, which should be used first. The solution should be diluted i : 10, or even more, for staining : HISTOLOGICAL METHODS. 247 Methylene-blue, i ; Carbonate of potassium, i ; Water, 100. 7. Unna's Polychrome Methylene-blue Solution. — The poly- chrome methylene-blue solution, much used by Unna in various staining methods, is an old alkaline solution of methylene-blue, of which the above is the original formula, in which, in consequence of oxidation, methyl-violet and methylene-red have formed. Months are required for the process of oxidation to take place. The ripened solution may be obtained from Griibler. 8. Sahli's Borax Methylene-blue Solution. — Saturated aqueous solution of methylene-blue, 24 ; 5 per cent, solution of borax, 16; Water, 40. Mix, let stand a day, and filter. Fuchsin. 1. Saturated alcoholic solution to be kept in stock. 2. Ziehl-Neelson' s Carbol-fuchsin. — Saturated alcoholic solution of fuchsin, 10 c.c. ; 5 per cent, carbolic-acid water, 90 " This solution is very powerful, stains quickly, keeps well, and can be employed for a variety of purposes. 3. Aniline-fuchsin. — Saturated alcoholic solution of fuchsin, 16 c.c. ; Aniline-water, 84 " Gentian- violet, r. Saturated alcoholic solution to be kept in stock. 2. EhrlicKs Aniline-gentian-violet. — Saturated alcohoHc solution of gentian- violet, 16 c.c; Aniline-water, 84 " During the first few hours after the solution is made con- siderable precipitation takes place, so that it is best not to 248 PATHOLOGICAL TECHNIQUE. use it for twenty-four hours. After about ten days it begins to lose its staining power. Zenker recommends a solution without alcohol : Dissolve the gentian-violet directly in the aniline-water. The color is said to be less easily removed from tissues when this solu- tion is used. 3. Stirling's Solution of Gentian-violet. — Gentian-violet, 5 grams; Alcohol, 10 c.c. ; Aniline, 2 " Water, 88 " This solution is said to keep remarkably well. 4. Carbol-gentian Violet. — Saturated alcoholic solution of gentian-violet, 10 c.c. ; 5 per cent, carbolic-acid water, 90 " Saft-anin. — Two of the many preparations by this name have been found especially useful : 1. Safranin O soluble in water. 2. Safranin soluble in alcohol. The three following solutions of safranin can be thoroughly recommended : 1. Saturated aqueous solution of " safranin O soluble in water" (to be made with the aid of heat). 2. A mixture of equal parts of — A saturated aqueous solution of " safranin O soluble in water." A saturated alcoholic solution of " safranin soluble in alcohol." 3. Babes' Aniline Safranin. — 2 per cent, aniline-water, 100; " Safranin O soluble in water," in excess. Saturate the solution by heating it in a flask set in hot water to 60-80° C. ; filter. This solution is extremely powerful, stains almost in- stantly, and will keep, about two months. Methyl-violet. — i. Aqueous solutions of various strengths, ■■/■ HISTOLOGICAL METHODS. 249 ■|- to 2 per cent, keep well and are used for staining nuclei, bacteria, and amyloid. 2. Methyl-violet can be used instead of gentian-violet in Ehrlich's solution. 3. For staining neuroglia-fibers Weigert employs a satu- rated solution made with the aid of heat in 70-80 per cent, alcohol. Bismarck Brown. — The most common solutions are the following:. 1. A I per cent, aqueous solution. 2. A saturated aqueous solution made by boihng (3-4 per cent.). 3. A saturated solution in 40 per cent, alcohol (2-2^ per cent.). Unhke other aniline colors, Bismarck brown will keep in glycerin mounts and can be fixed in nuclei by acid alcohol. The stain is not used so much as formerly, except for photo- graphic purposes.' Other basic stains less frequently used, and then generally in aqueous solutions, are dahlia, methyl- green, iodin-green, and thionin. Diffuse Stains. — i. Eosin is sold in two forms — as " eosin soluble in water," and as " eosin soluble in alcohol." The first is to be preferred, because a greater degree of differen- tiation in stain can be obtained with it. Keep on hand a saturated aqueous solution and dilute with water as needed. The strength of solution to be used varies somewhat with the tissue and the reagent in which it has been fixed, but generally Hes between -^ and ^ per cent, when the eosin is used after a hematoxylin stain. When it is employed before an aniline dye such as methylene-blue, a 5 per cent, or even a saturated solution should be taken. 2. Picric Acid. — Saturated alcoholic and aqueous solu- tions should be kept in stock, to be diluted as needed. 3. Van Gieson's Solution of Picric Acid and Acid Puch- sin. — This valuable solution was originally made by adding to a saturated aqueous solution of picric acid enough of a saturated aqueous solution of acid fuchsin to give to the fluid a deep garnet-red color. Freeborn has recently given more 2SO PATHOLOGICAL TECHNIQUE. precise directions for making up the solution according to the purpose for which it is to be used. For Connective Tissue, — (See page 300). I per cent, aqueous solution of acid fuchsin, 5 c.c. ; Saturated aqueous solution of picric acid, 100 " For the Nervous System. — (See page 306). I per cent aqueous solution of acid fuchsin, 1 5 c.c. ; Saturated aqueous solution of picric acid, 50 " Water, ' 50 " Picro-nigrosin {Martinotti). — Dissolve picric acid and nigrosin to saturation in 70 per cent, alcohol. Combination Stains. — Biondi-Heidenhain Staining Solution. — Saturated aqueous solution of orange G, 100; Saturated aqueous solution of acid fuchsin, 20 ; Saturated aqueous solution of methyl-green, 50. Make up the separate solutions and let them stand for seve- ral days with excess of coloring matter (shaking the bottles occasionally) until they are saturated. Then mix the solu- tions. For staining dilute the combined solution with water I : 100. The following tests are used for finding out if the proper combination has been obtained: The addition of acetic acid should make the solution redder ; a drop of the solution on filter-paper should make a blue spot with green in the center and orange at the periphery. If a red zone appears outside of the orange, then too much acid fuchsin is present. Pianese's Staining Solutions and Staining Meth- ods. — The following stains, devised by Pianese, are recom- mended by him particularly for the study of cancer, but will be found useful in many lines of histological investigation. The first two were used by him for tissues hardened in cor- rosive sublimate or in Zenker's fluid ; the others, only after his special fixative (given on page 225). The methods are intended for parafiin sections : HISTOLOGICAL METHODS. 25 I 1. Carmine and Picro-nigrosin. — i. Stain in neutral or lithium carmine. 2. Decolorize in acid alcohol. 3. Wash in water. 4. Absolute alcohol. 5. Aniline-gentian-violet, ten minutes. 6. lodin solution, two to three minutes. 7. Absolute alcohol, so long as any color is discharged. 8. Saturated aqueous solution of picric acid and of nigro- sin, five minutes. 9. Decolorize in a i per cent, alcoholic solution of oxalic acid. 10. Water, several minutes. 1 1 . Absolute alcohol. 12. Oil of bergamot. 13. Balsam. Nuclei, red ; cell-protoplasm, light olive-green ; connec- tive tissue, dark olive-green ; elastic fibers, bluish ; bacteria and blastomycetes, violet. II. Methylene-blue and Bosin in Borax Solution. — Keep three solutions on hand : (a) Saturated solution of methylene-blue in a saturated aqueous solution of borax. {B) \ per cent, solution of " bluish eosin " in 70 per cent, alcohol. (c) Saturated aqueous solution of borax. For use mix together 2 parts of the filtered solution a, i of b, and 2 of c. The different steps of the staining pro- cess are as follows : 1. Absolute alcohol. 2. Staining solution, ten to twenty minutes. 3. Decolorize in a i per cent, solution of acetic acid. 4. Wash in water. 5. Absolute alcohol. 6. Xylol. 7. Xylol balsam. Nuclei, blue ; red blood-globules, cell-protoplasm, granules of eosinophiles, connective tissue, etc., rose-red. 252 PATHOLOGICAL TECHNIQUE. III. a. Malachite-green, Acid Fuchsin, and Nigrosin. — Malachite-green, i. gram; Acid fuchsin, .4 " Nigrosin, .1 " Water, 50 c.c. ; Alcohol saturated with acetate of copper, 50 " 1. Absolute alcohol. 2. Stain in 20 drops of above solution diluted with 10 c.c. of distilled water for twenty-four hours. 3. Decolorize in a | per cent, aqueous solution of oxalic acid. 4. Wash in water. 5. Absolute alcohol. 6. Xylol balsam. Resting nuclei, light red; protoplasm, reddish yellow. In the karyokinetic figures, nuclein green ; fibrillae of the achro- matic spindle and of the mitoma, bright red; centrosome and polar bodies, red ; the rest of the cell-body, a reddish- yellow color. III. b. Malachite-green, Acid Fuchsin, and Martius Yellow. — Malachite-green, .5 gram ; Acid fuchsin, .1 " Martius yellow, .01 " Distilled water, 150 c.c; Alcohol, 96 per cent, 50 " 1. Stain in the solution without diluting, half an hour. 2. Absolute alcohol. 3. Xylol. 4. Xylol balsam. Nuclei of resting and dividing cells, green ; cell-protoplasm, connective tissue, etc., rose-colored ; " cancer-bodies," mainly red, but in masses of them some are red and some green. IV. Acid Fuchsin and Picro-nigrosin. — Saturated alcoholic solution of acid fuchsin, 6 drops ; Martinotti's picro-nigrosin, 8 " Distilled water, 10 c.c. HISTOLOGICAL METHODS. 253 1. 70 per cent, alcohol. 2. Stain in the solution six hours. 3. Decolorize in dilute acetic acid. 4. Absolute alcohol. 5. Xylol. 6. Xylol balsam. Resting nuclei, red ; nuclein of karyokinetic figures, yellow : cell-protoplasm, dark olive-green; "cancer-bodies," mainly olive-gray, but some or portions of them may be ruby-red. V. Light Green (Lichtgriin) and Hematoxylin. — Ehrlich's acid hematoxylin, 1 5 c.c. Saturated solution of Lichtgriin in 70 per cent. alcohol, 5 " Distilled water, 15 " 1. Distilled water. 2. Stain in above mixture half an hour. 3. Wash thoroughly in several waters. 4. Alcohol. 5. Oil of bergamot. 6. Balsam. Nuclei, green ; " cancer-bodies " take the hematoxylin stain. VI. Acid Fuchsin and Hematoxylin. — Ehrlich's acid hematoxylin, 15 c.c. I per cent, solution of acid fuchsin in 70 per cent, alcohol, 15 " Distilled water, 15 " Stain as in V. Nuclei, red ; protoplasm, brick-red ; " cancer-bodies " take the hematoxylin stain. Orcein, a vegetable dye obtained from certain tinctorial lichens, is used mainly for staining elastic fibers. It is soluble in alcohol, and is employed either in a neutral or acid (HCl) alcoholic solution. lodin is the oldest of the histological stains, but is now but little used for that purpose. The tincture of iodin, a saturated solution in alcohol, is 254 PATHOLOGICAL TECHNIQUE. used for getting rid of the precipitate of mercury formed in tissues fixed in corrosive sublimate or in Zenker's fluid ; a small amount of the tincture is added to the alcohol in which the tissue is preserved (see page 223). I/Ugol's solution, a solution of iodin in water containing iodid of potash, is of varying strength. Iodin in this form is much used as a test for starch, amyloid, glycogen, and corpora amylacea. In Gram's stain and its modifications iodin produces some chemical change in the coloring material em- ployed, in consequence of which, when appropriate decolor- izers are used, the stain remains fast in certain structures, while from others it is easily entirely extracted. The strength originally employed by Gram for his stain- ing method was — Iodin, I gram; Iodid of potash, 2 grams ; Water, 300 c.c. Weigert in his modification of this method employed a stronsfer solution : Iodin, I gram ; Iodid of potash, 2 grams ; Water, 100 c.c. Recently he has recommended the following strength both for fibrin and for neuroglia-fibers : Iodid of potash, 5 grams 1 ^ ^ , vi • j- „j ^ ' J t> > saturated with lodm. Water, 100 c.c. ) The only difference in the action of the various solutions probably is that the strong solution acts practically instan- taneously, while the weaker solutions require some little time. Acid Alcohol {Orth's Discharging Fluid). — Hydrochloric acid, i c.c. ; 70 per cent, alcohol, 99 " Aniline Water {Aniline-oil Water). — Shake together 5 HISTOLOGICAL METHODS. 255 parts of aniline with 95 parts of water, and filter the re- sulting milky fluid. It should come through perfectly clear. Carbolic-acid water is made in like manner by shaking together 5 c.c. of melted carbolic-acid crystals and 95 c.c. of water. The solution should be filtered. Mayer's glycerin-albumin mixture for attaching paraffin sections to slides is composed of equal parts of the white of egg and of glycerin. The mixture should be thor- oughly beaten and then filtered, or after standing for some time can be decanted. Add a little camphor or carbolic acid to prevent decomposition. Egg-albumin is dissolved by acids and alkalies, so that when such reagents are to be used the sections are best attached to the slide by some other substance. For this purpose Schdllibaum's solution, of celloidin i part in 3 or 4 parts of oil of cloves, is often useful. Cover the slide with a thin layer of the solution. Arrange the sections in order on the slide and place it in the thermostat at 54° to 60° C. for several hours, or heat for a few seconds to half a minute over the flame until the oil of cloves runs together in drops. After cooling, remove the paraffin with xylol, pass through origanum oil to 95 per cent, alcohol, and proceed as with other paraffin sections. Clearing Reagents. — The object of clearing reagents is to render certain tissue-elements more prominent than others. This result may be brought about by dilute acetic acid (2—5 : 100), which swells up the ground substance, so that nuclei, elastic fibers, fat, myelin, and micro-organisms are more distinct, or by alkalies, which destroy the cells and ground substance and leave only elastic fibers and bacteria but little changed. This method is used almost wholly for fresh tissues. The same result is more commonly obtained by soaking the tissues in substances which by reason of their high index of refraction render the tissues more or less transparent. Any structure which it is desirable to study is usually pre- viously stained and thus easily rendered prominent. This second method is most applicable to hardened tissues. For soaking and clearing the tissues a variety of reagents 256 PATHOLOGICAL TECHNIQUE. of different chemical properties are used. Glycerin and acetate of potash are not so much employed as formerly, because balsam mounts are more generally preferred. Of the other reagents (ethereal oils and coal-tar products), the the choice depends mainly on two factors — the kind of stain which has been employed, and the substance in which the sections have been imbedded. Many of the clearing re- agents either dissolve celloidin or will not clear it from 95 per cent, alcohol, and nearly all of them will extract aniline colors more or less rapidly. Most of the clearing reagents can be used after hema- toxylin and carmine stains. For celloidin or paraffin sections stained by either of them oleum origani cretici, oil of ber- gamot, or the mixture of the oils of cloves and thyme is recommended in the order given. After aniline stains the best clearing reagent is xylol, which, however, clears only from absolute alcohol. It is to be used, therefore, for unimbedded sections and for paraffin sections dehydrated in absolute alcohol. The most difficult problem in clearing is offered by cel- loidin sections stained with aniline dyes. It is, perhaps, best solved by clearing and dehydrating in oleum origani cretici or in oil of bergamot after 95 per cent, alcohol, and then washing out the oil with several changes of xylol before placing in balsam. For certain dyes other clearing reagents may be used, as aniUne after gentian-violet or fuchsin, but for methylene- blue, which seems to be one of the most sensitive of the aniline dyes, the above method is particularly recommended. Occasionally the decolorizing effect of certain of the clearing reagents is purposely made use of (see oil of cloves, page 279). Oleum Origani Cretici. — Light brown in color; clears readily from 95 per cent, alcohol without dissolving cel- loidin ; affects aniline colors slowly. Ordinary origanum oil is impure oil of thyme and should not be used. Oil of Bergamot. — Light green in color ; clears quickly from 95 per cent, alcohol ; does not dissolve celloidin, but HISTOLOGICAL METHODS. 2$ J after repeated use of the same lot of oil it will sometimes soften it a little. Affects aniline colors slowly, with the ex- ception of eosin, which it extracts very quickly. Oil of Cloves. — Straw-colored; clears quickly from 95 per cent, alcohol ; dissolves celloidin ; extracts aniline colors, especially methylene-blue. Oil of Thyme. — Colorless ; clears readily from 95 per cent, alcohol ; makes sections brittle ; does not dissolve cel- loidin ; affects aniline colors. Oil of Lavender. — Clears celloidin sections readily from 95 per cent, alcohol. Oil of Cedar-wood. — Pale straw-color ; clears from 95 per cent, alcohol, but, unfortunately, clears celloidin sections very slowly ; does not affect aniline colors. Aniline {Aniline Oil). — Colorless when perfectly pure and fresh, but soon oxidizes and turns brown ; does not dissolve celloidin ; clears readily from 70 per cent, alcohol ; will clear from water by Weigert's method; extracts aniline colors slowly. Xylol. — Colorless ; does not dissolve celloidin ; will clear from absolute alcohol only ; does not affect aniline colors ; shrinks sections considerably. Dunham's Mixture of the Oils of Cloves and Thyme. — Excellent for sections stained in hematoxylin or carmine. Not nearly so expensive as pure origanum or bergamot oil. Oil of cloves, I part; Oil of thyme, 4 parts. Filter if cloudy ; clears celloidin sections readily from 95 per cent, alcohol without dissolving the celloidin. Weigert's Mixture of Carbolic Acid and Xylol. — Carbolic-acid crystals, i part; Xylol, 3 parts. Recommended for clearing thick sections of the central nervous system after carmine and hematoxylin stains only. The next mixture is more used now-a-days. 17 258 PATHOLOGICAL TECHNIQUE. Weigert's Mixture of Aniline and Xylol. — Aniline, 2 parts ; Xylol, I part. Mounting Reagents. — The most generally used reagent for permanent mounts is Canada balsam. Damar and colo- phonium are only exceptionally preferred. Canada balsam occurs in commerce as a very thick, tena- cious, pale, straw-colored fluid. It should be evaporated over a water-bath to drive off" all volatile substances, which might affect aniline colors, until it becomes solid and brittle on cooling. Dissolve it then in xylol, which does not affect aniline colors, to a rather thin, syrupy consistency. In this condition it is often called xylol balsam. Canada balsam has a high index of refraction, so that tis- sues mounted in it become very transparent, and only those parts are visible which are stained. Other solvents of Canada balsam, such as chloroform and benzine, may be used, but cannot be recommended for sections stained with aniline colors. Damar has a lower index of refraction than Canada bal- sam ; is soluble in xylol, chloroform, etc. ; dries slowly, and is generally recommended for Golgi preparations. Colophonium dissolved in benzine is employed by Nissl for mounting stained preparations of gangUon-cells. METALLIC STAINS OR IMPREGNATIONS. Experimental investigation has shown that certain metals can be used for staining certain tissue-elements, either be- cause they are directly reduced from solutions of appropriate salts or because they are taken up and retained by certain tissue-elements, which are rendered prominent when the metallic salt is reduced later. The most valuable metals for this purpose are silver, gold, and osmium. Silver is used, generally in the form of silver nitrate, to stain of a brown or dark-brown color the cement substance HISTOLOGICAL METHODS. 259 between epithelial and endothelial cells and the ground sub- stance of connective tissue. The method finds its chief use in pathology in demonstrating the endothelial covering of a doubtful surface, in outlining the endothelial cells of patho- logically altered blood- and lymph-vessels, and in staining the ground substance of the connective tissue of the cornea when that organ is used experimentally for the study of in- flammation. In combination with certain other salts, espe- cially bichromate of potassium, nitrate of silver is much em- ployed in the Golgi methods to stain ganglion-cells and their processes in the central nervous system. The difficulty of the silver method lies in the fact that the salt forms with albuminous fluids granular and thread-like coagula which can easily give rise to false pictures. For this reason the method is limited almost entirely to natural surfaces, which should be washed off with water or a 2 per cent, solution of nitrate of sodium before the silver solu- tion is applied. It is generally advisable to use the nitrate of silver in a very dilute solution, i : 250 or 500. The solu- tion is allowed to act on the surface for about a minute, and is then washed off with water. The tissue is next exposed in water to the action either of sunlight or of diffuse light. The outlines of the cells soon appear as dark lines, brown to black in color. The tissue to be stained should be kept stretched, because a precipitation of the silver occurs wherever there is a fold in the surface. Although nitrate of silver penetrates but a slight distance, it is possible to stain the outlines of the endothelial cells of the lymphatics and blood-vessels as well as the ground substance of the connective tissue — in a rabbit's diaphragm, for instance — by treating the upper or lower surface with the silver solution. ' The thoracic organs should be removed, and then the upper surface of the tendinous portion of the diaphragm left in situ is exposed to the action of the silver salt in the manner already described. The outlines of the endothelial cells of blood-vessels are usually stained by injections of the silver salt through an artery. In the same way the limits of the epithelial cells 26o PATHOLOGICAL TECHNIQUE. of the alveoli of the lung can be stained by injections through a bronchus. Although generally employed in solution, nitrate of silver is sometimes used in the solid form, and for the cornea this method is preferable. Chloroform the animal, preferably a rabbit, deeply; rub the cornea with a stick of nitrate of silver hard enough to remove the surface epithelium. Allow the salt to act about ten minutes, then kill the animal, re- move the eye, cut out the cornea, wash it, and expose to diffuse daylight for half an hour. It is then placed in a mixture of glycerin and water, 30 parts to 70, very slightly acidulated with acetic acid (about -^-^ per cent.) for twenty- four hours, so as slightly to swell and to soften the tissues. Sections of the cornea are best made with the freezing microtome. Incise the periphery a little at four points equally distant from each other, so that the cornea will lie flat. A direct stain with alum-hematoxylin gives by all odds the best results. The sections may be mounted in glycerin or balsam. The latter method is perhaps the better. De- hydrate the sections in 50 per cent., then in 70 per cent, alcohol, clear in aniline oil, wash with xylol, and imbed in balsam. This method avoids the shrinkage which is caused by using strong alcohol. Gold, in the form of the simple or double chlorid, is em- ployed to stain the protoplasm of cells of connective tissue, and more particularly the axis-cylinders of nerve-fibers and their terminal processes. Like nitrate of silver, it acts as a fix- ing and hardening reagent as well as a stain. Unfortunately, it penetrates tissues but a very slight distance, and, so far as staining is concerned, is inconstant in action. Its chief use in pathology is in connection with experimental work on the cornea and in regeneration. The conditions under which the reduction of the gold salt takes place are not exactly understood, but both penetration and reduction are aided by the action of organic acids, such as formic, citric, and tartaric acids, on the tissues both before and after the treat- ment with the gold salt. Of the many methods proposed, the following are recommended: HISTOLOGICAL METHODS. 26 1 Lowit's Pormic-acid Method. — i. Place very small bits of fresh tissue in a mixture of formic acid i part, and water 1 to 2 parts, until they become transparent (a few seconds to several minutes). 2. Transfer to chlorid of gold, i to 1.5 parts to 100 of water, for fifteen minutes. 3. Formic acid, i part to water 3 parts, for twenty-four hours. 4. Concentrated formic acid twenty-four hours. Preserve in glycerin or balsam. All the steps except the first should be performed in the dark. Ranvier's Formic-acid Method. — i. Boil together 8 c.c. of a I per cent, solution of chlorid of gold and 2 c.c. of formic acid. When the solution is cold place very small bits of tissue in it for one hour, in the dark. 2. Wash quickly in water. 3. Expose to diffuse light in a mixture of formic acid 10 c.c. and water 40 c.c. Reduction takes place slowly (twenty- four to forty-eight hours). 4. Harden in 70 per cent., then 90 per cent., alcohol in the dark. Osmic Acid (perosmic acid, osmium tetroxid) is used as a fixing reagent and for staining fat and myelin, by which it is reduced. As osmic acid is quickly reduced by organic substances, care must be taken in making up the solution. Remove the label from the sealed tube in which the acid comes, and place the tube, after cracking off one end, in a glass-stoppered bottle containing enough water to make a 2 per cent, solution. If desired, the tube can be broken after it is in the bottle by violent shaking. It should be borne in mind that osmic acid is very-irritating to the bron- chial mucous membrane. In a I or 2 per cent, solution osmic acid is used to stain fat in teased preparations or frozen sections of fresh tissues. In Marchi's method it is used to stain fat in tissues which have been hardened for some time in Miiller's fluid. As a fixing reagent it is usually combined with other reagents, as in Flem- 262 PATHOLOGICAL TECHNIQUE. ming's solution, both for its property as a fixative and for the purpose of staining any fat present. STAINING riETHODS. The purpose of staining is to render prominent the differ- ent tissue-elements, so that they may be readily recognized and studied. The constant tendency now-a-days is toward selective or differential staining methods, by which but one tissue-element will be colored to the exclusion of all others, or at least of any element that might be confused with it morphologically. These selective stains, which really are micro-chemical color reactions, enable us to differentiate from each other with ease and accuracy cellular and inter- cellular elements, or pathological products which otherwise look alike. The list given on page 263 does not pretend to be either complete or perfect in arrangement, but will give some idea of the various elements which we wish to stain. Those for which we now possess more or less perfect differential stains are printed in italics. The simplest selective stain is, of course, that for nuclei, and it can be obtained with a great variety of staining re- agents. The most difficult element to stain differentially, although it can be done under certain conditions with a fair amount of success, is probably the axis-cylinder and its ter- minal processes. Tissue-elements and pathological products differ from each other, not only in form and consistency, but also in chemical properties. While perfect preservation of form is sufficient to distinguish certain cells or elements from each other — as, for instance, polynuclear leucocytes from lymph- oid cells — differentiation based on micro-chemical tests is always to be preferred when possible. A few of the tests employed are colorless, like the precipitation of mucin by acetic acid. Certain tests, like the methylene-blue or gold stain for axis-cylinders, can be applied to fresh tissues only. HISTOLOGICAL METHODS. 263 Cell. Nucleus. Cuticle. Protoplasm. ■ Nucleolus. Resting nucleus. Linin. I I. Do not stain by Gram. Bacteria. ] 2. Stain by Gram. l 3. Stain by tubercle-bacillus method. Nucleus of ameba coli. r Centrosome and polar bodies. Mastzellen. Plasma-cell of Unna. , Five kinds of gran- Leucocytes. \ ules described by I Ehrlich. Nissfs granules in ganglion-cells. Dendritic processes of ganglion- cells. Axis-cylinder and terminal processes. Contractile elements of striated muscle-fiber. Red blood-globules. Cilia of bacteria. Certain dots in ependymal cells. So-called cilia in certain renal cells. Bile-capillaries. Granules. Intercellular substances. Cement substance of endothelial cells. Ground substance of connective tissue. Connective-tissue fibrillce. Myxomatous tissue ; mucin. Elastic fibers. Intercellular substances of cartilage. Ground substance of bone. Myelin. Neuroglia-fibers. Clubs of actinomyces. Capsules of bacteria. ' Fibrin. Mucin. Amyloid. athological Hyaline substances. Glycogen. products. Hyalin. Fat. Colloid. Hemosiderin. Keratohyalin Hematoidin. ^ E lead in. . Hemoglobin. 264 PATHOLOGICAL TECHNIQUE. Others, like the various amyloid reactions, can be obtained with fresh or hardened tissues. Most of the micro-chemical reactions, however, can be employed only with tissues which have been properly preserved. It is exceedingly important, therefore, that a tissue-element be so fixed and hardened that its peculiar chemical properties be preserved intact, otherwise a differential stain for it is impossible. Each tissue-element is a law unto itself For example, the peculiar chemical properties of red blood-globules depend on the presence in them of hemoglobin. As a differential stain of the red blood-globules depends on fixing this substance in them, it is necessary to find out the chemical properties of hemo- globin, such as the fact that it is soluble in water or dilute alcohol, but not in salt solution, and that it is fixed in the red blood-globules by heat, absolute alcohol and ether equal parts, corrosive sublimate, formaldehyde, bichromate of po- tassium, etc. While differential stains depend in part on the chemical properties of the tissue-elements, they also depend to a cer- tain extent on the chemical properties of the staining re- agents and the decolorizers used. Some of the tissue-elements can be stained differentially in a number of ways, sometimes after one fixing agent, some- times after another. The simplest differential stains are those where certain tissue-elements stain directly in a given solu- tion after they have been properly fixed. Good examples are — Ehrlich's triple stain for certain protoplasmic granules in leucocytes, and the direct stain for elastic fibers with an acid alcoholic solution of orcein. Other differential stains depend on the property of certain elements to hold colors they have once taken up when treated with decolorizers. The best example of this is the tubercle bacillus, which holds certain stains through various acids, or aniline hydrochlorate, followed by alcohol, and, if necessary, by a contrast-stain. Still another varied group of elements (certain bacteria, fibrin, neuroglia-fibers, etc.) depends for a differential stain in part on changes produced in gentian- or methyl-violet by HISTOLOGICAL METHODS. 265 iodin, in part on the decolorizer employed for extracting the coloring reagent. Although the steps of the various staining methods differ considerably, they may be roughly arranged in the following order : 1. Staining. 2. Differentiating. 3. Decolorizing. 4. Dehydrating. 5. Clearing. 6. Mounting. Very often two or more of the steps are combined in one, as when aniline oil is used for decolorizing, dehydrating, and clearing sections stained for certain bacteria. Sometimes the staining process occupies more than one step, as in Weigert's myelin-sheath stain. In alum-hematoxylin the differentiating reagent, the excess of alum, is combined with the stain ; in Gram's method the differentiating reagent, iodin, forms a step by itself NUCLEAR STAINS. For general histological work few stains are more valua- ble or can be more highly recommended than alum-hema- toxylin, either alone or in contrast with eosin. Prop- erly made and used, the solution stains the nuclei sharply and of varying degrees of intensity, depending on- the cha- racter of the cells. Besides the nuclei, however, it stains other tissue-elements in delicate shades of blue, so that they are readily visible, and thus more or less differentiated from those structures which fail to stain. Of the carmine stains, lithium carmine, followed by picric acid, will be found the most brilliant, generally useful, and permanent. Safranin gives, perhaps, the most permanent stain of any of the basic aniline dyes, and confines itself very sharply to the nuclei. It is much used after certain fixing reagents, such as Flemming's and Hermann's solutions. Eosin, followed by methylene-blue, gives beautiful results, especially when 266 PATHOLOGICAL TECHNIQUE. Unna's alkaline solution of methylene-blue is used. The advantages of this solution are that it stains readily tissues hardened in Zenker's fluid and brings out nuclei and nuclear figures with great sharpness, while at the same time it stains the protoplasm of certain cells so that they are easily dis- tinguished from other cells. The Heidenhain-Biondi triple stain is useful after fixation in corrosive sublimate, but can- not be employed with celloidin sections, so that its field is limited. The other aniline dyes are used on occasion or for some definite purpose, but not so generally as those men- tioned above. Alum-hematoxylin Stains. — Most alum-hematoxylin solutions will over-stain if the sections are left too long in them. The proper time required depends on the fixing re- agent used and on the degree of ripeness of the staining solution. It is therefore advisable to wash a section in water occasionally and decide from the color it has acquired if it be sufficiently stained, or to mount it in water on a slide and examine with the low power of the microscope. The best results are obtained with alum-hematoxylin solu- tions by staining sections just deeply enough, washing them thoroughly in several changes of water, and leaving them in a large dish of water over night. This thorough washing is done to rid the tissues of every trace of alum or of acid, so that the color will become a clear blue and will keep in- definitely. Many microscopists prefer to stain deeply and diffusely in an old, quickly-staining alum-hematoxylin solution, and then to employ a decolorizer. The agents most used for the pur- pose are alum (i per cent, aqueous solution for one to two hours), hydrochloric acid (^Ig to \ per cent, aqueous solution, or even the ordinary acid alcohol), and acetic acid (i to 3 per cent, solution) for a few seconds only. After being suf- ficiently decolorized the sections must be thoroughly washed in water, preferably for a number of hours, otherwise the stain will fade. The objection to this method is that a pure nuclear stain only is obtained, because the acid removes the color completely from all the rest of the tissue. Under cer- HISTOLOGICAL METHODS. 267 tain circumstances, as when hematoxylin is used as a con- trast-stain to fuchsin in staining for tubercle bacilli, such a sharp limitation to the nuclei is desirable. Alum-hematoxylin stains well and quickly tissues hard- ened in alcohol, in corrosive sublimate, and in picric acid. It stains much more slowly tissues hardened in solutions containing chrome salts, such as Zenker's and Miiller's fluids. For counter-staining eosin will usually be found to give the most beautiful contrast, although picric acid. Van Gieson's mixture, and neutral carmine are often of service. A good alum-hematoxylin solution should have a bluish or purplish color, and should stain celloidin very faintly or not at all. Aqueous Alum-hematoxylin; Delafield's Hema- toxylin ; i^hrlicli's Acid Hematoxylin (see pages 241 and 242). 1. Stain in one of the above solutions two, five, or thirty minutes, or sometimes even longer. 2. Wash in several changes of water, and then leave sec- tions, if possible, for. several hours or over night in a large dish of water. 3. Contrast-stain, usually an aqueous solution of eosin, 3^ to J per cent, for one to five minutes. 4. Alcohol, 95 per cent., two or three changes to dehydrate and to remove excess of contrast-stain. 5. Clear in oleum origani cretici or in Dunham's oils-of- cloves-and-thyme mixture. 6. Canada balsam. The staining of the nuclei by Ehlrich's alum-hematoxylin solution is not so sharp as that obtained by the simple aque- ous solution. The more customary method of using Delafield's alum- hematoxylin solution is to filter a few drops of it into a dish of water and to stain sections for a long time, even over night, with the very dilute solution thus obtained. It is sometimes advisable to use the aqueous solution in the same way. 268 PATHOLOGICAL TECHNIQUE. Mayer's Hemalum (see page 242). — i. Stain three to five minutes or longer. 2. Wash out in i per cent, alum solution until the stain is precise. 3. Wash thoroughly in several changes of water. 4. Alcohol, 95 per cent. 5. Oleum origani cretici. 6. Canada balsam. The staining is rather diffuse, so that it has to be washed out to some extent with alum-water. Mayer's acid hemalum is more precise, and usually does not need to be decolorized, so that the second step can be omitted. Hemalum is used for staining tissues in bulk. Twenty- four hours are required for large pieces. Heidenhain's Hematoxylin Stain. — i. Stain twenty- four to forty-eight hours in a simple \ per cent, aqueous solution of hematoxylin dissolved by the aid of heat. 2. Transfer the sections directly to a ^ per cent, aqueous solution of simple chromate of potassium for twenty-four to forty-eight hours, changing the solution frequently until no more color is given off by the sections. 3. Wash thoroughly in water. 4. Alcohol. 5. Oil. 6. Canada balsam. Carmine Stains. — The ordinary carmine solutions give good nuclear stains, but of the finer details in a specimen they bring out much less than a direct alum-hematoxylin stain. They are much less used now than formerly, except as contrast-stains to bacteria and to fibrin in the methods of Gram and Weigert, for which purpose lithium carmine will usually give the best results. Alum Carmine; Alum Cochineal (see page 244). — I. Water. 2. Stain in either of the above solutions for five to twenty minutes. 3. Wash thoroughly in water. 4. Alcohol, 95 per cent. HISTOLOGICAL METHODS. 269 5. Oleum origani cretici. 6. Canada balsam. Over-staining does not occur. The solutions cannot be recommended for tissues which stain with difficulty. When used for staining in bulk, twenty-four to forty-eight hours are required. Lithium. Carmine (see page 244). — i. Water. 2. Stain two to five minutes. 3. Transfer directly to acid alcohol, one or more changes for several minutes or more, until the sections are well differ- entiated. 4. Wash in water. 5. Alcohol, 95 per cent. 6. Oleum origani cretici. 7. Canada balsam. This method gives an intense and permanent bright-red nuclear stain. Over-staining is impossible. A trace of picric acid added to the alcohol used for dehydration affords a beautiful contrast-stain. Aniline Dyes as Nuclear Stains. — Any of the basic anilme dyes may be used as nuclear stains after the following general method : 1. Stain celloidin sections in a strong solution of the dye preferred in water or in dilute alcohol for five to thirty minutes. 2. Wash in water. 3. Dehydrate in 95 per cent, alcohol. 4. Clear in oil of bergamot or origanum. 5. Wash out the oil thoroughly with several changes of xylol. 6. Xylol balsam. With paraffin sections use absolute alcohol and clear di- rectly in xylol. As a matter of fact, however, certain dyes and certain so- lutions are generally used in preference to the others. The use of aniline dyes as stains presents for solution one or two problems. Most of the colors are more or less affected by all clearing reagents except xylol. With paraffin sections 270 PATHOLOGICAL TECHNIQUE. and those from which the celloidin has been removed it is very easy to dehydrate in absolute alcohol and to clear in xylol. With celloidin sections, however, this is impossible, because the absolute alcohol will dissolve out the celloidin, and this is usually not desirable. For celloidin sections it will therefore generally be found advisable to dehydrate in 95 per cent, alcohol, to clear and further dehydrate in ber- gamot or origanum oil (the decolorizing effect of these oils is slight and slow), to wash out the oil with xylol, and then to mount in xylol balsam. In washing out the excess of color it is sometimes found advantageous to acidulate very slightly either the water or the first alcohol with acetic or hydrochloric acid. This pro- cess, if not carried too far, tends to make the nuclear stain sharper. Safranin is one of the very best nuclear-staining aniline dyes. Tissues may be hardened in alcohol, corrosive subli- mate, Flemming's, Hermann's, or Zenker's fluids. Any one of the solutions of safranin given on page 248 may be used. 1. Stain paraffin sections two to five minutes to twenty- four hours according to the staining solution and fixing re- agent used. 2. Wash in water. 3. Absolute alcohol, several changes, until the section appears properly differentiated. 4. Xylol. 5. Xylol balsam. For celloidin sections dehydrate in 95 per cent, alcohol, clear in bergamot or origanum oil, and wash out in xylol. To render the stain more precise a few drops of acid alcohol are sometimes added to the first alcohol. Bosin and Methylene-blue. — Harden in alcohol, Zenker's fluid, or corrosive sublimate. 1. Stain paraffin sections in a 5 per cent, or saturated aqueous solution of eosin for five to twenty minutes or longer. 2. Wash in water to get rid of excess of eosin. HISTOLOGICAL METHODS. 2J\ 3. Stain in Unna's alkaline methylene-blue solution (see page 246), diluted i-io with water, for one half to several hours. 4. Wash in water. 5. Differentiate and dehydrate in absolute alcohol until the pink color returns in the section. 6. Xylol. 7. Xylol balsam. For celloidin sections use 95 per cent, alcohol, clear in origanum oil, and wash out with xylol. It is important to get a deep stain with eosin, because the methylene-blue washes it out to a considerable extent. The eosin must be used first, because methylene-blue is readily soluble in an aqueous solution of eosin, and therefore is quickly extracted if the eosin is used after it, while on the other hand eosin is very slightly soluble in an aqueous solu- tion of methylene-blue which is precipitated by any excess of eosin. DiflEuse or contrast-stains are useful to make promi- nent certain of the tissue-elements left uncolored bjj the nuclear stain. A greater richness of detail is obtained with diffuse stains if, after rather deep staining, the sections be washed out for some time in alcohol, because certain struc- tures possess a greater affinity than others for certain diffuse stains, and by holding them are brought out sharply. Of the diffuse stains, eosin, picric acid, and acid-fuchsin in Van Gieson's mixture are the ones most frequently em- ployed. Eosin is most frequently used as a contrast to alum-hema- toxylin stains, but is often serviceable with alum-cochineal, methylene-blue, gentian-violet, etc. It brings out particu- larly well red blood-globules and smooth and striated muscle- fibers. The strength of the solutions used after hematoxy- lin varies from -^-^ to \ per cent, according to the tissue and the fixative used. Zenker's preparations stain intensely in eosin, so that for them a very dilute solution is advisable. When desired as a contrast-stain to basic aniline dyes, eosin should be used first in a 5 per cent, or even saturated solu- 2/2 PATHOLOGICAL TECHNIQUE. tion, because otherwise it is likely to be washed out by the nuclear stain. Picric acid is used for contrast with the carmine stains, more rarely with alum-hematoxylin. Striated muscle-fibers and cornified epithelium are rendered especially prominent by it. To stain with picric acid it is only necessary to add a few drops of a saturated aqueous solution to a dish of water, or of a saturated alcoholic solution to a little alcohol, and allow sections to remain in the solution for a few seconds. Van Gieson's stain (see p. 249), a mixture of picric acid and acid fuchsin, is excellent as a contrast-stain to alum- hematoxylin, especially when it is desirable to render promi- nent connective-tissue fibrillae or certain pathological prod- ucts. ^The nuclear stain with alum-hematoxyhn rnyst be rather deep, because the picric acid to some extent extracts or overpowers it. 1. Stain deeply in alum-hematoxylin. 2. Wash in water. 3. Stain in Van Gieson's solution three to five minutes. 4. Wash in water and dehydrate directly in 5. Alcohol, 95 per cent. 6. Oleum origani cretici. 7. Canada balsam. Neutral Carmine (see page 245). — Neutral carmine is a diffuse stain, and is employed more especially for the central nervous system and for bone. Filter one or two drops of the solution into 20 c.c. of dis- tilled water, and leave the sections in the dilute solution over night. It is advisable to place a piece of filter-paper on the bottom of the dish for the sections to rest on, otherwise they may be stained on the upper side only. In double stains with hematoxylin and carmine the sections should be stained first in the hematoxylin and then thoroughly washed in water for six to twelve hours before they are stained in the carmine. After the carmine they are again to be thoroughly washed in water. Combination Stains. — Biondi-Heidenhain Stain (see p. 250). — Tissues must be hardened in corrosive sublimate. HISTOLOGICAL METHODS. 273 1 . Stain paraffin sections six to twenty-four hours with the dilute solution. 2. Wash out a little in 90 per cent, alcohol. 3. Dehydrate in absolute alcohol. 4. Xylol. 5. Canada balsam. It is important to place the sections directly from the staining fluid into the alcohol, because water washes out the methyl-green almost instantly. Staining in Mass. — The staining of tissues in mass is a procedure much less employed in pathological than in nor- mal histology, but still occasionally useful. For patholog- ical tissues a variety of stains is generally necessary. It is therefore much better to make a series after one of the methods described, and then to stain the sections in what- ever way seems best. For staining in bulk only a limited number of solutions are available — either those, like alum-carmine and alum- cochineal, which do not stain beyond a certain point, or those, like lithium and borax-carmine and Heidenhain's hematoxylin, which may be decolorized so as to leave only the nuclei stained, The process of staining differs from that for sections only in the length of time required for each step. Tissues \ cm. thick will need from one to two days in the staining solution. MITOSIS. For the study of karyomitosis it is important that the tissue be perfectly fresh — that is, just removed from a living animal or from one just dead — and that it be fixed in a suitable reagent as quickly as possible. The best results cannot be obtained with tissues put into a hardening fluid over half an hour after removal from a living animal. On the other hand, mitotic figures can be demonstrated in tissues which have been dead for some time (twenty-four hours or more) before being put into a fixing reagent, but the details of the figures are not so perfect as those in perfectly fresh tissues, and the figures are not so numerous, because some 18 274 PATHOLOGICAL TECHNIQUE. of them have completed their changes and can no longer be recognized. It is therefore evident that mitosis can be studied much better in tissues from the lower animals, or in tissues removed by operation from the human body, than in the organs and tissues removed at post-mortem examinations. The choice of fixing reagents for the study of mitotic figures is important. The figures can often be demonstrated after hardening in alcohol or even in Miiller's fluid, but for their careful study quicker and more perfect fixing reagents must be used. Nearly all of the reagents employed pene- trate slowly, so that it is absolutely necessary for the best results that the tissue to be hardened be cut into very thin slices, rarely over 4 mm. in thickness and preferably not over 2 mm. The amount of fixing reagent used should always be at least ten to fifteen times as great as the volume of the tissue, and should be changed if it becomes cloudy. The most important fixing reagents are — 1. Flemming's solution. 2. Hermann's solution. 3. Pianese's solution. 4. Zenker's fluid. 5. Corrosive sublimate. 6. Orth's fluid. The first three solutions penetrate with much difficulty, so that tissues placed in them should be especially thin. The most generally useful stain for mitosis is probably safranin. The time of staining varies with the solution used. Babes' is the quickest. The mitotic figures should be stained deeply : then, when treated with alcohol slightly acidulated with hydrochloric acid, they will retain the color, while the resting nuclei will yield up most of theirs and become very pale or even colorless. In consequence of this intense stain mitotic figures can then be veiy readily found. Directions for Staining Karyomitotic Figures with Safranin. — i. Stain paraffin sections five minutes to twenty- four hours, according to solution used. 2. Wash in water. HISTOLOGICAL METHODS. 275 3. Wash in 95 per cent, alcohol to which are added a few drops of acid alcohol. 4. Wash in pure 95 per cent, alcohol, followed by absolute alcohol. 5. Xylol. 6. Xylol balsam. For celloidin sections, clear in oil of bergamot or oleum origani cretici after the 95 per cent, alcohol, wash out in xylol, and mount in xylol balsam. Safranin can be used after any of the above fixing reagents. Other useful stains are carbol-fuchsin and aniline-gentian- violet, used in the same way as the safranin. The Gram- Weigert method gives good results after Flemming's solu- tion. After fixing in corrosive sublimate mitotic figures can be demonstrated by the Biondi-Heidenhain solution, which stains resting nuclei blue-violet and mitotic figures green. After Pianese's solution his special staining mixtures should be used (see page 250). His methods are said to give beautiful results. THE STAINING OF BACTERIA IN TISSUES. Bacteria are demonstrated in sections of tissues almost entirely by means of the aniline dyes, of which three have thus far proven themselves to be particularly valuable — namely, methylene-blue, gentian-violet, and fuchsin. These dyes are employed in aqueous or dilute alcoholic solutions, of which the effective staining power is greatly increased by means of heat and by the addition to the solutions of certain chemical substances. The effect of moderate heat is obtained by placing the sections in the incubator for several hours, or greater heat for a short time is utiHzed by warming the staining solution on the slide over a small flame for a few seconds or minutes, keeping the fluid steaming, but not allowing it to boil. Of the various methods employed to increase the staining power of aniline dyes by means of chemical substances, the most successful have been the use of caustic potash with 2/6 PATHOLOGICAL TECHNIQUE. methylene-blue, of aniline oil with gentian-violet and fuchsin, and of carbolic acid with fuchsin and methylene-blue. For decolorizing sections after they have been stained the most commonly employed reagents are — 1. Acetic acid in dilute aqueous solutions i : lOO, i : looo. 2. Alcohol. 3. lodin in iodid-of-potash solution (with certain dyes only). 4. Mineral acids in various strengths. 5. Chlorid of aniline. 6. Acid aniline colors added to the alcohol to increase its extractive power. 7. Aniline and ethereal oils. The choice of a decolorizer varies with the staining solu- tion employed and with the organism that is to be stained. Sections which are to be stained for bacteria may be di- vided into two classes : 1 . Sections free from celloidin, subdivided into — a. Sections cut without an imbedding mass ; b. Paraffin sections ; c. Sections from which the celloidin has been removed. 2. Sections infiltrated with celloidin. Celloidin imbedding is to some extent a drawback to the stains for certain organisms, because the celloidin tends to hold the color, so that the bacteria are not so distinct as they otherwise would be. Still, it is so important to be able to stain bacteria in celloidin sections that particular care is de- voted in the following pages to methods which obviate most of the difficulties. Paraffin sections should, as a rule, be attached to the slide by means of Mayer's glycerin-albumin mixture. It will usually be found advisable to attach celloidin sec- tions to the slide by means of ether-vapor. They will then keep perfectly flat in any staining solution, ai;id may be heated without danger of wrinkling or contracting. The heat should never be applied directly under a section, but at one end of the slide. All bacteria yet known will stain when placed in appro- priate staining solutions. Some, however, are stained quickly, HISTOLOGICAL METHODS. 2// while others are stained with difficulty ; some give up the stain readily to decolorizers, while others retain it tenaci- ously. In consequence of their reactions to certain dyes and to certain decolorizers, bacteria, from the point of view of staining, niay be divided into three groups : 1. Bacteria which do not stain by Gram; 2. Bacteria which stain by Gram ; 3. Bacteria which stain by the tubercle bacillus method. Two at least of the organisms in the third group will also stain by Gram. The organisms of the second and third groups are much more easily demonstrated in tissues than those in the first group, because it is possible to stain them of one color and the nuclei of the cells of another color. In other words, it is possible to stain them so that they are differentiated from the tissue in which they lie, and hence stand out prominently. The organisms of the first group have no differential stain ; they take the same color as the nuclei of the tissue. More- over, although they stain easily, most of them do not stain deeply, and readily part with the color they have taken up. Pathogenic Bacteria which do not Stain by Gram. (See also page 91.) Gonococcus ; Diplococcus intracellularis meningitidis ; Typhoid bacillus; Glanders bacillus ; Bacillus of malignant edema ; Influenza bacillus ; Capsule bacillus ; Colon bacillus ; Spirillum of Asiatic cholera. Of these organisms certain ones deserve special mention on account of their frequent occurrence or on account of the difficulty of demonstrating them in tissues, and certain varia- tions in staining methods which have proved serviceable will be given. Loffler's methylene-blue solution is generally considered the most useful stain for this class of bacteria. 278 PATHOLOGICAL TECHNIQUE. but excellent results can also be obtained with Unna's alka- line methylene blue solution preceded by eosin, especially after fixation in Zenker's fluid. Loflfler's Methylene-blue Solution. — i. Stain paraffin sections twenty minutes to twenty-four hours. 2. Wash in weak acetic acid, i : 1000, for ten to twenty •seconds. 3. Absolute alcohol, two or three changes, to differentiate and dehydrate (as a rule, only a few seconds are required for this step). 4. Xylol. 5. Xylol balsam. For celloidin sections use 95 per cent, alcohol ; clear and finish the dehydration in oil of bergamot or origanum ; wash out the oil with several changes of xylol, and mount in xylol balsam. This solution of methylene-blue is extremely useful, be- cause it will stain all bacteria except the tubercle-bacillus group. Other solutions which may be used in the same way are — aniline-gentian-violet, Stirling's solution of gentian- violet, simple aqueous solutions of gentian- or methyl-violet, and Ziehl's carbol-fuchsin. Gonococcus. — Loffler's solution gives good results. Touton recommends staining sections in carbol-fuchsin and wa.shing out in alcohol. Typhoid Bacillus.— Typhoid bacilli in stained sections are generally best hunted for with a low power. The cha- racteristic colonies which they form are easily recognized. Good results in staining can be obtained with Loffler's methylene-blue solution used in the manner already de- scribed, but the stain is never very intense. For rendering the bacilli rather more prominent, so that small groups of them may be recognized, Flexner has recently advised the two following methods : A. — I. Stain paraffin sections in Loffler's methylene-blue solution for two hours. 2. Acetic-acid solution, i : looo, for several minutes. 3. Dehydrate in absolute alcohol. HISTOLOGICAL METHODS. 2"]^ 4. Oil of cloves to clear and differentiate. 5. Xylol, several changes. 6. Xylol balsam. B. — I. Stain sections in Stirling's gentian-violet solution for ten minutes. 2. Acetic-acid solution, i : 1000, for some minutes. 3. Dehydrate quickly in 95 per cent, alcohol. 4. Transfer to slide, blot, add oil of cloves to clear, and differentiate. Change the oil several times until the desired differentiation is obtained. 5. Wash off section several times with xylol. 6. Xylol balsam. Influenza Bacillus. — i. Harden in alcohol. 2. Stain half an hour or more in carbol-fuchsin diluted with 20 parts of water. 3. Wash out in a watch-glass of water to which is added a drop of glacial acetic acid until the section appears gray- violet in color. 4. Alcohol, oil, balsam. Glanders Bacillus. — The bacilli are usually not numer- ous, and are scattered about in a mass of deeply-staining fragmented nuclei, so that they are recognized with great difficulty. LoflBer's Method for Sections. — i. Stain paraffin sections twenty minutes in Loffler's methylene-blue solution or in equal parts of aniline-gentian-violet and i : 10,000 KOH solution. 2. Place for five seconds in the following solution : Distilled water, 10 c.c. ; Concentrated sulphuric acid, 2 drops ; 5 per cent, oxalic acid, i drop. 3. Wash out quickly in distilled water. 4. Absolute alcohol. 5. Xylol. 6. Xylol balsam. It is recommended to place the section for a few minutes before staining in the i : 10,000 caustic-potash solution. 280 PATHOLOGICAL TECHNIQUE. Schiitz's Method. — i. Stain twenty-four hours in equal parts of concentrated alcoholic solution of methylene-blue and caustic potash, i : 10,000. 2. Wash in acidified water. 3. 50 per cent, alcohol for five minutes. 4. Absolute alcohol for five minutes. 5. Xylol. 6. Canada balsam. Nonie-wicz's Method. — i. Stain in Loffler's methylene- blue solution two to five minutes. 2. Wash in water. 3. Decolorize one to five seconds in \ per cent, acetic acid, 75 parts ; \ per cent, aqueous solution of tropeolin, 25 4. Wash in water. 5. Dehydrate section on slide with filter-paper; then in the air; finally, over small flame. 6. Clear by dropping xylol on it repeatedly. 7. Xylol balsam. Friedlander's Capsule-bacillus. — The following meth- od is recommended for staining the capsules in sections : 1. Stain for twenty-four hours in the incubator in the fol- lowing solution : Concentrated alcoholic solution of gentian-violet, 50 ; Distilled water, lOO; Glacial acetic acid, 10. 2. Wash out in a I per cent, solution of acetic acid. 3. Alcohol. 4. Oil. 5. Canada balsam. If the process of decolorization is stopped at the right moment, the capsules will be pale blue, while the bacilli will be stained deep blue. Pathogenic Bacteria which Stain by Gram. Micrococcus lanceolatus ; Streptococcus pyogenes ; HISTOLOGICAL METHODS. 281 Staphylococcus pyogenes aureus, albus, citreus, and flavus ; Micrococcus tetragenus ; Anthrax bacillus ; Bacillus of rhinoscleroma ; Diphtheria bacillus ; Tetanus ; Actinomyces ; Tubercle bacillus ; \ Leprosy bacillus. J These organisms, with the exception of the tubercle- bacillus group, are all readily stained by the general methods employed for staining under Group i. For staining most of them in sections, however, the differential Gram-Weigert method will be found to give the most satisfactory results. The Gram Staining Method. — Directions for staining paraffin sections : i. Stain in aniline-gentian-violet five to twenty minutes. 2. Wash in normal salt solution or water. 3. lodin solution (1:2: 300) one minute. 4. Wash in water. 5. Absolute alcohol, several changes, until no more color is given off and the section is apparently decolorized. 6. Xylol. 7. Xylol balsam. This method is not suited for celloidin sections, because the alcohol does not decolorize the celloidin sufficiently. In fact, it is better to reserve Gram's method for cover-slip work alone, and to use instead of it, for sections of all kinds, Weigert's modification. This consists simply in the use of aniline oil instead of alcohol as a decolorizer. The method is easily acquired, is perfectly adapted to celloidin sections, and the results are more perfect than after Gram. The Gram-Weigert Staining Method. — Directions for staining celloidin sections: i. Stain sections with lithium carmine in the ordinary way (see page 244). 2. After dehydrating in 95 per cent, alcohol stick the sec- tion to the slide with ether- vapor. 3. Stain in aniline-gentian-violet five to twenty minutes. 282 PATHOLOGICAL TECHNIQUE. 4. Wash off excess of stain in normal salt solution. 5. lodin solution (1:2: 100) one minute. 6. Wash off in water. 7. Blot section with filter-paper to remove as much of the moisture as possible. 8. Aniline oil, several changes, to dehydrate and to re- move all excess of color. 9. Xylol, several changes to remove the aniline oil com- pletely. 10. Xylol balsam. Bacillus of Rhinoscleroma. — Method of staining cap- sules in sections of tissues hardened in alcohol (Wolko- witsch) : I. Stain twenty-four to forty-eight hours in aniline- gentian-violet. 2. Wash off in water. 3. lodin solution one to four minutes. 4. Absolute alcohol. 5. Oil of cloves, which removes still more of the color. 6. Xylol. 7. Canada balsam. According to Wolkowitsch, the hyaline masses in rhino- scleroma stain intensely with methyl-violet, gentian-violet, methylene-blue, and fuchsin ; less with safranin, and not at all with hematoxylin. Eosin stains them well. Double staining with hematoxylin and eosin is therefore to be recommended highly. Actinomyces. — In staining actinomyces it is important to stain not only the filaments and other forms of the organ- ism, but also the hyaline swollen sheaths which surround the ends of the filaments. It is believed that the two fol- lowing methods will give better results than can be obtained by any of the methods previously published for this purpose. The first is, perhaps, the better and surer, although the clubs are sometimes brought out more intensely by the second method. Method No. 1 (Mallory). — i. Stain sections deeply in a saturated aqueous solution of eosin for at least ten min- utes. HISTOLOGICAL METHODS. 283 2. Wash off in water. 3. Stain in aniline-gentian-violet two to five minutes. 4. Wash off with normal salt solution. 5. lodin solution (1:2: 100) one minute. 6. Water. Blot with filter-paper. 7. Aniline oil till section is clear. 8. Xylol, several changes. 9. Xylol balsam. A light preliminary stain with alum-cochineal will often be found useful. Method No. 2 (Mallory). — i. Stain lightly in alum-coch- ineal three to five minutes. 2. Wash in water. 3. Dehydrate in 95 per cent, alcohol. 4. Fasten section to slide with ether-vapor. 5. Aniline-gentian-violet five to twenty minutes. 6. Wash off with water. 7. Dry with filter-paper. 8. Aniline saturated with fuchsin one to three minutes. 9. Wash out the fuchsin with pure aniline until the clubs are sharply differentiated : watch the process under the low power of the microscope. 10. Xylol, several changes. 11. Xylol balsam. The polymorphous bacterium is stained blue, the swollen membrane (the club), light to dark pink. Alum-cochineal furnishes a better contrast to the actinomyces than either alum-carmine or alum-hematoxylin. By these methods it is possible to demonstrate in sections containing young colonies the ends of the threads stained blue surrounded by the swollen cell-membrane stained pink. Bacteria that Stain by the Tubercle Bacillus Method. Tubercle bacillus ; Leprosy bacillus ; Smegma bacillus ; Syphilis bacillus. The important point about staining tubercle bacilli is to 284 PATHOLOGICAL TECHNIQUE. stain them deeply enough in the beginning ; then there is little danger of their fading in the subsequent steps of con- trast-staining. It is probable that carbol-fuchsin, used hot, is the most powerful stain we have for this purpose. If the solution is steamed, generally on the slide, one to five min- utes are probably sufficient for all purposes. Tubercle bacilli stain well, not only after alcohol, but also after most of the other fixing reagents, such as corrosive sublimate, Zenker's fluid, Flemming's solution, etc. Ehrlich's Method. — i. Stain paraffin sections in aniline- fuchsin or gentian-violet for half an hour to twenty-four hours, or for one to five minutes if solution is heated to steaming. 2. .Wash in water. 3. Decolorize in 20 per cent, nitric acid one-half to one minute. 4. Wash in 70 per cent, alcohol until no more color is given off 5. Contrast-stain in a saturated aqueous solution of meth- ylene-blue or of Bismarck brown one to two minutes. 6. Wash in water. 7. Dehydrate in absolute alcohol. 8. Xylol, xylol balsam. Ziehl-Neelson-Gabbet Method. — i. Stain paraffin sec- tions in carbol-fuchsin solution, warming the solution so that it steams one to three minutes. 2. Wash in water. 3. Decolorize and stain for contrast in sulphuric-acid- methylene-blue solution one minute (see page 246). 4. Wash in water. 5. Absolute alcohol. 6. Xylol. 7. Xylol balsam. This method is not suited to celloidin sections, because the celloidin retains too deep a blue stain. Kiihne's Method. — i. Stain paraffin sections lightly in alum-hematoxylin. 2. Wash in water. HISTOLOGICAL METHODS. 285 3. Stain in carbol-fuchsin one to five minutes if warmed; longer if cold. 4. Wash in water. 5. Aniline hydrochlorate, 2 per cent, aqueous solution, fif- teen seconds. 6. Wash in water. 7. Absolute alcohol. 8. Xylol. 9. Xylol balsam. To Stain Tubercle Bacilli in Celloidin Sections. — i. Stain rather lightly in alum-hematoxylin. 2. Wash in water. 3. Carbol-fuchsin five to twenty minutes cold ; one to five minutes steaming. 4. Water. 5. Orth's discharging fluid (acid alcohol) one-half to one minute. 6. Wash in several changes of water to remove acid thor- oughly and to bring back blue color to nuclei. 7. Alcohol 95 per cent, until fuchsin is entirely discharged. 8. Aniline. 9. Xylol, several changes. 10. Xylol balsam. The advantages of this method are — that the celloidin is colorless : the nuclei are stained blue ; the rest of the tissue is colorless ; the tubercle baciUi stand out in sharp contrast. It is sometimes an advantage to bring out the cell-proto- plasm and the intercellular substance by staining the sec- tions, after decolorization in alcohol, in an aqueous solution of orange G or methyl-orange for a few seconds. The bacillus of leprosy stains more easily than the tubercle bacillus. Simple aqueous solutions of the aniline dyes are sufficient. The same methods can be employed as for tubercle bacilli. Baumgarten gives the following differ- ential stain for leprosy bacilli : I. Stain six to seven minutes in a dilute solution of fuch- sin (5 drops of a concentrated alcoholic solution to a watch- glass of water). 286 PATHOLOGICAL TECHNIQUE. 2. Discharge one-quarter minute in nitric-acid alcohol (nitric acid i, alcohol lo). 3. Wash in water. 4. Contrast-stain in a saturated aqueous solution of meth- ylene-blue. 5. Alcohol. 6. Xylol. 7. Balsam. While leprosy bacilli stain readily by this method, tubercle bacilli will not stain in so short a time. Syphilis Bacillus. — Lustgarten's Method. — i. Stain twenty-four hours at room-temperature and two hours in the thermostat at 40° in aniline-gentian-violet. 2. Wash off in absolute alcohol three to five minutes. 3. Decolorize (a) in a ^ per cent, aqueous solution of per- manganate of potassium, and then [B) a few seconds in an aqueous solution of pure sulphurous acid (strength not given). 4. Wash in water. 5. Alcohol. 6. Oil of cloves. 7. Canada balsam. If the section is not entirely decolorized when the section is put into water, then the third step must be repeated until decolorization is complete. If desired, the sections can be stained in safranin after the fourth step. Giacomi's Method. — i. Stain several minutes in hot ani- line-fuchsin. 2. Wash out in very dilute aqueous solution of chlorid of iron. 3. Decolorize in concentrated solution of chlorid of iron. 4. Wash out in absolute alcohol. 5. Xylol. 6. Xylol balsam. The smegma bacillus is stained by Lustgarten's method, but not by the methods given for tubercle bacilli in tissues, because in all of them alcohol is used as well as an acid to effect the decolorization. HISTOLOGICAL METHODS. 287 METHODS OF EXAMINATION OF ANIMAL PARASITES. Protozoa. — Of the rhizopoda, the malarial organisms and the ameba coli are of much pathological interest. Malarial Organisms. — Three varieties of the plasmodium malarise have been described — namely, the tertian, quartan, and estivo-autumnal parasites. They develop within the red corpuscles and cause the destruction of the corpuscles af- fected. The earliest forms of the parasite appear in the blood during the latter part of the malarial paroxysm or shortly after it. At this time they appear as small, color- less, disc-shaped hyaline bodies which occupy but a small portion of the blood-corpuscles. They possess a varying degree of ameboid movement, the amount depending upon the type of the organism. These ameboid movements are best observed on the warm stage. During the process of development the parasites increase in size and more or less completely fill the red corpuscles containing them. Small particles of reddish-brown pigment are produced, during their growth, from the hemoglobin of the corpuscles in which the organisms are developing. These granules show varying degrees of motion, probably imparted to them by the movements of the parasites. At first the pigment ap- pears to be scattered about in the corpuscle, but it is in real- ity in the extremities of the pseudopodia. Later it appears more evenly spread about in the periphery. Toward the end of the cycle of development the pigment collects in the center of the parasite ; at this time the ameboid movements have ceased, indications of segmentation occur, and the parasite nearly or completely fills the corpuscle. Oftentimes at this stage only a small portion of the corpuscle is visible at some point on the edge of the parasite. The beginning of segmentation is indicated by a number of radial lines extending from the periphery of the parasite toward the central clump of pigment. Segmentation takes place, and the pigment is surrounded by a number of dis- tinct segments which vary with the type of the organism. Each of these segments shows a central refractive spot 288 PATHOLOGICAL TECHNIQUE. which probably is the nucleus. At this time one notices small hyaline bodies, like those of the early stage in the de- velopment of the parasite, in some of the red blood-cor- puscles. Oftentimes such a regular process of segmentation is not observed, but enough has been said to indicate the manner in which reproduction occurs. Segmentation is the indication of an approach of a paroxysm. Extra-cellular forms of the parasites are not infrequently seen. They may be fully-grown organisms which have destroyed the cor- puscles that contained them, or they may be partly-grown organisms which have left the corpuscles. These free para- sites are indistinct in outline and contain pigment. They possess ameboid movements, and may be considerably larger than a red blood-corpuscle. Various changes are observed in them : 1. They may increase in size until they become nearly as large as polymorphonuclear leucocytes. With the increase in size there is a gradual cessation in the movement of the pigment-granules until finally the organisms present the ap- pearance of misshapen masses of protoplasm containing motionless pigment-granules. 2. They may undergo fragmentation and give off several small circular pigmented bodies. 3. Vacuolization may occur. 4. Flagellate forms may develop. One or more thread- like processes are thrust out from the organisms. These flagella may contain pigment, and may break away from the organism and move about among the corpuscles, looking not unlike the spirilla of relapsing fever. The three varieties of parasites differ from each other in a number of ways. The chief differences are the length of the cycle of development ; the size of the full-grown organ- isms ; the difference in the refractibility of the organisms ; the quantity, size, and color of the pigment-granules ; the degree of ameboid movement ; and the number and shape of the segments into which the full-grown organisms divide. In the earliest stage the varieties of organisms cannot be dis- tinguished from each other. HISTOLOGICAL METHODS. 289 The tertian parasite completes its cycle of development in about forty-eight hours. When it has attained its fullest growth it almost fills the corpuscle, which has become larger than normal. This organism is less refractive than either of the other two. The pigment-granules are more numerous, finer, and more reddish-brown in color ; the ame- boid movements are much more active ; the segments are more irregular in shape and more numerous than those of the quartan parasite, varying from twelve to twenty in number. The quartan appears to complete its cycle of development in from sixty-four to seventy-two hours. The full-grown organism does not fill completely the corpuscle, and the latter is not increased in size. The organism is more refractive than the tertian parasite. The pigment-granules are fewer in number, coarser, and have a darker-red color. The ame- boid movements are slower ; the segments are pear-shaped, more symmetrical, and less numerous than those of the ter- tian parasite, varying from six to twelve in number. Seg- menting organisms are more numerous in the peripheral circulation than in the case of the tertian parasite. The estivo-autumnal parasite cannot be studied so thor- oughly in the peripheral circulation, because the later develop- ment and segmentation take place in the internal organs. The length of time required to complete its cycle of development is not so definitely settled. It appears to require from twelve to twenty-four hours, more or less. The full-grown organ- ism is smaller than the tertian parasite, and the corpuscle which contains it is often smaller than normal and more or less distorted. The parasite is quite refractive. The pig- ment-granules are few in number and coarse. The ameboid movements are slow. After the duration of fever for from five days to a week or more, elongated, ovoid, or crescent- shaped bodies make their appearance. They are sometimes as large or larger than a red corpuscle. These bodies are not a result of segmentation, but appear to be a further de- velopment of the round hyaline bodies. They are highly refractive and contain granules of coarse pigment m the center. They lie usually at one side of the red corpuscles, 19 290 PATHOLOGICAL TECHNIQUE. the latter more or less completely filling the concavity be- tween the two horns of the crescent. They may lie in the center of the corpuscles. Some of the apparently free ovoid bodies are turned in such a way as to present a convex sur- face toward the observer. Double infections occur quite frequently in both tertian and quartan fever, and in the latter not infrequently triple infections occur. In the double infections two groups of parasites reach maturity on successive days and cause daily febrile paroxysms. In the triple infection of quartan fever three groups of organisms mature on successive days and cause corresponding paroxysms. Methods of Examining the Blood for Malarial Organisms. — The organisms of malaria can be detected in fresh speci- mens of blood or in specimens of blood which have been fixed and stained. The examination of fresh specimens of blood is simpler and more reliable, because the development of the parasites can be observed. The method employed in making cover-glass preparations of the blood has been thoroughly described (see preparation of cover-glass specimens in the Examination of the Blood, page 336). In examining a fresh specimen of the blood for the malarial organisms a glass slide is substituted for one of the cover- glasses, and the cover-glass which has the drop of blood on its surface is dropped lightly upon the glass slide and allowed to remain there. The first four or five drops of blood should be quickly wiped away from the ear until a very small drop is obtained. Great care must be exercised to touch only the tip of the drop with the cover-glass, so as to avoid smearing the blood. If the blood is smeared on the cover-glass, the edges of the blood-drop will dry before the cover-glass can be transferred to the slide, and the blood will not spread. It is necessary that the blood should spread in a thin layer in order to study satisfactorily the individual corpuscles. If one desires to study the preparation for several hours, the edges of the cover-glass can be sur- rounded by melted paraffin or vaselin to exclude the air. HISTOLOGICAL METHODS. 29I The examination should be made with an oil-immersion lens. It should be remembered that the action of cold inhibits the ameboid movements of the parasites ; it may be necessary, therefore, at times to warm the slide before examining the specimen. Evaporation not infrequently occurs, caused by the air penetrating beneath the cover-glass. This produces changes in many of the corpuscles which may be mistaken for hyaline bodies : the central depression becomes paler and less refractive than the periphery of the corpuscles ; later a number of corpuscles contain small glistening points, and still later the corpuscles become crenated. The method of procuring permanent cover-glass prepara- tions is the same as that elsewhere described (see Cover- glass Preparations, page 336). Specimens may be fixed by heating or by a mixture of absolute alcohol and ether, equal parts. Numerous methods of staining have been employed. The following methods are simple and satisfactory : The fixed cover-glass preparation is stained from one to two minutes in a concentrated aqueous solution of methylene- blue. The staining solution should be filtered before using. The specimen is thoroughly washed in water, dried, and mounted in Canada balsam. This stains the organisms and th« nuclei of the white corpuscles blue. The red corpuscles are unstained. A good contrast-stain can be obtained with eosin and methylene-blue. The fixed specimens are stained for from thirty seconds to five minutes in a J per cent, solution of eosin in 60 per cent, alcohol, washed in water, dried, and placed for from thirty seconds to one minute in a concen- trated aqueous solution of methylene-blue, washed, dried, and mounted in Canada balsam. The red corpuscles are stained a bright red by the eosin, and the organisms and the nuclei of the white corpuscles are stained blue. Excellent results are obtained by Romanowsky's method. This method is as follows : A saturated aqueous solution of methylene-blue and a i per cent, aqueous solution of eosin are kept separately. The older the methylene-blue solution the better the results. The specimen is then heated 292 PATHOLOGICAL TECHNIQUE. not less than thirty minutes at a temperature of from 105° to 1 10^ C. The staining mixture is made just before it is to be used. To i part of the filtered methylene-blue solu- tion are added about 2 parts of the eosin solution. This mixture is carefully stirred with a glass rod, but not filtered, and poured into a watch-glass. The cover-glass prepara- tions are allowed to float upon the top of the fluid with the blood surface down. The specimens are covered by another inverted glass, and the whole by an inverted cylinder which is moistened upon the inside. Good specimens are obtained in from one-half to three hours. Romanowsky believes that he obtains thus three colors : the red corpuscles stained red by the eosin, the malarial parasites a Prussian blue by the methylene-blue, and the nuclear chromatin a violet color (a neutral stain). Thayer and Hewitson claim to have ob- tained their best results by means of a slight modification of Romanowsky's method. The modification consists in fixing the specimens in absolute alcohol for from ten to twenty minutes instead of employing heat. In the fixed and stained specimens there is always a possi- bility of mistaking small particles of methylene-blue, which have passed through the filter-paper, for malarial organisms. Sometimes these particles lie on the red corpuscles, and it is difficult to determine whether they are inside of the cor- puscles or lying on the surface. Note. — The de.scription of the development of the parasites is abstracted from Thayer and Hewitson's The Malarial Eevers of Baltimore. Ameba Coli. — In cases of dysentery suspected of being due to the ameba coli (Fig. 88) the stools are best examined as soon as voided, although the amebae will sometimes re- main active in stools even over twenty-four hours old. A warm stage during the examination is an advantage, but not a necessity. A drop of the fluid material, preferably that containing mucus or blood, is placed on a slide and lightly covered with a cover-glass. If the slide is cold and the or- ganisms do not move, warm the slide gently and the move- ments of the amebse will often start up. Pus from abscesses due to the amebse is examined in the same way. A positive HISTOLOGICAL METHODS. 293 diagnosis rests on the presence of the characteristic large, pale cells, consisting of nucleus, granular endosarc, and hyaline ectosarc, and on the movements of the protoplasm, which projects itself more or less actively in the form of pseudopodia. In hardened preparations the nuclei of the amebae do not stain with the ordinary nuclear stains, such as alum-hema- toxylin and methylene-blue. The following method ofstain- FlG. 88.- -Amebne coli in intestinal mucus {after Losch). ing them has been found to give very satisfactor)' results and to render the recognition of the organisms easy : Differential Stain for the Aineba Coli [Mallory). — i . Harden in alcohol. 2. Stain sections in a saturated aqueous solution ofthionin three to five minutes. 3. Differentiate in a 2 per cent, aqueous solution of oxalic acid for one-half to one minute. 4. Wash in water. Dehydrate in alcohol. Clear in oleum origani cretici. Wash off with xylol. X}'lol balsam. The nuclei of the amebae and the granules of the mast- 7X-llen are stained brownish red : the nuclei of the mastzellen and of all other cells are stained blue. Excellent results were obtained by this method with bits 294 PATHOLOGICAL TECHNIQUE. of the purulent discharge from a so-called amebic abscess of the liver. After hardening in 95 per cent, alcohol, small fragments the size of a pin-head and less were stained as above directed, and teased apart after they were in the balsam. The reddish nuclei stood out so sharply in the bluish background of fragmented nuclei and granular de- tritus that they were easily picked out with the high dry power. The results obtained with feces examined in the same way or after imbedding in celloidin were much less satisfactory, for the reason that various substances in the feces precipitate the thionin in the form of reddish crystals and give rise to deceptive pictures. A similar differential stain can be ob- tained by Unna's method for staining the granules of mast- zellen (see page 300). Other protozoa, such as the cercomonas and trichomonas, are best examined in fresh preparations. Sporoijoa. — The coccidium oviforme should be examined both fresh in cover-slip preparations and in sections after hardening. The cyst-forms often stain well by the tubercle- bacillus method. For studying all the stages in the develop- ment of the organism Pianese recommends highly his special fixing reagent and stains (see p. 225). Other sporozoa should be studied in the same manner. ,Cover-slip prep- arations are often useful. Round-worms. — The embryos of the filaria sanguinis hominis (Fig. 89) are examined for in suspected cases by mounting a drop of the fresh blood or of the chylous or bloody urine on the slide and examining under a low power. They are readily detected when present on account of their FIG. s^Fiiaria sl^guinis hominis ^^'7 ^^tive movements. They (after von jaksch). should be looked for during the resting hours of the pa- tient, as at night for day-workers and during the day for night-workers. HISTOLOGICAL iVETIIODS. 295 TricliinEe (Figs, 90, 91 ) are obtained from the fresh muscle by means of teasing. A quick method is to squeeze small bits of tissue between two slides and examine with a low power. Pieces of muscle nearest the insertion of the ten- FlG. 90. — Living embryos (Heller). Flo 91 — Encapsulated trichina (Leuckart). don are chosen from the diaphragm or from the muscles of the jaws. Encjipsulated and calcified trichina; are cleared up by means of acids. Fig. 92. — Taenia solium : A, head enlarged ; B, ripe joint, X 6 ; C egg of tasnia solium ( Heller). In hardened tissues the trichinae are best studied in longi- tudinal sections of the muscle-fibers. The other round-worms which sometimes occur in the 296 PATHOLOGICAL TECHNIQUE. intestinal tract can be recognized with the naked eye. Their eggs must be looked for with the microscope. Tape-worms. — It is not always easy to recognize the kind of tape-worm by a single segment passed with the feces, because the uterus, which furnishes the most characteristic Fig. 93. — Taenia mediocanellata : A, liead darkly pigmented ; B, ripe joint, X 6 ; C, ^g2^ of t;^nia mediocanellata. points of difference, is not developed in the young segments and is atrophied in the old ones. When the whole worm is obtained the problem is much simpler. The uterus is best made out by squeezing a segment between two slides and f 10 y Fig. 94. — Echinococctis ; solices, hooks (Heller). holding it up to the light. The heads are examined under the microscope in water, salt solution, or glycerin. Tsenia Soliuin (Fig. 92). — Head has four suckers and a HISTOLOGICAL METHODS. 297 circle of booklets ; uterus is noticeably but little branched. The genital tract opens laterally. The eggs de\-elop into the cysticcrci ccllulosa;, which are not infrequently found in man. Fu;. 95.— Bothriocephalus latus ; A, head ; /?, ripe joint, X 6 ; C (^-g-g of bothrio- ccphalus latus (Heller) ; D. egg with developed embryo (Leuckart). The scolex is obtained for examination by tearing open the cyst and examining the inner wall. The suckers and hook- FlG. 96. — Segments of t^t'nia saginata (after Stem). Fic. 97. — Segments of FiG. 98. — Segments bothriocephalus latus of taenia solium (after (after Stein). Stein). lets are best studied after mounting fresh and pressing under a cover-glass 298 PATHOLOGICAL TECHNIQUE. Taenia Mediocanellata s. Saginata (Fig. 93). — Head has four strong suckers, but no booklets ; uterus is very much branched, segments show marked muscular development. The genital tract opens laterally. The eggs develop into cysticerci, which do not occur in man. Taenia Bchinoooccus (Fig. 94) occurs in dogs. The echin- ococcus cysts which occur in man are recognized by the very characteristic laminated structure of the cyst-wall. The heads of the scolices have four suckers and a double circle of booklets. Bothriocephalus Latus (Fig. 95). — The opening of the a b c d e Fig. 99. — Comparative size of eggs of intestinal parasites : a, taenia solium ; ^, taenia mediocanellata ; c, ascaris lumbricoides ; plus one-third to one-half as much absolute alcohol until the sections become gray to black in color. If too much alcohol is added, the carbonate of potassium will be pre- 312 PATHOLOGICAL TECHNIQUE. cipitated, but will redissolve on the addition of a little more developer. 2. 70 per cent, alcohol for ten to fifteen minutes. 3. Hyposulphite of sodium (20 per cent, aqueous solu- tion). 4. Wash thoroughly in a large amount of water for twenty- four hours. 5. Alcohol, oil, Canada balsam ; cover-glass. Cox's Modification of Golgi's Corrosive-sublimate Method. — The same black pictures are obtained by this method as by Golgi's, but with this difference, that nearly all of the cells in the section are impregnated. This is an ad- vantage when the topographical arrangement of the cell- layers is desired, but a disadvantage when it comes to the study of individual cells, because on account of the luxuri- ance of the impregnation such a study is rendered impos- sible. Small pieces of nervous tissue are placed in the fol- lowing solution : Bichromate-of-potassium 5 per cent, solution, 20 ; Corrosive-sublimate 5 per cent, solution, 20 ; Distilled water, 30-40; Simple chromate-of-potassium 5 per cent, solution, 16. The time required for impregnation is a month in summer and two to three months in winter. The after-treatment is the same as for Golgi preparations. Axis-cylinders and their Terminal Processes. — The three methods most in use for the study of central and peripheral nerve-fibers and their terminations are the gold, the Golgi, and the methylene-blue method. All three may give beautiful results, but, as a rule, they are very unreliable. Their use is confined almost wholly to the study of normal tissues. Gold Stain for Nerve-fibers. — For the application of the gold method to fresh tissues see p. 260. Various attempts have been made to devise a reliable method of employing chlorid of gold for staining nerve- fibers in sections of hardened tissues. The results have not HISTOLOGICAL METHODS. 313 been altogether successful. The best results can probably be obtained by — A. Gerlach's Method. — i. Harden tissues in a 1-2 per cent, solution of bichromate of ammonium for one to three weeks ; cut sections without passing through alcohol, which must be avoided. 2. Place the sections in a very dilute solution (y^ per cent.) of the double chlorid of gold and potassium very slightly acidulated with hydrochloric acid, for ten to twelve hours, until they become slightly violet in color. 3. Wash in a solution of hydrochloric acid i, to water 2000-3000. 4. Place for ten minutes in a ^ per cent, solution of hy- drochloric acid in 60 per cent, alcohol. 5. Absolute alcohol, oil of cloves, Canada balsam. Another method frequently recommended is the following : B. Freud's Gold Stain for Nerve-fibers. — i. Harden tissues in Erlicki's or Miiller's fluid, followed by alcohol. Imbed in celloidin. 2. Stain sections three to five hours in i per cent, solution of chlorid of gold, and 95 per cent, alcohol, equal parts. 3. Wash in water. 4. Reduce in — Caustic soda, i ; Distilled water, 6, for two to three minutes. 5. Wash in water. 6. Place for five to fifteen minutes in a 10 per cent, solu- tion of iodid of potassium. 7. Wash in water. 8. Alcohol, oil, Canada balsam. C. Stroebe's Aniline-blue Stain for Nerve-fibers in Hard- ened Sections. — Harden tissues in Miiller's fluid. i. Stain one-half to one hour in a saturated aqueous solution of ani- hne-blue. 2. Wash in water. 3. Transfer to a small dish of alcohol to which are added 314 PATHOLOGICAL TECHNIQUE. 20 to 30 drops of a I per cent, alcoholic solution of caustic potash (caustic potash i to alcohol 100 : let the mixture stand for twenty-four hours ; then filter). In one to several minutes the sections become bright brownish-red and trans- parent. 4. Transfer to distilled water for five minutes. The sec- tion becomes bright blue again. 5. Stain in a half-saturated aqueous solution of safranin one-quarter to one-half hour long. 6. Wash out and dehydrate in absolute alcohol. 7. Xylol, Canada balsam. D. Chlorid-of-iron and dinitroresorcin method for the study of degenerated peripheral nerves : 1. Place fresh pieces of peripheral nerves for several days in a solution of — Chlorid of iron, i part; Distilled water, 4 parts. 2. Wash out thoroughly in water. 3. Transfer to a saturated solution of dinitroresorcin in 75 per cent, alcohol for several weeks. 4. Wash, dehydrate, imbed, etc. A permanent green color is formed which stains the nerves green and brings out the green axis-cylinders very sharply. The stain will succeed with preparations which have been hardened in Flemming's solution or Miiller's fluid. Golgi's methods are sometimes employed for the study of the terminal processes of nerve-fibers (for directions see p. 308). Methylene-blue Stain for Nerve-flbers. — The methylene- blue method is due to Ehrlich. Many modifications of the original procedure have been suggested with a view to mak- ing the results surer or the specimens more permanent. Tissues can be stained either by injection or by immersion. The methylene-blue used should be Griibler's " rectified methylene-blue for vital injection." For injection in the blood- or lymph-vessels of live or dead animals a i to 4 per cent, solution in normal salt solu- HISTOLOGICAL METHODS. 315 tion is recommended. The injected organs are exposed to the air until a bluish tint is visible. As soon as the greatest intensity of stain is reached (five minutes to two hours) the color in the preparation is fixed by placing small bits of the tissue in a freshly-filtered, cold, saturated, aqueous solution of picrate of ammonium, or, better still, in the solution given below, recommended by Bethe. Very small or thin pieces of tissue intended for staining by immersion (the method employed for human tissues) are placed in a very dilute solution (iVtV P^*" cent.) of methyl- ene-blue in normal salt solution. Lavdowski recommends very highly a solution of methylene-blue in egg-albumin, either alone or combined with chlorid of sodium or ammo- nium. The white of egg is freed from the thicker portions or filtered. When the experiment is to last some time, add to the egg-albumin an equal part of a J per cent, solution of chlorid of sodium or of a J per cent, solution of chlorid of ammonium. The tissue, protected by a large dish, is exposed to the air for fifteen minutes to twelve hours, until the maxi- mum stain is obtained. The stain may then be fixed by the method already given, or, better still, in the following manner : Bethe' s Method of Fixing Methylene-blue Stains of Nerve- fibers. — I. Wash off excess of color with normal salt solu- tion. 2. Place in — Molybdate of ammonium, i gr. ; Distilled water, 10 c.c. ; Peroxid of hydrogen, I " Hydrochloric acid, I drop. A precipitate forms on making up the solution, but disap- pears on shaking. The solution will keep eight days, but is best made up fresh each time. It should be used as cold as possible, preferably surrounded by a mixture of ice and salt. Leave the tissue in the cold solution for from two to five hours, and then for a while longer at the room-temperature. 3. Wash one half to two hours in running water. 3l6 PATHOLOGICAL TECHNIQUE. 4. Dehydrate and harden as quickly as possible (not over twelve to twenty-four hours) in cold absolute alcohol. (The color is soluble in warm alcohol.) 5. Clear in xylol. 6. Imbed in paraffin. The sections may be mounted directly or brought into water and stained with alum-cochineal for contrast. If a little osmic acid be added to the fixing solution after the speci- mens have been in it for a while, a more permanent methyl- ene-blue stain is obtained. Stains for the Myelin-sheatli. — The myelin-sheath of nerve-fibers is a form of fat, and like it possesses the property of reducing osmic acid, by means of which a selec- tive sheath stain can be obtained. Unfortunately, however, the osmic acid penetrates to but a very slight depth. The differential hematoxylin stain, originated by Weigert, and ordinarily used, depends on some chemical reaction which takes place between the myelin and a chrome salt, in consequence of which the myelin is fixed so that it will not later be dissolved out by alcohol or ether, and at the same time is so mordanted that it can be deeply stained with hematoxylin, to which it clings when treated with certain decolorizers. This reaction between the myelin and the chrome salt takes place very slowly at the ordinary temper- ature ; six weeks to several months are usually required. Weigert has lately published a method depending on the interaction of two chrome salts in the same solution, in consequence of which the time needed for this reaction or mordanting is reduced to four days. The solution may be used alone, but is best combined with formaldehyde or used after it. Large masses of nervous tissue, Hke the medulla and pons or the basal ganglia, should be hardened in formal- dehyde for one to three weeks, and then cut into parallel slices not over i cm. thick for mordanting by Weigert's quick method. A. Weigert's Quick Method for Mordanting Myelin- sheaths. — i. Fix the tissues in a 4 per cent, solution of formaldeyhde (10 per cent, solution of formaline) for four HISTOLOGICAL METHODS. 317 days to several weeks. Four days are sufficient for pieces not over i cm. thick. The solution, of which several times the volume of the specimen must be taken, is changed at the end of twenty-four hours. The tissue may remain in- definitely in it. 2. Mordant the myelin-sheaths in the following solution ; Bichromate of potassium, 5 ; Chrome alum, 2 ; Water, lOO, for four to eight days. The tissues should not exceed i cm. in thickness, and should not be left in the solution longer than eight days, because they will become brittle. 3. Transfer directly to alcohol (80 per cent.), and keep in the dark until wanted for imbedding, changing the alcohol occasionally as it becomes colored. Steps I and 2 may be combined by adding 4 per cent, of formaldehyde to the mordanting solution and placing fresh tissues directly in the mixture. In place of Weigert's quick mordant, Miiller's fluid or a 5 per cent, solution of bi- chromate of potassium may be used after fixation in formal- dehyde, but the process of mordanting will require as long (six to eight weeks) as though the tissues had originally been placed in them. Weigert's quick mordant can be highly recommended, both on account of the short time it requires and because the stains obtained after it are very satisfactory. The great- est objection to it is that exposure for over four days to the mordant renders the perebral cortex very brittle. Weigerfs Myelin-sheath Stain. — i. Fix and mordant tlie tissues in one of the ways already described, or, if preferred, in Miiller's fluid. 2. Dehydrate in alcohol without washing out in water. 3. Imbed in celloidin. 4. Place sections for twenty-four hours in the following solution, which Weigert now recommends in place of the simple saturated solution of acetate of copper formerly ad- vised : 3l8 PATHOLOGICAL TECHNIQUE. Acetate of copper, 5. Acetic acid, 36 per cent, solution, 5. Clirome alum, 2.5 Water, ad 100. (For method of preparation see page 323). 5. Stain fifteen minutes to twenty -four hours in — Hematoxylin, i ; Absolute alcohol, 10; Saturated aqueous solution of carbonate of lithium, 7 ; Water, 90. Keep a 10 per cent, solution of hematoxylin in alcohol on hand, so that it will be ripe. Combine with the carbonate of lithium and the water at the time of using. 6. Wash thoroughly in water. 7. Decolorize in — Borax, 4 ; Ferrocyanide of potassium, 5 ; Water, 200, until the gray substance is distinctly yellow. 8. Wash thoroughly in water. 9. Dehydrate in 95 per cent, alcohol. 10. Clear in aniline oil 2, xylol I. 1 1 . Canada balsam. Weigert has given up his direct myelin-sheath stain be- cause the preparations do not keep. B. Pal's Modiflcation of Weigert's Myelin-sheath Stain. — I. Fixation as for Weigert's method. 2. Place sections for several hours in a J per cent, aqueous solution of chromic acid, or for a longer time in a 2—3 per cent, solution of bichromate of potassium. This step is often omitted, especially when the tissues have been but recently mordanted. 3. Transfer sections to Weigert's hematoxyhn solution for twenty-four to forty-eight hours (if necessary for an hour in the incubator at 37° C). HISTOLOGICAL METHODS. 319 4. Wash in water plus i to 3 per cent, of a saturated aqueous solution of carbonate of lithium until the sections appear of a uniform deep-blue color. 5. Differentiate for twenty seconds to five minutes in a J per cent, aqueous solution of permanganate of potassium until the gray matter looks brownish-yellow. 6. Transfer to the following solution : Oxalic acid, i ; Sulphite of potassium, i ; Water, 200, for a few seconds until the gray matter is colorless or nearly so. 7. Wash thoroughly in water. 8. Dehydrate in 95 per cent, alcohol. 9. Oil, Canada balsam. Steps 5 and 6 sometimes have to be repeated when the differentiation has not been complete. Of all the numerous modifications of Weigert's original myelin-sheath stain, the only one that has found general acceptance is Pal's. It has the following advantages : It gives very clear pictures ; everything except the sheaths is completely decolorized, so that contrast-stains are possible ; it is more successful with thick sections than Weigert's method ; the separate steps are quicker. On the other hand, the danger of decolorizing the sheaths of the finer fibers is greater. C. Bxner'a Method of Demonstrating Myelin-sheaths. — The tissue should be obtained as soon as possible after death, although the method will succeed with tissues even over twelve hours old. 1 . Place pieces of brain or cord not over \ cm. thick in a I per cent, aqueous solution of osmic acid, using at least ten times as much fluid as the volume of the specimen. 2. Change the osmic-acid solution on the second day. 3. After five or six days wash thoroughly in water. 4. Dehydrate, imbed, etc. 320 PATHOLOGICAL TECHNIQUE. 5. Examine sections in glycerin rendered slightly ammoni- acal. The myelin-sheaths appear gray to black. The prepara- tions are not permanent. This procedure has been almost entirely replaced by Wei- gert's method, which has numerous advantages. Lately, however, it has been brought forward again by Heller, who uses a photographic developer to reduce the osmic acid and to make possible permanent mounts. He has lately pub- lished the following method for sections, but it cannot be unconditionally recommended : D. Heller's Method for Staining Myelin-sheaths with Osmic Acid. — i. Harden as for the Weigert method (Heller used Muller's fluid). 2. Imbed in celloidin. 3. Place sections in a i per cent, aqueous solution of os- mic acid for ten minutes in thermostat or for half an hour at room-temperature. 4. Wash in water. 5. Reduce in the following developer: Sulphate of sodium, 125 ; Carbonate of sodium, 70; Water, 500; Pyrogallic acid, 15. 6. Wash in water. 7. Differentiate in an aqueous solution of permanganate of potassium, \ per cent, or less. 8. Remove the brown of the permanganate of potassium in a I per cent, aqueous solution of oxalic acid. 9. Wash in water. 10. Alcohol, oil, Canada balsam. By a modification of Heller's method Robertson claims to get better results. I. Harden in Weigert's chrome-alum-copper solution plus 4 per cent, of formaldehyde ; in other words, use the mor- dant for neuroglia-fibers (page 323) eight to ten days. HISTOLOGICAL METHODS. 32 1 2. Wash off in water. 3. Alcohol ; imbed in celloidin. 4. Stain sections in a i per cent, solution of osmic acid half an hour in the dark. 5. Place in a 5 per cent, aqueous solution of pyrogallic acid for half an hour. 6. Differentiate in a J per cent, aqueous solution of per- manganate of potassium one to four minutes. 7. Remove brown color in i per cent, oxalic acid three to five minutes. 8. Alcohol, oil, balsam. It is important to wash carefully in water between each of the staining steps. Stains for Neuroglia-fibers. — It is possible to obtain a differential stain of the neuroglia-fibers in man by three different methods, provided the first two of the following steps are complied with : 1. The tissue must be as fresh as possible. The best re- sults are obtained with tissues placed in the fixing solution within one hour after death. After four to six hours the re- sults are only fair ; after twenty-four hours they are practi- cally nil. The chemical property in the neuroglia-fibers on which the differential stain depends has undergone some chemical change or has disappeared. It is retained longest where the fibers are most numerous, as about the central canal. 2. Formaldehyde must be used as the fixative, either alone or combined with certain other reagents mentioned below. No other fixatives yet known penetrate so rapidly and pre- serve so well the chemical properties of the neuroglia-fibers. According to Weigert, who first fully recognized the value of formaldehyde in preserving neuroglia-fibers, the best strength to use is a 4 per cent, solution («'. ^. a lO per cent, solution of formaline, etc.). The pieces of tissue in which it is desired to stain the neurogha-fibers must be cut very thin, never over \ cm. thick, and preferably thinner. With thicker pieces only the surface sections are of any value. 3. The tissues after fixation must be mordanted, so as to 21 322 PATHOLOGICAL TECHNIQUE. render the staining more intense ; without mordanting only a few of the fibers will stain. 4. Staining may be performed by modifications of Wei- gert's fibrin-stain or with phosphotungstic-acid hematoxylin. The methods of staining neuroglia-fibers are given in the order of their publication. The second method (Weigert's) gives a more intense stain than the first, but has the draw- back of staining degenerated nerve-fibers. The third method is not so selective as the other two, but is useful because it brings out all the tissue-elements. All of the methods are unsuccessful with the neuroglia-fibers of animals other than man. Differential Stain for Neuroglia-fibers {Mallory). — i. Fix in a 4 per cent, aqueous solution of formaldehyde four days or more. 2. Place in a saturated aqueous solution of picric acid four to eight days. Steps I and 2 may be combined by adding 10 parts of the 40 per cent, formaldehyde solution to 90 parts of the satu- rated aqueous solution of picric acid. 3. Transfer to a 5 per cent, aqueous solution of bichro- mate of ammonium for four to six days in the incubator at 37° C, or for three to four weeks at room-temperature. Change the solution on the second day. 4. Place directly in alcohol. 5. Imbed in celloidin. 6. Fasten sections to slide by means of ether-vapor. 7. Stain in aniline-gentian-violet fifteen to twenty minutes. 8. Wash off with normal salt solution. 9. lodin solution, 1:2: lOO, for one minute, or a stronger solution for a few seconds. 10. Wash off with water. 11. Dry with filter-paper. 12. Decolorize in equal parts of aniline and xylol. 13. Wash off thoroughly with xylol. 14. Xylol balsam. The neuroglia-fibers, fibrin, nuclei, and, to some extent, the red blood-globules, are stained blue. The other tissue- HISTOLOGICAL METHODS. 323 elements are colorless. By verj'^ faintly tinting the aniline- and-xylol mixture with fuchsin, which is readily soluble in anihne, the other tissue-elements are easily brought out, but the finer neuroglia-fibers are likely to lose their blue color. Weigert's Method for Neurog-lia-flbers. — A. Fix thin pieces of tissue, not over J cm. thick, in a 4 per cent, solu- tion of formaldehyde for at least four days. B. Mordant in the following solution for four to five days in the incubator or for eight days at room-temperature : Acetate of copper, 5 gr. ; Acetic acid, 36 per cent, solution, 5 c.c. ; Chrome alum, 2.5 gr. ; Water, ad lOO c.c. Boil the chrome alum in a covered dish (it turns green in color), turn out the gas, add the acetic acid and then the acetate of copper; stir until the latter is dissolved, then cool. The solution remains clear. If the directions and order of procedure are not followed exactly, a green pre- cipitate will form. (Steps I and 2 may be combined by adding 4 per cent, of formaldehyde to the above solution ; change on the second day ; harden eight days.) C. Wash off in water; dehydrate in alcohol; imbed in celloidin. D. Reduction of copper salt in sections : 1. Place the sections, which must not be over .02 mm. thick, in a J per cent, aqueous solution of permanganate of potassium for ten minutes. 2. Wash off with water. 3. Decolorize and reduce for two to four hours in the fol- lowing solution, carefully filtrated : Chromogen, 5 gr.; Formic acid (sp. gr. 1.20), 5 c.c. ; Water, ad 100 " to 90 c.c. of which are added just before using 10 c.c. of a 10 per cent, solution of sulphite of sodium. 324 PATHOLOGICAL TECHNIQUE. The sections lose their color in a few minutes, but are best kept in the solution as long as above dirfected. The sections can now be stained in the manner to be de- scribed, but the color of the fibers will be more intense if the following steps are added, and a slight yellowish con- trast-stain is obtained for the ganglion and ependymal cells and for the larger nerve-fibers. E. Further reduction of copper salt : 1. Wash twice in water. 2. Place sections in a carefully filtered saturated (5 per cent.) aqueous solution of chromogen over night. 3. Wash in water. 4. The sections are now ready for staining or may be pre- served until wanted in — 80 per cent, alcohol, 90 c.c. 5 per cent, oxalic acid, 10 " F. Staining of neuroglia-fibers : 1. Lift section from large dish of water on slide freshly cleaned with alcohol ; blot with filter-paper (method recom- mended by Weigert for attaching sections to slide). 2. Stain in the following mixture : Saturated solution of methyl-violet in 70-80 per cent, alcohol, 100 c.c; (saturated with aid of heat ; decanted when cold). 5 per cent, aqueous solution of oxalic acid, 5 " The oxalic acid is added to render the preparations more lasting. The staining is practically instantaneous. 3. Wash off with normal salt solution. 4. lodin solution : 5 per cent, iodid-of-potassium solution saturated with iodin. It is simply poured on and then off, as the reaction is instantaneous. 5. Wash off with water and blot with filter-paper. 6. Decolorize thoroughly in equal parts of xylol and aniline oil. HISTOLOGICAL METHODS. 325 7. Wash repeatedly with xylol or the stain will not keep. 8. Canada balsam. The sections keep better if exposed for from two to five days to diffuse light before being put away. Differential Stain of Neuroglia-flbers by means of Phosphotungstic-acid Henaatoxylin (Mallory). — i. Fix and mordant tissues in exactly the same way as in the first method given for neuroglia-fibers — viz. : [a) Fix in 4 per cent, aqueous solution of formaldehyde four days. {J}) Saturated aqueous solution of picric acid four days. (c) 5 per cent, aqueous solution of bichromate of ammonium four to six days in incubator or three to four weeks at room-temperature. (d) Alcohol, celloidin, etc. 2. Stain sections in phosphotungstic-acid hematoxylin four to twenty-four hours. 3. Wash in water. 4. Alcohol. 5. Oleum origani cretici. 6. Xylol balsam. Neuroglia-fibers, if properly preserved, deep blue ; nuclei, blue; connective tissue, deep pink; axis-cylinders, fight pink ; myelin-sheaths, yellow if thoroughly mordanted with the bichromate of ammonium, otherwise with prolonged staining they may take on a greenish-blue color : proto- plasm of ganglion-cells and the dendritic processes, purplish or bluish gray. It will sometimes be found advantageous to stain sections lightly in Van Gieson's mixture first, so that the axis-cylinders may have a deeper red stain, and the con- trast in color between the dendritic processes and the neurog- lia-fibers may be greater. Degenerations of the Nervous System. — The same methods apply to the study of degenerations in nervous tis- sue that apply elsewhere, except in the demonstration of fat. Both myelin and fat reduce osmic acid, so that the or- dinaiy test for fat in the hardened tissues fails. Marchi and Algeri, however, have shown that after myelin has been mor- 326 PATHOLOGICAL TECHNIQUE. danted for eight days or over in Miiller's fluid or other solu- tion of the bichromates, it loses the property of reducing the osmic acid, while fat retains the property unimpaired. On this peculiarity is based their method for differentiating fat from myelin. Marchi and Algeri's Method for Staining Patty De- generated Myelin-sheaths of Nerve-flbers. — i. Harden in Miiller's fluid or in formaldehyde, followed by Miiller's fluid, for eight days to three months. 2. Transfer tissue for five to eight days directly into the following solution : Miiller's fluid, 2 parts ; I per cent, osmic-acid solution, i part. 3. Wash out thoroughly in water. 4. Dehydrate in alcohol. 5. Imbed in celloidin. 6. Mount in thick Canada balsam (which must not be dis- solved in chloroform). EXAMINATION OF THE BLOOD. The specific gravity of the blood varies but slightly and averages 1055. For cHnical purposes the method of Ham- merschlag is the best for estimating the specific gravity. The method depends upon the physical law that a body which re- mains suspended in a fluid must have the same specific grav- ity as that fluid. The fluid selected is a mixture of chloro- form (specific gravity 1526) and benzol (specific gravity 0.889). A drop of blood does not mix with either fluid. A small test-tube holding about 10 c.c. is half filled with a mixture of benzol and chloroform. This mixture should have the specific gravity of from 1050 to 1060. A drop of freshly-drawn blood is allowed to fall into this mixture, care being exercised that the drop falls directly into the fluid. Chloroform or benzol is added according as the drop of blood sinks to the bottom or floats on the surface of the fluid. It HISTOLOGICAL METHODS. 327 is necessary in adding either of the fluids to thoroughly mix them by gentle rotating movements without breaking the blood-drop. If the drop floats on the surface, it is better to add sufficient benzol to make it sink to the bottom, and then add chloroform until it becomes suspended in the fluid. Too large a drop of blood is liable to be broken up in mix- ing the fluids, and this must be avoided. When the drop re- mains suspended in the thoroughly mixed fluids the latter is filtered and the specific gravity tested. Apparatus "Used in the i^xamination of the Blood. — An accurate examination of the blood can be made only by the strictest observance of cleanliness and attention to de- tails. It has been deemed wise, therefore, to devote the greater part of this article to a description of the various steps of the process for the benefit of those who are unfa- miliar with the technique. With this object in view it is not necessary to describe the numerous instruments which have been devised for examin- ing blood or to refer to many of the staining fluids. The Thoma-Zeiss hemocytometer, or blood-counting instrument (Fig. 100), is the one which is generally employed to count the red and white blood-corpuscles, and consists of a glass slide on which the blood-corpuscles are counted and a pi- pette for mixing the blood and diluting fluid. The counting slide has a thin square plate of glass cemented on its surface : a circular opening in the center of this plate is nearly filled by a glass disc ^^5- mm. thinner than the square plate which surrounds it. A series of horizontal and vertical lines on the surface of the disc divides it into squares, the sides of which are -^ mrh. long. Additional lines placed close together divide this surface into quadrants. Each quadrant contains sixteen of the small squares. The pipette consists of a capillary tube which expands into an ovoid chamber above. The chamber contains a glass pearl, which assists in mixing the blood and diluting fluid. The capillary tube has a capacity of i mm. and is graduated to tenths. Above the ovoid chamber is a line marked loi mm. A dilution of i to 100 or 1 to 200 is obtained by sucking 328 PATHOLOGICAL TECHNIQUE. Of a -Q)- O c % isr u _£l_ 0= ^-c^ — q o O— o TT o O ■4»- -^ ^i^SJ- c C5 o -Q- o _ ■^ ^ -€^ ^ ^ 0^^ Q. C "^ o T5 cS ^ O D Fig. ioo. — Thoma-Zeiss blood-counting apparatus (Limbeck) : A, melangeur ; a, capillary tube in which the blood is taken ; i, chamber for mixing the blood with the diluting solution ; c, glass ball to aid in mixing the blood with the dilut- ing solution ; B, cross-section of the chamber in which the blood is counted ; C, section of the field on which the blood is counted, showing thirty-six squares ; D, diagram of the whole field. HISTOLOGICAL METHODS. 329 the blood up to the mark i. or 0.5, and the diluting fluid up to the mark loi. A second pipette for counting the white corpuscles is grad- uated in such a way as to give a dilution of i to 10 or i to 20. For clinical purposes the Von Fleischl hemometer (Fig. loi) Fig. ioi. — Von Fleischl's hemometer : a, stand ; b, narrow wedge-shaped piece of colored glass fitted into a frame (c) which passes under the chamber ; d, hol- low metal cylinder, divided into two compartments, which holds the blood and water; e, white plate from which the light is reflected through the chamber; f, screw by which the frame containing the colored glass is moved ; g, capillary tube to collect the blood ; h, pipette for adding the water ; i, opening through which may be seen the scale indicating percentage of hemoglobin. is generally used to estimate the hemoglobin. This instru- ment is fairly accurate, but for percentages of hemoglobin below 50 per cent, it is safe to allow 5 per cent, for error in the apparatus. The instrument consists of a metal stand {a), a narrow wedge-shaped piece of colored glass {B), a hollow metal cylinder {d^, and capillary tubes (^g). The metal stand 330 PATHOLOGICAL TECHNIQUE. {a) is fitted with a flat stage above, in which is an aperture for holding the cylinder (d\ On the under surface of this stage are two metal rims in which the glass wedge (b") fits. These rims are arranged so as to allow the glass wedge to move across the aperture by means of an adjustment screw. The glass wedge (B) is colored red, the degree of color becoming more intense toward the thick end. The glass is held in a metal frame, on one of whose sides parallel with the glass there is a scale of percentages from O. to 1 20, corresponding to the intensity of the color in the glass. The metal cylinder {d) is divided into two equal com- partments by a thin metal partition which extends from one end of the cylinder to the other. One end of the cylinder is closed by a piece of glass. The blood is collected in the capillary tube (^). Preparation of Apparatus. — Three things are essential in order to make a satisfactory examination of the blood : the apparatus must be absolutely clean ; the various stages in the process must be performed rapidly, because a very slight coagulation of the blood will interfere with any of the tests ; and the work must be done accurately. Many of the details which follow may appear trivial to one who is unfami'iar with the methods employed in making blood-examinations. Experience will convince such an one to the contrary. All apparatus should be thoroughly cleaned and dried immediately after completing a blood-ex- amination. The " mixing pipette " for estimating the blood- corpuscles can be readily cleaned by removing the rubber tubing and fixing it to the other end of the pipette, or a double atomizer bulb, such as is used on the Paquelin cau- tery, may be substituted. The pipette should be cleaned with water, alcohol, and finally with ether, until it is dry in- side. If the alcohol fails to remove all of the staining fluid from the inside of the pipette and from the glass pearl, acid alcohol (hydrochloric acid i part, alcohol (70 per cent.) 100 parts) may be used. This should be followed by alcohol, and then by ether. The blood-counting slide may be cleaned with water. If any of the staining fluid adheres to the slide, HISTOLOGICAL METHODS. 331 it may be removed with alcohol. If alcohol is used to clean the counting slide, it must be used rapidly and the slide washed with water, because alcohol dissolves the cement by means of which the thin square of glass and the disc are fastened to the slide. The capillary tubes used for estimat- ing the hemoglobin should be cleaned in the same way as the " mixing pipette." It has been found that the blood fills the capillary tube more readily if the latter is cleansed a second time with water directly before making the blood- examination. If this is done, it is necessary to thoroughly dry the tube afterward. This may be accomplished by re- moving most of the water and drying the remaining moist- ,ure by passing the tube rapidly through an alcohol flame. Care must be taken to avoid breaking it. The metal cylinder is cleaned by unscrewing the bottom and removing the glass. In this way the cylinder can be easily and thor- oughly washed and dried. The cover-glasses should be kept in a solution of acid alcohol (see above). Before begin- ning a blood-examination a dozen or mofe of these cover- glasses should be cleaned with water, and then with alcohol, and dried ; then they should be passed through an alcohol flame and spread on a piece of paper. The cover-glasses should not be touched with the fingers after cleaning them, but should be handled with forceps. New cover-glasses are covered with an oily glaze which prevents the blood from spreading. Moisture from the fingers may alter the shape of the red blood-corpuscles. Never begin an examination of the blood until everything required for the entire examination is ready for use. A suf- ficient quantity of the diluting solutions must be poured into small dishes and placed, with' the apparatus and cover- glasses, close at hand. If the preserving fluid contains a staining fluid, the solution must be filtered before using. To obtain the blood the part from which it is taken should be cleaned with soap and water, alcohol, and finally with ether, and thoroughly dried The blood is usually ob- tained from the lobe of the ear or from the end of the finger. The ear possesses distinct advantages over the end of the 332 PATHOLOGICAL TECHNIQUE. finger. It is not necessary to hold the ear to prevent its withdrawal ; the operation is less painful, and the blood is obtained more easily because the epidermis is thinner. These advantages are especially evident in infants and young chil- dren. In children it is almost impossible to keep the fingers extended without exerting pressure. Pressure on the part from which the blood is withdrawn must be avoided. It has been ascertained that moderately firm pressure on the finger is capable of diminishing the number of red coriDuscles in a cubic millimeter by increasing the quantity of serum. The free border of the lobe of the ear is preferable to the anterior surface, because the border is convex. This is of advantage in making cover-glass preparations from small drops of blood. A narrow-bladed knife, similar to a tenotomy-knife, makes a relatively wide and shallow opening, and is prefer- able to a needle. The liemog-lobin test must be performed in a dark room by means of a yellow light — lamp, gas, or candle light. Before obtaining the blood fill one of the divisions of the cylinder one-fourth full with distilled water ; then partly fill the pipette provided for this purpose, and place it close at hand ready to wash out any blood which may remain in the capillary tube, and thus prevent coagulation. The capillary tube is held horizontally, and touched slightly on the side of ^. freshly drawn drop of blood. The end of the tube should never be dipped deeply beneath the surface of a blood-drop. If the tube is perfectly clean and no coagulation has occurred in the drop of blood, the latter will instantly fill the tube. Sometimes it is necessary to slightly withdraw the end of the tube, without, however, permitting it to entirely leave the surface of the blood. Observe that the tube is exactly filled with blood and that the ends show neither convexity nor concavity. Any blood adhering to the end of the tube or to its outer surface must be quickly wiped off. Rapidly submerge the tube in the water and move it quickly to and fro several times, and with as little delay as possible wash out the remaining blood by means of the pipette. Fill the chamber three-fourths full with water and stir briskly with HISTOLOGICAL METHODS. 333 the metal handle of the capillary tube. Be careful to stir in the corners where the partition joins the sides. The blood should be equally diffused throughout the fluid when the mixing has been well done, and there should be no coagula in it. When this has been done fill the cylinder to the level of the diaphragm ; the other division of the cylinder is then filled to the level of the diaphragm with distilled water. The surface of the fluid in each side must be perfectly flat. This can be readily ascertained by looking across the surface of the fluid with the eyes on a level with the top of the cylinder. A convexity or concavity of the water side can be easily obviated. A convexity on the side which contains the blood should be avoided. When the cylinder is in posi- tion, the division containing the distilled water is over the colored glass. The reflector should be arranged to throw as much light as possible when the percentage of hemoglobin is high ; but when the hemoglobin is much diminished, less light will give more exact results. To perform the test one eye should be closed and the face shielded from direct light by holding a piece of paper between the face and the lamp. The instrument should be arranged so that the observer stands at the side of it and looks directly down into the fluid. This is done so that the images of both divisions of the cylinder are thrown upon the retina side by side, and not one above the other. This permits a more accurate comparison of colors to be made. By means of the adjust- ment screw move the colored glass from a point where the glass is paler than the diluted blood to a point where the color of the glass and of the blood match. Then read the percentage on the scale, and control the result by moving the colored glass from above the point where the colors match downward, to see if the same result is obtained ; pause a moment and repeat the test. More accurate results are obtained by resting the eye several times than by looking for a long time. In order to estimate the number of red corpuscles in a cubic millimeter of blood it is necessary to use a dilut- ing fluid. It is desirable that this fluid should be of such a 334 PATHOLOGICAL TECHNIQUE. character that the corpuscles may not change their form or lose their color. Various fluids are used. Toison's fluid possesses the advantage of staining the white corpuscles. The formula of this fluid is as follows : Distilled water, 160 c.c. ; Neutral glycerin, 30 Sodium sulphate, pur.. 8 grams Sodium chlorid. I gram ; Methyl-violet, 5 B, 0.025 This fluid must be filtered each time before using. A drop of blood is required of sufficient size to more than fill the capillary tube of the " mixing pipette." If the drop is too small, it is difficult to prevent the entrance of air and coagu- lation of the blood in the tube in the interval of obtaining a second drop of blood. It is advisable, therefore, to perform this test before estimating the hemoglobin or making the cover-glass preparations. While filling the capillary tube of the pipette care must be exercised to keep the point of the pipette beneath the surface of the blood-drop to prevent the entrance of air. The blood is sucked up to the mark .5 or I., and then the tip of the tongue is pressed firmly over the hole in the mouth-piece. This prevents the blood-column from sinking or air from entering below while the tip of the pipette is being wiped and immersed in the diluting fluid. This part of the test requires the utmost precision and avoid- ance of delay. It is necessary, therefore, to keep the eyes constantly fixed on the capillary tube in order to note any variation in the blood-column. Rapidly wipe the tip of the pipette to remove the blood from the outside, and then im- merse the tip in the diluting fluid. Suck the fluid up to the mark loi, close the ends of the pipette with the thumb and middle finger, and shake the pipette for two minutes. If the ends of the pipette are not completely closed during this process, some of the fluid will escape. At the end of two minutes allow two drops to escape from the pipette before examination, because the fluid in the capillary tube is unmixed with blood. Then allow a drop to escape upon the HISTOLOGICAL METHODS. 335 central part of the counting slide. This drop should com- pletely fill the depression after the cover-glass has been applied. A little practice is necessary in order to estimate the size of the drop required. A moderately thick cover- glass should be slid over or carefully laid upon the square raised surface, and pressure applied to the edges until the Newton color-zone can be seen between the cover-glass and the square raised surface beneath. Never press on the center of the cover-glass. Allow the blood-corpuscles to settle a minute or two before counting. The corpuscles are estimated as follows : One side of a small square is ^ mm. long ; the enclosed square surface is -^^ mm. The distance between the cover-glass and the disc is .1 mm. which gives a cubic capacity of -^^^^ c.mm. for each square. To estimate the number of corpuscles in I c.mm. of blood, multiply the number of corpuscles counted by 4000, and then by the number representing the amount of dilution, 100 or 200 as the case may be, and divide the result by the number of squares counted. Corp. X dilution X 4000 , • , c •± = corpuscles m i c.mm. Squares counted To avoid counting any of the corpuscles twice, always begin at the upper left-hand square of a quadrant and count four squares downward. Count all the corpuscles which touch the upper and left-hand lines of a square, together with the corpuscles in the square. Never count the cor- puscles touching the right-hand or lower double lines of a quadrant. In order to make an accurate count it is neces- sary to count at least 1200 red corpuscles. If air-bubbles are present when the cover-glass is applied, it is necessary to clean the slide and use a fresh drop of the diluted blood. Before beginning the count examine the various quadrants with a low-power objective, to see if the corpuscles are evenly distributed. If they are not, it will be because the blood is not thoroughly mixed, and the slide should be washed and the pipette well shaken. Before ex- amining a second drop of the diluted blood shake the 336 PATHOLOGICAL TECHNIQUE. pipette for two minutes as before. The results of three drops should be averaged. There is a special pipette for counting the white corpuscles, graduated so as to give a dilution of i to 10 or i to 20. The white corpuscles are estimated in the same way as the red corpuscles, except that the dilution 10 or 20 is sub- stituted for 100 or 200. This necessitates a fresh drop of blood. For a diluting fluid for counting the white corpuscles a 1^ or J per cent, solution of acetic acid may be used. This solution destroys the red corpuscles. A little gentian-violet solution may be added to the acetic acid if it is desired to stain the white corpuscles. It is possible to estimate both the white and red corpuscles in the same drop of blood quite accurately. In order to do this it is necessary to calculate the number of squares which would be contained in the entire ruled surface outside of the portion which is divided into quadrants. This may be done by means of a micrometer eye-piece and a movable stage. The number has been found to be about 2000, although there may be some slight variation in each slide. This enables one to count 50 white corpuscles or more in each drop by counting all the corpuscles within the lines. For calculating the number of corpuscles one would divide by the estimated number of squares counted, just as in determining the red corpuscles. The average of three drops gives quite accurate results and saves much time and trouble. Cover-glass Preparations. — The blood must be spread extremely and uniformly thm. If this is done, the blood dries very quickly, and the red blood-corpuscles retain their shape and are not crowded together and lying over one an- other. To obtain such a result it is essential that the cover- glasses should be absolutely clean ; that there should be no delay in bringing the cover-glass which has the drop of blood on its surface in contact with a second cover-glass ; and that the drop of blood should be quite small. The following method gives the best results : The procedure is rendered much easier if some one is present to assist. This person places a finger beneath the lobe of the ear in order to raise HISTOLOGICAL METHODS. 337 it slightly without pressing upon it, and with a clean com- press wipes away the blood as fast as it flows with a quick motion of the hand. This is done to prevent coagulation, which occurs very quickly, and prevents the drop of blood from spreading between the cover-glasses. It takes a little time for the blood to spread, the cover-glasses to be sepa- rated and laid down, and fresh ones picked up ; and if, during Fig. I02. — Forms of forceps for holding cover-glasses. this time, some one wipes away the blood as fast as it flows, much better preparations are obtained. If, in spite of this, as often happens, the blood coagulates about the opening, one end of the compress can be slightly moistened with water and passed over the opening and the surface dried quickly. The blood then flows freely again. A drop of blood a little larger than a pin-head is sufficient. Grasp the edge of the cover-glass with a pair of spring forceps, pick up a second cover-glass with a pair of plain forceps. Both pairs of forceps must be especially prepared by having the inner surfaces of the points ground smooth. The cover-glass in the spring forceps is held horizontally just below the ear, and the other cover-glass, held with the other forceps, is touched lightly on the blood and immediately dropped on the first one. If the cover-glasses are dry and clean and the 338 PATHOLOGICAL TECHNIQUE. blood has not begun to coagulate, it spreads at once in a thin film between the glasses. The glasses are then drawn apart with a rapid sliding motion by means of the forceps, waved in the air a few seconds, and laid down with the blood- surface uppermost. The layer of blood cannot be too thin, but it can easily be too thick. The cover-glasses should never be pressed together to make the blood spread. Con- siderable practice is required before one becomes proficient. The specimens may be fixed by heat or by a mixture of al- cohol and ether, equal parts. The best method is to put the cover-glasses for twelve hours in a thermostat at a tempera- ture between iio° and 120° C. (Ehrlich's method). This is objectionable on account of the time and apparatus required. A practical modification of this method is to heat the cover- glasses on a brass plate for an hour at a point on the plate where water boils. The plate should be about \ of an inch thick and from 1 5 to 18 inches long. It should be heated from one end to a constant temperature. Test the degree of heat with drops of water and select a part where the water boils. At a point nearer the flame it will be found that the water sputters and rolls about, indicating too high a temper- ature. After putting the cover-glasses, with the blood-side uppermost, upon the selected point, it is necessary to test the degree of heat from time to time, and perhaps to shift the cover-glasses. A shorter and easier method, not so satisfactory, is to fix the specimens by immersing them for from thirty minutes to an hour in a mixture of absolute alcohol and ether, equal parts. This gives good results, as a rule. The iElements of the Blood. — Red corpuscles, white corpuscles, and hematoblasts. The red blood-corpuscles (ery- throcytes) are biconcave discs. The diameter of a red blood- corpuscle in human blood is the same for male and female, and averages 7.8/i. The red corpuscles have a pale-yellow color with a faint tinge of green ; they are homogeneous, highly refractive, and have no nuclei (normally). Outside the cir- culating blood the corpuscles rapidly undergo alterations in their shape and size. These changes may be prevented or HISTOLOGICAL METHODS. 339 hindered, for purposes of study, by appropriate methods of fixation and preservation. The average number of red cor- puscles in I cubic millimeter of human adult blood is from four million to five million. Under abnormal conditions the red corpuscles vary in size, shape, and number, and a small proportion of them may have nuclei. 1. Variations in the shape {poikilocytosis) are of frequent occurrence. Some of the corpuscles may be pear-shaped or bottle-shaped; others may be shaped like saddle-bags or have projections. Variations in the size of the erythrocytes are also common. They all retain the central depression, notwithstanding the variations in shape and size. 2. Abnormally large erythrocytes, varying from 10 to 14// in diameter and without nuclei, are called megalocytes. Very small erythrocytes, varying from 2 to 5/^ in diameter, are called microcytes. The microcytes contain hemoglobin, but do not have the central depression. 3. Nucleated red corpuscles (erythroblasts) occur in two forms, the so-called normoblasts and the megaloblasts or gigantoblasts. A normoblast is the same size as a normal corpuscle, but contains a single relatively large nucleus. The nucleus is commonly situated in the centre of the cor- puscle, but it may lie in the periphery, and takes a more intense stain than the nucleus of any other corpuscle. Mitosis is observed frequently. Free nuclei are found often. A megalo- or gigantoblast is from three to five times as large as a normal red corpuscle, and contains a relatively large round nucleus in the center. The intensity with which this nucleus stains is midway between that of the normoblast nucleus and the nuclei of the white cor- puscles. 4. The so-called anemic degeneration of the red corpuscles is less commonly met with than the preceding changes. When cover-glass-preparations are stained with eosin-hema- toxylin or eosin-methylene-blue, the red corpuscles, instead of staining a bright eosin-red, take a muddy-looking, darker stain. This appearance is assumed to be due to degenera- tive changes in the stroma of the corpuscles, which cause 340 PATHOLOGICAL TECHNIQUE. the hemoglobin to become stained to some extent by the nuclear stain in addition to the eosin. The white corpuscles (leucocytes) differ in size, in the size and shape of their nuclei, and in the granules which are con- tained in the protoplasm. They have been classified in vari- ous ways, depending upon their supposed origin and upon the granules which they contain. Ehrlich differentiates five different kinds of granules found in the human blood : the a or eosinophilic granules ; the ^ or amphophilic ; the 8 and y or basophilic granules ; and the e or neutrophilic granules. The a or eosinophilic granules are coarse, round or oval, highly refractive granules which have an affinity for the acid aniline stains, especially eosin. The /3 or amphophilic granules have an affinity for both acid and basic stains. They occur very rarely in human blood, but are common in the blood of certain animals. The b and y granules are finer and less refractive granules which have an affinity for basic stains. The y granules are nearly as large as the eosinophilic granules, but are not so refractive. The 8 granules are much finer and not very re- fractive, and are very numerous in the cells in which they occur. The e or neutrophilic granules are much smaller, more numerous, and less refractive than the eosinophilic granules, and have an affinity for neutral stains. They occur only in human blood. Morphologically, there are five varieties of white cor- puscles. They are — the small mononuclear cell or lympho- cyte ; the large mononuclear cell ; the transitional large mononuclear cell; the neutrophile; and the eosinophile. The small mononuclear cell or lymphocyte is slightly larger than a red corpuscle, and has a single round nucleus sur- rounded by a narrow strip of protoplasm which contains no granules. The nucleus stains more intensely than the nuclei of the other white corpuscles. The large mononuclear cell is much larger than the lymphocyte, and contains a large, oval or round nucleus HISTOLOGICAL METHODS. 34 1 surrounded by a wide zone of protoplasm. The nucleus takes a fainter stain than that of the lymphocyte. The transitional large mononuclear cell differs from the large mononuclear only in having a saddle-bag-shaped nucleus. Variations in size are frequently observed in the small mononuclear as well as in the large mononuclear cells, so that under some circumstances it is difficult to distinguish to which type of cell a given cell belongs. The neutrophile or polymorpho-nuclear leucocyte is about three times as large as a red corpuscle. This cell is the so- called pplynuclear leucocyte found in pus. The nucleus, as the name implies, is more or less subdivided, and often re- sembles various letters, as S, Z, V, M, etc. The nucleus is surrounded by a relatively large amount of protoplasm which is more or less completely filled with fine neutrophilic granules. The normal eosinophile resembles the neutrophile in size and in the chai-acter of its nucleus. It differs from the neutrophile, at least chemically, by containing the eosinophilic a granules. The large mononuclear, transitional, and neutrophilic cells are regarded as the same cell in different stages of develop- ment. The younger form is the large mononuclear, and the oldest is the neutrophile. During the process of " ripen- ing," as it is called, the fine e granules are formed in the pro- toplasm, and the nucleus becomes more or less subdivided. The so-called mastzellen — that is, cells which contain baso- philic granules — were supposed by Ehrlich to originate from fixed connective-tissue corpuscles and from the spleen, and not to be present normally in the blood. Later investiga- tions show that an occasional mastzelle may be found in normal blood. Markzellen, or myelocytes, is the name applied to large mononuclear cells containing neutrophilic, and sometimes eosinophilic, granules. These cells are present in the medullary cavity of the long bones, but are never found in the blood normally. Hematoblasts (blut-pliittcheji) are small round or oval discs 342 PATHOLOGICAL TECHNIQUE. from 1.5 to 3.5;« in diameter. They do not contain hemo- globin and have no central depression. They are so rapidly disintegrated on exposure to the air that it is necessary to use special precautions in order to preserve them. The best way to study them is to put a drop of some preserving fluid on the skin from which the blood is to be obtained, and then puncture the skin through the fluid. A i per cent, aqueous solution of osmic acid can be used, or, if it is desired to stain them, i part of methyl-violet in 5000 parts salt solution may be used. Methods of Staining. — Of the many staining fluids which have been employed to differentiate the white cor- puscles, it is necessary to mention only those which are com- monly used and which have been found to answer all pur- poses. Ehrlich's triple stain possesses the advantage of staining both the eosinophilic and neutrophilic granules in addition to the nuclei. The formula is as follows : Orange G, Distilled water. Acid fuchsin, Distilled water. Absolute alcohol. 135 gms, 100 " 6s " 100 " 100 " Methyl green, Distilled water. 125 " 100 " Absolute alcohol. Glycerin, 100 " 100 " The various ingredients are prepared separately as indi- cated by the dotted lines, and are afterward mixed gradually. The mixture must stand for several weeks before using. It is advisable to withdraw by means of a pipette some of the staining fluid from the middle portion without disturbing the bottom. The cover-glass preparations should be stained from six to HISTOLOGICAL METHODS.' 343 eight minutes, washed thoroughly with water, dried, and mounted in Canada balsam. The neutrophilic granules are stained violet ; the eosinophilic, a bright red ; the nuclei of the neutrophilic and the eosinophilic cells are a greenish- blue ; the nuclei of the lymphocytes, a deep blue ; the nu- clei of the large mononuclear cells, a pale blue ; the red cor- puscles, copper color : and the nuclei of the red corpuscles, if any be present, a more intense blue than the nuclei of the lymphocytes. For some unexplained reason this stain is not always uniform in its action. It is sometimes difficult to distinguish a nucleated red cor- puscle from a lymphocyte. It is well to remember, there- fore, that the nuclei of red corpuscles stain more intensely than other nuclei, and have very sharply defined outlines, and by careful focussing it is seen that the surrounding stroma is stained the same color as the other red corpuscles. Bhrlioh's Hematoxylin-eosin Stain. — Distilled water. Alcohol, Glycerin, da. 100 grams ; Hematoxylin, 4-5 " Acetic acid, 20 " Alum in excess. The mixture remains from four to six weeks in the sun, and then about i per cent, of eosin is added. Stain for from twelve to twenty-four hours in a covered glass dish in the sun. Wash thoroughly in water, dry, and mount in Canada balsam. The red corpuscles are stained red, with at times a tinge of orange ; the nuclei of the red corpuscles, a deep black ; the protoplasm of the leucocytes, a light lilac ; the nuclei, a dark lilac ; the eosinophilic granules, a bright red ; the nu- clei of the lymphocytes, black, with not quite so deep a tinge as the nuclei of the red corpuscles. The protoplasm scarcely stains. Eosin and Methylene-blue Stains. — Separate stain. The eosin solution, a \ per cent, alcoholic solution, is heated and 344 PATHOLOGICAL TECHNIQUE. the cover-glasses are stained in it from two to three minutes, and are then stained in a saturated aqueous solution of methylene-blue for from two to three minutes ; thoroughly washed, dried, and mounted in Canada balsam. The red corpuscles are stained eosin red ; the nuclei of the red corpuscles, a deep-blue; the nuclei of the leucocytes are stained a light blue ; the eosinophilic granules, a bright red. Chenzinsky-Plein Solution. — Saturated aqueous solution of methylene-blue, 40 c.c. ; J per cent, (in 70 per cent, alco- hol) eosin solution, 20 c.c. ; distilled water, 40 c.c. The best results are obtained by staining the specimens for twenty-four hours. A fairly good stain may be obtained by heating the solution fifteen minutes. The red corpuscles are stained eosin red ; the eosinophilic granules, a bright red ; and the nuclei, blue. The basic granules may be stained by a concentrated so- lution of methylene-blue. The specimens should remain in the solution from five to ten minutes. METHODS OF FIXING AND EXAMINING SPECIAL OR= CANS AND TISSUES. Tissues which are to be hardened should be obtained as fresh as possible. For this reason autopsies rarely furnish such perfect material as is obtainable from experimental le- sions in animals or from surgical operations. Still, most of the pathological material comes from autopsies, and it is en- couraging to know that very good work can often be done with tissues not fixed until twenty-four hours or even more after death. The most valuable autopsies are those which are freshest, and in which but one etiological factor has been concerned, so that the relation between the cause and the le- sion produced is uncomplicated and can be readily grasped and understood. The choice of the proper fixing reagent varies with the tissue, the lesion, and the use to which the material is to be HISTOLOGICAL METHODS. 345 put. For simple diagnosis alcohol is usually sufficient. For special investigations other fixatives may be required. In general it may be said that alcohol is best suited for bacteria and for many micro-chemical color reactions ; Zenker's or Orth's fluids for red blood-globules, nuclear figures, and pro- toplasm ; formaldehyde for the central nervous system. More attention will be paid to this point in the consideration of the separate organs and tissues. It is important that pieces of tissue for histological study should be placed in the proper fixative as soon after the re- moval of the organs from the body as possible, so that the surface will not dry or the blood and other fluids escape from the vessels. Do not wash off" the surface with water. The tissues should almost invariably be cut into thin slices, not over J to I cm. thick. For the finer fixatives 2 mm. should not be exceeded. Frozen sections of fresh tissues will often show whether the material is worth saving, and suggest what fixatives had best be used — such as Flemming's solution, for instance, if fat is present. Acute Inflammatory l^xudations; Granulation- tissue. — The elements in acute inflammatory exudations which require preservation are polynuclear leucocytes, serum, fibrin, and red blood-globules. The best general fixative for them all is Zenker's fluid. It not only pre- serves perfectly the characteristic nuclei of the leucocytes, but also the protoplasm which stands out sharply in con- trast-staining with eosin. The albumin of the serum is coagulated into a finely granular material. The fibrin and red blood-globules stain brilliantly with eosin. Alcohol is gen- erally preferred for the fixation of any organisms associated with the exudation, but after fixation in Zenker's fluid they can be perfectly stained with Unna's alkaline methylene-blue solution. Occasionally other fixatives, such as Flemming's or Pianese's, will be found useful, especially for the study of any attendant degenerations. Zenker's fluid will also be found invaluable for fixing granulation-tissue where a similar exudation is generally 346 PATHOLOGICAL TECHNIQUE. combined with new-formed blood-vessels and connective tissue. For the study of the plasma-cells which early make their appearance in granulation-tissue hardening in alcohol is gen- erally advisable, although Zenker's fluid is often to be pre- ferred, and for eosinophiles is indispensable. I/ting. — In the preservation of the lungs it is important to save portions that have not been squeezed, so that the relations of the exudations may not have been changed or the alveoli compressed. Thin slices are usually preferable to cubical pieces, and should be cut with a very sharp knife, so as not to compress the tissue, and dropped immediately into the fixing fluid, before the contents of the bronchi and of small cavities have had time to run out. An emphysem- atous lung is so delicate that it is usually better to inject a whole lung through the bronchi with the fixing fluid or to snip out small pieces with scissors. Zenker's fluid and alcohol are the most useful fixatives. Spleen. — The spleen-pulp may be examined by means of dried cover-slip preparations in the same way as described for the blood. Material can be obtained immediately after death by means of a long trocar thrust into the spleen. Teased preparations can be examined in salt solution. The spleen-tissue is well preserved in Zenker's fluid if cut into thin pieces not over \ cm. thick. Paraffin sections are to be preferred when convenient, so that the thinnest possible sec- tions may be obtained. Bone-marrow may be examined in the same way as spleen-plup in cover-slip preparations, or small bits may be hardened in Zenker's fluid or some other fixative for study by means of sections. Kidney. — The choice of fixing reagents varies largely with the lesions present. Zenker's fluid is advisable for general histological purposes, for blood, and for hemo- globin ; alcohol for bacteria, amyloid, hemosiderin ; Flem- ming's for fat; boiling water for albuminous exudations. The pieces of tissue preserved should include both cortex and pyramids. HISTOLOGICAL METHODS. 347 In cases of chronic nephritis the capsule should not be peeled from those parts kept for microscopical pur- poses. Paraffin imbedding is generally to be preferred for the kidney, especially when lesions of the glomeruli are present. For the simple demonstration of fat, teased preparations or frozen sections of fresh material can be mounted in water and treated with acetic acid. Gastro-intestinal Tract. — Portions of the stomach or intestine should be hardened as soon after death as possible for satisfactory study, because the gastro-intestinal tract so rapidly undergoes post-mortem changes. It has been recom- mended in appropriate cases, where an autopsy is allowable, to inject the stomach with the desired fixing solution by means of a rubber tube as soon after death as is permissible. Under no circumstances should the surface of the intestine or stomach be washed with water. Use either normal salt solution or some of the fixing solution. It is important to keep the tissue flat while hardening. This can usually be done by laying it with the peritoneal surface down on thick filter-paper, to which it readily sticks. Sometimes it is necessary to pin the specimens down at the edges on flat pieces of cork. Do not let the surface dry before the speci- men is placed in the fixing solution. Zenker's fluid can be highly recommended as a fixative, but alcohol is sometimes to be preferred. I/iver. — For fat the liver is examined fresh in teased prep- arations or frozen sections, or after hardening in Flemming's or Marchi's solution, by means of celloidin or paraffin sec- tions. For obtaining the iron reaction with hemosiderin in cases of pernicious anemia, and for the reactions of amyloid, harden in alcohol. For general histological study Zenker's fluid will be found exceedingly useful. The bile-capillaries may be demonstrated by means of Golgi's silver method. Boehm's directions for it are as follows : 348 PATHOLOGICAL TECHNIQUE. 1 . Harden pieces of liver i cm. in diameter for seventy- hours or more in 3 per cent, solution of bichromate of potassium, 4 parts ; I per cent, solution of osmic acid, i part. 2. Transfer for twenty-four to forty-eight hours to a | per cent, solution of nitrate of silver. 3. Wash in water. 4. Harden and cut. The bile-capillaries appear dark brown on a yellow ground. Bone and Cartilage. — Excellent work can be done after hardening in alcohol, and fixation in it is generally recom- mended for all infectious processes in bone. The histological structure is, however, better preserved in Zenker's or Orth's fluid. In decalcifying bone, after proper fixation, thin pieces should be taken, not more than 2 to 4 mm. thick, so that the process may be finished as quickly as possible. While tu- bercle bacilli will stain readily after being twenty-four or even forty-eight hours in 5 per cent, nitric acid, it is impossible to stain them after they have been subjected to the same strength of nitric acid for four days. (For details in regard to decalcification see page 228.) Celloidin is preferable to parafifin for imbedding; Besides a simple stain with alum-hematoxylin, double stains of the latter with neutral carmine or eosin are sometimes advanta- geous. The best pictures with carmine as the contrast-stain are obtained by staining first in alum-hematoxylin, washing twelve to twenty-four hours, and then staining in the neutral carmine. The carmine stains decalcified bone and osteoid tissue red. Phosphotungstic-acid hematoxylin will some- times be found useful, especially when cartilage is present, because it stains the intercellular substance both of bone and of cartilage pink, while the nuclei are stained blue. The ground substance of cartilage, especially in new-growths, often stains so intensely with alum-hematoxylin that the nuclei are quite obscured. The following method is recommended for dififerentiating cartilage from bone : HISTOLOGICAL METHODS. 349 SchafFer's Safranin Method. — Decalcify with nitric acid. 1 . Stain sections a half to one hour in an aqueous solution of safranin, i : 2000. 2. Wash in water. 3. Place for two to three hours in a j^ per cent, solution of corrosive sublimate. 4. Examine in glycerin, or, if permanent specimens are desired, pass very quickly through alcohol, blot with filter- paper, further dehydrate, and clear for a long time in berga- mot or clove oil, and mount in xylol balsam. This is a double stain : cartilage, orange ; bone, uncolored ; connective tissue and marrow, red. None of the methods above given have proved reliable in the study of rickets and of osteomalacia for differentiating os- teoid from true bone-tissue. In important cases, therefore, it is advisable to use an old knife, and to cut sections of the undecalcified tissue after imbedding thoroughly in celloidin. Skin. — Much of the material for the study of lesions of the skin is obtained during life by means of a Mixter punch or with the knife or scissors. Fixation in absolute alcohol is often advisable, especially when it is desired to stain bacteria, mastzellen, plasma-cells, and elastic fibers. The staining methods for these tissue-elements will be found on pages 300, 302. For Unna's innumerable stains for degenerated connective-tissue fibers, elastic fibers, etc. the reader is re- ferred to his numerous articles on technique in the Monat- sheft. f. prakt. Dermatologie during the last half-dozen years. For many skin-lesions, especially those in which blood- vessels play a more or less prominent part, Zenker's fluid is advisable. For finer histological work Pianese's fixatives and special stains are recommended. In the examination of hairs or scales of epidermis for bacteria and fungi it is important first to remove the fat from them by means of equal parts of alcohol and ether. They are then examined in 40 per cent, caustic potash, which, by clearing up the cells, brings out the organisms and spores quite distinctly. Heating the potash over a small flame hastens the process, but is a somewhat risky proceeding; 3SO PATHOLOGICAL TECHNIQUE. soaking in the solution over night is better. Examine the preparation with most of the light excluded. Preparations may be made in certain cases by touching the cover-slip to the surface of the lesion, drying, and passing through the flame. After removing the fat by means of al- cohol and ether, stain as with ordinary cover-slip preparations. Unna's method is to rub up the scales of epidermis in a little glacial acetic acid between two slides, which are then drawn apart and quickly dried over the flame. After re- moving the fat by means of alcohol and ether the slide preparations are stained in borax-methylene-blue. For staining the various vegetable parasites of the skin Malcolm Morris recommends the following method, which he claims is the best one yet devised, as it avoids the use of the hydrate of potash : 1. Ether, or alcohol and ether equal parts. 2. Stain in a solution of 5 per cent, gentian-violet in 70 per cent, alcohol, five to thirty minutes. 3. lodin solution, one minute. 4. Aniline, or aniline plus 2 to 4 drops of nitric acid. 5. Aniline. 6. Xylol. 7- Xylol balsam. The most suitable medium for the growth of the various ringworms is the following, due to Sabourand : Agar-agar, 1.30; Peptones, .50 ; Maltose, 3.80; Water, 100. Instead of test-tubes, Ellenmeyer flasks are used, so as to get a large flat surface for the growth to spread over from the point of inoculation in the center. The most favorable temperature for growth is 30° C. Museum Preparations.— Specimens intended to be preserved for the museum should generally be gotten into pretty good shape by trimming and dissecting before they are placed in the hardening reagent. Of the liver or other HISTOLOGICAL METHODS. 35 I large organs and tumors sections several cm. thick are gen- erally preferable to the whole specimen. The usual custom in the past has been to wash the specimen for a number of hours or over night in running water, to get rid of the blood, and then to preserve in 80 per cent, alcohol. This method preserves form and relations well, but is nearly valueless for preserving colors. Since the introduction of formaldehyde, from which at first much was expected in the way of faithful fixation of the normal colors of gross preparations, numerous attempts have been made to improve on the results obtainable with formaldehyde alone. Of the methods advocated, the follow- ing fr6m Virchow's laboratory seems the most promising, and can be highly recommended : Kaiserling's Method of Preserving the Natural Colors in Museum Preparations. — i. Fixation for one to five days in — Formaldehyde, 200 c. c. ; Water, looo " Nitrate of potassium, 1 5 grams ; Acetate of potassium, 30 " Change the position of the specimen frequently, using rubber gloves to protect the hands from the injurious effect of the formaldehyde. The time of fixation varies with the tissue or organ and size of the specimen. 2. Drain and place in 80 per cent, alcohol one to six hours, and then in 95 per cent, alcohol for one to two hours, to re- store the color, which is somewhat affected in the fixing solution. 3. Preserve in — Acetate of potassium, 200 grams ; Glycerin, 400 c.c. Water, 2000 " Exposure to light gradually affects the colors. The process of fixation should be performed in the dark, and the speci- mens when preserved should be kept in the dark except when on exhibition. 352 PATHOLOGICAL TECHNIQUE. If it seem desirable to cut a thin slice from the face of a specimen, this should not be done until the preparation has been in the preservative fluid two weeks. The specimen may then be placed in alcohol for one to two hours to brighten up the colors. PATHOLOQICAL PRODUCTS. Cloudy Swelling; Albuminous Degeneration. — The increase in the relative number of the albuminous granules of the protoplasm of the various tissue-cells in pathological processes is usually determined by examination of the fresh material, either macroscopically from the appear- ances on section, or microscopically from teased preparations or frozen sections mounted in salt solution. The organ as a whole (and therefore the individual cells) usually shows some increase in size. The nucleus is generally more or less obscured if the process is at all marked. According to Israel, the cloudiness must be recognizable with low powers and in places where the cells are massed together. The diagnosis should not be based on the appearances of single cells. The chemical properties of the albuminous granules are the following : they disappear on treatment with dilute acetic acid (i-2 per cent, solution usually); they are not dissolved by chemical substances which dissolve fat (absolute alcohol, ether, chloroform, etc.) ; and they do not stain with osmic acid. The acetic-acid test is the one usually employed. Albuminous degeneration can also be studied in sections of tissues hardened in certain of the fixatives and stained with diffuse colors. For this purpose hardening in Zenker's fluid and staining in alum-hematoxylin and eosin can be highly recommended. Fatty Degeneration. — Fatty degeneration can be studied both in fresh and in properly hardened tissues. The recognition of fat in fresh specimens depends, aside from its .physical peculiarities, on the following chemical properties : I. It does not disappear on the addition of acetic acid. HISTOLOGICAL METHODS. 353 2. It resists the action of dilute caustic potash or soda. 3. It is blackened by osmic acid. 4. It is dissolved (after dehydration in alcohol) by chloro- form, ether, etc. The tests usually employed with teased preparations or frozen sections of fresh tissues are the reactions with acetic and osmic acids. The acetic acid is usually employed in a I or 2 per cent, solution, of which a few drops are placed at the edge of the cover-slip and drawn under by means of a bit of filter-paper placed at the opposite edge. The osmic acid in a i per cent, solution may be used in the same manner for teased preparations, or the tissue may be teased apart in a small quantity of the fluid. Frozen sections can be placed directly in it. Fat is unaffected by formaldehyde or by solutions of the chrome salts, so that tissues preserved in them may be ex- amined like fresh tissues in teased preparations or frozen sec- tions mounted in water, so long as they remain in those fluids. This no longer holds true, however, after the tissues have once been transferred to alcohol. The examination of fat in hardened tissues depends on its property of reducing osmium and thereby being stained black. Although osmic acid may be used alone for harden- ing tissues and staining fat, it is generally employed in com- bination with certain other fixatives. The two solutions generally selected are Flemming's and Marchi's. Flemming's solution should be allowed to act from two to four days if the tissue is from 2-3 mm. thick, and then the pieces of tissue should be thoroughly washed in running water for twenty-four hours before being placed in alcohol. Marchi's method was intended for differentiating fat from myelin (see page 228), but the solution employed by him may be used for staining fat in ordinary tissues. Place small pieces of tissue in it for five to eight days, wash thoroughly in running water, and harden in alcohol. Marchi's method, carried through in the manner just de- scribed, succeeds perfectly with tissues hardened in formal- dehyde. 23 354 PATHOLOGICAL TECHNIQUE. Fat stained with osmium is soluble in ether, turpentine, xylol, toluol, and very slowly in chloroform, but is not dis- solved by alcohol or oil of cloves. Imbedding in celloidin is not contraindicated, as the alcohol probably protects the fat from the injurious action of the ether. For the paraffin method, chloroform or a mixture of oil of cloves and xylol equal parts will give good results if it is not allowed to act too long. Mount in thick xylol or chloroform balsam. Cholesterin crystals are recognized by their shape. On the addition of concentrated sulphuric acid the crystals turn yellow, and then rose-color. Treated with a little iodin, fol- lowed by concentrated sulphuric acid, they become colored violet, changing gradually to blue, green, and red. Necrosis. — Necrosis in tissues is generally recognized by two features : either by the disappearance of the nuclei, al- though the cell-outhnes may be visible, so that the nuclear stain is no longer possible, or by the presence of irregular, larger or smaller masses, generally supposed to be due to a fragmentation or breaking up of the chromatin, which stain intensely with nuclear stains. The disappearance of the nu- cleus is not synchronous with the death of the cell, but be- gins some twenty-four hours later, so that it is really evidence of changes following necrosis. It follows from the above that the microscopic evidence of necrosis is best studied in sections of tissues hardened in fixatives which favor nuclear staining, such as alcohol, Zenker's fluid, etc. Teased prep- arations and frozen sections of fresh tissue are much less useful. For the study of sections from hardened material double stains with alum-hematoxylin and eosin, or with eosin fol- lowed by Unna's alkaline methylene-blue solution, are very useful, for the reason that the necrotic areas usually stain rather deeply with the diffuse stain, and are thereby brought out sharply. For rendering the fragmented nuclei prominent the same methods may be followed as for mitosis. A fuchsin stain washed out by picric acid in the alcohol will often give ex- cellent results. HISTOLOGICAL METHODS. 355 Caseation is probably a tissue-change following local necrosis. Macroscopically and microscopically it resembles harder or softer cheese. Under the microscope it appears as coarsely or finely granular masses which have more or less completely lost the original tissue-structure. The chem- ical changes which have taken place have not been studied. Fibrin is rarely present. Caseous tissue possesses no peculiar staining reactions. Fragmented nuclei are frequently pres- ent in it, especially in the peripheries of the areas. Demonstration of Fibrin. — Fibrin usually appears as delicate, transparent, slightly refractive threads which are often closely matted together so as to form large masses. More rarely it appears as coherent masses of the finest gran- ules, as homogeneous glassy lumps, or as thin sheets. The characteristic reaction for fresh fibrin is that it quickly swells up and optically dissolves in very dilute acetic acid. Fibrin is well brought out in sections of hardened tissues by a double stain of alum-hematoxylin and eosin, or of eosin followed by Unna's alkaline methylene-blue solution, especially if the specimens have been fixed in Zenker's fluid. Phosphotungstic-acid hematoxylin is often useful for staining fibrin after any of the hardening reagents. Of the differential stains for fibrin, the most important and useful is Weigert's : 1. Harden in alcohol. 2. Stain celloidin sections in lithium carmine. 3. Fasten sections to slide with ether-vapor (see page 234). 4. Stain in aniline-gentian-violet five to twenty minutes. 5. Wash off with normal salt solution. 6. lodin solution i : 2 : 100 one minute. 7. Wash off with water. 8. Decolorize in — Aniline, 2 parts ; Xylol, I part. 9. Wash off with three changes of xylol. 10. Canada balsam. The fibrin and those bacteria which are stained by Gram 356 PATHOLOGICAL TECHNIQUE. are stained blue. The nuclei are red if the decolorization is carried far enough. It can easily be watched under the low power of the microscope. The method is not always suc- cessful, especially with tissues which are old. It is some- times advisable to increase the proportion of xylol in the decolorizing solution, so that the extraction of the color may not be so rapid. With paraffin sections fastened to the slide with egg-albumin use alum carmine as the contrast- stain, because the acid used with the lithium-carmine method will dissolve the albumin and loosen the sections. Besides the fibrin, certain forms of hyaline are often stained by this method. Fibrin can be stained by the above procedure in sections of tissiies hardened in Miiller's fluid if the speci- mens are first placed for several hours in a 5 per cent, aque- ous solution of oxalic acid to reduce the bichromate of potassium. Mucin. — The term " mucin " is applied to a proteid sub- stance having certain chemical reactions, and also to certain other substances which give the same reactions, but do not belong to the proteids. These various substances of secre- tory and degenerative origin cannot be distinguished micro- scopically, and have been investigated but little chemically. The reactions in common are the following : they dissolve in water to form a slimy fluid ; they are precipitated from slightly alkaline solutions by acetic acid ; the fresh precipi- tate dissolves in alkalies and in neutral salt solutions. Acetic acid, usually employed for this purpose in a i or 2 per cent, solution, precipitates mucin in the form of threads or granules. This reaction with fresh tissues has long been the main test for mucin. The acetic acid is drawn under the cover-slip by means of filter-paper placed at the opposite edge. The preparation should be mounted in water, not in salt solution, which may hinder or entirely prevent the reac- tion from taking place. Of late certain color reactions have become prominent. Mucin is coagulated into threads by alcohol or corrosive sublimate, and in this form can be stained by a number of staining reagents. Alum-hema- toxylin under certain conditions will stain mucin. Accord- HISTOLOGICAL METHODS. 357 ing to p. Mayer, these conditions depend on a certain degree of ripeness of the solution, on the presence of enough alum to keep the nuclei from staining deeply, and, most important of all, on the absence of any free acid. This is difficult to manage, unless the solution is carefully neutralized, on ac- count of the acid properties of alum. Mayer therefore recommends staining the sections in muchematein (see page 243). Various aniline dyes have been recommended for staining mucin : those most favorably spoken of are methylene-blue (Orth), Bismarck brown (P. Mayer), thionin (Hoyer), poly- chrome methylene-blue (Unna), and toluidin-blue. The drawback to most of the aniline stains is that they are quickly extracted by the alcohol used for dehydrating. On this account P. Mayer highly recommends Bismarck brown, because permanent mounts can be easily made with it. It is not extracted by alcohol, and it does not fade in Canada balsam like many of the others. Hardening in corrosive sublimate and imbedding in pa- raffin are generally recommended as preferable to hardening in alcohol and imbedding in celloidin. Stain sections for five to fifteen minutes in a rather dilute aqueous solution of the dye chosen. Of Bismarck brown use a saturated aque- ous solution, and stain, if necessary, twenty-four hours. With thionin, toluidin-blue, and polychrome methylene-blue metachromatic stains are obtained ; the mucin is colored red, the rest of the tissue blue. Two special methods for stain- ing mucin are given in detail : Hoyer's Method with Thionin. — Mucin, red; everything else, blue. i. Harden in corrosive sublimate, followed by alcohol. 2. Paraffin sections are passed through xylol, chloroform, and 95 per cent, alcohol to free them from paraffin, and are then placed in a 5 per cent, aqueous solution of corrosive sublimate for three to five minutes. 3. Stain in a dilute solution of thionin for ten to fifteen minutes. 4. Alcohol. 358 PATHOLOGICAL TECHNIQUE. 5. Clear in the mixture of the oils of cloves and thyme. 6. Turpentine oil or oil of cedar. 7. Balsam. Before the staining the sections must not be treated with iodin solution to get rid of the precipitate of mercury, be- cause it spoils the staining. Unna's Method with Polychrome Methylene-blue. — i. Stain paraffin or celloidin sections hardened in alcohol in polychrome methylene-blue five to ten minutes or longer. 2. Wash in acidulated water. 3. Fix in 10 per cent, solution of bichromate of potassium half a minute. 4. Wash in water. 5. Dry on slide with filter-paper. 6. Decolorize in aniline plus i per cent, hydrochloric acid (a few seconds only). 7. Wash off with oil of bergamot. 8. Balsam. Pseudo-mucin dissolves jn water to form a slimy material, and is precipitated from its solutions by alcohol in thread- like masses which are again soluble in water. It is not affected by acetic acid. Pseudo-mucin is found in certain ovarian and other tumors. Colloid and Hyaline. — The terms colloid and hyaline are not yet sharply limited to definite chemical substances. The term colloid was originally applied to the homogeneous substance found in the thyroid gland, but has been broad- ened to include various substances of a similar appearance. The term hyaline is still more indefinite, but its use may be said to be applied most generally to those homogeneous substances which stain deeply with various stains, in contra- distinction to those which like colloid show no marked af- finity for staining reagents after ordinary fixatives. Unquestionably, numerous substances of different chemi- cal composition and of varying origin have been grouped under these two titles because of their physical and optical characteristics — namely, that they occur as glassy, refractive, homogeneous, occasionally colored gelatinous or firm masses. HISTOLOGICAL METHODS. 359 Chemically, very little that is definite is known about them, and they possess no peculiar chemical reactions. Several at- tempts have been made to classify them in accordance with their reactions to various staining reagents. Von Recklinghausen applied the term colloid to all the homogeneous, transparent-looking substances, including mu- cin, amyloid, etc., and reserved the term hyaline for a special group, which, according to him, is characterized by the fol- lowing peculiarities : it resembles amyloid in physical charac- teristics, but does not react to iodin ; it stains deeply with acid dyes, such as eosin and acid fuchsin. Ernst has recently endeavored to differentiate two groups of hyaline substances, colloid and hyaline, by means of their reaction to Van Gieson's picro-acid fuchsin solution. Ac- cording to him, true hyaline stains with acid fuchsin alone, and appears of a deep-red color, while colloid, of which the typical example is found in the thyroid gland, stains with both picric acid and acid fuchsin, so that it appears of an orange or yellowish-brown color. He has also tried to prove that all colloid is derived from epithelial cells, while all hya- line comes from connective tissue or from blood-vessels. According to Von Kahlden, these differential staining re- actions with Van Gieson's mixture claimed by Ernst for col- loid and hyaline are by no. means justifiable, because true colloid often stains a deep red. Furthermore, Unna has shown that in the skin connective-tissue cells can give rise to the so-called true hyaline, of which part is acidophilic and part basophilic, while the intercellular substance gives rise to colloid. The last attempt to classify the various homogeneous sub- stances on the basis of their reactions to dyes, apparently the only method possible at present, has been made by Pianese as a result of his studies of the various degenerative processes occurring in cancer-cells. He used a special fixa- tive (see p. 225) and five different staining methods (see p. 250, methods HI. A and B, IV., V., and VI.). Of these methods, III. B is the best, because it gives a characteristic color to each substance — hyaline, brick-red ; colloid, bright 360 PATHOLOGICAL TECHNIQUE. green ; mucin, clear sky-blue ; and a substance resembling amyloid, a dark reddish-violet. Besides these distinct re- actions for colloid, hyahne, mucin, and a substance resem- bling amyloid, he found others less definite ; one of these he calls pseudo-mucin and another pseudo-colloid. As a basis for his studies he took the reactions of amyloid, mucin (in- testine), colloid (thyroid gland), and hyaline (hyaline remains of ovarian follicles, hyaline degeneration of renal glomeru- li), with the same stains after fixation in his own hardening mixture. The above brief historical statement is considered neces- sary to show the present views in regard to these various, more or less indefinite, homogeneous, transparent substances. For demonstrating them after the usual hardening reagents, of which alcohol and corrosive sublimate are perhaps the best, a double stain with alum-hematoxylin and eosin is very useful. Certain of the homogeneous substances stain deeply with eosin ; others, like the transparent drops and masses oc- casionally found in the walls of the blood-vessels of the brain, stain with hematoxylin. Sometimes good results can be obtained with Weigert's fibrin stain or with carbol-fuch- sin. The most generally useful stain, aside from alum-hema- toxylin and eosin, is probably Van Gieson's mixture. 1. Stain deeply in alum-hematoxylin. 2. Wash in water. 3. Stain three to five minutes in a saturated aqueous solu- tion of picric acid, to which is added enough of a saturated aqueous solution of acid fuchsin to give it a deep-red color. The effect of various proportions is sometimes useful. 4. Wash in water. 5. Alcohol. 6. Oleum origani cretici. 7. Balsam. The transparent homogeneous substances usually stain from orange to deep red in color; connective tissue, red. Unna's Method for Hyaline and Colloid Material. — A. Harden in alcohol, i. Acid fuchsin (2 per cent, aqueous solution) five minutes. HISTOLOGICAL METHODS. 36 1 2. Saturated aqueous solution of picric acid two minutes. 3. Saturated alcoholic solution of picric acid two minutes. 4. Wash off in alcohol. 5. Oil, balsam. Hyaline and connective-tissue fibers, red; colloid of thy- roid gland, yellow ; protoplasm, yellow. B. To show acidophilic and basophilic hyaline : i . Water- blue (2 per cent, aqueous solution) twenty to thirty seconds. 2. Water. 3. Carbol-fuchsin one to two minutes. 4. Water. 5. Alcohol slightly tinged with iodin. 6. Pure alcohol. 7. Oil, balsam. Nuclei, keratin, and large hyaline masses, cherry red ; con- nective-tissue fibrillze, protoplasm, and small hyaline bodies, blue. For finer work the methods of Pianese should be used. Keratohyalin {Unna). — i. Stain sections in a fairly old alum-hematoxylin solution until they are over-stained. 2. Place in a very weak solution of permanganate of potassium (about i : 2000) for ten seconds. 3. Dehydrate and decolorize in alcohol. 4. Oil, balsam. An isolated stain of the granules of keratohyalin is ob- tained, blue-black in color. In like manner a 33 per cent, solution of sulphate of iron acting for ten minutes, or a 10 per cent, solution of chlorid of iron for a few seconds, will produce the same effect. Or- dinarily, sections are stained deeply in alum-hematoxylin, and decolorized with acetic acid and alcohol or with hydro- chloric acid and alcohol. Glycogen Infiltration. — Glycogen is a carbohydrate of slightly varying composition, occurring in cells, more rarely in the intercellular tissue, either diffusely or more commonly in the form of larger and smaller masses and granules of a transparent homogeneous appearance. It is demonstrated micro-chemically by means of its reaction with 362 PATHOLOGICAL TECHNIQUE. iodin, which stains it brown. It is easily differentiated from amyloid by the fact that with the exception of the glycogen from certain sources, such as cartilage-cells, it is readily soluble in water and does not give the iodin-sulphuric-acid reaction. In consequence of its property of dissolving readily in water the aqueous Lugol's solution of iodin cannot be em- ployed for staining glycogen in fresh tissues. Instead, a thick solution of gum arable containing i per cent, of Lugol's solution must be used, or, better still, equal parts of glycerin and Lugol's solution, in which the sections are more per- fectly cleared. For sections hardened in absolute alcohol the same methods may be used, but better results, and practically permanent mounts, can be obtained by the method of Lang- hans. Lugol's solution is used for staining the sections, be- cause after hardening in alcohol the glycogen is much less soluble in water than in the fresh state. The iodin-glycerin mixture would probably be better. 1. Stain in Lugol's solution. 2. Dehydrate in i part of tincture of iodin to 3 or 4 parts of absolute alcohol. 3. Clear in oleum origani cretici. The sections are to be preserved in oil. Even a ring of balsam around the cover-slip will cause the color to fade. Other oils are not so good. Amyloid Infiltration. — Amyloid is insoluble in water, alcohol, ether, and dilute acids, and is not digested by pepsin and hydrochloric acid. It is distinguished from the other homogeneous substances, except glycogen, by the fact that it is stained mahogany-brown by iodin in solution. If a section containing amyloid be quickly and lightly stained in iodin solution and then transferred to sulphuric acid, the color of the amyloid will usually change at once or in a few minutes from red, through violet, to blue. Sometimes the color turns simply of a deeper brown. Several of the aniline dyes give almost as perfect characteristic color-reac- tions for amyloid as iodin, and are perhaps better for the HISTOLOGICAL METHODS. 363 purposes of histological study. Any of these differential stains may be used with fresh or hardened tissues. Alcohol as a hardening reagent gives the best results, but the other fixatives may be employed. Unfortunately, good permanent mounts cannot be made with any of the characteristic stains, so that the ordinary double stains of alum-hematoxylin with eosin or Van Gieson's mixture will often be found of the greatest help in studying the distribution of amyloid. lodin Reaction for Amyloid. — i. Stain sections in a weak solution of iodin (Lugol's solution diluted until of a clear yellow color) for three minutes. 2. Wash in water. 3. Mount and examine in water or glycerin. If the tissue reacts strongly alkaline, a condition which may result from post-mortem decomposition, the color reac- tion with iodin will not take place. In such cases the tissue or the sections of it should be treated with dilute acetic acid before applying the test. The normal reaction of amyloid with iodin may be increased by treating the section after staining with dilute acetic acid. Langhans' Method for Obtaining Permanent Mounts with Iodin. — i. Harden in alcohol. 2. Stain sections in Lugol's solution. 3. Dehyhrate in i part of tincture of iodin to 3 or 4 parts of absolute alcohol. 4. Clear and mount in oleum origani cretici. The color is said to keep remarkably well. Other oils or balsam cause it to fade quickly. The staining in Lugol's solution may be omitted, as the tincture of iodin usually stains the amyloid sufficiently deeply. Iodin and Sulphurio-acid Reaction. — i. Stain quickly and lightly in dilute Lugol's solution. 2. Treat with sulphuric acid, either concentrated or dilute (i to 5 per cent.), on the slide or in the staining dish. Strong hydrochloric acid may be used in the same way. The change of colors from red to blue already spoken of usually occurs within a few minutes, but occasionally does not take place at all. 364 PATHOLOGICAL TECHNIQUE. The following substances give reactions with the above iodin tests : 1. Cholesterin crystals are stained rather dark with dilute iodin solution, and turn a beautiful blue color at the edges on the addition of strong sulphuric acid. 2. The corpora amylacea in the prostate and central nerv- ous system stain brown with the dilute iodin solution. 3. Starch-granules stain blue with dilute iodin solution. 4. Cellulose stains yellow with iodin. If washed and treated with strong sulphuric acid, it turns blue where the acid touchs it. For the reactions with the aniline dyes the sections must be free from celloidin. Eeaotion with Methyl- of Gentian- violet. — i. Stain in I per cent, methyl-violet three to five minutes. 2. Wash in w^ter plus i per cent, of hydrochloric acid. 3. Examine in water or in glycerin. The stain will keep for some time if mounted in a satu- rated solution of acetate of potash or in levulose. Other methods are to stain in aniline-gentian-violet and to wash out in a I or 2 per cent, solution of acetic acid, or to stain in a strong solution of methyl-violet to which acetic acid is added, and to wash out in water. The amyloid is stained violet-red, the tissue blue. Reaction with lodin-green. — i. Stain fresh or hardened sections in a J per cent, aqueous solution of iodin-green for twenty-four hours. 2. Wash in water. 3. Mount in water or glycerin. Amyloid, a violet-red ; tissue, green. Stilling claims that the reaction is surer than with methyl-violet. Reaction with Bismarck Brown and Gentian-violet (Biroh-Hirschfeld). — i. Stain in a 2 per cent, alcoholic solu- tion of Bismarck brown five minutes. 2. Wash in absolute alcohol. 3. Wash in distilled water ten minutes. 4. Stain in a 2 per cent, solution of gentian-violet five to ten minutes. HISTOLOGICAL METHODS. 365 5. Wash in dilute acetic-acid solution. 6. Mount in levulose. Amyloid, red ; tissue, brown. Pigmentation. — The various pigments found in the human body under normal and pathological conditions may be divided into three groups : 1. Hematogenous pigments, derived from the coloring matter of the blood. (a) Hemoglobin and methemoglobin : soluble in water and alcohol not absolute ; occur as yellowish to yellowish-brown granules and droplets ; stain deeply with eosin after proper fixation; occur in hemoglobinuria, etc. ip) Parhemoglobin : a form of hemoglobin; crystallizes like it, but is insoluble in alcohol. {c) Hematoidin=bilirubin : contains no iron; is insoluble in water, alcohol, and ether ; dissolves in chloroform ; oc- curs as yellow or brown amorphous material or as crys- talline rhombic plates and needles. Is found in extravasa- tions of blood. ifT) Hemosiderin : occurs as bright-colored, yellowish- brown and brown granules and masses ; gives iron reac- tion ; is insoluble in water, alcohol, and ether ; is found in extravasations of blood, in the liver in pernicious anemia, etc. (e) Melanin : occurs as dark-brown or black granules and masses ; does not give iron reaction ; is found in malaria in the red blood-globules and in the tissues of the spleen, liver, and brain, but not of the lungs. (/) Bile-pigment=bilirubin=hematoidin: insoluble in water, ether, and alcohol ; occurs as yellowish granules and masses which are often greenish if old ; is found in jaundice. 2. Autochthonous pigments, formed by cells from color- less elements of nutrition. They all occur microscopically as lighter or darker brown granules ; are insoluble in water, alcohol, dilute caustic potash, etc., and contain no iron. They are found in the iris, retina, skin, ganglion-cells, Addi- son's disease, melanotic sarcomata, etc. 3. Extraneous pigments, entering the body from with- 366 PATHOLOGICAL TECHNIQUE. out. The most common examples are carbon in anthra- cosis pulmonum, iron in siderosis pulmonum, silver in argyria. Pigments are recognized microscopically, partly by their color and form, partly by their chemical reactions, and partly, though less accurately, by the lesions or pathological pro- cesses in connection with which they occur. They usually show best in contrast to red nuclear stains, such as alum or lithium carmine, but alum-hematoxylin often gives excellent results. The pigments of the second and third groups are perfectly preserved by all of the ordinary fixatives, of which alcohol, corrosive sublimate, and Zenker's fluid can be particularly recommended. Of the first group, melanin and hematoidin are preserved in any fixative. Hemoglobin and methemo- globin must be fixed in the solutions recommended for red blood-globules — namely, Zenker's fluid, corrosive sublimate, and Mijller's fluid. Pai'hemoglobin and hemosiderin should be preserved in alcohol. Bile-pigment is turned green, accord- ing to Ziegler, by fixation in corrosive sublimate, and is thereby rendered more prominent. In alcohol it preserves its yellow color. Carbon may be distinguished from melanin or any of the other pigments by the fact that it is insoluble in concentrated sulphuric acid. The only pigment for which micro-chemical color reactions are generally employed is hemosiderin, which really represents a group of pigments containing iron instead of one definite compound. Most of them will show the iron reaction after a shorter or longer time, but others, like the hemoglobin from which they are all derived, refuse to give it. The iron compounds present are usually ferric salts, but occasionally ferrous. Both groups react in the same way to sulphate of ammonium, but only the ferric salts react to ferrocyanide of potassium. For the ferrous salts, which oc- cur much more rarely, the ferricyanide of potassium must be used. Dr. E. S. Wood suggests that a mixture of ferro- and ferricyanide of potassium be employed, so as to demonstrate at once both groups of iron compounds, as with the sulphate- HISTOLOGICAL METHODS. 367 of-ammonium method. In performing the iron reactions steel needles must be avoided. Reactions for Iron in Hemosiderin. — Tissues should be hardened in alcohol. I. Reaction for ferric salts with ferro- cyanide of potassium and hydrochloric acid. A. I. Place sections for five to twenty minutes or longer in a 2 per cent, aqueous solution of ferrocyanide of potassium. 2. Transfer to acid alcohol (HCl i c.c. to 70 per cent, al- cohol 100 c.c.) for five to ten minutes, or to glycerin plus \ per cent, hydrochloric acid. The iron appears bright blue in color. If desired, the sections can be washed out after the acid al- cohol, and passed through alcohol and oil to balsam. After the iron reaction has been performed the nuclei may be stained in either alum or lithium carmine, or a little ferrocy- anide of potassium may be added to the lithium carmine, and the reaction and nuclear stain thus effected by the same step. B. H. Stieda's Method for Permanent Mounts with Nuclear Stains. — i. Stain several hours in lithium carmine. 2. Wash off quickly in water. 3. Place from four to six hours in a 2 per cent, aqueous solution of ferrocyanide of potassium. 4. Transfer to acid alcohol for six to twelve hours. 5. Wash quickly in water. 6. Alcohol, oil, Canada balsam. II. The reaction for the ferrous salts is performed in the same way as for ferric salts, with the exception that ferri- cyanide of potassium is used instead of the ferro- compound. III. Reactions for Ferric and Ferrous Salts. — A. Use a mixture of ferro- and ferricyanide of potassium (i gram each to 100 c.c. of water), followed by acid alcohol. Nuclear stains and permanent mounts may be made as above. B. I. Place sections in a freshly prepared solution of sul- phate of ammonium for five to twenty minutes, until they are dark or black-green in color. 2. Wash quickly in water. 3. Examine in glycerin or pass through alcohol and oil to Canada balsam. 368 PATHOLOGICAL TECHNIQUE. The iron appears in the form of black or dark-greenish granules. Sulphate of ammonium causes similar precipi- tates with other metal salts, such as the nitrates of silver, lead, and mercury. Petrifaction. — Calcification, the more common form of petrifaction, is the term applied to the infiltration of tissues with phosphate and carbonate of lime. The salts appear microscopically as small, very refractive granules which may be mistaken for fat, or as large masses due to the fusion of granules. They are dissolved by hydrochloric or nitric acid (S per cent, solution). If carbonate of Hme is present, bubbles of carbon-dioxid are set free. Phosphate of lime dissolves without effervescence. To differentiate between lime-salts and other substances soluble in hydrochloric acid use con- centrated sulphuric acid to form sulphate of Hme (gypsum), which appears as fine, short, radiating needles. On dissolv- ing out the lime-salts a matrix of dead tissue or of hyaline material will usually be found left behind. As a rule, this hyahne material stains deep blue in alum-hematoxylin or red in Van Gieson's mixture. Another form of petrifaction is that found in gout, due to the infiltration of certain tissues with uric-acid salts, of which urate of sodium is the most common. The crystals are sol- uble with difficulty in cold water, insoluble in alcohol and ether. CLINICAL PATHOLOGY. The material received at the pathological laboratory from clinical cases consists largely of new growths and lesions produced by infectious, chemical, or traumatic agents — to a less extent of fluids from various natural or artificial cavities or secretions and eliminations from the respiratory, genito- urinary, and gastro-intestinal tracts. In the following pages a brief synopsis is given of what may be expected in the various fluids and eliminations, and the more important chemical tests required for the recognition of certain sub- stances are added. HISTOLOGICAL METHODS. 369 Gross specimens removed at operations should be treated as parts of autopsies, and dissected or otherwise examined bacteriologically and histologically in an equally careful manner. For the finer study of cancers and other new- growths the recently published methods of Pianese should be employed (see page 250). iExamination of Tissues from Clinical Cases for Diagnosis. — Tumors of any size or large pieces of tissue present no difficulties. There is plenty of material to ex- amine fresh or after fixing in a variety of ways. The small bits removed with a Mixter punch or cut from the edge of a growth, of which a diagnosis is wanted immediately, some- times prove troublesome. Frozen sections of the fresh tissue are often sufficient. Sometimes it is better to harden for an hour or more in formaldehyde and then to make frozen sections. Often it is wisest to harden in strong alco- hol and then to make razor sections or to imbed in celloidin. The whole process of hardening, imbedding, and sectioning can easily be carried through in twenty-four hours with small pieces of tissue. Imbedding in paraffin is sometimes preferred. In cutting sections of small pieces it is important to mount them if possible, so that the cut sections will show proper relations — i. e. vertical sections through the skin, uterine mucous membrane, etc. — otherwise confusing pict- ures will often be presented. It is important to know, in regard to pieces of tissue sent for diagnosis, from what part of the body they come, because what is normal in one situa- tion may be pathological in another. A hematoxylin-and-eosin stain will be found the most generally useful for hardened sections. Uterine Scrapings. — Small pieces may be examined fresh in frozen sections or after hardening for one or more hours in formaldehyde. Better results are obtained by hard- ening in alcohol and imbedding in celloidin or paraffin. Where the fragments are small, it is advisable to mass them together on a small piece of filter-paper and to harden in strong alcohol. The mass can then be imbedded in celloidin and cut as one piece of tissue. A hematoxylin-and-eosin 24 370 PATHOLOGICAL TECHNIQUE. stain is the best, because the eosin brings out the smooth muscle-fibers prominently, so that any invasion of the mus- cular coat by a malignant growth is more readily made out — a valuable help in the diagnosis of malignant adenoma. :^xamination of Fluids obtained by Puncture. — The transudations obtained largely from the serous cav- ities are non-inflammatory in origin. They are ' usually of a transparent, pale-yellow color with slightly greenish tint, alkaline in reaction, and deposit on standing a slight floccu- lent coagulum. The specific gravity, to be taken at room-temperature, varies according to the origin of the fluid. According to Reuss, it is below 1015 in hydrothorax; below 1012 in ascites; below loio in anasarca; below 1008.5 i" hydro- thorax. The amount of albumin in hydrothorax is always under 2.5 per cent., and in ascites between 1.5 and 2 per cent. Microscopically, a few leucocytes, usually fatty degenerated and rarely desquamated endothelial cells, are found. The exudations are of inflammatory origin, and are also generally obtained from the serous cavities. From their various microscopic appearances they are divided into serous (fibrino-serous), hemorrhagic, purulent, and gangrenous. The specific gravity of all is over 1018 ; the reaction is always alkaline. On standing they deposit a varying amount of sediment. Examinations for organisms should always be made (see page 89). Occasionally a peculiar opalescent layer, due to cholesterin crystals, forms on the surface of fluids which come from old cases of pleurisy. Serous Exudations. — The fluid, which immediately after • removal is slightly cloudy and yellowish in color, deposits more or less quickly a flocculent or dense coagulum. Micro- scopically, the coagulum shows a dense meshwork of fibrin and numerous polynuclear leucocytes. Hemorrhagic Exudations. — The sero-fibrinous exudation is colored a lighter or darker red according to the amount of blood present. Microscopically, the same elements are found as in the serous exudations, plus a marked increase HISTOLOGICAL METHODS. 37I of red blood-globules, which are usually well preserved, but in old exudations may be more or less decolorized. Aside from injuries, hemorrhagic exudations are most com- mon in connection with tuberculosis and new growths, so that their microscopic examination is of much diagnostic and prognostic value. For the examination of tubercle bacilli see page 91. In this form of exudation it is rarely possible to demonstrate them. On the other hand, it is not infrequently possible to make the diagnosis of a malignant growth, especially of cancer, from the examination of the sediment. No cell is significant of cancer or other neoplasm, but the occurrence of numerous cells which vary greatly in form is suspicious. The cells from new growths are often unusually large, up to 120//, frequently contain one or more vacuoles, and usually lie in clumps. Large drops of fat are also considered sus- picious. A positive diagnosis can only be made by obtaining bits of tissue which show the structure of the new growth, such as the atypical alveolar arrangement of the cells in cancer. Purulent exudations appear more or less thick and yel- low, and deposit a corresponding layer of pus. Microscopic- ally, they present no peculiarities other than the organisms to which they are due. Among the etiological factors acti- nomyces must always be thought of in puzzling cases. Putrid exudations occur in the pleural and peritoneal cavities in consequence of gangrenous masses breaking into them and from stomach or intestinal ulcerations, from new growths, occasionally from no clear cause. The fluid result- ing from the perforation of a gastric ulcer may show yeast- cells and sarcinse, and give an acid reaction. I/Umbar Puncture. — The diagnostic value of lumbar puncture has been sufficiently demonstrated. Not only is it possible to diagnosticate inflammation of the meninges, but the character and cause of the inflammation may usually be demonstrated if the examination of the fluid is properly performed. In a number of cases of general infection in which there was no inflammation of the meninges a diag- 372 PATHOLOGICAL TECHNIQUE. nosis has been made by means of cultures taken from the cerebro-spinal fluid. Finally, a number of cases of hemor- rhage into the brain and spinal canal have been diagnosticated by lumbar puncture. The operation and the subsequent examination of the fluid should be as carefully performed as any other bacteriological investigation in order to obtain accurate results. The back of the patient and the operator's hands should be made sterile. The needle should be boiled for ten minutes. The patient should lie on the right side, with the knees drawn up, and with the uppermost shoulder so depressed as to present the spinal column to the operator. This position permits the operator to thrust the needle directly forward rather than from the side. An antitoxin needle 4 cm. in length, with a diameter of i mm., is well adapted for infants and young children. A longer needle is necessary for adults and chil- dren over ten years of age. Aspiration of the fluid is not necessary, but some ope- rators prefer to attach a hypodermic syringe to the needle to afford a better grasp for the hand. In this case the syringe would have to be detached to allow the fluid to flow. The additional manipulation, and possibly the defective steriliza- tion of the syringe, might impair the subsequent bacterio- logical examination. The puncture is generally made between the third and the fourth lumbar vertebrae ; sometimes between the second and third. The thumb of the left hand is pressed between the spinous processes, and the point of the needle is entered about I cm. to the right of the median line and on a level with the thumb-nail, and directed slightly upward and in- ward toward the median line. Care must be exercised to prevent the point of the needle from passing to the left of the median line and striking on the bone. At a depth of 3 or 4 cm. in children and 7 or 8 cm. in adults the needle en- ters the subarachnoid space, and the fluid flows usually by drops. If the point of the needle meets with abony obstruc- tion, it is advisable to withdraw the needle somewhat, and to thrust again, directing the point of the needle toward the HISTOLOGICAL METHODS. 373 median line, rather than to make lateral movements, with the danger of breaking the needle or causing a hemorrhage. The smallest quantity of blood obscures the macroscopic appearance of the fluid by rendering it cloudy. The fluid is allowed to drop into an absolutely clean test-tube which pre- viously has been sterilized by dry heat to 150° C. and stop- pered with cotton. The fluid should be allowed to drop into the tube without running down the sides. From 5 to 1 5 c.c. of fluid is a sufficient quantity for examination. Cultures on blood-serum should be made at once from the fluid. The test-tube is then held toward the light and gently shaken ; by this means the slightest cloudiness can be de- tected. In meningitis there is always an exudation of cells which makes the fluid more or less cloudy. The degree of cloudi- ness is to some extent proportionate to the amount and cha- racter of the exudation. In tubercular meningitis the amount of cellular exudation is sometimes so slight that the fluid ap- pears clear unless examined carefully. A guinea-pig should be inoculated with some of the fluid as a control-experiment. The inoculation should be made a short time after withdraw- ing the fluid before the formation of fibrin occurs, because the web-like coagulum of fibrin holds the tubercle bacilli in its meshes. After standing for several hours more or less fibrin forms in the fluid, and contains the cells in its meshes. The supernatant fluid is left clear. Cover-glass preparations should be made from this sediment, dried by passing through the flame of an alcohol lamp or Bunsen burner, and stained with methylene-blue, which stains the nuclei of the cells and any pathogenic bacteria which may be present. In tubercular meningitis the predominant cell is the lymph- oid cell. In purulent meningitis the polynuclear leucocyte predominates. Cover-glass preparations may also be stained for tubercle bacilli by appropriate methods of staining. It is often necessary to examine twenty or more prepara- tions, however, before finding the bacilli. The albumin should be quantitated. Normally, the cerebro-spinal fluid contains from -^ to -^ of i per cent, or less. In menin- 374 PATHOLOGICAL TECHNIQUE. gitis the amount is increased from ^^ to -jlj^ of i per cent., or often more. Percentages of albumin down to y^j of i per cent, can be estimated by the ferrocyanide-of-potassium-and acetic-acid test and a centrifugal machine. Take 3^ c.c. of a 20 per cent, solution of ferrocyanide of potassium, \\ c.c. of acetic acid, and 10 c.c. of the fluid in which the albumin is to be quantitated. The mixture of reagents and fluid is poured into a conically-shaped graduated glass vessel de- signed for the purpose and centrifugalized. Sugar is rarely present and has no diagnostic value. Ovarian and Parovarian Cysts. — The simple cysts of the ovary due to distention of Graafian follicles or to cystic change of corpora lutea, and the parovarian cysts contain a thin, clear, serous fluid of low specific gravity. The contents of the multilocular and papillary adeno-cys- tomata of the ovary are usually tenacious and mucous, of very varying specific gravity, from 1005-1050, but usually between 1020 and 1024. The fluid generally contains much albumin and is rich in metalbumin, which is precipitated by alcohol, but not by acetic acid, nitric acid, or boiling, so that it can readily be distinguished from mucin. Before making the test the albumin must be removed. The cyst-contents are usually yellowish, but sometimes may be dark-red or chocolate-colored. Microscopically, red and white blood-globules, occasionally blood-pigment and cholesterin crystals, often fat-granules and large vacu- olated cells, are found in the cyst fluid. Bizzozero considers cylindrical epithelial cells, ciliated and beaker cells, and col- loid concretions especially important from a diagnostic point of view. Pancreatic Cyst or Fistula.— The fluid obtained from a permanent fistula or large cyst of the pancreas contains much less solids than the normal pancreatic juice, and the trypsin ferment may be present in very small amount or possibly be entirely wanting. The fluid is colorless, alkaline in reaction, and has a specific gravity of about loii. It is characterized by three distinct properties on which its re- cognition depends — namely : HISTOLOGICAL METHODS. 375 1. It splits up fat into fatty acids and glycerin. Mix to- gether equal parts of neutral olive oil and the alkaline fluid. Test with litmus-paper. Place the mixture in the incubator '^t 37° C, and test from time to time. If the fluid is pan- creatic, an acid reaction will be obtained in twelve to eighteen minutes. 2. It transforms starch into sugar. Place in the incubator equal parts of a i per cent, aqueous solution of starch and of the fluid to be tested. In ten to twenty minutes test for sugar with Fehling's solution. 3. It digests fibrin in an alkaline solution (trypsin ferment). Place some fibrin in the alkaline fluid and set it in the in- cubator. In one-half to one hour examine for peptones by the biuret test. Add caustic potash or soda and a few drops of a dilute solution of sulphate of copper. If peptones are present, a beautiful reddish-violet color will be produced. Dropsy of the Gall-bladder. — Puncture is generally not advisable. The fluid is usually colorless and mucoid or serous in character. All trace of biliary constituents may have disappeared. According to Lenhartz, numerous colon bacilli are usually present. Hydronephrosis and Renal Cysts. — The fluid is almost always clear as water, rarely reddish or yellow. Specific gravity always under 1020 (usually between loio and 1015). Urea and uric acid are generally present, but may be absent. (Small amounts of urea are sometimes present in ovarian cysts.) Albumin is slight in amount. Microscopically, almost nothing is found. Bchinococcus Cysts. — The fluid is perfectly clear, free from albumin, and contains a little succinic acid and much chlorid of sodium. The specific gravity varies between 1 008 and 1013. Microscopically, often no traces of morphological ele- ments can be found. Occasionally, however, hemosiderin granules or chlolesterin crystals occur, or the characteristic structures from which a positive diagnosis can be made — - namely, scolices, booklets, or pieces of cyst-membrane. 376 PATHOLOGICAL TECHNIQUE. A positive diagnosis from a chemical examination depends on showing — 1. The absence of albumin. 2. The presence of chlorid of sodium. Evaporate a drop of the fluid slowly on a sUde, so as to get the characteristic crystals of chlorid of sodium. 3. The presence of succinic acid. Acidify a little of the fluid with hydrochloric acid and evaporate to dryness. Extract the residue with ether. The crystallized material left on the evaporation of the ether, if dissolved in water, will give a rust-colored, gelatinous pre- cipitate with sesquichlorid of iron if succinic acid be present. Bxamiaation of the Sputum. — The secretion raised from the air-passages by coughing is almost invariably con- taminated with the secretion of the naso-pharynx and with particles of food from the mouth. In examinations of sputum these contaminations must always be borne in mind. The amount raised varies from a few c.c. to one or even several liters in twenty-four hours. The macroscopic appearances of the sputum depend on the varying proportions of mucus, pus, blood, and serum present. The tenacity is mainly due to the mucus. The reaction is usually alkaline. The general color, consistency, and separation into layers is best seen after the sputum has stood for some time in a tall glass. For more careful macroscopic examination small portions of the sputum are transferred to flat glass dishes, where they are spread out thinly by needles and examined over black or white paper. Porcelain plates painted black or black paper itself can be used. The latter method is con- venient, because the sputum can be burned up with the paper. The constituents of the sputum which may be recognized macroscopically are few in number, and not so important as those which may be found microscopically. Macroscopic Examination. — i. Caseous Masses. — In the sputum from tubercular cases small, opaque, yellowish-white masses from the size of a pin-head to that of a small pea can occasionally be found, which spread out beneath ^ HISTOLOGICAL METHODS. 377 cover-glass like a bit of cheese. They are small caseous masses which are valuable for microscopic examination be- cause they usually contain tubercle bacilli and elastic fibers. 2. Fibrinous casts of the bronchioles can usually be found in the sputum from the third to the seventh day in cases of acute lobar pneumonia. They appear as yellowish-white or reddish-yellow threads, 2 to 3 mm. thick and \ to several cm. long, and are often branched. The large ones are often rolled into balls, and show best after being shaken in water. Casts of the bronchi are found in" cases of fibrinous bronchitis. 3. Curschmanri s spirals (Fig. 103) of twisted threads of JSm^ ^B Fig. 103. — Curschmann's spirals ; a, central fiber. mucus enclosing epithelial cells and leucocytes occur rarely except in bronchial asthma. They appear macroscopically as grayish-white or whitish-yellow masses or threads, J to ij mm. thick and J to 8 cm. long, and often show a visible spiral arrangement. 4. DittricKs Plugs. — These are whitish-yellow masses from the size of a pinhead to that of a bean, which are formed in cases of putrid bronchitis and of gangrene of the lung. They have a very fetid odor, a cheesy consistency, and are rather easily compressed. Besides organisms they contain numerous fat-crystals. 5. Shreds of tissue are found almost solely in gangrene of the lung, and are best recognized with the microscope. 6. Concretions, portions of cysticercus membrane, etc. are rare in the secretion from the lungs. 378 PATHOLOGICAL TECHNIQUE. Microscopic Examination. — Microscopically, the sputum may show various kinds of cells, fragments of tissue, includ- ing elastic fibers, vegetable and animal parasites, and crystals. They will be taken up in order : 1. Red Blood-globules. — In fresh hemorrhages they appear normal, often in rouleaux. In old sputa many have lost their color. 2. White blood-globules are almost invariably polynuclear, and the majority of them contain neutrophilic granules. In asthma, however, numerous" eosinophilic and rather numer- ous basophilic leucocytes are regularly found. The leuco- cytes often contain pigment- or fat-granules. 3. Epithelial Cells. — Pavement, cylindrical, and ciliated cells are found. The first come from the naso-pharynx ; the others usually from the trachea and bronchi, but may come from the nose. Desquamated alveolar epithelium is difficult to demonstrate. The pigmented cells found almost wholly in chronic passive congestion of the lungs are in small part alveolar epithelium, in larger part leucocytes. The pigment appears as yellowish, yellowish-red, or brownish-red granules or as yellow diffuse pigmentation. Occasionally, however, it is black, and then is less easily distinguished from carbon. The pigment is derived from the blood, and will usually give the iron reaction (see page 367), but very young or old pig- ment will not. 4. Fatty Detritus. — Fat-drops are frequently found, due to the fatty degeneration of cells. 5. Elastic fibers (Fig. 104) occur singly, but more often as a network. They are recognized by their sharp, dark out- lines, due to their high degree of refractiveness, and by their marked degree of resistance to acids and alkalies by which other like tissues, such as connective-tissue fibers, are de- stroyed. Elastic fibers are most abundant in the caseous masses above mentioned. When these masses cannot be found, the thicker portions of the sputum are squeezed be- tween a slide and cover-glass or between two slides, and ex- amined with a low power. The examination is rendered easier by mixing a little sputum with a 10 per cent, solution HISTOLOGICAL METHODS. 379 of caustic potash or soda. In certain cases it is necessary to mix together equal parts of the sputum and 10 per cent, caustic potash or soda, and to boil the mixture until the sputum is dissolved. The solution is then mixed with four times its own volume of water and allowed to stand for twenty-four hours, when the sediment can be examined for the elastic fibers. Vegetable and Animal Parasites. — Of the vegetable parasites, the most important is the tubercle bacillus (for its examination see page 91). Other bacteria sometimes ex- amined for are the pneumococcus, the influenza bacillus, and actinomyces. Of the animal parasites, the ameba coli is sometimes Fig. 104. — Elastic fibers (after Striimpell). found secondary to an hepatic abscess which has perforated into the lung (see page 292). Portions of the membrane of an echinococcus cyst or the booklets from the head may be found in the sputum, but infection with this parasite is very rare in this country. Of the crystals which occur in sputa, the most important are the Charcot-Leyden crystals, found mainly in bronchial asthma, and the crystals of the fatty acids, of cholesterin, and of hematoidin. Tyrosin and leucin are much more rare. The Charcot-Leyden crystals are colorless, elongated octahedra of varying size, soluble with difficulty in cold water, insoluble in alcohol, ether, chloroform, and dilute saline solution. Hematoidin crystals occur as ruby-red rhombic plates or columns. 380 PATHOLOGICAL TECHNIQUE. Cholesterin crystals (Fig. 105) occur as the well-known small and large rhombic plates. The fatty-acid crystals occur as long, pointed needles, either singly or in groups. They are easily soluble in ether or hot alcohol, insoluble in water and acids. Examination of the Gastric Contents. — The micro- scopic examination of the contents of the stomach is much less important than the chemical. Fresh blood is easily recognized by the microscope. Disintegrated blood must be examined for chemically by the hemin test, as follows : Mix a little of the suspected material with a crystal or two of common salt, or place it on the thin layer of salt formed Fig. 105. — Crystals of cholesterin (after Striimpell). by slowly evaporating a small drop of normal salt solution on a slide. Cover with a cover-glass, and run in enough glacial acetic acid to fill up the space between slide and cover. Warm the slide over a flame for three-quarters to one minute until bubbles arise, adding more glacial acetic acid as evaporation takes place, until a faint reddish-brown tint appears. Then let the acetic acid evaporate entirely, and run glycerin in from the edge of the cover-glass. Micro- scopic examination will show dark-brown rhombic plates or columns of hemin if blood is present. Shreds of tissue or bits of mucous membrane are some- times found in the vomitus or removed by means of a stomach-tube. Examination of them in the fresh condition, or, more satisfactorily, in stained sections after hardening and HISTOLOGICAL METHODS. 38 1 imbedding, will sometimes give definite information in regard to the condition of the mucous membrane, or render possible the diagnosis of a malignant growth. Examination for Free Hydrochloric Acid. — Of the fol- lowing tests, that with Congo-paper is the quickest and easiest, but shows only that a free acid is present. To prove that the free acid is hydrochloric acid the phloroglucin-vanillin test or one of the others is necessary. 1. Congo-paper is turned blue by free acids only. Free hydrochloric acid turns it of a cornflower-blue, a tint obtained with lactic acid only when in much greater concentration than is ever present in the stomach. Congo-paper is used simply by dipping it into the stomach-contents, preferably after filtration. 2. Gunzburg's Test with Phloroglucin-vanillin. — The solu- tion consists of — Phloroglucin, 2 ; Vanillin, i j Absolute alcohol, 30. Three or four drops of this solution are placed with an equal amount of the filtrate from the stomach-contents in a porce- lain dish and carefully heated over a small flame. Keep the dish in constant motion, and do not allow the mixture to boil, because boiling prevents the reaction from taking place. If free hydrochloric acid is present, a rose-red mirror is pro- duced. The phloroglucin-vanillin solution does not always keep well, so that it is best to keep alcoholic solutions of \ phloroglucin and of vanillin in separate bottles, and to mix together one or two drops of each when required. 3. Boas' Resorcin Test. — The solution consists of — Resublimed resorcin, 5 ; Cane-sugar, 3 ; Alcohol, 94 per cent, ad 100. It is used in the same manner as the phloroglucin-vanillin test. A similar but more transient mirror is produced. Topfer's Diinethyl-aniido-azo-benzol test is highly recom- 382 PATHOLOGICAL TECHNIQUE. mended by Simon as superior to the phloroglucin-vanillin test. "One or two drops of a 0.5 per cent, alcoholic solu- tion is added to a trace of the gastric contents, which need not be filtered ; in the presence of free HCl a beautiful cherry- red color develops,, which varies in intensity according to the amount of free HCl present." iExamination of the Feces. — In examining for tape- worms and their eggs it is often best to dilute the feces with water, and then to examine the sediment both macroscop- icaily and under the microscope. (For ameba coli see p. 292.) The other protozoa are best looked for in fresh slide preparations. For the cholera vibrio see p. 195 ; for the typhoid bacillus see p. 198. The membranous casts sometimes found in feces consist almost wholly of mucus, cylindrical epithelial cells, and leucocytes. Bits may be examined fresh, or the casts may be hardened and sections made and stained after imbedding in celloidin. Bxaminatiou of the "Urine. — Only those points are mentioned which come within the province of the patholo- gist. Of the animal parasites, the echinococcus and the filaria sanguinis hominis are the only important ones, and they are very rare (see pp. 294, 298). Of the vegetable parasites, tubercle bacilli and gonococci are the most common ; actinomycetes are very rare. New growths in the kidneys are accompanied with hemor- rhage in less than half of the cases, while new growths in the bladder almost invariably give rise to it. Fragments from new growths in the bladder are rare. A diagnosis of malig- nant disease from cells only is impossible. Pieces of tissue which show on microscopic examination the characteristic structure of cancer or other neoplasm must be obtained in order to render a diagnosis possible from the pathological (but not from the clinical) standpoint. histological methods. 383 Partial List of Works Consulted. Friedlander : Mikroskopische Technik. Israel : Practicum der Pathologischen Histologic. V. Jaksch : Klinische Diagnostik. Innerer Krankheiten. V. Kahlden : Technik der Histologischen Untersuchung. Lee : The Microtomisf s Vade-Mecum. Lenhartz : Mikroskopie und Chemie am Krankenbett. V. Lenhoss^k : Der Feinere Bau des Nervensy stems. Simon : Clinical Diagnosis. Thoma : Lehrbuch der Pathologischen Anatomic. Weigert : Beitrage zur Kcnntniss der normalen vicnschlichen Neuroglia. INDEX. Abbe's illuminating apparatus, 205 camera lucida, 206 Abbott's method of staining spores, 100 Abdominal cavity, 40 fluid in, 29 gas in, 29 inspection of, 29 opening of, 27 Acetic acid, 217 Acid, acetic, 217 chromic, 218 hydrochloric, 217 nitric, 229 osmic, 217, 261 picric, as a decalcifying reagent, 230 as a stain, 272 Acid-alcohol, 254 Acid-fuchsin, 249 Actinomyces, 176 method of staining in sections, 282 Adhesions, pleural, 32 Adrenals, 95 removal of the, 46 Agar-agar, glucose, 79 glycerin, 80 lactose-litmus, 80 plain, 77 slant culture of, 79 stab culture of, 79 Albuminous degeneration, 352 Alcohol, 222 Ranvier's one-third, 218 Algeri, method of staining fatty degenerated nerve - fibers, 326 Alum-carmine, 244, 268 25 Alum-cochineal, 244, 268 Alum-hematoxylin, aqueous, 241, 267 Delafield's, 241, 267 Ehrlich's, 242, 267 stains, 266 Ameba coli, 292 differential stain for, 293 Amputating knives, 18 Amyloid, stains for, 362 stain with Bismarck brown and gentian-violet, 364 stain with iodin, 363 stain with iodin-green, 364 stain with methyl-violet, 364 Anaerobic cultures, method of Buchner, 119 method of Esmarch, 119 method of Frankel, 121 method of Liborius, 117, 118 methods of preparation of, 117 Aniline, 257 Aniline and xylol, Weigert's mix- ture of, 258 Aniline dyes, 245 as nuclear stains, 269 Aniline oil, 257 Aniline water, 254 Aniline-blue, Strobe's stain with. for nerve-fibers, 313 Aniline-fuchsin, 247 Animal parasites, 287 Animals, care of, 117 food of, 117 inoculations of, 112-117, i8i at autopsy, 96 with the bacillus tuberculosis, 97 with the pneumococcus, 97 385 386 INDEX. Anterior incision, the long, 28 Antlirax, bacillus of, 56 Aorta, incision of the, 50 Apparatus used in the examination of the blood, 327 preparation of, 330 Appendicitis, 181 Autopsies, bacteriological examina- tions at, 89 general rules in regard to, 21 organs from, from which cultures are taken, 96 private, 24 Autopsy-knife, 18 Autopsy-needle, 21 Autopsy-record, 22 Autopsy-table, 18 Axillary glands, 29 Axis-cylinders and their terminal processes, stains for, 312 Babes' aniline-safranin, 248 Bacillus aerogenes capsulatus, 173 coli communis, 145, 181 diphtherise, 137 mallei, 164 mucosus capsulatus, 168 of anthrax, 156 of glanders, 164 of green pus, 160 of influenza, 162 of leprosy, 285 / of malignant edema, 175 of syphilis, 286 of tetanus, 171 of typhoid fever, 141 differential diagnosis of, 143 serum or clump reaction with, 144 of rhinoscleroma, 282 proteus, 167 pyocyaneus, 160 tuberculosis, 92 animal inoculations with, 97 differentiation of, from smegma bacillus and bacillus of lep- rosy, 92 Gabbet's stain for, 92 lesions produced by, 97 Bacteria, method of studying in cultures, 98 not stained by Gram's method, 92, 277 stained by Gram's method, 92, 280 by tubercle bacillus method, 283 staining of, in tissues, 275 Bacteriological apparatus, 70 diagnosis, 121 examinations, 70 at autopsies, 89 collection of material for, 178 Balsam, Canada, 258 xylol, 258 Band-saw, 18 Bergamot oil, 256 Bethe's method of fixing methy- lene-blue stains of nerve- fibers, 315 Bile-pigment, 365 Bilirubin, 365 Biondi-Heidenhain stain, 250, 272 Birch-Hirschfeld's stain for amy- loid, 364 Bismarck brown, 249 Bladder, incision of the, 48 Blood, apparatus used in the ex- amination of, 327 cover-glass preparations of, 336 elements of the, 338 estimation of number of red cor- puscles of, 333 examination of, 326 hypostasis of, 26 methods of staining, 342 test for hemoglobin in, 332 Blood-coloring matter, diffusion of, 26 Blood-serum, coagulation of, 82 coagulator, 70 collection of, 82 method of preparing cultures on, 94 old method of preparation of, 84 preparation of, 81 Boards, 21 INDEX. 387 Boas' resorcin test for hydrochloric acid, 381 Body, external examination of, 26 internal examination of 27 length of, 26 restitution of, 67 special inspection of the different parts of, 27 Body-cavity, method of sewing up, 68 Boiling, 227 Bone, methods of fixing and ex- amining, 348 Schaffer's method of staining, 349 Bone-cutter, 21 Bone-marrow, methods of fixing and examining, 346 Bothriocephalus latus, 298 Bottles, patent T. K., dropping-, 216 Bouillon, preparation of 75 Brain, external examination of, 55 Pitre's method of sectioning, 59 removal of 50, 53 section of, 56 Virchow's method of sectioning, 58 weight of 55 Breast, incision of 29 Buchner's method for anaerobic cultures, 1 19 Bunge's method of staining fla- gella, 103 Camera lucida, 206 Canada balsam, 258 Capsules and flagella, staining of 104 Carbol-fuchsin, 247 Carbol-gentian-violet, 248 Carbolic acid and xylol mixture, 257 Carbolic-acid water, 255 Carbon, 366 Corpora amylacea, reaction of with iodin, 364 Carmine, alum-, 244, 268 lithium, 244, 269 neutral, 245, 272 Cartilage, methods of fixing and examining, 348 Cartilage-knife, 19 Caseation, 355 Costotome, 21 Catheter, 21 Caustic potash, 218 Cedar-wood, oil of 257 Celloidin, 231 imbedding, 231 microtome, 209 sections, method of attaching to slides, 234 Cellulose, reaction of with iodin, 364 Central nervous system, methods of staining, 303 Charcot-Leyden crystals, 379 Chenzinsky-Plein stain for blood, 344 Chisel, 21 Cholera, spirillum of 152 Cholesterin-crystals, 354. reaction of with iodin, 364 Chromic acid, 218 Cleaning of slides and cover-slips, 214 Clearing reagents, 255 Clinical bacteriology, 178 pathology, 368 Cloudy swelling, 352 Cloves, oil of 257 Cochineal, alum-, 244 Colloid, stains for, 358 Colophonium, 258 Combination stains, 252 Comma bacillus, 152 Congo-paper test for hydrochloric acid, 381 Connective-tissue fibrillae, 300 Ribbert's stain for, 301 Unna's stain for, 301 Van Gieson stain for, 300 Contrast-stains, 271 Cornet cover-glass forceps, 71 Coronary arteries, opening of 35 Corrosive sublimate, 225 Cover-glass preparations, 89, 180 from cultures, 98 388 INDEX. Cover-glass preparations, import- ance of, 90 mounting of, in water, 90 of blood, 336 staining of, 91 Cover-slips, 214 Cox's modification of Golgi's method, 312 Cultivation of bacteria vvfithout oxygen, 117, 121 method of Buchner, 119 of Esmarch, 119 of Frankel, 121 of Liborius, 117, 118 Culture-media, preparation of, 75 agar-agar, plain, 77 bouillon, 75 blood-serum, 81 gelatin, plain, 80 glucose agar-agar, 79 glucose bouillon, 76 glucose gelatin, 81 glycerin agar-agar, 80 lactose-litmus agar-agar, 80 litmus milk, 85 Lubinski's medium for the bacil- lus tuberculosis, 151 peptone solution, Dunham, 86 potato cultures, Bolton, 85 serum agar-agar, 131 sterilization of, 87 urine-serum agar-agar, 132 Cultures, cover-glass preparations from, 98 examination of, 94 from organs at autopsy, 96 method of diluting, 95 method of preparing on blood- serum, 94 method of studying bacteria in, 98 Curschmann's spirals, 377 Cysts, echinococcus, 375 ovarian and parovarian, 374 pancreatic, 374 renal, 375 Damar, 258 Darkschewitsch's method for serial sections, 238 Delafield's alum-hematoxylin solu- tion, 241, 267 Decalcification, 228 Degenerations, albuminous, 352 fatty, 352 of the nervous system, 325 Development of the skeleton, 26 Diaphragm, position of, 30 Diffuse stains, 249, 271 Diffusion of blood-coloring matter, 26 Diluting cultures, method of, 95 Dilutions, 108 Dimethyl-amido-azo-benzol test for hydrochloric acid, 381 Diphtheria, bacillus of, 137 Diplococcus intracellularis menin- gitidis, 135 pneumoniae. See Pneumococcus. Director, 21 Discolorations, greenish, 26 post-mortem, 26 Dishes, oblong, rectangular, Petri, 215 staining, 214 Steinach's sieve, 215 Stender, 215 Dittrich's plugs, 377 Drop-bottle for microtome, 209 Dropping-bottles, T. K. patent, 71 Dunham's mixture of oils of cloves and thyme, 257 peptone solution, 86 Duodenum, incision of, 43 Dura, inspection of, 53 removal of, 53 Ear, removal of middle, 63 Echinococcus cysts, 375 Ehrlich's acid-hematoxylin solu- tion, 242, 267 aniline -gentian -violet solution, 247 hematoxylin-eosin stain for blood, 343 method of staining mastzellen, 298 tubercle bacilli, 284 Elastic fibers, 301 INDEX. 389 Elastic fibers, Herxheimer's method of staining, 303 in sputum, 378 phosphotungstic acid-hema- toxylin stain for, 302 Unna's orcein stain for, 302 Embolism of pulmonary artery, 35 Enterotome, 20 Eosin, 249, 271 Eosin and methylene-blue, 270 Erlicki's fluid, 228 Erysipelas, 181 Esmarch's method for anaerobic cultures, 119 of roll cultures, no Esophagus, incision of, 40 Exner's method of staining myelin- sheaths, 319 Exudations, 370 hemorrhagic, 370 purulent, 371 putrid, 371 serous, 370 Eye, examination of, 62 Fatty degeneration, 352 Feces, examination of 382 Fetus, measurements of, 66 weight of, 66 Fibrin, stains for, 355 Weigert's stain for, 355 Filaria sanguinis hominis, 294 Fining of test-tubes, 86 Filtering apparatus, bacterial, 72 Fiocca's method of staining spores, 100 Fixing reagents, 220 Flagella, staining of 100-105 Bunge's method, 103 Lbffler's method, 102 Pitfield's method, 103 Van Ermengem's method, 104 Flemming's solution, 224 Fluid in abdominal cavity, 29 Fluids, examination of, 218, 370 indifferent, 217 macerating, 218 Food for animals, 117 Forceps, 21 Forceps, Cornet cover-glass, 71 Formaldehyde, 226 Fractional sterilization, 88 Fracture of the skull, 52 Freezing microtome, 206 Fresh material, examination of, 216 tissues, sections of, 216 teased preparations of, 216 Freud's gold stain for nerve-fibers, 313 Fuchsin, 247 aniline-, 2^7 carbol-, 247 Gabbet's method of staining the bacillus tuberculosis, 92 methylene-blue solution, 246 Gall-bladder, dropsy of, 375 removal of, 45 Ganglion-cells, Lenhossek's method of staining, 308 Nissl's method of staining, 307 Gas in abdominal cavity, 29 in pleural cavity, 31 Gas stove, 71 Gastric contents, examination of, 380 Gastro-intestinal tract, 41 methods of fixing and exam- ining, 347 Gelatin, glucose, preparation of, 81 plain, preparation of 80 General rules in regard to autopsies, 21 Generation of hydrogen, 120 Gentian-violet, 247 aniline-, 247 carbol-, 248 Stirhng's, 248 Gerlach's method of staining nerve- fibers, 313 Giacomi's method of staining the bacillus of syphilis, 286 Glanders, bacillus of 164 Lbffler's method of staining in sections, 279 Noniewicz's method of staining in sections, 280 390 INDEX. Glanders, bacillus of, Schutz's method of staining in sec- tions, 280 Glands, axillary, 29 Glucose agar-agar, preparation of, 79 bouillon, preparation of, 176 gelatin, preparation of, 81 Glycerin ag^r-agar, preparation of, 80 Glycerin-albumin mixture, Mayer's, 25s Glycogen, 361 Gold, 260, 312 Freud's stain ■ for nerve-fibers with, 313 Gerlach's stain for nerve-fibers with, 313 Lowit's stain for nerve-fibers with, 261 Ranvier's stain for nerve-fibers with, 261 Golgi's method of staining the nervous system, 308 Golgi stains. Cox's modification of, 312 Kalhus' method of fixing, 311 Gonococcus, 130, 278 special culture-media for, 131 Gout, 368 Graduates, 21 Gram's method of staining, 91-93, 99, 227, 280, 281 Gram-Weigert stain, 281 Granulation-tissue, methods of fix- ing and examining, 345 Gunzburg's test for hydrochloric acid, 381 Hammer of soft iron, 2 1 of steel, 21 Hanging drop, method of exami- nation of 112 of preparation of, in, 112 Harke's operation for exposing the naso-pharynx, 64 Hatchet-chisel, 21 Head, holder for, 21 Heart, external inspection of, 32 Heart, incisions of, 33 measurements of, 36 removal of, 33 valves, water test of, 35 weight of, 36 Heidenhain-Biondi stain, 250 Heidenhain's hematoxyhn stain, 268 Heller's method of staining myehn- sheaths, 320 Hemalum, acid, Mayer's, 242 Mayer's, 242, 268 Hematein, Mayer's glycerin-alum, 242 Hematoidin, 365 Hematoxylin, aqueous, 241, 267 Delafield's, 241, 267 Ehrlich's acid, 242, 267 Heidenhain's, 268 phosphomolybdic acid, 243, 306 phosphotungstic acid, 243, 325 stains, 239 Weigert's alcohol, 243 Hemin test, 380 Hemocytometer, Thoma-Zeiss, 327 Hemoglobin, 365 test for, in blood, 332 Hemometer, Von Fleischl's, 329 Hemosiderin, 365 Hermann's solution, 225 Herxheimer's method of staining elastic fibers, 303 Histological methods, 204 Holder for head, 21 Hollow shde, in Hoyer's method of staining mucin, 357 Hyalin, stains for, 358 Hydrochloric acid, 217 test for, in gastric contents, 381 Hydrogen, bouillon cultures under, iig, 120 cultivation under, 119-121 generation of, 120 plate cultures under, 121 Hydronephrosis, 375 Hypodermic syringe, 71 Hypostasis of blood, 26 INDEX. 391 Illuminating apparatus. Abbe's, 205 Imbedding in celloidin, 231 in paraffin, 234 processes, 230 Indifferent fluids, 217 Influenza, bacillus of, 162 method of staining in sections, 279 Injection of bacteria into mesen- teric vein, 114, IIS Injection-masses, 219 Injections, 218 Inoculation of animals, 112-117 at autopsies, 96 of culture-tubes, 105, 106 of guinea-pigs, 112, 113 of mice, 115-117 of rabbits, 113-115 with the bacillus tuberculosis, 97 with the pneumococcus, 97 Instruments, autopsy, 18 for histological purposes, 215 Intestine, incision of, 43 methods of fixing and examin- ing, 347 Intraperitoneal inoculation, 113, 116 Intravenous inoculation, method of, 114 lodin, 253 Lugol's solution of, 254 tincture of, 253 Iron, reactions for, 367 Isolation of a bacterium in pure culture, method of, 107, 108 Kaiserling's method of preserv- ing the natural colors of mu- seum preparations, 351 Kallius' method of fixing Golgi's stains, 311 Keratohyalin, Unna's stain for, 361 Kidneys, 45 examination of, 47 measurements of, 47 methods of fixing and examin- ing, 346 removal of, 46 Kidneys, weight of, 47 Kipp's hydrogen generator, 120 Knife, amputating, 18 autopsy, 18 cartilage, 19 for freezing microtome, 208 for microtomes, 212 sharpening of microtome, 213 Kiihne's method of staining the tubercle bacillus, 284 methylene-blue solution, 246 Laboratory outfit, 204 Lactose-litmus agar-agar, 80 Langhans' method for obtaining mounts of iodin stains of amyloid, 363 Larynx, incision of, 40 Lavender, oil of, 257 Length of body, 26 Lenhossek's method of staining ganglion-cells, 308 Leprosy, bacillus of, 285 differentiation of, from bacillus tuberculosis, 93 Liborius, method of, 117, 118 Lithium carmine, 244, 269 Litmus-milk, preparation of, 85 Liver, 44 incision of, 44 measurements of, 45 methods of fixing and examin- ing, 347 removal of, 44 weight of, 45 Loffler's blood-serum mixture, 81 method of staining bacteria, 278 method of staining flagella, 102 method of staining the bacillus of glanders in sections, 279 methylene-blue solution, 246, 278 Lowit's gold method, 261 Lubinski's special culture-medium for the bacillus tuberculosis, 151 Luer's double rachiotome, 20 Lumbar puncture, 371 Lungs, incisions of, 38 392 INDEX. Lungs, methods of fixing and ex- amining, 346 removal of, 37 Lustgarten's method of staining the bacillus of syphilis, 286 Macerating fluids, 218 Malarial organisms, 218 method of examining blood for, 290 Malignant edema, bacillus of, 175 Mallory's stain for actinomyces in sections, 282 for amebffi coli, 293 for neuroglia-fibers, 322 by means of phospho- tungstic-acid hematox- ylin, 325 stain for elastic fibers, 302 phosphomolybdic-acid hematox- ylin, 243 phosphotungstic-acid hematox- ylin, 243 Mamma, incision of, 29 Marchi's fluid, 228 method for staining fatty degen- erated nerves, 326 Martinotti's picro-nigrosin, 250 Mastzellen, 298 Ehrlich's methods of staining, 298 Unna's methods of staining, 299, 300 Mayer's acid hemalum, 242 glycerin-albumin mixture, 255 glycerin-alum-hematein solution, 242 hemalum, 242, 268 muchematein, 243 Mechanical stage, 206 Mediastinum, anterior, 32 Melanin, 365 Mesenteric veins, inoculation into, 114, IIS Mesentery, removal of, 43 Metallic stains or impregnations, 258 Methemoglobin, 365 Method of diluting cultures, 95 Method of distributing material over surface of culture medium, 95 of fixing and examining special organs and tissues, 344 of preparing cultures on blood- serum, 94 of sterilizing surface of organs, 95 Methods, histological, 204 of studying bacteria in cultures, 98 Methylene-blue, 246 eosin and, 270 Gabbet's solution of, 246 Kiihne's solution of, 246 Loffler's solution of, 246 method of staining bacteria with, 278 Sahli's solution of, 247 stain for nerve-fibers, 314 Unna's alkaline solution of, 246, 270 polychrome solution of, 247 Methyl-violet, 248 Micrococcus lanceolatus. See Pneumococcus. pneumoniae crouposae. See Pneumococcus. of sputum septicemia. See Pneumococcus. Micrococcus tetragenus, 133 Microscopes, 204 Microtome, celloidin, 209 freezing, 206 knives, 212 knives, sharpening of, 213 paraffin, 210 Mitosis, 273 Moller's method of staining spores, 100 Motility of bacteria, 1 1 1 method of determining, 1 1 1 Mounting reagents, 258 Mouse-holder, 116 Muchematein, Mayer's, 243 Mucin, Hoyer's method of staining, 357 stains for, 356 INDEX. 393 Mucin, Unna's method of staining, 358 Miiller's fluid, 227 Museum preparations, 350 Kaiserling's method of pre- serving the natural colors of, 351 Myelin-sheaths, Exner's method of staining, 319 Heller's method of staining, 320 Marchi and Algeri's method of staining fatty degenerated, 326 Pal's modification of Weigert's stain for, 318 Robertson's method of stain- ing, 320 stains for, 316 Weigert's quick method of mordanting, 316 Weigert's stain for, 317 Myelotome, 18 Naso-pharynx, examination of, by Harke's operation, 64 Neck, organs of, 39 Necrosis, 354 Nerve-fibers, Bethe's method of fix- ing methylene-blue, stains of, 315 chlorid of iron and dinitro-resor- cin method, 314 degenerations of, 325 gold stains for, 312 Marchi and Algeri's method of staining fatty degenerated, 326 methylene-blue stain for, 314 Strobe's aniline-blue stain for, 313 Nervous system, general stains for, 305 Golgi's method of staining, 308 methods of staining, 303 stains for the ganglion-cells, 307 stains for the myelin-sheaths, 316 Nervous system, stains for the neu- roglia-fibers, 321 Neuroglia-fibers, Mallory's stains for, 322 Weigert's stain for, 323 Neutral carmine, 245, 272 New-born child, examination of, 65 weight of organs of, 67 Nigrosin, as a stain for the nervous system, 307 picro-, 250 Nissl's method of staining gan- glion-cells, 307 Nitric acid, 229 Noniewicz's method of staining the bacillus of glanders in sec- tions, 280 Nuclear stains, 265 Nutrition, general, 26 Oil of bergamot, 256 of cedar-wood, 257 of cloves, 257 of lavender, 257 of origanum, 256 of thyme, 257 Oil-immersion lens, 205 Oleum origani cretici, 256 Omentum, removal of, 42 Orcein, 253 Organs from which cultures are taken at autopsies, 96 Origanum oil, 256 Orth's discharging fluid, 254 fluid, 224 lithium carmine, 244 Osmic acid, 217, 261 stain for myelin-sheaths, 319 Ovarian cysts, 374 Ovaries, incision of, 48 weight of, 48 Pacchionian granulations, 53 Pal's modification of Weigert's myelin-sheath stain, 318 Pancreas, incision of, 44 removal of, 43 weight of, 44 Pancreatic cyst or fistula, 374 394 INDEX. Paraffin, 210 bath, 210 imbedding, 234 microtome, 210 sections, method of attaching to the shde, 236 Parhemoglobin, 365 Parovarian cysts, 374 Pathological products, 352 Pelvic organs, removal of, 47 Penis, incision of, 48 removal of, 49 Pepton solution, preparation of, 86 Pericardium, opening of, 32 Peritonitis, 181 Perosmic acid. See Osmic Acid. Petri dish, 108 oblong rectangular, 2 1 5 Petrifaction, 368 Phloroglucin and nitric acid, 229 -vanillin test for hydrochloric acid, 381 Phosphomolybdic-acid hematoxy- lin, 243, 306 Phosphotungstic-acid hematoxylin, 243, 302, 306 Pia, removal of, 56 Pianese's fixing solution, 225 staining solutions, 250 Picric acid, 230, 249, 272 Picro-nigrosin, 250 Pigmentation, 365 Pigments, autochthonous, 365 extraneous, 365 hematogenous, 365 Pitfield's method of stainingflagella, 103 Pitre's method of sectioning the brain, 59 Plasma cells, Unna's stains for, 300 Plate cultures, 108 Plate method of Petri, 108 Platinum needle for handling sec- tions, 216 Platinum wire, 72 Platinum-wire loop, 72 Pleural cavity, adhesions in, 32 gas in, 31 Pleural cavity, inspection of, 31 Pneumococcus, 128—130 animal inoculations with, 97 Curry's stain for capsule of, 93 Gram stain for capsule of, 94 Welch's stain for capsule of, 93 Poikilocytosis, 339 Portal vein, incision of, 41 Post-mortem discolorations, 26 examinations, 17 rigidity, 26 Potato cultures, preparation of, 85 Private autopsies, 24 Probes, 21 Protozoa, 287 Pseudo-mucin, 358 Pulmonary artery, embolism of, 35 Pure cultures, method of obtaining, 105 Quantity of bacteria for inocula- tion, 116, 117 Rabl's chromoformic-acid solu- tion, 225 Rachiotome, Luer's, 20 Ranvier's gold method, 261 one-third alcohol, 218 Record of autopsies, 22 Rectum, incision of, 48 Renal cysts, 375 Resorcin test for hydrochloric acid, 381 Restitution of the body, 67 Rhinoscleroma, bacillus of, 282 Ribbert, method of staining con- nective-tissue fibrillae, 301 Rigidity, post-mortem, 26 Robertson's method of staining myelin-sheaths, 320 Roll cultures, no Round-worms, 294 Rubber gloves, 23 Running water, 213 Sahli's borax methylene-blue solu- tion, 247 Safranin, 248 Babes' aniline, 248 INDEX. 39S Saw, 20 I Scales, 18 Scalpel, 19 Schiitz's method of staining the bacillus of glanders, 280 Schaffer's safranin stain for bone, 349 Schallibaum's solution, 255 Scissors, 19 Semilunar ganglia, 49 Serial sections by celloidin method, 237 by paraffin method, 239 Serum agar-agar, 13 J Serum reaction with the bacillus of typhoid fever, 144 Sharpening of microtome knives, 213 Sieve-dish, Steinach's, 215 Silver, nitrate of, 258 Skeleton, development of, 26 Skin, general condition of, 26 methods of fixing and examin- ing, 349 Skull, fracture of, 52 incision of, 51 of infant, opening of, 52 Slee's method of fastening the cal- varia, 68 Slides, 213 cleaning of, 214 Smegma bacillus, 286 differentiation of, from the bacillus tuberculosis, 92 Spatula, 215 Spinal cord, method of incising, 62 removal of, 60 Spine, removal of, 50 Spirillum of Asiatic cholera, 1 52 Spleen, incision of, 41 measurements of, 41 methods of fixing and examin- ing, 346 removal of, 41 weight of, 41 Sponges, 21 Spore-formation, study of, 112 Spores, staining of, 100 Spores, Abbott's method of stain- ing, 100 Fiocca's method of staining, 100 Holler's method of staining, 100 sterilization of, 8g Sporozoa, 294 Sputum, examination of, 376 for tubercle bacilli, 186 Staining in mass, 273 methods for sections, 262 for cover-glass preparations, 91 solutions, 239 Staining-dishes, 214 Starch-granules, reaction of, with iodin, 364 Staphylococcus cereus albus, 124 cereus flavus, 124 epidermidis albus, 124 pyogenes albus, 124 pyogenes aureus, 121, 181 pyogenes citreus, 124 Steinach's sieve-dish, 215 Stender dishes, 215 Sterilization, fractional, 88 of culture-media, 87 of gelatin tubes, 81 of organs, 95 time required for, 88 Sterilizer, hot-air, 70 steam, 70 Stieda's stain for iron and nuclei, 367 Stirling's solution of gentian-violet, 248 Stomach, incision of, 43 methods of fixing and examin- ing, 347 removal of, 43 Strobe's aniline-blue stain for nerve-fibers, 313 Streptococcus pyogenes, 124, 181 Subcutaneous inoculations, 112, 113, 115 of guinea-pigs, 112 of mice, 115 of rabbits, 1 1 3 Suggestions to beginners, 23 396 INDEX. Suppurative processes, i8l Suprarenal capsule. See Adrenal. Syphilis, bacillus of, 286 Syracuse solid watch-glasses, 215 Swab, 179 TjENIA echinococcus, 298 mediocanellata s. saginata, 298 solium, 296 Tape-worms, 296 bothriocephalus latus, 298 taenia echinococcus, 298 tania mediocanellata, 298 taenia solium, 296 Teased preparations of fresh tis- sues, 216 Test-tubes, 71 fining of, 86 new, 74 old, 74 preparation of, 74 Testes, incision of, 48 weight of, 48 Tetanus, bacillus of, 171 Thermo-regulator, 73 Thoma-Zeiss hemocytometer, 327 Thoracic duct, 49 Thorax, opening of, 30 Thyme, oil of 257 Thymus gland, 32 Thyroid gland, incision of, 40 Tin cups, 73 Tonsils, incision of, 40 Topfer's dimethyl-amido-azo-ben- zol test for hydrochloric acid, 381 Touton's method of staining gono- cocci in sections, 278 Trachea, incision of, 40 Transudations, 370 Trichinae, 295 Trichloracetic acid, 230 Tuberculosis, bacillus of, 148 diagnosis of, from animal in- oculations, 97 Ehrlich's method of staining, 284 Kiihne's method of staining, 286 Tuberculosis, bacillus of, method of staining in celloidin sec- tions, 285 Ziehl-Neelsen-Gabbet method of staining, 284 Twine, 21 Typhoid fever, bacillus of, 141 method of staining in sec- tions, 278 Unna's alkaline methylene-blue solution, 246, 270 orcein stain for elastic fibers, 302 polychrome methylene-blue solu- tion, 247 stain for connective-tissue fibril- Ise, 301 stain for hyalin and colloid, 360 stain for kerato-hyalin, 361 stain for mastzellen, 299, 300 stain for mucin, 358 Urine, examination of 382 Urine-serum agar-agar, 132, 133 Uric-acid salts in gout, 368 Uterine scrapings, examination of, 369 Uterus, incision of, 48 Vagina, incision of, 48 Valves of heart, water test for, 35 Van Ermengem's method of stain- ing flagella, 104 Van Gieson's stain, 249, 272 stain for central nervous sys- tem, 306 stain for connective-tissue fibril- lae, 300 Von Fleischl's hemometer, 329 Vena cava, incision of, 49 Virchow's method of sectioning the brain, 58 Vulcanized fiber, 211 Watch-glasses, Syracuse solid, 215 Water, running, 213 test of valves of heart, 35 Weigert's alcohol hematoxylin, 243 INDEX. 397 Weigert's differential stain for neu- roglia-fibers, 323 method for serial sections, 238 mixture of aniline and xylol, 258 mixture of carbolic acid and xylol, 257 modification of Gram's stain, 281 quick method of mordanting myelin-sheaths, 316 stain for fibrin, 355 stain for myelin-sheaths, 317 Welch's method of staining the capsule of the pneumococ- cus, 93 Wire baskets, 72 Xylol, 257 Xylol balsam, 258 Zenker's fluid, 223 Ziehl-Neelsen-Gabbet method of staining the bacillus tuber- culosis, 284 Ziehl-Neelsen's carbol-fuchsin, 247 Si I I I ^ •IS I PUBLISHED BY W. g. gaLindep^, Q2r Walnut ^beet, aDl^ilaslelpya. MR. SAUNDERS, In presenting to the profession the fol- lowing list of publications, begs to state that the aim has been to malectaior, London, England. THE PICTORIAL ATLAS OF SKIN DISEASES AND SYPH- ILITIC AFFECTIONS (American Edition). Translation from the French. Edited by J. J. Pringle, M. B., F. R. C. P., Assistant Physician to, and Physician to the department for Diseases of the Skin at, the Middle- sex Hospital, London. Photo-lithochromes from the famous models of der- matological and syphilitic cases in the Museum of the Saint-Louis Hospital, Paris, with explanatory wood-cuts and text. In 12 Parts, at I3.00 per Part. Parts I to 8 now ready. " The plates are beautifully executed." — ^Jonathan Hutchinson, M. D. (London Hospital). " I strongly recommend this Atlas. The plates are exceedingly well executed, and will be of great value to all studying dermatology." — Stephen Mackenzie, M. D. (London Hospital). " The plates in this Atlas are remarkably accurate and artistic reproductions of typical ex- amples of skin disease. The work will be of great value to the practitioner and student." — William Anderson, M. D. (St. Thomas Hospital). " If the succeeding parts of this Atlas are to be similar to Part I, now before us, we have no hesitation in cordially recommending it to the favorable notice of our readers as one of the finest dermatological atlases with which we are acquainted." — Glasgow Medical Journal, Aug., 1895. " Of all the atlases of skin diseases which have been published in recent years, the present one promises to be of greatest interest and v.ilue, especially from the standpoint of the general practi- tioner." — American Medico- Surgical Bulletin, Feb. 22, 1896. " The introduction of explanatory wood-cuts in the text is a novel and most important feature which greatly furthers the easier understanding of the excellent plates, than which nothing, we ven- ture to say, has been seen better in point of correctness, beauty, and general merit." — Nezv York Medical Journal, Feb. 15, 1896. " An interesting feature of the Atlas is the descriptive text, which is written for each picture by the physician who treated the case or at whose instigation the models have been made. We pre- dict for this truly beautiful work a large circulation in all parts of the medical world where the names St. Louis and Baretta have preceded it." — Medical Record, N. Y., Feb. I, 1896. CATALOGUE OF MEDICAL WORKS. 13 PRACTICAL POINTS IN NURSING. For Nurses in Private Practice. By Emily A. M. Stoney, Graduate of the Training-School for Nurses, Lawrence, Mass. ; Superintendent of the Training-School for Nurses, Carney Hospital, South Boston, Mass. 456 pages, handsomely illustrated with 73 engravings in the text, and 9 colored and half-tone plates. Cloth. Price, ^1.75 net. In this volume the author explains, in popular language and in the shortest possible form, the entire range of private nursing as distinguished from hospital nursing, and the nurse is instructed how best to meet the various emergencies of medical and surgical cases when distant from medical or surgical aid or when thrown on her own resources. An especially valuable feature of the work will be found in the directions to the nurse how to improvise everything ordinarily needed in the sick-room, where the embarrassment of the nurse, owing to the want of proper appliances, is fre- quently extreme. The work has been logically divided into the following sections : I. The Nurse : her responsibilities, qualifications, equipment, etc. II. The Sick-Room : its selection, preparation, and management. III. The Patient : duties of the nurse in medical, surgical, obstetric, and gyne- cologic cases. IV. Nursing in Accidents and Emergencies. V. Nursing in Special Medical Cases. VI. Nursing of the New-born and Sick Children. VII. Physiology and Descriptive Anatomy. The Appendix contains much information in compact form that will be found of great value to the nurse, including Rules for Feeding the Sick ; Recipes for Invalid Foods and Beverages ; Tables of Weights and Measures ; Table for Com- puting the Date of Labor ; List of Abbreviations ; Dose-List ; and a full and com- plete Glossary of Medical Terms and Nursing Treatment. " There are few books intended for non-professional readers which can be so cordially endorsed by a medical journal as can this one." — Therapeutic Gazette, Aug. 15, 1896. " This is a well-written, eminently practical volume, which covers the entire range of private nursing as distinguished from hospital nursing, and instructs the nurse how best to meet the various emergencies which may arise and how to prepare everything ordinarily needed in the illness of her patient." — -American Journal of Obstetrics and Diseases of Women and Children, Aug., 1896. " It is a work that the physician can place in the hands of his private nurses with the assurance of benefit." — Ohio Medical Journal, Aug., 1896. A TEXT- BOOK OF BACTERIOLOGY, including the Etiology and Prevention of Infective Diseases and an account of Yeasts and Moulds, Hsematozoa, and Psorosperms. By Edgar M. Crook- shank, M. B., Professor of Comparative Pathology and Bacteriology, King's College, London. A handsome octavo volume of 700 pages, illustrated with 273 engravings in the text, and 22 original and colored plates. Price, %(y.^o net. This book, though nominally a Fourth Edition of Professor Crookshank's "Manual of Bacteriology," is practically a new work, the old one having been reconstructed, greatly enlarged, revised throughout, and largely rewritten, forming a text-book for the Bacteriological Laboratory, for Medical Officers of Health, and for Veterinary Inspectors. 14 W. B. SAUNDERS' ILLUSTRATED DISEASES OF THE EYE. A Hand-Book of Ophthalmic Practice. By G. E. DE ScHWEiNiTZ, M. D., Professor of Ophthalmology in the Jeffer- son Medical College, Philadelphia, etc. A handsome royal-octavo volume of 679 pages, with 256 fine illustrations, many of which are original, and 2 chromo-lithographic plates. Prices: Cloth, ^4.00 net; Sheep or Half- Morocco, ^5.00 net. The object of this work is to present to the student, and to the practitioner who is beginning work in the fields of ophthal- mology, a plain description of the optical defects and diseases of the eye. To this end special attention has been paid to the clinical side of the question; and the method of examination, the symptoma- tology leading to a diagnosis, and the treatment of the various ocular defects have been brought into prominence. SECOND EDITION, REVISED AND GREATLY ENLARGED. ~ ~ ~ ' The entire book has been thoroughly specimen Illustration. revised. In addition to this general re- vision, special paragraphs on the following new matter have been introduced : Filamentous Keratitis, Blood-staining of the Cornea, Essential Phthisis Bulbi, Foreign Bodies in the Lens, Circinate Retinitis, Symmetrical Changes at the Macula Lutea in Infancy, Hyaline Bodies in the Papilla, Monocular Diplopia, Subconjunctival Injections of Germicides, Infiltra- tion-Anaesthesia, and Sterilization of Collyria. Brief mention of Ophthalmia Nodosa, Electric Ophthalmia, and Angioid Streaks in the Retina also finds place. An Appendix has been added, containing a full description of the method of deter- mining the corneal astigmatism with the ophthalmometer of Javal and Schiotz, and the rotations of the eyes with the tropometer of Stevens. The chapter on Operations has been enlarged and rewritten. "A clearly written, comprehensive manual. . . . One which we can commend to students as a reliable text-book, written with an evident knowledge of the wants of those entering upon the study of this special branch of medical science." — British Medical Journal. " The work is characterized by a lucidity of expression which leaves the reader in no doubt as to the meaning of the language employed. . . . We know of no work in which these diseases are dealt with more satisfactorily, and indications for treatment more clearly given, and in harmony with the practice of the most advanced ophthalmologists." — Maritime Medical News. " It is hardly too much to say that for the student and practitioner beginning the study of Ophthalmology, it is the best single volume at present published." — Medical Ne^vs. " The latest and one of the best books on Ophthalmology. The book is thoroughly up to date, and is certainly a work which not only commends itself to the student, but is a ready reference for the busy practitioner." — International Medical Magazine. FEEDING IN EARLY INFANCY. By Arthur V. Meigs, M. D. Bound in limp cloth, flush edges. Price, 25 cents net. Synopsis : Analyses of Milk^ — Importance of the Subject of Feeding in Early Infancy — Proportion of Casein and Sugar in Human Milk — Time to Begin Arti- ficial Feeding of Infants— Amount of Food to be Administered at Each Feeding — Intervals between Feedings— Increase in Amount of Food at Different Periods of Infant Development — Umuitableness of Condensed Milk as a Substitute for Moth- er's Milk — Objections to Sterilization or "Pasteurization" of Milk — Advances made in the Method of Artificial Feeding of Infants. CA TALOGUE OF MEDICAL WORKS. 15 A TEXT-BOOK OF HISTOLOGY, DESCRIPTIVE AND PRAC- TICAL. For the Use of Students. By Arthur Clarkson, M. B., C. M., Edin., formerly Demonstrator of Physiology in the Owen's College, Manchester; late Demonstrator of Physiology in the Yorkshire College, Leeds. Large 8vo, 554 pages, with 22 engravings in the text, and 174 beautifully colored original illustrations. Price, strongly bound in Cloth, ^6.00 net. The purpose of the writer in this work has beeri to furnish the student of His- tology, in one Volume, with both the descriptive and the practical part of the science. The first two chapters are devoted to the consideration of the general methods of Histology ; subsequently, in each chapter, the structure of the tissue or organ is first systematically described, the student is then taken tutorially over the specimens illustrating it, and, finally, an appendix affords a short note of the methods of preparation. TEXT-BOOK UPON THE PATHOGENIC BACTERIA. Spe- cially written for Students of Medicine. By Joseph McFarland, M. D., Professor of Pathology and Bacteriology in the Medico-Chirurgical College of Philadelphia, etc. 359 pages, finely illustrated. Cloth. Price, ^2.50 net. The book presents a concise account of the technical procedures ^lecessary in the study of Bacteriology. It describes the life-history of pathogenic bacteria, and the pathological lesions following invasions. The work is intended to be a text-book for the medical student and for the practitioner who has had no recent laboratory training in this department of med- ical science. The instructions given as to needed apparatus, cultures, stainings, microscopic examinations, etc. are ample for the student's needs, and will afford to the physician much information that will interest and profit him. " The author has succeeded admirably in presenting the essential details of bacteriological technics, together with a judiciously chosen summary of our present knowledge of pathogenic bac- teria. . . . The work, we think, should have a wide circulation among English-speaking students of medicine." — N. Y. Medical Journal, April 4>, 1896. A MANUAL OF PHYSIOLOGY, with Practical Exercises. For Students and Practitioners. By G. N. Stewart, M. A., M. D., D. Sc, lately Examiner in Physiology, University of Aberdeen, and of the New Museums, Cambridge University; Professor of Physiology in the Western Reserve University, Cleveland, Ohio. Handsome octavo volume of 800 pages, with 278 illustrations in the text, and 5 colored plates. Price, Cloth, $3.50 net. " It will make its way by sheer force of merit, and amply deserves to do so. It is one of the ■very best English text-books on the subject.^' — Lancet. " Of the many text-books of physiology published, we do not know of one that so nearly comes up to the ideal as does Prof. Stewart's volume." — British Medical Journal.' WATER AND WATER SUPPLIES. By John C. Thresh, D. Sc, M. B., D. P. H., Lecturer on Public Health, King's College, London; Editor of the "Journal of State Medicine," etc. i2mo, 438 pages, illus- trated. Handsomely bound in Cloth, with gold side and back stamps. Price, I2.25 net. i6 W. B. SAUNDERS' ILLUSTRATED ARCHIVES OF CLINICAL SKIAGRAPHY. By Sydney Rowland, B. A., Camb., late Scholar of Downing College, Cambridge, and Shuter Scholar of St. Bartholomew's Hospital, London ; Special Commissioner to "British Medical Journal" for the Investigation of the Applications of the New Photography to Medicine and Surgery. A series of collotype illustra- tions, with descriptive text, illustrating the applications of the New Photog- raphy to Medicine and Surgery. Price, per Part, ^i.oo. Parts I. to III. now ready. The object of this publication is to put on record in permanent form some of the most striking applications of the new photography to the needs of Medicine and Surgery. ' The progress of this new art has been so rapid that, although Prof. Rontgen's discovery is only a thing of yesterday, it has already taken its place among the approved and accepted aids to diagnosis. ESSENTIALS OF ANATOMY AND MANUAL OF PRACTICAL DISSECTION, containing "Hints on Dissection." By Charles B. Nancrede, M. D., Professor of Surgery and Clinical Surgery in the Uni- versity of Michigan, Ann Arbor; Corresponding Member of the Royal Academy of Medicine, Rome, Italy; late Surgeon Jefferson Medical Col- lege, #tc. Fourth and revised edition. Post 8vo, over 500 pages, with handsome full-page lithographic plates in colors, and over 200 illustrations. Price : Extra Cloth (or Oilcloth for the dissection-room), ^2.00 net. No pains nor expense has been spared to make this work the most exhaustive yet concise Student's Manual of Anatomy and Dissection ever published, either in America or in Europe. The colored plates are designed to aid the student in dissecting the muscles, arteries, veins, and nerves. The wood-cuts have all been specially drawn and engraved, and an Appendix added containing 60 illustrations representing the structure of the entire human skeleton, the whole being based on the eleventh edition of Gray's Anatomy. A MANUAL OF PRACTICE OF MEDICINE. By A. A. Stevens, A. M., M. D., Instructor of Physical Diagnosis in the University of Pennsyl- vania, and Demonstrator of Pathology in the Woman's Medical College of Philadelphia. Specially intended for students preparing for graduation and hospital examinations. Post 8vo, 512 pages. Illustrated. Price, ^2.50. FOURTH EDITION, REVISED AND ENLARGED. Contributions to the science of medicine have poured in so rapidly during the last quarter of a century that it is well-nigh impossible for the student, with the limited time at his disposal, to master elaborate treatises or to cull from them that knowledge which is absolutely essential. From an extended experience in teach- ing, the author has been enabled, by classification, to group allied symptoms, and by the elimination of theories and redundant explanations to bring within a com- paratively small compass a complete outline of the practice of medicine. TEMPERATURE CHART. Prepared by D. T. Laine, M. D. Size 8x 13^ inches. Price, per pad of 25 charts, 50 cents net. A conveniently arranged chart for recording Temperature, with columns for daily amounts of Urinary and Fecal Excretions, Food, Remarks, etc. On the back of each chart is given in full the method of Brand in the treatment of Typhoid Fever. CATALOGUE OF MEDICAL WORKS. i7 MANUAL OF MATERIA MEDICA AND THERAPEUTICS. By A. A. Stevens, A. M., M. D., Instructor of Physical Diagnosis in the Uni- versity of Pennsylvania, and Demonstrator of Pathology in the Woman's Medical College of Philadelphia. 445 pages. Price, Cloth, ^2.25. SECOND EDITION, REVISED. This wholly new volume, which is based on the last edition of the Pharma- copceia, comprehends the following sections : Physiological Action of Drugs ; Drugs ; Remedial Measures other than Drugs ; Applied Therapeutics ; Incom- patibility in Prescriptions ; Table of Doses ; Index of Drugs ; and Index of Dis- eases ; the treatment being elucidated by more than two hundred formulae. NOTES ON THE NEWER REMEDIES; their Therapeutic A ppli- cations and Modes of Administration. By David Cerna, M.D., Ph.D., Demonstrator of and Lecturer on Experimental Therapeutics in the Univer- sity of Pennsylvania. Post 8vo, 253 pages. Price, ;?i.2S. SECOND EDITION, RE-WRITTEN AND GREATLY ENLARGED. The work takes up in alphabetical order all the newer remedies, giving their physical properties, solubility, therapeutic applications, administration, and chem- ical formula. SAUNDERS' POCKET MEDICAL FORMULARY. By William M. Powell, M. D., Attending Physician to the Mercer House for Invalid Women at Atlantic City. Containing 1750 Formulae, selected from several hundred of the best-known authorities. Forming a handsome and convenient pocket companion of nearly 300 printed pages, with blank leaves for additions; with an Appendix containing Posological Table, Formulae and Doses for Hypodermic Medication, Poisons and their Antidotes, Diameters of the Female Pelvis and Foetal Head, Obstetrical Table, Diet List for Various Dis- eases, Materials and Drugs used in Antiseptic Surgery, Treatment of Asphyxia from Drowning, Surgical Remembrancer, Tables of Incompatibles, Eruptive Fevers, Weights and Measures, etc. Third edition, revised and greatly enlarged. Handsomely bound in morocco, with side index, wallet, and flap. Price, ^1.75 net. " This little book, that can be conveniently carried in the pocket, contains an immense amount of material. It is very useful, and as the name of. the author of each prescription is given is unusually reliable." — New York Medical Record. SAUNDERS' POCKET MEDICAL LEXICON; or. Dictionary of Terms and 'Words used in Medicine and Surgery. By John M. Keating, M. D., Editor of "Cyclopaedia of Diseases of Children," etc.; Author of the "New Pronouncing Dictionary of Medicine," and Henry Hamilton, Author of " A New Translation of Virgil's ^neid into English Verse;" Co- Author of a "New Pronouncing Dictionary of Medicine." A new and revised edition. 32mo, 282 pages. Prices: Cloth, 75 cents; Leather Tucks, ^i.oo. " Remarkably accurate in terminology, accentuation, and definition." — Journal of American Medical Association. W. B. SAUNDERS' ILLUSTRATED DISEASES OF WOMEN. By Henry J. Garrigues, A. M., M. D., Pro- fessor of Obstetrics in the New York Post- Graduate Medical School and Hos- pital ; Gynaecologist to St. Mark's Hospital, and to the German Dispensary, etc., New York City. One octavo volume of nearly 700 pages, illustrated by 300 wood-cuts and colored plates. Prices: Cloth, ^4.00 net; Sheep, I5.00 net. A PRACTICAL work on gynaecology for the use of students and practitioners, written in a terse and concise manner. The importance of a thorough knowledge of the anatomy of the female pelvic organs has been fully recognized by the author, and considerable space has been devoted to the subject. The chapters on Operations and on Treatment are thoroughly modern, and are based upon the large hospital and private practice of the author. The text is elucidated by a large number of illustrations and colored plates, many of them being original, and forming a complete atlas for studying embryology and the anatomy of the female genitalia, besides exemplifying, whenever needed, morbid conditions, instruments, apparatus, and operations. EXCERPT OF CONTENTS. Development of the Female Genitals. — Anatomy of the Female Pelvic Organs. — Physiology. — Puberty. — Menstruation and Ovulation. — Copulation. — P'ecundation. — The Climacteric. — Etiology in General. — Examinations in General. — Treatment in General. — Abnormal Menstruation and Me- trorrhagia. — Leucorrhea. — Diseases of the Vulva. — Diseases of the Perineum. — Diseases of the Vagina. — Diseases of the Uterus. — Diseases of the Fallopian Tubes. — Diseases of the Ovaries. — Diseases of the Pelvis. — Sterility. The reception accorded to this ■work has been most flattering. In the short period -which has elapsed since its issue, it has been adopted and recommended as a text-book by more than 60 of the Medical Schools and Universities of the United States and Canada. " One of the best text-books for students and practitioners which has been published in the English language ; it is condensed, clear, and comprehensive. The profound learning and great clinical experience of the distinguished author find expression in this book in a most attractive and instructive form. Young practitioners, to whom experienced consultants may not be available, will find in this book invaluable counsel and help." Thad. a. Reamy, M. D., LL.D., Professor of Clinical Gynecology, Medical College of Ohio ; Gynecologist to the Good ■ Samaritan and to the Cincinnati Hospitals. ESSENTIALS OF PHYSICAL DIAGNOSIS OF THE THO- RAX. By Arthur M. Corwin, A. M., M. D., Demonstrator of Physical Diagnosis in the Rush Medical College, Chicago ; Attending Physician to the Central Free Dispensary, Department of Rhinology, Laryngology, and Diseases of the Chest. 200 pages. Illustrated. Cloth, flexible covers. Price, ^1.25 net. This book was originally published for the use of students, but its rapid absorp- tion by the practitioner made it appear that a wider field had been reached. In this edition the author has added to his revision of the text a section setting forth the signs found in each of the diseases of the chest, thereby increasing its value to the general practitioner for post-graduate study. " It is excellent. The student who shall use it as his guide to the car.eful study of physical exploration upon normal and abnormal subjects can scarcely fail to acquire a good working know- ledge of tlie subject." — Philadelphia Polyclinic. CA TALOGUE OF MEDICAL WORKS. i9 SYLLABUS OF OBSTETRICAL LECTURES in the Medical Department, University of Pennsylvania. By Richard C. Norris, A. M., M. D., Demonstrator of Obstetrics in the University of Pennsylvania. Third edition, thoroughly revised and enlarged. Crown 8vo. Price, Cloth, interleaved for notes, ^2.00 net. "This work is so far superior to others on the same subject that we take pleasure in calling attention briefly to its excellent features. It covers the subject thoroughly, and will prove invaluable both to the student and the practitioner. The author has introduced a number of valuable hints which would only occur to one who was himself an experienced teacher of obstetrics. The subject-matter is clear, forcible/and modern. We are especially pleased with the portion devoted to the practical duties of the accoucheur, care of the child, etc. The paragraphs on antiseptics are admirable ; there is no doubtful tone in the directions given. No details are regarded as unimportant ; no minor matters omitted. We venture to say that even the old practitioner will find useful hints in this direction which he cannot afford to despise. ' ' — Medical Record. A SYLLABUS OF GYNECOLOGY, arranged in conformity with " An American Text-Book of Gynecology." By J. W. Long, M. D., Professor of Diseases of Women and Children, Medical College of Virginia, etc. Price, Cloth (interleaved), Ji.oo net. Based upon the teaching and methods laid down in the larger work, this will not only be useful as a supplementary volume, but to those who do not already possess the Text-Book it will also have an independent value as an aid to the prac- titioner in gynecological work, and to the student as a guide in the lecture-room, as the subject is presented in a manner systematic, succinct, and practical. A SYLLABUS OF LECTURES ON THE PRACTICE OF SUR- GERY, arranged in conformity with "An American Text-Book of Surgery." By Nicholas Senn, M. D., Ph. D., Professor of Surgery in Rush Medical College, Chicago, and in the Chicago Polyclinic. Price, ^2.00. This excellent work of its eminent author, himself one of the contributors to "An American Text-Book of Surgery," will prove of exceptional value to the advanced student who has adopted that work as his text-book. It is not only the syllabus of an unrivalled course of surgical practice, but it is also an epitome of, or supplement to the larger work. AN OPERATION BLANK, with Lists of Instiniments, etc. re- quired in Various Operations. Prepared by W. W. Keen, M. D., LL.D., Professor of Principles of Surgery in the Jefferson Medical College, Philadelphia. Price per pad, containing Blanks for fifty operations, 50 cents net. SECOND EDITION, REVISED FORM. A convenient blank (suitable for all operations), giving complete instructions regarding necessary preparation of patient, etc., with a full list of dressings and medicines to be employed. On the back of each blank is a list of instruments used — viz. general instruments, etc., required for all operations; and special in- struments for surgery of the brain and spine, mouth and throat, abdomen, rectum, male and female genito-urinary organs, the bones, etc. The whole forming a neat pad, arranged for hanging on the wall of a surgeon's office or in the hospital operating-room. JV. B. SAUNDERS' ILLUSTRATED LABORATORY EXERCISES IN BOTANY. By Edson S. Bastin, M. A., Professor of Materia Medica and Botany in the Philadelphia Col- lege of Pharmacy. Octavo volume of 536 pages, with 87 plates. Price, Cloth, ^2.50. This work is intended for the beginner and the advanced student, and it fully covers the structure of flowering plants, roots, ordinary stems, rhizomes, tubers, bulbs, leaves, flowers, fruits, and seeds. Particular attention is given to the gross and microscopical structure of plants, and to those used in medicine. The illus- trations fully elucidate the text, and the complete index facilitates reference. Trailing Arbutus (Epigea repens). Specimen Illustration. LABORATORY GUIDE FOR THE BACTERIOLOGIST. By Langdon Frothingham, M. D. V., Assistant in Bacteriology and Veterinary Science, Sheffield Scientific School, Yale University. Illustrated. Price, Cloth, 75 cents. The technical methods involved in bacteria-culture, methods of staining, and microscopical study are fully described and arranged as simply and concisely as possible. The book is especially intended for use in laboratory work. OBSTETRIC ACCIDENTS, EMERGENCIES, AND OPERA- TIONS. By L. Ch. Boisliniere, M. D., late Emeritus Professor of Ob- stetrics in the St. Louis Medical College. 381 pages, handsomely illustrated. Price, ^2.00 net. " For the use of the practitioner who, when away from home, has not the opportunity of consulting a library or of calling a friend in consultation. He then, being thrown upon his own resources, will find this book of benefit in guiding and assisting him in emergencies." CA TALOGUE OF MEDICAL WORKS. HOW TO EXAMINE FOR LIFE INSURANCE. By John M. Keating, M. D., Fellow of the College of Physicians and Surgeons of Phila- delphia; Vice-President of the American Psediatric Society; Ex-President of the Association of Life Insurance Medical Directors. Royal 8vo, 211 pages, with two large half-tone illustrations, and a plate prepared by Dr. McClellan from special dissections ; also, numerous cuts to elucidate the text. Price, in Cloth, ^2.00 net. " This is by far the most useful book which has yet appeared on insurance examination, a sub- ject of growing interest and importance. Not the least valuable portion of the volume is Part II., which consists of instructions issued to their examining physicians by twenty-four representative companies of this country. As the proofs of these instructions were corrected by the directors of the companies, they form the latest instructions obtainable. If for these alone the book should be at the right hand of every physician interested in this special branch of medical science." — The Medical News, Philadelphia. THE CARE OF THE BABY. By J. P. Crozer Griffith, M. D., Clftii- cal Professor of Diseases of Children, University of Pennsylvania ; Physician to the Children's Hospital, Philadelphia, etc. 392 pages, with 67 illustrations in the text, and 5 plates. i2mo. Price, ^1.50. A reliable guide not only for mothers, but also for medical students and prac- titioners whose opportunities for observing children have been limited. " The whole book is characterized by rare good sense, and is evidently written by a master hand. It can be read with benefit not only by mothers, but by medical students and by any prac- titioners who have not had large opportunities for observing children." — American yournal of Obstetrics, July, 1895. " The best book for the use of the young mother with which we are acquainted. . . . There are very few general practitioners who could not read the book through with advantage." — Archives of Pediatrics, Aug., 1895. " No better book of its kind has come under our notice for some time. Although intended primarily for mothers and nurses, it will well repay perusal by medical students." — Birmingham Medical Review, Oct., 1895. " This is one of the best works of its kind that has been presented to the people for many a day." — Maryland Medical yournal, Aug. 13, 1895. NURSING: ITS PRINCIPLES AND PRACTICE. By Isabel Adams Hampton, Graduate of the New York Training School for Nurses attached to Bellevue Hospital ; Superintendent of Nurses, and Principal of the Training School for Nurses, Johns Hopkins Hospital, Baltimore, Md. ; late Superin- tendent of Nurses, Illinois Training School for Nurses, Chicago, 111. In one very handsome i2mo volume of 484 pages, profusely illustrated. Price, Cloth, ;g2.oo net. This original work on the important subject of nursing is at once compre- hensive and systematic. It is written in a clear, accurate, and readable style, suit- able alike to the student and the lay reader. Such a work has long been a deside- ratum with those intrusted with the management of hospitals and the instruction of nurses in training-schools. It is also of especial value to the graduate nurse who desires to acquire a practical working knowledge of the care of the sick and the hygiene of the sick-room. 22 IV. B. SAUNDERS' ILLUSTRATED CATALOGUE. NURSE'S DICTIONARY of Medical Terms and Nursing Treat- ment, containing Definitions of the Principal Medical and Nursing Terms and Abbreviations ; of the Instruments, Drugs, Diseases, Accidents, Treat- ments, Physiological Names, Operations, Foods, Appliances, etc. encountered in the ward or in the sick-room. Compiled for the use of nurses. By HoNNOR Morten, Author of "How to Become a Nurse," "Sketches of Hospital Life," etc. i6mo, 140 pages. Price, Cloth, ^i.oo. This little volume is intended merely as a small reference-book which can be ' onsulted at the bedside or in the ward. It gives sufficient explanation to the i.urse to enable her to comprehend a case until she has leisure to look up larger and fuller works on the subject. DIET IN SICKNESS AND IN HEALTH. By Mrs. Ernest Hart, formerly Student of the Faculty of Medicine of Paris and of the London School of Medicine for Women ; with an Introduction by Sir Henry Thompson, F. R. C. S., M. D., London. 220 pages; illustrated. Price, Cloth, $1.50. Useful to those who have to nurse, feed, and prescribe for the sick. ... In each case the accepted causation of the disease and the reasons for the special diet prescribed are briefly described. Medical men will find the dietaries and recipes practically useful, and likely to save them trouble in directing the dietetic treatment of patients. " We recommend it cordially to the attention of all practitioners ; . . . . both to them and to their patients'it may be of the greatest service." — Medical Journal, New York. DIETS FOR INFANTS AND CHILDREN IN HEALTH AND IN DISEASE. By Louis Starr, M. D., Editor of "An American Text- Book of the Diseases of Children." 230 blanks (pocket-book size), per- forated and neatly bound in flexible morocco. Price, $1.25 net. The first series of blanks are prepared for the first seven months of infant life ; each blank indicates the ingredients, but not the quantities, of the food, the latter directions being left for the physician. After the seventh month, modifications being less necessary, the diet lists are printed in full. Formula for the prepara- tion of diluents and foods are appended. DIET LISTS AND SICK-ROOM DIETARY. By Jerome B. Thomas, M. D., Visiting Physician to the Home for Friendless Women and Children and to the Newsboys' Home ; Assistant Visiting Physician to the Kings County Hospital ; Assistant Bacteriologist, Brooklyn Health Department. Price, ^1.50. Send for sample sheet. There is here offered, in portable form, as an efficient aid to the better practice of Therapeutics, a collection of detachable Diet Lists and a Sick-room Dietary. It meets a want, for the busy practitioner has but little time to write out Systems of Diet appropriate to his patients, or to describe the preparation of their food. Compiled from the most modern works on dietetics, the Dietary offers a variety of easily-digested foods. "A convenience that will be appreciated by the physician.'' — Medical yournal. New York. " The work is an excellent one, and ought to be welcomed by physician, patient, and nurse alike." — Indian Lancet, Calcutta. Practical, Exhaustive. Authoritative. SAUNDERS' NEW AID SERIES OF MANUALS. FOR STUDENTS AND PRACTITIONERS. Mr. Saunders is pleased to announce the successful issue of several volumes of his NEW AID SERIES OF MANUALS, which have received the most flattering commendations from Students and Practitioners and the Press. As publisher of the Standard Series of Question Compends, and through intimate relations with leading members of the medical profession, Mr. Saunders has been enabled to study progressively the essential desiderata in practical "self-helps " for students and physicians. This study has manifested that, while the published "Question Compends" earn the highest appreciation of students, whom they serve in reviewing their studies preparatory to examination, there is special need of thoroughly reliable handbooks on the leading branches of Medicine and Surgery, each subject being compactly and authoritatively written, and exhaustive in detail, without the intro- duction of cases and foreign subject-matter which so largely expand ordinary text- books. The Saunders Aid Series will not merely be condensations from present literature, but will be ably written by well-known authors and practitioners, most of them being teachers in representative American Colleges. This new series, therefore, will form an admirable col- lection of advanced lectures, which will be invaluable aids to students in reading and in comprehending the contents of " recommended " works. Each Manual will further be distinguished by the beauty of the new type ; by the quality of the paper and printing ; by the copious use of illustrations ; by the attractive binding in cloth; and by the extremely low price at which they will be sold. 23 Saunders' New Aid Series of Manuals. VOLUMES PUBLISHED. PHYSIOLOGY, by Joseph Howard Baymond, A. M., M. D., Professor of Physi- ology and Hygiene and Lecturer on Gynecology in the Long Island College Hos- pital ; Director of Physiology in the Hoagland Laboratory ; formerly Lecturer on Physiology and Hygiene in the Brooklyn Normal School for Physical Education ; Ex- Vice- President of the American Public Health Association ; Ex-Health Commis- sioner, City of Brooklyn, etc Illustrated. $1.25 net. SURGERY, General and Operative, by John Chalmers DaCosta, M.D., Demon- strator of Surgery, Jefferson Medical College, Philadelphia; Chief Assistant Sur- geon, Jefferson Medical College Hospital ; Surgical Begistrar, Philadelphia Hospital, etc. 188 illustrations and 13 plates. (Double number.) $2.50 net. DOSE-BOOK AND MANUAL OF PRESCRIPTION-WRITING, by E. Q. Thornton, M. D., Demonstrator of Therapeutics, Jefferson Medical CoUege, Phila- delphia. Illustrated. Price, cloth, 11.25 net. SURGICAL ASEPSIS, by Gael Beck, M. D., Surgeon to St. Mark's Hospital and to the New York German Poliklinik, etc. Illustrated. Price, cloth, $1.25 net. MEDICAL JURISPRUDENCE, by Henry C. Chapman, M. D., Professor of Insti- tutes of Medicine and Medical Jurisprudence in the Jefferson Medical College of Philadelphia; Member of the College of Physicians of Philadelphia, of the Acade- my of Natural Sciences, of the American Philosophical Society, and of the Zoologi- cal Society of Philadelphia. Illustrated. JSi-So net. SYPHILIS AND THE VENEREAL DISEASES, by James Nevins Hyde, M.D., Professor of Skin and Venereal Diseases, and Frank H. Montgomery, M. D., Lecturer on Dermatology and Genito-Urinary Diseases, in Bush Medical College, Chicago. Profusely Illustrated. (Double number.) $2.50 net. PRACTICE OF MEDICINE, by George Bob Lockwood, M. D., Professor of Practice in the Woman's Medical College of the New York Infirmary ; Instructor of Physical Diagnosis of the Medical Department of Columbia College ; Attending Physician to the Colored Hospital ; Pathologist to the French Hospital ; Member of the New York Academy of Medicine, of the Pathological Society, of the Clinical Society, etc. Illustrated. (Double number.) $2.50 net. MANUAL OF ANATOMY, by Irving S. Haynes, M. D., Adjunct Professor of Anatomy and Demonstrator of Anatomy, Medical Department of the New York University, etc. Beautifully Illustrated. (Double number.) Price, ^2.50 net. MANUAL OF OBSTETRICS, by W. A. Newman Dorland, M. D., Asst. Demon- strator of Obstetrics, University of Pennsylvania ; Chief of Gynecological Dispen- sary, Pennsylvania Hospital ; Member of Philadelphia Obstetrical Society, etc. Profusely illustrated. (Double number.) Price, $2.50 net. DISEASES OF WOMEN, by J. Bland Sutton, F. E. C. S., Asst. Surgeon to Mid- dlesex Hospital, and Surgeon to Chelsea Hospital, London ; and Arthur E. Gile.s, M. D., B. So. Lond., F. R. C. S. Edin., Asst. Surgeon to Chelsea Hospital, London. 436 pages, handsomely illustrated. (Double number.) Price, $2.50 net. VOLUMES IN PREPARATION. NOSE AND THROAT, by D. Beaden Kyle, M.D., Chief Laryngologist of the St. Agnes Hospital, Philadelphia; Bacteriologist of the Orthopsedic Hospital and Infirmary for Nervous Diseases ; Instructor in Clinical Microscopy and Assistant Demonstrator of Pathology in the Jefferson Medical College, etc. NERVOUS DISEASES, by Charles W. Burr, M. D., Qinical Professor of Nervous Diseases, Medico-Chirurgical College, Philadelphia ; Pathologist to the Orthopsedic Hospital and Infirmary for Nervous Diseases ; Visiting Physician to the St. Joseph Hospital, etc. *:i,* There will be published in the same series, at close intervals, carefully-prepared works on various subjects, by prominent specialists. 24 SAUNDERS' QUESTION COMPENDS. Arranged in Question and Answer Form. THE LATEST, CHEAPEST, AND BEST ILLUSTRATED SERIES OF OOMPENDS EVER ISSUED. Now the Standard Authorities in Medical Literature students and Practitioners in every City of the United States and Canada. THE REASON A7VHY They are the advance guard of "Student's Helps"— that do help; they are the leaders in their special line, well and authoritatively written by able men, who, as teachers in the large col- leges, know exactly what is wanted by a student preparing for his examinations. The judgment exercised in the selection of authors is fully demonstrated by their professional elevation. Chosen from the ranks of Demonstrators, Quiz-masters, and Assistants, most of them have become Pro- fessors and Lecturers in their respective colleges. Each book is of convenient size (5x7 inches), containing on an avei'age 250 pages, profusely ■ illustrated, and elegantly printed in clear, readable type, on fine paper. The entire series, numbering twenty-three volumes, has been kept thoroughly revised and enlarged when necessary, many of them being in their fourth and fifth editions. TO SUM UP. Although there are numerous other Quizzes, Manuals, Aids, etc. in the market, none of them approach the " Blue Series of Question Compends ;" and the claim is made for the following points of excellence : 1 . Professional distinction and reputation of authors. 2. Conciseness, clearness, and soundness of treatment. 3. Size of type and quality of paper and binding. *^ Any of these Compends will be mailed on receipt of price (see over for List). 25 26 W. B. SAUNDERS' ILLUSTRATED Saunders' Question-Compend Series. J8®° Price, Cloth, $1.00 per copy, except when otherwise noted. 1. ESSENTIALS OF PHYSIOLOGY. 3d edition. Illustrated. Revised and enlarged. By H. A. Hare, M. D. (Price, ;^i.oo net.) 2. ESSENTIALS OF SURGERY. Sth edition, with an Appendix on Antiseptic Surgery. 90 illustrations. By Edward Martin, M. D. 3. ESSENTIALS OF ANATOMY. 5th edition, with an Appendix. l8o illustrations. By Charles B. Nancrede, M. D. 4. ESSENTIALS OF MEDICAL CHEMISTRY, ORGANIC AND INORGANIC. 4th edition, revised, with an Appendix. By Lawrence Wolff, M. D. 5. ESSENTIALS OF OBSTETRICS. 3d edition, revised and enlarged. 75 illustrations. By W. Easterly Ashton, M. D. 6. ESSENTIALS OF PATHOLOGY AND MORBID ANATOMY. 6th thousand. 46 illustrations. By C. E. Armand Semple, M. D. 7. ESSENTIALS OF MATERIA MEDICA, THERAPEUTICS, AND PRE- SCRIPTION-WRITING. 4th edition. By Henry Morris, M. D. 8. 9. ESSENTIALS OF PRACTICE OF MEDICINE. By Henry Morris, M. D. An Appendix on Urine Examination. Illustrated. By Lawrence Wolff, M. D. 3d edition, enlarged by some 300 Essential Forniulse, selected from eminent authorities, by Wm. M. Powell, M. D. (Double number, price ;J2,oo.) 10. ESSENTIALS OF GYN.ffiCOLOGY. 3d edition, revised. With 62 illustrations. By Edwin B. Cragin, M. D. 11. ESSENTIALS OF DISEASES OF THE SKIN. 3d edition, revised and enlarged. 71 letter-press cuts and 15 half-tone illustrations. By Henry W. Stelwagon, M. D. (Price, ^l.oo net.) 12. ESSENTIALS OF MINOR SURGERY, BANDAGING, AND VENEREAL DISEASES. 2d edition, revised and enlarged. 78 illustrations. By Edward Martin', M. D. 13. ESSENTIALS OF LEGAL MEDICINE, I'OXICOLOGY, AND HYGIENE. 130 illustrations. By C. E. Armand Semple, M. D. 14. ESSENTIALS OF DISEASES OF THE EYE, NOSE, AND THROAT. 124 illustrations. 2d edition, revised. By Edward Jackson, M. D., and E. Baldwin Gleason, M. D. 15. ESSENTIALS OF DISEASES OF CHILDREN. 2d edition. By William M. Powell, M. D. 16. ESSENTIALS OF EXAMINATION OF URINE. Colored " Vogel Scale," and numerous illustrations. By Lawrence Wolff, M. D. (Price, 75 cents.) 17. ESSENTIALS OF DIAGNOSIS. By S. Solis-Cohen, M. D., and A. A. Eshner, M. D. 55 illustrations, some in colors. (Price, ^1.50 net.) 18. ESSENTIALS OF PRACTICE OF PHARMACY. By L. E. Sayre. 2d edition, revised and enlarged. 20. ESSENTIALS OF BACTERIOLOGY. 3d edition. 82 illustrations. By M. V. Ball, M. D. 21. ESSENTIALS OF NERVOUS DISEASES AND INSANITY. 48 illustrations. 2d edition, revised. By John C. Shaw, M. D. 22. ESSENTIALS OF MEDICAL PHYSICS. 155 illustrations. 2d edition, revised. By Fred J. Brockway, M. D. (Price, |!i.oo net.) 23. ESSENTIALS OF MEDICAL ELECTRICITY. 65 illustrations. By David D. Stewart, M. D., and Edward S. Lawrance, M. D. 24. ESSENTIALS OF DISEASES OF THE EAR. By E. B. Gleason, M. D. 89 illustrations. CATALOGUE OF MEDICAL WORKS. 27 JUST PUBLISHED. A TEXT-BOOK OF MATERIA MEDICA, THERAPEUTICS, AND PHARMACOLOGY. By George F. Butler, Ph. G., M. D., Professor of Materia Medica and of Clinical Medicine in the College of Physicians and Surgeons, Chicago ; Professor of Materia Medica and Thera- peutics, Northwestern University, Woman's Medical School, etc. 8vo, 858 pages. Illustrated. Prices; Cloth, ^4.00 net; Sheepor Half- Morocco, J5. 00 net A clear, concise, and practical text-book, adapted for permanent reference no less than for the requirements of the class-room. The arrangement (embodying the synthetic classification of drugs based upon therapeutic affinities) is believed to be at once the most philosophical and rational, as well as that best calculated to engage the interest of those to whom the academic study of the subject is wont to offer no little perplexity. Special attention has been given to the Pharmaceutical section, which is exceptionally lucid and complete. LECTURES ON RENAL AND URINARY DISEASES. By Robert Saundby, M. D. Edin., Fellow of the Royal College of Physicians, London, and of the Royal Medico- Chirurgical Society; Physician to the General Hospital; Consulting Physician to the Eye Hospital and to the Hospital for Diseases of Women ; Professor of Medicine in Mason College, Birmingham, etc. 8vo, 434 pages, with numerous illustrations and 4 colored plates. Price, Cloth, J 2. 50 net. In these Lectures, which are a re-issue in one volume of the author's well- known works on Bright' s Disease and Diabetes, there is given, within a modest compass, a review of the present state of knowledge of these important affections, with such additions and suggestions as have resulted from the author's thirteen years' clinical and pathological study of the subjects. The lectures have been carefully revised and much new matter added to them. There has also been added a section dealing with "Miscellaneous Affections of the Kidney," making the book more complete as a work of reference. ELEMENTARY BANDAGING AND SURGICAL DRESSING, with Directions concerning the Immediate Treatment of Cases of Emergency. For the use of Dressers and Nurses. By Walter Pye, F. R. C. S., late Surgeon to St. Mary's Hospital, London. Small i2mo, with over 80 illus- trations. Cloth, flexible covers. Price, 75 cents net. This little book is chiefly a condensation of those portions of Pye's " Surgical Handicraft ' ' which deal with bandaging, splinting, etc. , and of those which treat of the management in the first instance of cases of emergency. Within its own limits, however, the book is complete, and it is hoped that it will prove extremely useful to students when they begin their work in the wards and casualty rooms, and useful also to surgical nurses and dressers. " The directions are clear and the illustrations are good." — London Lancet. " The author writes well, the diagrams are clear, and the book itself is small and portable, although the paper and type are good." — British. Medical Journal. " One of the most useful little works for dressers and nurses. The author truly says that it is ' r very little book,' but it is large in usefulness." — Chemist and Druggist. JUST ISSUED. SOLD BY SUBSCRIPTIO^. ANOMALIES AND CURIOSITIES OF MEDICINE. BY GEORGE M. GOULD, M. D., AND WALTER L. PYLE, M. D. Several years of exhaustive research have been spent by the authors in the great medical libraries of the United States and Europe in col- lecting the material for this work. Medical literature of all ages and all languages has been carefully searched, as a glance at the Bibliographic Index will show. The facts, which will be of extreme value to the author and lecturer, have been arranged and anno- tated, and full reference footnotes given, indicating whence they have been obtained. In view of the persistent and dominant interest in the anomalous and curious, a thorough and systematic collection of this kind (the first of which the authors have knowledge) must have its own peculiar sphere of usefulness. As a complete and authoritative Book of Reference it will be of value not only to members of the medical profession, but to all persons interested in general scientific, sociologic, and medico-legal topics ; in fact, the general interest of the subject and the dearth of any complete work upon it make this volume one of the most important literary innovations of the day. An especially valuable feature of the book consists of the Indexing. Besides a complete and comprehensive General Index, containing numerous cross-references to the subjects discussed, and the names of the authors of the more important reports, there is a convenient Bibliographic Index and a Table of Contents. The plan has been adopted of printing the topical headings in bold-face type, the reader being thereby enabled to tell at a glance the subject-matter of any particular paragraph or page. Illustrations have been freely employed throughout the work, there being 165 relief cuts and 130 half-tones in the text, and 12 colored and half-tone full-page plates — a total of over 320 separate figures. The careful rendering of the text and references, the wealth of illus- trations, the mechanical skill represented in the typography, the print- ing, and the binding, combine to make this book one of the most attractive medical publications ever issued. Handsome Imperial Octavo Volume of 96S Pages. PRICES: Cloth, $6. GO net; Half Morocco, $7. GO net. JUST ISSUED. PENROSE'S DISEASES OF WOMEN. A Text=Book of Diseases of Women. By Charles B. Penrose, M. D., Ph.D., Pro- fessor of Gynecology, University of Pennsylvania; Surgeon to the Gynecean Hospital, Phil- adelphia. Octavo volume of 529 pages, handsomely illustrated. Price, $3.^0 net. MALLORY AND WRIGHT'S PATHOLOGICAL TECHNIQUE. Pathological Technique. By Frank B. Mallory, A. M., M. D., Asst. Professor of Pathology, Harvard University Medical School; and James H.Wright, A.M., M.D., Instructor in Pathology, Harvard University Medical School. Octavo volume of 396 pages, handsomely illustrated. SENN'S GENITO=URINARY TUBERCULOSIS. Tuberculosis of the Qenito= Urinary Organs, Male and Female. By Nicholas Senn, M. D., Ph.D., LL.D., Professor of the Practice of Surgery and of Clinical Surgery, Rush Medical College, Chicago. Handsome octavo volume of 320 pages. Illustrated. SUTTON AND GILES' DISEASES OF WOMEN. Diseases of Women. By J. Bland Sutton, F. R. C. S., Asst. Surgeon to Middlesex Hospital, and Surgeon to Chelsea Hospital, London; and Arthur E. Giles, M. D., B. Sc. Lond., F. R. C. S. Edin., Asst. Surgeon to Chelsea Hospital, London. 436 pages, hand- somely illustrated. Price, ji!2.5o net. « IN PREPARATION. ANDERS' PRACTICE OF MEDICINE. A Text=Book of the Practice of Medicine. By James M. Anders, M. D., Ph.D., LL.D., Professor of the Practice of Medicine and of Clinical Medicine, Medico-Chirurgical College, Philadelphia. In press. AN AMERICAN TEXT=BOOK OF GENITO=URINARY AND SKIN DISEASES. Edited by L. Bolton Bangs, M. D., Late Professor of Genito-Urinary and Venereal Dis- eases, New York Post-Giaduate Medical School and Hospital, and William A. Hard- away, M. D., Professor of Diseases of the Skin, Missouri Medical College. AN AMERICAN TEXT=BOOK OF DISEASES OF THE EYE, EAR, NOSE, AND THROAT. Edited by G. E. de Schweinitz, M. D., Professor of Ophthalmology in the Jefferson Medical College, and B. Alexander Randall, M. D., Professor of Diseases of the Ear ■ in the University of Pennsylvania and in the Philadelphia Polyclinic. MACDONALD'S SURGICAL DIAGNOSIS AND TREATMENT. Surgical Diagnosis and Treatment. By J. W. Macdonald, M. D., Graduate of Medicine of the University of Edinburgh; Licentiate of the Royal College of Surgeons, Edinburgh; Professor of the Practice of Surgery and of Clinical Surgery, Minneapolis College of Physicians and Surgeons. HIRST'S OBSTETRICS. A Text=Book of Obstetrics. By Barton Cooke Hirst, M. D., Professor of Obstet- rics, University of Pennsylvania. MOORE'S ORTHOPEDIC SURGERY. A Manual of Orthopedic Surgery. By James E. Moore, M. D., Professor of Orthopedics and Adjunct Professor of Clinical Surgery, University of Minnesota, College of Medicine and Surgery. HEISLER'S EMBRYOLOGY. A Text=Book of Embryology. By John C. Heisler, M. D., Prosector to the Pro- fessor of Anatomy, Medical Department of the University of Pennsylvania. NOW READY— VOLUMES FOR 1896 AND 1897. SAUNDERS' American Year-Book of Medicine and Surgery COLLECTED AND ARRANGED BY EMINENT AMERICAN SPECIALISTS AND TEACHERS, . UNDER THE EDITORIAL CHARGE OF GEORGE M. GOULD, M. D. Notwithstanding the rapid multiplication of medical and surgical works, still these publications fail to meet fully the requirements of the general physician, inasmuch as he feels the need of something more than mere text-books of well- known principles of medical science. Mr. Saunders has long been impressed with this fact, which is confirmed by the unanimity of expression from the profession at large, as indicated by advices from his large corps of canvassers. This deficiency would best be met by current journalistic literature, but most practitioners have scant access to this almost unlimited source of informa- tion, and the busy practiser has but little time to search out in periodicals the many interesting cases, whose study would doubtless be of inestimable value in his practice. Therefore, a work which places before the physician in convenient form an epitomieation of this literature by persons competent to pronounce upon The Value of a Discovery or of a Method of Treatment cannot but command his highest appreciation. It is this critical and judicial ftinction that will be assumed by the Editorial stafi" of the " American Year-Book of Medicine and Surgery." It is the special purpose of the Editor, whose experience peculiarly qualifies him for the preparation of this work, not only to review the contributions to American journals, but also the methods and discoveries reported in the leading medical journals of Europe, thus enlarging the survey and making the work characteristically international. These reviews will not simply be a series of uhdigested abstracts indiscriminately run together, nor will they be retro- spective of "news" one or two years old, but the treatment presented will be synthetic and dogmatic, and will include only what is new. Moreover, through expert condensation by experienced writers, these discussions will be Comprised in a Single Volume of about 1200 Pages. The jvork will be replete with original and selected illustrations skilfully reproduced, for the most part, in Mr. Saunders' own studios established for the purpose, thus ensuring accuracy in delineation, afibrding efficient aids to a right comprehension of the text, and adding to the attractiveness of the volume. Prices : Clotli, $6.50 net ; Half Morocco, $7.50 net. W. B. SAUNDERS, Publisher, 925 "Walnut Street, Philadelphia*