THE LIBRARY OF THE UNIVERSITY OF CALIFORNIA PRESENTED BY PROF. CHARLES A. KOFOID AND MRS. PRUDENCE W. KOFOID LEWIS'S PRACTICAL SERIES. MEDICAL MICROSCOPY LEWIS'S PRACTICAL SERIES. MEDICAL MICROSCOPY. A GUIDE TO THE USE OF THE MICROSCOPE IN MEDICAL PRACTICE. By FRANK 7. WETHERED, M.D., M.R.C.P., Medical Registrar to the Middlesex Hospital and Demonstrator of Practical Medicine to the Middlesex Hospital Medical School. With Illustrations, crown 8vo. [Now ready. MEDICAL ELECTRICITY. A PRACTICAL HANDBOOK FOR STUDENTS AND PRACTITIONERS. By W. E. STEA VENSON, M.D., and H. LEWIS JONES, M.A., M.D., M.R.C.P., Medical Officer in Charge of the Electrical Department in St. Bartholomew's Hospital. With Illustrations, crown 8vo, gs. [Now ready. HYGIENE AND PUBLIC HEALTH, By LOUIS C. PARKES, M.D., D.P.H. (Land. Univ.), Assistant Professor of Hygiene and Public Health at University College. Third Edition, with Illustra- tions, crown 8vo. [Nearly ready. A PRACTICAL TEXTBOOK OF THE DISEASES OF WOMEN. By ARTHUR H. N. LEWERS, M.D., M.R.C.P., Obstetric Physician to the London Hospital. Third Edition, with Illustrations, crown 8vo, los. 6d. ANESTHETICS, THEIR USES AND ADMINISTRATION. By DUDLEY W. BUXTON. M.D., B.S., Administrator of Anesthetics in University College Hospital. Second Edition, with Illustrations, crown 8vo, 55. [Now ready. MANUAL OF OPHTHALMIC PRACTICE. By C. HIGGENS, F.R.C.S., Ophthalmic Surgeon to Guy's Hospttal. With Illustrations, crown 8vo, 6s. TREA TMENT OF DISEA SE IN CHILDREN. By A NGEL MONE Y, M.D., F.R.C.P., late Assistant Physician to the Hospital for Sick Children, Great Ormond Street. Second Edition, with Plates, crown 8vo, los. 6d. ON FEVERS, THEIR HISTORY, ETIOLOGY, DIAGNOSIS, PROG- NOSIS, AND TREATMENT. By ALEXANDER COLLIE, M.D. (Aberd.), M.R.C.P. (Lond.) With Coloured Plates, crown 8vo,8s.6d. HANDBOOK OF DISEASES OF THE EAR. By URBAN PRITCHARD, M.D. (Edin.), F.R.C.S. (Eng.), Professor of Aural Surgery at King's College; Aural Surgeon to King's College Hospital. Second Edition, Illustrated, crown 8vo, 55. A PRACTICAL TREATISE ON DISEASES OF THE KIDNEYS AND URINARY DERANGEMENTS. By C. H. RALFE, M.A., M.D., F.R.C.P., Assistant Physician to the London Hospital. With Illustrations, crown 8vo, IDS. 6d. DENTAL SURGERY FOR GENERAL PRACTITIONERS AND STUDENTS OF MEDICINE. By ASHLEY W. BARRETT M.D., M.R.C.S., L.D.S., Dental Surgeon to the London Hospital. Second Edition, Illustrated, crown 8vo, 35. 6d. BODILY DEFORMITIES AND THEIR TREATMENT. A Hand- book of Practical Orthopedics. By H. A. REEVES, F.R.C.S. (Edin.), Senior Assistant Surgeon to the London Hospital. With 228 Illustrations, crown 8vo, 8s. 6d. LONDON: H. K. LEWIS, 136 GOWER STREET, W.C. J- V L- s Fig. 1. TUBERCLE BACILLI IN THE SPUTUM Stained by Neelsen-Ziehl method (from Jaksch). Fig. 2. LEPTOTHRIX BUCCALIS. Stained with iodine solution (from Jaksch) ^W MEDICAL MICEOSCOPY A GUIDE TO THE USE OF THE MICROSCOPE IN MEDICAL PRACTICE BY FRANK J. WETHERED, M.D. (LOND.) MEMBER OF THE ROYAL COLLEGE OF PHYSICIANS; MEDICAL REGISTRAR TO THE MIDDLESEX HOSPITAL AND DEMONSTRATOR OF PRACTICAL MEDICINE TO THE MIDDLESEX HOSPITAL MEDICAL SCHOOL; LATE ASSISTANT PHYSICIAN TO THE CITY OF LONDON HOSPITAL FOR DISEASES OF THE CHEST, VICTORIA PARK WITH ILLUSTRATIONS LONDON H. K. LEWIS, 136 GOWER STREET, W.C. 1892 PRINTED BY H. K. LEWIS, 136 GOWER STREET, LONDON, W.C. TO KICHAllD DOUGLAS POWELL, M.D., F.B.O.P. PHYSICIAN EXTRAORDINARY TO HER MAJESTY THE QUEEN; CONSULTING PHYSICIAN TO THE HOSPITAL FOR CONSUMPTION AND DISEASES OF THE CHEST, BROMPTON ; PHYSICIAN TO THE MIDDLESEX HOSPITAL ; WITH THE SINCERE RESPECT AND GRATITUDE OF THE AUTHOR. P REFACE. WITHIN recent years the microscope has gradually been brought more and more into use as an aid to diagnosis. In certain classes of diseases, notably those of the chest and urinary organs, it has become almost a routine practice, in hospitals at any rate, to examine the sputa and deposits, and the use of the microscope is rapidly being extended to private practice. In this work an effort has been made to lay before practitioners and students the most simple methods of preparing microscopic sections, and of the examination of urinary deposits, sputa, blood, &c. At the same time, for those who wish to extend their studies further, more elaborate methods have been detailed, and chap- ters added on the examination of food and bacteriological methods. It would have been beyond the scope of the book to have given descriptions of all the pathological changes found in the different organs, but a chapter on " Tumours " has been inserted, as for purposes of diagnosis, such examinations are frequently made in the operating theatre or post-mortem room. Many of the illustrations are original, but I have also to thank Profs. Ziegler, von Jaksch and Crookshank, Vlll PREFACE. Drs. Troup, Parkes and Crocker, for permission to use many of their figures. I have also to thank Prof. Stirling, Drs. Vincent Harris and Sims Woodhead, for allowing me to copy many formulae from their works, and especially Dr. Wynter for consenting to my making use of the material collected for our joint work on " Clinical and Practical Pathology." Also my thanks are due to Mr. Mansell Moullin, Mr. Eve, Dr. A. Garrod, and Dr. Herman, for their kind advice and help, also to Mr. Lewis for his aid in the matter of illustrations, and to the various firms who have provided cliches of apparatus. Queen Anne Street, July, 1892. CONTENTS. CHAPTER I. THE MICROSCOPE AND ACCESSORIES. PAGE Choice of an Instrument Compound Microscope Requisites of a good microscope : stage, diaphragm Qualities of a good objective : defining power, flatness of field, penetrating power, angle of aperture, resolving power Nose-piece Oculars Camera-lucida Makers of microscopes Use of the microscope Abbe's Condenser Microscope lamp Drawing objects Micrometer Accessory apparatus Cath- cart's microtome Swift's microtome Williams' freezing microtome Reichert's microtome Rocking microtome Valentin's knife Glass slides Cover-glasses Normal saline solution Iodine in iodide of potassium (Gram's solu- tion) i CHAPTER II. HARDENING AND DECALCIFYING. General remarks Choice of hardening fluid Absolute alcohol-- Methylated spirit Miiller's fluid Bichromate of potash- Chromic acid Chromic acid and spirit Ammonium chro- mate Bichromate of ammonia Corrosive sublimate Platinum chloride Osmic acid Picric acid Decalcifying fluids Nitric acid Perenzi's solution Hydrochloric acid Von Ebner's solution Picric acid , . . . 34 X CONTENTS. CHAPTER III. EMBEDDING. PAGE General remarks Freezing media Gum Dextrin Simple em- bedding Infiltration methods Embedding in celloidin : and in paraffin 47 CHAPTER IV. CUTTING SECTIONS. General remarks Cutting with Cathcart's microtome Cutting with Swift's microtome Use of Williams' ice freezing microtome Cutting sections embedded in celloidin Cut- ting with Reichert's microtome Collodionisation of sec- tions Cutting sections in paraffin Cutting in series Cut- ting with rocking microtome 55 CHAPTER V. STAINING. General Remarks Choice of a stain Staining with haema- toxylin and eosin Staining with carmine and picric acid Lithium carmine Acidulated alcohol Picro-carmine Alum-carmine Ammonium carmine Borax carmine Staining in bulk Safranin Iodine green General stains : Bismarck brown, rubin, orseille Multiple staining Treble staining 68 CHAPTER VI. SELECTIVE STAINS. General remarks Metallic stains : nitrate of silver, chloride of gold, osmic acid Staining micro-organisms : methylene CONTENTS. XI PAGE blue, Loffler's method, Kiihne's method, Mast-zellen, Gram's method, Weigert's modification of Gram's method, Neelsen-Ziehl method Methods of staining actinomyces: amyloid reaction, karyokinetic figures 86 CHAPTER VII. CLEARING AND MOUNTING. Fluid media : glycerine, glycerine jelly, glycerine mounting fluid, Farrants' solution, iodine mounting fluid Cementing materials Mounting in solidifying media Clearing agents: Canada balsam, dammar varnish Breaking down old speci- mens 105 CHAPTER VIII. COMPLETE PROCESSES FOR THE PREPARATION OF SECTIONS. First method Second method 117 CHAPTER IX. METHODS OF PREPARING SECTIONS OF THE CENTRAL NERVOUS SYSTEM. Hardening Cutting Staining: Weigert's method, Pal's method, Pal-Exner method, aniline blue-black, Jolgi's sublimate method 120 CHAPTER X. INJECTION OF TISSUES. Cold injection Carmine gelatine injection mass . . . 130 Xll CONTENTS. CHAPTER XL EXAMINATION OF FRESH TISSUES. PAGE Normal saline solution Serous fluid Iodised serum Teasing Softening Landois's fluid 134 CHAPTER XII. PREPARATION OF INDIVIDUAL TISSUES AND ORGANS. Epithelium Areolar tissue White fibrous tissue Elastic tissue Adenoid tissue Adipose tissue Gelatinous or embryonal tissue Cartilage Bone Teeth Muscle Nervous sys- tem Circulating system Glandular tissue The eye In- ternal ear Digestive system Lungs Thyroid gland Liver, spleen, &c. Genito-urinary system .... 139 CHAPTER XIII. THE EXAMINATION OF TUMOURS. Fibromata Myxomata Enchondromata Osteomata Myo- fibromata Sarcomata Papillomata Adenomata Epithe- liomata Carcinomata 148 CHAPTER XIV. EXAMINATION OF URINARY DEPOSITS. Method of examination Organised deposits : epithelium, red blood corpuscles, leucocytes, mucus, fat, casts, spermatozoa, fragments of morbid growth Pathogenic micro-organisms : tubercle bacilli, erysipelas Septic micro-organisms Sar- CONTENTS. Xlll PAGE cinas, bilharzia haematobia, filaria sanguinis hominis, eu- strongylus gigas, echinococci Non-pathogenic micro-or- ganisms Unorganised deposits Sediments occurring in acid urine : uric acid, amorphous urates, urate of soda, oxalate of lime, sulphate of lime, cystin, xanthin, tyrosin, leucin, hippuric acid, haematoidin and bilirubin, soaps of lime and magnesia Sediments occurring in alkaline urine : triple phosphates, neutral phosphate of lime, basic mag- nesium phosphate, amorphous phosphate of lime, carbonate of lime, urate of ammonia, cholesterin, indigo Gravel and calculi Extraneous matters Preservation of urinary de- posits 162 CHAPTER XV. THE EXAMINATION OF THE F/ECES. Method of examination Macroscopic examination Foreign bodies Mucus and fibrinous shreds Portions of bowel separated from intussusceptions Intestinal concretions Gall-stones Microscopical examination : food matters, crystals, red blood corpuscles, leucocytes, epithelium Vege- table parasites : bacillus of tubercle, bacillus of typhoid fever, bacillus of cholera, bacillus of cholera nostras Non- pathogenic organisms Animal parasites : amoeba coli, in- fusoria Taenia mediocanellata Taenia solium Taenia nana Bothriocephalus latus Distoma hepaticum Dis- toma lanceolatum Distoma sineuse Ascaris lumbri- coides Ascaris mistax Oxyuris vermicularis Tricho- cephalus dispar Anchylostoma duodenale Strongylus gigas Anguillula intestinalis Trichina spiralis . . . 215 XIV CONTENTS. . CHAPTER XVI. EXAMINATION OF SPUTUM. PAGE Method of examination : quantity, consistence, colour, odour, bronchial casts Microscopical examination: leucocytes and mucus corpuscles, compound granule cells, red blood cells, epithelium, elastic tissue, Curschmann's spirals, ton- sillar casts, corpora amylacea, portions of membrane, crys- tals, Charcot-Leyden crystals, concretions, foreign bodies, accidental ingredients 247 CHAPTER XVII. THE MICRO-ORGANISMS OF SPUTUM. Tubercle bacilli : Neelsen-Ziehl method, Ehrlich's method, Gabbet's method, Gibbes' method Pneumococci Acti- nomyces Micrococcus tetragonus Septic micro-organ- isms Fungi Infusoria Entozoa 281 CHAPTER XVIII. EXAMINATION OF VOMIT. Method of examination Food matters Epithelium Red blood cells Leucocytes Sarcinse Yeasts Micro-organisms . 295 CHAPTER XIX. EXAMINATION OF DISCHARGES AND CONTENTS OF CAVITIES. Serous exudations Hasmorrhagic exudations Chylous exuda- tions Sero-purulent exudations Putrid exudations Puru- CONTENTS. XV PAGE lent exudations : micro-organisms Tubercle bacilli Acti- nomyces Bacillus of leprosy Bacillus of anthrax Bacillus of syphilis Gonococcus Bacillus of glanders Trichina spiralis Cysticercus cellulosae Hydatid cysts Ovarian cysts Dermoid cysts Hydro-nephrosis Sperma- tocele 299 CHAPTER XX. DISCHARGES FROM THE GENITAL ORGANS. Discharges from the male genital organs: seminal fluid, sper- matic stains Discharges from the female genital organs : vaginal secretion, Trichomonas vaginalis Membranes : mucus, fibrinous clots, vaginal casts, membranous dys- menorrhoea, membranes of early abortion .... 313 CHAPTER XXI. EXAMINATION OF THE BLOOD. Red corpuscles White corpuscles Blood plates Counting the corpuscles: Gowers' haemocytometer, Thorn a-Zeiss haemo- cytometer Estimation of the amount of hasmoglobin in the blood : Gowers' haemoglobinometer, v. Fleischl's haemo- meter Olygocythaemia Polycythaemia Leucocythaemia Leucocytosis Microcythaemia Poikilocytosis Melanae- mia Vegetable psrasites : bacillus of anthrax, tubercle, typhoid fever, glanders and tetanus, spirillum of relapsing fever, haematozoa of malaria Animal parasites : Filaria sanguinis hominis, Bilharzia haematobia Examination of blood stains 322 XVI CONTENTS. CHAPTER XXII. CUTANEOUS PARASITES. PAGE Vegetable parasites: Trichophyton tonsurans, Microsporon fur- fur, Achorion Schonleinii Animal parasites: pediculi, Sar- coptes hominis, Acarus folliculorum 35 CHAPTER XXIII. EXAMINATION OF FOOD AND WATER. Wheat Barley Rye Oatmeal Maize Rice Beans Peas- Sago Tapioca Arrowroot Potato Turmeric Bread Flour Tea Coffee Cocoa Examination of drinking water . . . . 358 CHAPTER XXIV. BACTERIOLOGICAL METHODS. General considerations Apparatus required : steam-steriliser, hot-air steriliser, hot-water filter, incubator, accessories Cultivating media Solid media : potato cultivations, nu- trient gelatine, nutrient agar-agar, glycerine agar-agar, sterilised blood serum Liquid media: bouillon, liquid blood serum, Pasteur's fluid, Cohn's fluid Mode of using the solid nutrient media : test-tube cultivations, plate culti- vations Mode of using the liquid media: drop cultures Examination of micro-organisms : cover-glass preparations, cover-glass impressions Experiments on animals . . 373 LIST OF ILLUSTRATIONS. 1. Diagram of optical principle of the microscope ... 2 2. Baker's new student's microscope 5 3. Beck's pathological microscope 7 4. Swift's bacteriological microscope ...... 9 5. Nose-piece 10 6. Bausch and Lamb's universal microscope .... 13 7. Abbe chromatic condenser 16 8. Microscopic lamp 18 9. Prismatic camera-lucida 20 10. Abbe's camera-lucida . . . . . . . .20 11. Eye-piece micrometer 21 12. Frazer's modification of Cathcart's microtome ... 24 13. Clamp of Cathcart's microtome 25 14. Williams' ether microtome (" Swift's microtome ") . . 26 15. Williams' ice and salt microtome 27 16. Reichert's microtome 29 17. Rocking microtome 30 1 8. Valentin's knife * . fyi 19. Diagram for embedding box 49 20. Cells from a periosteal myxoma of the thigh . . . 150 21. Section from an osteoid chondroma of the humerus . . 151 22. Section through the margin of a sarcoma affecting the in- termuscular connective tissue 152 23. Section from a giant-celled sarcoma showing also spindle- shaped cells 154 XV111 LIST OF ILLUSTRATIONS. FIG. PAGE 24. Fibro-adenoma of mamma ; 157 25. Section from an epithelioma of skin 159 26. Carcinoma of the mamma .160 27. Vaginal epithelium . . . . . . .165 28. Epithelium from the bladder, ureter, and pelvis of the kidney .... 166 29. Renal epithelium, healthy and fatty 167 30. Blood corpuscles in urine 168 31. Pus cells in urine affected by acetic acid and unaltered . 169 32. Urinary casts . . . 179 33. Spermatozoa . . .. v . . . . 184 34. Uric acid . . . * 195 35. Amorphous urates . . . . . . . 197 36. Urate of soda 198 37. Oxalate of lime . . . ... . . . 199 38. Cystin 202 39. Tyrosin 203 40. Leucin ......... . 205 41. Triple phosphates . . 207 42. Stellar phosphates 208 43. Cholesterin 210 44. Head of Taenia mediocanellata 236 45. Head of Tasnia solium . . . . . . 238 46. Ova of intestinal parasites . 242 47. Trichocephalus dispar . 245 48. Bronchial stolon 256 49. Various cells in sputum ....... 261 50. Elastic tissue 265 51. Elastic tissue partially encrusted with lime salts . . . 268 52. Curschmann's spirals 269 53. Cast of a tonsillar crypt 271 54. Charcot-Leyden crystals 275 55. Silk fibres 278 LIST OF ILLUSTRATIONS. XIX FIG. 56. Cotton fibres . . . ,. . . . .278 57. Linen fibres .......... 279 58. Wool fibres . . ........ 279 59. Bacterium pneumonia crouposa ...... 289 60. Oi'dium albicans ......... 293 61. Vomit ....... .... 296 62. Trichina spiralis in muscle ....... 308 63. Head of cysticercus cellulosae ...... 308 64. Hydatids .......... 309 65. Wall of hydatid cyst ........ 310 66. Normal semen ......... 315 67. Trichomonas vaginalis ....... 318 68. Gowers' haemocytometer ....... 325 69. Pipette (Thoma-Zeiss haemocytometer) . . . . 327 70. Counting slide (Thoma-Zeiss apparatus) .... 328 71. Cross-section of Thoma-Zeiss apparatus .... 328 72. Counting scale (Thoma-Zeiss apparatus) .... 329 73. Gowers' haemoglobinometer ...... 332 74. Von Fleischl's hsemometer ....... 333 75. Poikilocytosis ......... 340 76. Filaria sanguinis hominis ....... 346 77. Bilharzia haematobia ........ 347 78. Trichophyton tonsurans ....... 351 79. Achorion SchSnleinii . . . . . . . . 352 80. Wheat-starch grains ........ 358 81. Barley-starch grains ........ 359 82. Oatmeal-starch grains ....... 360 83. Maize-starch grains ........ 361 84. Rice-starch grains . ....... 361 85. Bean-starch grains ........ 362 86. Pea-starch grains ........ 362 87. Sago-starch grains ........ 363 88. Tapioca-starch grains ....... 363 XX LIST OF ILLUSTRATIONS. FIG. PAGE 89. Arrowroot-starch grains 364 go. Potato-starch grains 364 91. Acarus farinae ......... 366 92. Coffee, cells of testa and cellular structure . . . 368 93. Chicory, dotted ducts and cellular structure . . . 369 94. Cocoa-starch cells 369 95. Koch's steam steriliser 375 96. Hot-water filtering apparatus with ring burner . . . 376 97. Babes' incubator 378 98. Platinum needles ; straight, hooked, looped . . . 379 99. Method of inoculating a test-tube containing sterile nutrient jelly . . . ... . ..... .387 100. Method of inoculating test-tubes in the preparation of plate cultivations 390 101. Damp chamber for plate cultivations .... 391 MEDICAL MICROSCOPY. CHAPTER I. THE MICROSCOPE AND ACCESSORIES. THE first question that naturally arises when preparing to study the use of the microscope in medicine is as to the choice of an instrument. The number of dif- ferent forms of microscopes now in the market is legion, and it is an exceedingly difficult matter to make a selection of any particular one. In order to do so, an intimate knowledge of its structure and mode of working is necessary, and it is very advisable that an inex- perienced person, instead of choosing for himself, should seek the advice of a more expert friend to help him in his search, for nothing is more annoying, after expend- ing a large sum on a large showy stand, than to find that for practical scientific work it is almost useless. Before stating in detail the requisites of a good microscope, it will be well to give a short description of the instrument as sold by all modern makers. Microscopes are distinguished as simple or com- pound. In the former, the rays which enter the eye of the observer come from an object brought near to it after refraction through either a simple lens, or a com- bination of lenses, acting as a simple lens, its action as a " magnifier " depending on its enabling the eye to B 4 MEDICAL MICROSCOPY. diaphragms, and by means of screws can be moved laterally, or upwards and downwards. A second "condenser" is usually supplied for pur- poses of looking at opaque objects by reflected light ; this, however, is seldom, if ever, required for medical work, all the objects being transparent and examined by transmitted light obtained from below. The column is connected with the base by means of a stiff hinge, so that the tube can be fixed either in a per- pendicular or horizontal position, or at any angle. What then are the requisites of a good microscope ? For useful practical work, it is desirable not to have too large an instrument. Especially should a binocular be avoided, it is cumbersome, the number of screws around the stage becomes bewildering, and no advan- tage is to be gained by it for medical work over a well made mon-ocular, with a simple sub-stage condenser. Steadiness is essential, this is secured by a wide base ; in nearly all microscopes the pedestal is either of the horse-shoe, or some modification of the tripod pat- tern ; it should be heavy enough to allow of both adjust- ments being turned, and of the object being freely moved on the stage without shaking the instrument. The column must of course be perfectly rigid, and this property is secured by the use of good stout metal. Both adjustments should turn easily and regularly, especially the fine one, for if this be at all stiff, or if the thread be in the least irregular, great annoyance will be experienced. The stage is usually three inches long by two and a half wide. It is not advisable to have a " movable " stage. By means of the fingers the glass slide can be moved more readily, and by practice quite as smoothly as by any mechanical contrivance, whilst extra screws and levers are apt to be in the way. THE MICROSCOPE AND ACCESSORIES. 5 There should be plenty of room between the mirror and the stage, so as to allow the insertion of a con- denser, if sufficient space be not allowed the full effect FIG. 2. Baker's new student's microscope. of this contrivance is not experienced. It is most cer- tainly desirable to have a condenser; the search for micro-organisms has now become almost a matter of D MEDICAL MICROSCOPY. routine in certain cases of doubtful diagnosis, and for their sure detection a special illumination is absolutely necessary. The diaphragm is made of several different patterns. The most ordinary form consists of a "wheel" of dia- phragms of various sizes, and should be examined to see whether the apertures are concentric with the axis of the instrument. Another form consists of small blocks in which the openings are pierced, all fitting into a common receptacle, which is inserted below the stage. The most important feature is that the diaphragm must be placed immediately beneath the object, in order to secure the best results. There are several more complicated forms such as the "iris," but for a description of them the reader is re- ferred to larger treatises on microscopy. Although the choice of a " stand " is of no mean im- portance, the chief point is of course the selection of the objectives. For ordinary histological work, an ' inch ' lens and a ' quarter-inch' is sufficient, but if it be intended to under- take thb examination of sputum, &c., a twelfth-oil- immersion is necessary, as the micro-organisms cannot be seen with any certainty without this power. According to the Continental systems, the first named lenses are represented by Nos. 3 and 7 of Hartnach, or A and D of Zeiss. It should be borne in mind that the term " one-inch objective," &c., indicates only that such a lens should possess the same magnifying power as a single lens of one-inch focus, and that it does not refer to the distance which the front of the lens focusses from the object, this varying greatly with the different makers. The qualities of a good objective are: de- THE MICROSCOPE AND ACCESSORIES. 7 fining power, flatness of field, penetrating power, and resolving power. Defining power. Expressed in more exact Ian- FIG. 3. Beck's pathological microscope. guage this means that the objective has been pro- perly corrected for spherical and chromatic aberra- tion. 8 MEDICAL MICROSCOPY. " Spherical aberration " causes the lines of the object to appear blurred and foggy, instead of being distinctly defined and clearly separated from one an- other. If the glass be not free from chromatic aberration, coloured fringes appear around certain spots, blue, if the lens be under-corrected, red, if over- corrected. " Defining power " may be tested by exam- ining a stage-micrometer and observing if each division can be clearly distinguished. Flatness of field. If an object be moved about from one portion of the field to another, it ought to appear equally well-defined in all parts. Thus, taking the stage-micrometer again as a test, the lines should be sharp and clear, and perfectly parallel to one another in every part of the field. Penetrating power. By this term is signified the power of an objective to view several planes of an object without altering the focus. A section of liver is a good test for judging this properly. The penetrating power of a lens is dependent on its " angle of aperture." This is the angle made by two lines drawn from opposite sides of the aperture of the object-glass with the principle focus of lens. If the system of lenses has a "low angle," its pene- trating power is greater than one which possesses a " high angle " of aperture. In connection with this subject it may be mentioned that low-power objectives with a high-angle and conse- quent shorter working distance will define much better than those with smaller apertures. Resolving power. This again depends entirely upon the aperture, supposing the glass to be perfect in other respects ; for good effects the angle ought to be large. Some authorities object to the term " resolving THE MICROSCOPE AND ACCESSORIES, FIG. 4. Swift's bacteriological microscope. 10 MEDICAL MICROSCOPY. power" as being synonymous with " defining power." An objective, however, may have perfect defining power and yet by reason of its want of aperture, it will be un- able to distinguish certain minutiae. It defines all that it can take up, but cannot define what is not imaged by it. In choosing an objective, it is well to select one with an universal screw, so that it can be used with any stand, otherwise an " adapter" will become necessary. A " nose-piece " (fig. 5) carrying two or three ob- jectives is a useful adjunct, saving a great deal of time FIG. 5. Nose-piece. when working with more than one power. This little piece of apparatus should be tested to ensure of there being sufficient space for the oil-immersion to revolve between the tube and the stage, and some minute object should be selected by means of the low power and arranged so that it occupies the centre of the field ; the high power should then be turned round, and lowered on to the glass, when this particular object should still THE MICROSCOPE AND ACCESSORIES. II be in the centre of the field ; if this be not the case, the nose-piece is not correctly " centred " and should be rejected. Not more than two oculars, a high and low, will be required ; their function, as already stated, is to magnify the image formed by the objective ; but enlargement is better obtained by drawing out the tube, or by substi- tuting a stronger lens, than by using a high eye-piece, which is trying to the eyes. A Camera-lucida is also of service for drawing the outline of specimens ; one need not be bought, however, for it is easy to manufacture one by bending a small piece of tin-foil round the upper end of the tube, and arranging it in such a fashion as to support a cover- glass at a proper angle to the eye-piece ; the angle is readily ascertained by experiment, and this simple contrivance has the advantage over the more compli- cated ones, in that the glass being so thin, only one image of the pencil instead of two is seen, and it is consequently much easier to use. This is the principal of Beale's " neutral tint reflector." It would be invidious and beyond the function of this book to recommend any particular maker from whom a microscope should be bought. There are many excel- lent dealers both in London and the Provinces. It is presumed that those who make use of these directions will not be desirous of possessing a large and expensive instrument. Very good " student's microscopes " are now made by many of ths London firms, which can be highly recommended. They are fitted with all the apparatus necessary for simple bacteriological work, and the cost is from 16 to 20. Any of the following makers may be fully trusted to supply good instruments, and any omissions that occur 12 MEDICAL MICROSCOPY. do not imply that therefore other firms are not equally as reliable : C. Baker, 243 and 244 High Holborn. Beck, 68 Cornhill. Crouch, 66 Barbican. Pillischer, 88 New Bond Street. Powell and Lealand, 170 Euston Road. Swift, 8 1 Tottenham Court Road. Parkes, 5 St. Mary's Row, Birmingham. Ross, 164 New Bond Street. In America very similar instruments, possessing everything that can be desired, and excellent both in manufacture and in the qualities of the lenses, may be obtained from Joseph Zentmayer, 209 South Eleventh Street, Philadelphia, and Bausch and Lamb, Optical Company, 48 and 50 Maiden Lane, New York, and at 543 North St. Paul Street, Rochester, N. Y. These firms also supply all microscopical apparatus and ac- cessories. If a suitable stand be already to hand, the extra apparatus may of course be added. This applies more particularly to an oil-immersion lens and Abbe's con- denser. The best immersions are perhaps those of Zeiss, but they are very expensive. A very good lens is made by Leitz, and costs about 5, whilst a con- denser can be fitted for about ^"3. Both the lens and condenser may be obtained from any of the makers above mentioned. Having now detailed the form of the microscope and the essentials of a good and practical one, we now pass to the use of the instrument. This may seem somewhat superfluous, but a few hints to the inexperi- eficed may save them not only various petty annoy- ances, but probably also the loss of valuable lenses. THE MICROSCOPE AND ACCESSORIES. 13 In removing the instrument from its box it should be grasped by the column, not by its foot, as thereby much FIG. 6. Bausch and Lamb's universal microscope. 14 MEDICAL MICROSCOPY. strain on its bearings is prevented. Any jarring of the adjustments soon puts them out of order. For this reason when in constant use it is preferable to keep it under a bell-jar rather than to be constantly lifting it out of and into its case. Scrupulous cleanliness cannot be too strongly insisted upon. Each time the lenses are used they should be carefully wiped with a soft piece of chamois leather. This applies more par- ticularly to the oil-immersion lens, for if the oil be allowed to remain on any length of time it is apt to become hard, and great trouble is experienced in re- moving it. It not infrequently happens when working with the high powers that these are accidentally forced through the cover-glasses and so become covered with balsam ; this is best removed by moistening the chamois leather with a little alcohol or xylol, rapidly passing it over the lens and immediately drying it. This must be done very cautiously as the lenses are " set " in balsam, and there is some danger of this being dissolved by the spirit, and if such be the case, the damage is ex- tremely difficult to rectify. Having settled the microscope on a firm table or stand (for any vibration is very disturbing) and inclined it at an angle to suit the convenience of the observer, without causing him to adopt an uncomfortable or tiring posture, the mirror is turned so as to reflect the light up the tube. The best illumination is obtained with a North light, so as to avoid the direct glare of the sun which is trying to the eyes ; a very good light is ob- tained by reflection from a white cloud. If a low power is being used the flat side of the mirror should be em- ployed, and if a high one, the concave side. In conjunction with the mirrors, various diaphragms THE MICROSCOPE AND ACCESSORIES. 15 are used to regulate the quantity of light passing to the specimen. In examining unstained sections, or objects such as are found in urinary deposits, the diaphragm with the smallest aperture that can illuminate the whole of the freld is applied. With stained specimens no regulation of the amount of light is necessary. With regard to the selection of a power to view any particular specimen, the rule is, never use a higher power than is absolutely necessary for clearly bringing out the minutiae of the object under examination. The inch lens should therefore first be used to gain a general idea of its structure and nature, and then the quarter- inch may be substituted ; for ordinary medical work a higher power will not often be needed, except for the examination of micro-organisms. In using the high powers, in order to avoid breaking the cover-glass by injudicious use of the coarse adjustment, it is a good plan before applying the eye to the tube to lower the lens until it just touches the cover-glass, and then to work it upwards by means of the fine adjustment whilst looking down the microscope ; if the structure be very fine, it is advisable to slightly move the slide up and down with the left hand, as a moving object is more easily caught sight of than one which is still. It should be remembered that the magnifying power may be augmented: (i) by increasing the power of the object-glass ; (2) by using a higher eye- piece ; (3) by increasing the distance between the object-glass and the eye-piece, that is by drawing out the tube. In using the oil-immersion a drop of thick cedar oil is placed on the cover-glass, and the lens lowered until it comes into contact with the oil. The fine adjustment is then cautiously turned until the object comes into focus. At ths same time care must be taken that the l6 MEDICAL MICROSCOPY. oil does not flow over the cover-glass and dissolve the balsam in which the section is mounted ; to prevent this it is a good plan to ring the specimen with gold size. The Abbe's condenser (fig. 7) must be focussed so as to produce the brightest possible illumination. The principle of the condenser is this : It is of such a form that the rays of light reflected from the mirror pass through it in such a direction that their convergence forms a cone of light as highly concentrated as possible. FIG. 7. Abbe chromatic condenser. Under these circumstances the outlines of unstained pre- parations appear blurred, and this illumination is conse- quently unsuited for fresh specimens. Also, with stained preparations the structure of the tissue or whatever may be under examination cannot be distinctly made out, but the portions which are more deeply stained, such as the nuclei, are brought clearly into view. It is THE MICROSCOPE AND ACCESSORIES. IJ for this reason that the condenser is so valuable in searching for micro-organisms ; after the use of suitable stains the small rods and micrococci stand prominently out from the indistinctly denned back-ground and thus can readily be seen and recognised. If diaphragms are placed between the mirror and condenser the characteristic action is cut off, and a good illumination is obtained for histological work and also for the examination of unstained micro-organisms, as in " drop cultures." The same effect is also produced by lowering the condenser by means of screws provided for the purpose. A caution may be given here on the use of the nose- piece. In causing this mechanism to revolve, care must be taken to raise the tube high enough to allow the high powers to travel without touching the glass, otherwise, of course, the object will be moved, and thus the great advantage of the nose-piece frustrated. This is par- ticularly liable to occur after searching the specimen for any one spot with a lower power, which it is desired to investigate with the oil-immersion lens, as in exam- ining tissues for tubercle bacilli, &c. The two clips may be removed for ordinary work, but when using the immersion lens, it is a good plan to take out the left-hand one only, leaving the right to fix one end of the slide whilst the other is moved about with the fingers. The chief use of the clips is to fix the slide in one position either for purposes of demonstration or when drawing with or without the camera-lucida. It is not always possible to work by daylight, and consequently some form of artificial light must be em- ployed. Nothing equals an oil-lamp. Various kinds of " microscopic lamps " (fig. 8) are sold, which have in c l8 MEDICAL MICROSCOPY. themselves no special advantages except in conjunction with very high powers, when by means of a slit in the shade, only a thin stream of light is allowed to fall on the mirror, so producing more intense but localised illumin- ation. For purposes of laboratory work at night, as well as for use with the microscope, " The Queen's " reading lamp can be confidently recommended. The FIG. 8. Microscopic lamp. common colza or any mineral oil may be burnt ; it is excellent as regards brilliancy and steadiness of light. With the modern improvements in electric-lighting it is probable that this mode of illuminating dwelling- houses will soon become more general, and this pure white light is very good for microscopic work, although it is often rather difficult to avoid the reflection of the incandescent wires. THE MICROSCOPE AND ACCESSORIES. 19 A good deal of discussion has taken place as regards the correct manipulation of the mirror, especially when working with the condenser as reflections of the window or of the lamp are extremely apt to appear in the field. The only way to remedy this is to turn the mirror so as just to cause these images to escape the circle of the lens, this giving the next best light to the centre of con- vergence of the rays. The next point to be considered in connection with the use of the microscope is the representation on paper of what is seen through the tube. This is naturally an important matter, as it is almost impossible to give such a graphic description of the microscopical specimen as shall enable everyone who reads it to make a mental picture of what the writer means. Time is not wasted, therefore, which is spent practising drawing objects, for the pains taken will be amply repaid. The most simple method is to place a sheet of paper on the right of the microscope, and then to look down the tube with the left eye, keeping the right open gazing at the paper ; an image of the object will then appear to be thrown on the paper, and the outlines may be traced with a pencil. If the paper be placed on a level with the stage, the picture when drawn will be of the same size as the image seen through the microscope. If it be placed higher, the representation will be smaller, whilst if lower, then the drawing will give an enlarged outline of the field. A more exact copy is obtained by aid of the camera-lucida. There are several different forms of this instrument ; the cheapest and most simple has already been described on p. n, and this contrivance will answer all the purposes of the more complex ones as sold by the different makers. In using it, the glass c 2 20 MEDICAL MICROSCOPY. slide is fixed in position by means of the clips, and the object accurately focussed. The tube is then inclined at right angles to the stand, and the mirror arranged so that the object is illuminated, but not too brightly. The paper, placed so that the projected image shall FIG. g. Prismatic camera-lucida. FIG. 10. Abbe's camera-lucida. fall upon it, should not be too much in the shade. Only the principle outlines need be traced, the shad- ing and minutiae being filled in freehand. The drawing is best made with a hard pencil. THE MICROSCOPE AND ACCESSORIES. 21 Two other forms of the camera-lucida are shown in the accompanying figures. Fig. 9 is a simple prismatic arrangement, but quite effectual. Fig. 10 is the instru- ment introduced by Abbe, and possesses the advantage that it may be used with the microscope in an upright condition. Another operation which is, however, but rarely re- quired in ordinary medical work, is ascertaining the size of an object. An appliance is employed known as a " micrometer." The form in most common use is the "stage-micrometer" which consists of a glass slip on which is ruled a series of lines ^^ of an inch apart, one of these divisions being again divided into ten parts. FIG. ii. Eye-piece micrometer. The most simple method of measuring an object is by means of a cover-glass made to act as a camera-lucida as above described. A stage-micrometer is placed under the microscope and focussed ; the instrument being horizontal, a piece of paper is laid on a level with the stage, and the lines magnified by a quarter-inch lens are carefully traced. The micrometer is now re- moved and replaced by the object whose size it is desired to ascertain. The object is traced, and com- pared with the scale by the aid of compasses. Some microscopes are provided with an " eye-piece micrometer" (fig. n), a flat piece of glass having a 22 MEDICAL MICROSCOPY. scale ruled upon it inserted in an ordinary ocular. The value of the divisions of the scale must first be deter- mined for each objective by observation of a stage micro- meter, and this being once done, a note should be made for future use. In subsequently using it for measure- ment all that is necessary is to see how many divisions of the scale the specimen under examination covers. A table is usually provided with every microscope designating the magnifying power of each objective with the different eye-pieces. Should this have been mislaid, or a new lens been added, the magnifying power may be determined as follows : The stage-micrometer is used, and its image cast by means of the camera-lucida on a sheet of paper, placed exactly ten inches from the eye. Two lines, which are in reality TG^H? of an inch from each other, are then traced, and the distance between them on the paper accurately measured, then the magnifying power is as- certained by calculating how many times the T o^o f an inch will go into the distance on the paper. Thus, sup- posing with an inch lens the lines of the image are -f$ of an inch apart, then, ^ -^ -^-^ = 200 ; therefore the system (objective and eye-piece) magnifies two hundred times. This completes a description of the use of the micro- scope and we now pass to consider the accessory apparatus which is required. It need not be supposed that a separate room is need- ful for microscopic work. A laboratory is of course to be preferred, but all that is really requisite is a firm table and a good light. The table is best when made of deal; it should stand on four steady legs, and be of such a height that the operator may sit comfortably before it while at work. It is essential that the edges THE MICROSCOPE AND ACCESSORIES. 23 of the table should be square, for if they are bevelled it will be found impossible to affix the microtome to the bench by means of a clamp. For convenience in manipulating specimens it will be found a good plan to paint on the table close by the worker, two oblong patches, one black, and the other white, each being about a foot long, and six inches wide ; the former to be used when dealing with un- stained sections, and the latter with coloured. Convenient drawers or shelves should be close at hand in which to place the specimens to preserve them from dust and guard them from possible injury. If in the room chosen for this work there be no tap and sink, a large jug for water and a receptacle for waste must be provided. Vessels made of metal should be avoided as they rapidly become corroded by acid, and therefore earthenware ones should be selected. It is almost impossible to prepare sections without the use of some special instrument. After considerable practice, sections indeed may be cut with a razor alone, but they are not so satisfactory, and even the most skilful worker can only succeed in procuring small ones, whilst by the aid of a microtome large and regular specimens may be obtained. Of these microtomes there are several kinds varying in price from a few shillings to several pounds. For ordinary use either Frazer's modification of Cathcart's microtome, or the instrument introduced by Swift may be safely recommended. Cathcart's microtome (fig. 12) consists of a brass plate supported on a micrometer screw. The plate is set between two parallel wooden uprights on which are fixed thick glass slides. These supports are about 6 inches long and three inches high. By means of pro- 24 MEDICAL MICROSCOPY. jections the apparatus may be firmly fastened to the table by clamps provided for the purpose. The cutting instrument is a large plane-like knife set in a wooden handle. It looks very clumsy but answers every pur- pose admirably. FIG. 12. Frazer's modification of Cathcart's microtome. When it is used for preparing frozen specimens a spray apparatus is fixed beneath the clamp, the free tube being connected with a bottle of ether which is hung on the side of the microtome. THE MICROSCOPE AND ACCESSORIES. 25 When required for " embedded " specimens the brass disk is removed, and in its place a clamp (fig. 13) is substituted for the reception of the cork, on which the substance to be cut is fixed. Swift's microtome (fig. 14) has a large metal clamp for affixing it to the table surmounted by a thick round plate of glass in the centre of which is a small brass disk roughened on its upper surface. The spray apparatus which is also connected with a bottle for con- taining ether is fixed below this, and Swift has lately devised an arrangement by which a clamp may be sub- stituted when embedded sections are being dealt with. FIG. 13. Clamp of Cathcart's microtome. With the microtome is supplied a " plough " for hold- ing the razor. This is a tripod metal stand supported by screws one at each angle. The one at the apex acts as a micrometer screw, and when turned, raises or lowers the edge of the knife. At the sides of the tri- angle are two smaller screws with notches at the lower ends. In the base of the tripod is another small screw, to which is attached a grooved block. This is for grasping the back of the razor, whilst its edge fits into the notches already mentioned. By tightening these three screws the razor is firmly fixed. A freezing microtome in common use, especially 26 MEDICAL MICROSCOPY. where a number of specimens are required for demon- stration purposes, is known as Williams' freezing microtome (fig. 15). It consists of a round wooden FIG. 14. Williams' ether microtome (" Swift's microtome.") box, provided with a drain pipe, in which the freezing mixture is placed. In the centre of the box is a vertical THE MICROSCOPE AND ACCESSORIES. 27 brass pillar, provided at the top with a roughened brass plate. The box is covered with a lid formed of a thick plate of glass, in the centre of which is a hole through which projects the plate for the reception of the speci- mens. The cutting instrument used in connection with this microtome is Swift's " plough " just described. FIG. 15. Williams' ice and salt microtome. Other microtomes are those of Katsch, Roy, Schanze, Zeiss, Jung, &c. If it be intended to do much section- cutting one cannot do better than at once purchase the more complete instrument made by Reichert It is more expensive costing about 6, but is far better adapted for cutting " embedded" specimens. The knife is fixed on a carriage which runs in a groove and its movement is therefore very steady. The 28 MEDICAL MICROSCOPY. micrometer screw is worked automatically. Its general form will be sufficiently appreciated by referring to the accompanying illustration (fig. 16). M is the razor fixed to the block K, by means of the clamp F. By knocking against the screw c, fixed by the milled head b to the carriage by means of a spring arrangement, the wheel Z is turned round and so raises the clamp g in which the specimen to be cut, is held. This clamp is fixed by the screw e. An indicator i marks the amount which the object is raised each time the lever h is set in action. a is a catch regulating the movement of the wheel Z, while W is a metal trap for catching the waste alcohol. A very capital instrument has been introduced by the Cambridge Scientific Instrument Co., known as the rocking microtome (fig. 17). Its chief use is for cutting a series of sections but for ordinary purposes it has no advantage over the instruments already de- scribed. Swift has recently introduced an ingenious modifica- tion of the rocking microtome, in which clamps are pro- vided so that it may also be used for cutting specimens embedded in celloidin and also as a freezing microtome. It is often useful to be able to cut sections of fresh material without any preliminary preparation, such as during an operation for the recognition of the different forms of tumour. For this purpose nothing is better than a Valentin's knife (fig. 18). It is formed of two narrow blades lying parallel to each other, their dis- tance being regulated by a fine screw. The method of using the above instruments will be described when directions are being given for cutting sections. A certain number of accessories for convenience in THE MICROSCOPE AND ACCESSORIES. MEDICAL MICROSCOPY. microscopic work will be wanted, but these, however, require little explanation beyond enumerating those ab- solutely needed. Objects are nearly always examined on glass slides about three inches long and one inch THE MICROSCOPE AND ACCESSORIES. 3! wide. The edges are usually ground, but in a cheaper make they are rough. It is scarcely necessary to say that the glass should be quite free from specks and flaws of all kinds. The specimens are covered by thin squares or circles of glass known as cover-glasses. These are made in varying thicknesses ; the most use- ful are the squares, J- of an inch wide, and the thickness known as " No. 2." These glasses are sufficiently thin to be used with a J inch lens and the oil immersion ; if however, .an eighth dry power be used, thickness " No. i " must be employed. Great care must be exercised in cleaning the cover glass, as being extremely brittle they are easily damaged. When first purchased they are often covered with a thick film of grease which is difficult to remove. The best mode of doing this is to place them for a short FIG. 18. Valentin's knife. time in strong hydrochloric acid, then swill them in water, and finally transfer them to a covered capsule containing methylated spirit ; and here they should be kept until required. Before use they are dried with a soft cloth, being rubbed between the finger and thumb, or a corner of the cloth is laid on the table, the cover- glass placed on it, and the glass is gently wiped on either side. " No. i " cover-glasses being very thin, a good plan is to swill them in absolute alcohol as a final stage in the cleaning, this evaporates quickly, and does away with the need of a cloth. Cover-glasses which have been used may be recleaned in the manner above described. A tumbler or beaker 32 MEDICAL MICROSCOPY. covered with a ground glass plate should be kept on the bench into which all waste alcohol should be thrown. Specimens which have been mounted in balsam, but which have not been considered worth preserving can be placed in this tumbler, and cover-glasses dissolved off. Some fine sewing needles should be mounted in wooden handles, and must always be kept clean and sharp. Special holders are sold in which the needles may be conveniently changed when they are blunted or spoilt. Excellent substitutes for needles are glass rods drawn out to a very fine point. These are superior to needles in that they do not corrode in acids, and are not so apt to tear the specimens. Two pairs of forceps will also be required ; those should be selected which have broad blades so that cover-glasses can safely be grasped by them and the student should avoid the long slender forceps which are usually sold with cases of microscopic instruments, as they are apt to chip and break the glasses. A scalpel and pair of scissors are also necessary as also a couple of section lifters. The last named are preferably made of some malleable material such as stout tin foil. Of glass apparatus the following are required. About half a dozen capsules of different sizes, the same number of watch glasses with ground bottoms, to ensure their steadiness, and two glass funnels. For holding the stains when in use nothing answers better than small glass salt-cellars, they do not easily upset, and are of a convenient depth. A packet of filter paper and a filter stand will complete the apparatus for ordinary microscopic work, THE MICROSCOPE AND ACCESSORIES. 33 whilst for the examination of urinary deposits, &c., test-tubes, some conical glasses and pipettes will be required. For the reception of the reagents, bottles with glass stoppers are preferable. A large supply of stains and other reagents is not necessary, and the following is a list of those absolutely needful. Those required for special purposes will be described as the need for them occurs, or they can always be added from time to time. Absolute alcohol. Methylated spirit. Glycerine. Farrants' solution. Canada balsam dissolved in xylol. Normal saline solution. This is made in the following manner (Woodhead) : Sodium chloride is heated to redness, and cooled over sulphuric acid ; 7^- parts by weight are then dissolved in 1000 parts by measure of distilled water. Iodine dissolved in iodide of potassium. The best formula for this is that known as " Gram's solution." This not only acts as a useful testing fluid but is also an essential part of the process for staining certain micro-organisms. It is made as follows : Iodine I part, Iodide of potassium ... 2 parts, Distilled water 300 parts. 34 MEDICAL MICROSCOPY. CHAPTER II. HARDENING AND DECALCIFYING. FOR examination by the microscope sections may be prepared in two ways, either directly from fresh speci- mens, or from those which have been duly "hardened." The former method is practically confined to the inves- tigation of tumours in the operating theatre, or for pur- poses of immediate diagnosis in the post-mortem room. When prepared in this manner, however, the sections cannot be permanently mounted, and hence are only of temporary service. A knowledge of how to prepare such sections is nevertheless greatly to be desired, and a chapter will be devoted to a consideration of this subject. The processes by which specimens may be mounted, and kept for future reference, or for purposes of demonstration, will be first described. In selecting the portions of an organ or tissue for microscopical purposes, some care has to be exercised. They should be obtained as soon after death as possible, for if decomposition has set in the sections will not properly take up the stain, and thus many important characteristics may be lost. In choosing the parts of a diseased organ, the liver for example, it is always pre- ferable to take a particle from near the surface, so as to include the capsule, as this affords great aid in recog- nising the minute anatomy of the organ. Again, in HARDENING AND DECALCIFYING. 35 preparing sections of the intestine or stomach small por- tions should be cut off transversely to the axis of the viscus, and about a quarter of an inch in length so as to show the condition of its various coats. When investigating a morbid growth invading an organ at least two pieces should be cut out ; one to be taken from the centre of the tumour, and one from its circumference, so as to include a portion of the organ as well as of the growth. The size of the pieces should be nearly an inch in length and breadth, and a little less than a quarter of an inch in thickness. They should be removed by a sharp scalpel, the cuts being cleanly made so as to avoid .any tearing, and when completely freed should be lifted upon the knife, and dropped into a small wide-mouthed bottle, which should be at once labelled with the name of the organ from which the specimen was taken, the date, and supposed morbid condition. Too much stress cannot be laid upon the importance of immediately labelling all preparations whether completed or other- wise, as from postponing this simple precaution many errors are apt to arise, and cause waste of time and much annoyance. Added to which a good specimen is of little value, unless its source in addition to its nature is known . The bottle into which the cuttings have been placed should be filled with the hardening solution, and the choice of such a solution must now be considered. The reagents in most common use are : Absolute alcohol. Methylated spirit. Miiller's fluid ; and bichromate of potash. The selection of one of these depends upon many points. D 2 36 MEDICAL MICROSCOPY. a. The organ or tissue which is to be hardened. The most useful reagent is undoubtedly absolute alcohol. It is suitable for nearly all tissues, except those which owing to their complex structure do not contract re- gularly. Thus, the central nervous system, and the eye should not be subjected to its action. Thin tissues and membranes also are best hardened by some other fluid on account of the manrer in which in the alcohol they curl up ; for instance sections of the intestines cannot be satisfactorily obtained after hardening in this reagent. For all such exceptions Miiller's fluid is preferable. b. The nature of the morbid change. Alcohol owing to its solvent power on fat should not be used when it is desired to demonstrate fatty changes ; on the other hand it is most suitable when micro-chemical reactions are to be exhibited, such as the amyloid reaction. When specimens are hardened in Miiller's fluid or bichromate of potash, such reactions are marred. c. It is occasionally desired to investigate certain particular elements of the tissue, such as the epithelial structure, or the division of nuclei. For these purposes it is advisable to use the chrome salts. d. For bacteriological purposes absolute alcohol or methy- lated spirit should be used, as the other reagents although they do not entirely destroy yet greatly interfere with the properties which the various bacteria possess of re- taining the aniline dyes. e. The time which the different hardening reagents occupy before the specimens are ready for cutting is occasionally of importance. If it be desired to secure a rapid and energetic action, provided there are no other objections, absolute alcohol should be chosen ; whereas if a more cautious course is advisable, Miiller's fluid or bichromate of potash should be selected. HARDENING AND DECALCIFYING. 37 Before describing seriatim the method of using the above named hardening reagents, a few general hints may be given. A relatively large volume of the liquid should be employed and frequently changed ; the periods for so doing depending upon the fluid used. As before stated the specimens should be placed in the reagent as soon as possible after removal from the body. They may either be suspended by means of threads in a tall cylindrical vessel, each thread having attached to it a label, by means of which it may be easily recognized ; or a layer of cotton-wool may be placed in the jar. The reason for these precautions being, that the speci- mens may be entirely surrounded by the hardening reagent. If Muller's fluid or bichromate of potash are the re- agents selected, the jars or bottles should be kept in a cool dark place, as these fluids are very apt to develop moulds. Absolute alcohol. This acts as a hardening re- agent by means of the withdrawal of water and coagula- tion of albumin. Specimens placed in it usually shrink somewhat, but this is of very little moment, for when the sections obtained from them are placed in water, they soon resume their natural appearance. As previously mentioned it is suitable for nearly all structures, the exceptions being those which owing to their varied composition shrink unequally, such as the spinal cord and eye, but even these with a little manipu- lation may be hardened with this reagent. Thus, if it be desired to harden an eye-ball, this can be effected by injecting alcohol into the vitreous by means of a hypo- dermic needle before the specimen is placed in the jar, no inequalities will then be produced. Again, lungs, muscles, and old spirit preparations are 3 MEDICAL MICROSCOPY. sometimes difficult to harden. This may be accom- plished by placing the specimens for twenty-four hours in equal parts of glycerine and mucilage of gum arable, and transferring them once more to the alcohol, when an excellent consistence for cutting will be obtained. The portions to be hardened should always be small, as owing to the rapidity with which alcohol acts, the outer surfaces of the specimens become so set as to prevent the alcohol penetrating to the interior. The liquid generally becomes turbid a few hours after the fresh tissues are immersed in it, and therefore after twenty-four hours it is better to transfer them to a fresh lot of alcohol, but subsequently no further change need be made, as there will be no tendency to over-harden. In this respect alcohol gives far less trouble than Miiller's fluid or bichromate of potash, and the hardening process is moreover more easily controlled, for the action of the last named reagents greatly depends upon time and temperature, and very often produces cloudiness and discolourations. As regards the time required for the completion of process, this greatly depends upon the size of the speci- mens. Small ones will be ready in twenty-four hours, whilst larger ones will require about four days. Methylated spirit. This is suitable for the same class of objects as absolute alcohol, and its effects are practically the same. Its action, however, is not so rapid, and the specimens should remain immersed for about fourteen days. The spirit should be changed after the first twenty-four hours, and again after the first week. If time be no object it is very suitable for hardening tissues in which it is intended to search for micro- organisms. HARDENING AND DECALCIFYING. 39 Miiller's fluid. This has the following composi- tion : Bichromate of potash ... 2 parts, Sulphate of sodium .... i part, Distilled water 100 parts. This reagent fixes the protoplasm of the cells rather than hardens them, and so unlike alcohol causes but little shrinking. It may be used for any tissue except those in which micro-organisms are to be sought for, or in which some micro-chemical reactions may have to be made use of such as in staining amyloid material. There is one other rare exception which may be mentioned, a deposit of lime salts is dissolved by this medium, and conse- quently chalky deposits such as occasionally occur in the glomeruli of the kidneys are destroyed if the organs be hardened in this way. Miiller's fluid is especially adapted for hardening the central nervous system, and indeed is essential when Weigert's or Pal's methods of staining are adopted. Portions of intestine or other hollow organs are also best hardened by this fluid. Specimens of any size, even whole brains, may be prepared in this manner, but as the penetrating power of the liquid is very small, a considerable time must elapse before the process is complete. Even the smallest portions of any organ require about six weeks, whilst a brain must remain about a year. Moulds are likely to form, and to pre- vent this a small piece of camphor should be kept floating in it. The process of hardening may be hastened by placing the jars in an oven kept at a temperature of 30 to 40 C. (80 to 1 00 F.). The fluid has to be frequently changed, every second day for the first week, and then each week 4O MEDICAL MICROSCOPY. until the hardening is complete. In order to ascertain when this stage has been reached, the specimens should be removed from the jars and gently pressed with the ringers, when if the desired result has been obtained, they will be found to be tough but not brittle. They are next placed in water, which should be frequently re- newed until the washings are colourless, and are finally put into methylated spirit until an opportunity is found for cutting them. Bichromate of potash. This salt is usually em- ployed in the strength of from two to five per cent, watery solution, commencing with the former and gradu- ally increasing the amount of the potash salt until the latter strength is obtained. It is used for the same class of specimens as Miiller's fluid, but its action is rather quicker ; three weeks being required for specimens of ordinary size, but six weeks for a spinal cord, and six months for an entire brain. The after-treatment is the same with both reagents. Another form in which bichromate of potash is used is Erlicki's solution. The composition of this is as follows : Bichromate of potash ... 2-5 parts, Sulphate of copper .... i part, Water 100 parts. This reagent owing to the presence of the sulphate of copper hardens very much more rapidly than the previ- ously described chrome preparations. A spinal cord may be hardened in it in ten days at ordinary tempera- tures, or in four days at a temperature of about 100 F. To give a complete list of chromic acid salts which are used for hardening purposes the following must be mentioned. Chromic acid. This hardens extremely rapidly, HARDENING AND DECALCIFYING. 4! most specimens being ready in a few days. The strength is gradually increased ; at first it should not exceed J per cent., and this should be gradually raised to \ per cent. It is very important that small pieces of tissue should be placed in it, and large quantities of the solution employed. The fluid must be changed at the end of the first, second and third days, and then every third day until the hardening is complete, this being tested at frequent intervals as already described, for if allowed to remain too long the specimens become brittle and almost useless. This may in part be pre- vented by adding a little glycerine to the reagent. Care must be taken to thoroughly wash out the acid with water before placing them in alcohol for preservation. Chromic acid and spirit. This mixture, first introduced by Urban Pritchard, is occasionally em- ployed instead of chromic acid. It is made thus : Chromic acid i part, Water 20 parts, Rectified spirit 180 parts. The chromic acid must be dissolved in the water, and then the spirit added, otherwise a too violent action will ensue. This mixture is especially suited for hardening speci- mens of the eye or internal ear. Ammonium chromate and bichromate of ammonia. Both these salts are occasionally used as hardening reagents, and in a similar manner. They are suitable for all tissues, but the former is especially useful for preparing the kidney and other secretory glands. The strength of each is the same, i.e., five per cent. The portions of tissues should be very small, and placed in about fifteen times their bulk of the solution. With ammonium chromate the process is complete in 42 MEDICAL MICROSCOPY. forty-eight hours, and the specimens are then thoroughly washed in water, and either cut at once or removed to spirit for preservation. With bichromate of ammonia the time occupied is much longer. The fluid must be changed at the end of the first, third and seventh days, and at the end of each week until the sixth, when the tissue will be ready for cutting. It will be seen from the above descriptions that the chrome salts usually require a long time before the hardening process is complete, and as they penetrate slowly, this depends in a great measure upon the size of the specimens. Frequent changes are necessary and therefore much more attention is required than when alcohol is used. Various alterations are also produced by their action in the interstices of the tissues. A peculiar network due to coagulation of albuminous substances very often takes place which may lead to errors if not rightly understood. Another disadvantage is that if the pieces are allowed to remain too long in the fluid they will be over-hardened and become brittle. Corrosive sublimate. This, though seldom used, is a capital hardening reagent. It is especially useful for glandular structures. The best strength is a half- saturated alcoholic solution, this is made by dissolving in one portion as much of the salt as 70 per cent, alco- hol will take up, and then adding to it an equal quantity of alcohol of the same strength. The specimens must not remain in it too long as it hardens with great rapidity. A quarter of an hour to two hours (depending on the size of the specimens) is quite sufficient. At the end of the process the corrosive sublimate must be washed out with alcohol, otherwise the sections will be marred by the presence of black spots. Platinum chloride. This reagent is particularly HARDENING AND DECALCIFYING. 43 suitable for delicate objects such as the retina. It is usually made up in what is known as Merbrel's solution. This consists of equal volumes of 1-4 solution of chromic acid, and 1-4 solution of platinum chloride. Its action is fairly rapid, the process being complete in from three to four days. No change is necessary, and the reagent should be washed out with dilute alcohol (60 per cent.). Osmic acid Owing to its expense, and the irritat- ing property of its vapour, this substance is rarely used for hardening purposes, except in the Pal-Exner method of staining the central nervous system, and for portions of retina. It is sold as a i per cent, solution, and must be kept in opaque bottles in the dark. Specimens should be submitted to its action for about ten hours, and then thoroughly washed in water. One of the most important uses of osmic acid is the selective power which it has on fat cells, these becoming blackened. A good plan in demonstrating fatty changes is first to harden the specimens in Miiller's fluid and then to place them for a few hours in osmic acid. Another point which may be mentioned here is that turpentine and some other essential oils will decolourise particles of fat black- ened by this reagent, and that therefore when sections have been cut and stained, the process of ''clearing" by these oils must be rapidly conducted. Picric acid. A saturated solution may be used. The albuminates are gradually transformed into the insoluble form, so that hardening occurs without any shrinking. At the same time the specimens are stained a deep yellow which is very advantageous as a counter- stain to carmine. It is especially useful for epithelial structures, intestine, and the various tumours. After a stay of forty-eight hours in the acid, the specimens are washed in water, and preserved in alcohol. Picric acid 44 MEDICAL MICROSCOPY. is occasionally employed as Kleinenberg's solution. This is prepared as follows : Saturated watery solution of picric acid . 100 parts, Strong sulphuric acid 2 ,, Filter and add distilled water .... 300 ,, This hardens more quickly than picric acid alone (three to twelve hours) and is especially adapted for the rapid hardening of various soft structures such as sar- coma or myxoma. The sections should afterwards be stained in carmine. DECALCIFYING. In order to prepare sections of bone or of tissues in which lime salts are deposited it is necessary to remove these in order that the substances may be cut with a razor, for the process of grinding down such specimens until they are thin enough to be properly mounted is far too tedious, and the results which are produced are unsatisfactory. The process of decalcification is carried out in much the same way as the process of hardening, except that of course the fluids employed are not quite the same. Only the principal reagents will be described here. Nitric acid. This is used in two strengths, for large bones one part of pure nitric acid is diluted with ten parts of water, whilst for smaller bones and for specimens containing calcareous deposits, this must be further diluted to one per cent. Specimens are first treated with absolute alcohol for three days, and are then placed in the dilute nitric acid for eight or ten days, the fluid being changed daily. They must be frequently examined to ascertain if the HARDENING AND DECALCIFYING. 45 process of decalcification is complete, this being tested by piercing them with a needle. When ready they are washed for some hours in water and then placed in alcohol for preservation. A modification of this pro- cess, and a very excellent one consists in making a mixture of equal parts of 3 per cent, of nitric acid, and 70 per cent, alcohol. This acts more slowly than the previous one, requiring several days or even weeks. Perenzi's solution. This is perhaps about the best decalcifying fluid. Its composition is as follows : \ per cent, chromic acid .... 3 parts, Absolute alcohol 3 ,, 10 per cent, nitric acid 4 ,, Its action is very rapid, the specimens being ready in one week.. The after-treatment is the same as for nitric acid alone. The advantage of the three fluids just men- tioned over other decalcifying fluids is that no swelling is caused, whilst at the same time no injury is suffered by the tissue elements. Hydrochloric acid. The strength of this acid for decalcifying purposes is 10 per cent. The process is much slower than with nitric acid, requiring a month before it is completed, in addition the acid causes the soft tissues to swell, so that their structure becomes almost unrecognisable. To avoid this a little chromic acid or alcohol may be added. Yon Ebner's solution. This is a modification of the preceding, and has the following composition : Hydrochloric acid .... 5 parts, Alcohol 1000 ,, Distilled water 100 Chloride of sodium . . . f 5 ,, A large quantity of the fluid must always be used and frequently changed, the specimens being also occasion- 46 MEDICAL MICROSCOPY. ally tested to ascertain if they are soft enough for cutting ; they are afterwards thoroughly washed in water. If it be desired to examine the fibrillar structure of bone, the sections should be mounted in 10 per cent, salt solution, but if this is not a matter of importance, the ordinary mounting media may be employed. Picric acid. A saturated watery solution of picric acid may be used, but is not altogether satisfactory. Its action is weak and very slow, in addition it causes considerable shrinking of the tissues. A large volume of the reagent must be employed, and a few crystals of the acid should always be kept at the bottom of the jar to ensure the strength being maintained. When decal- cification is accomplished, after washing in water, the specimens are preserved in spirit. This process is only suitable for very small pieces of young bone. EMBEDDING. 47 CHAPTER III. EMBEDDING. IN order that thin and uniform sections may be made it is not sufficient that the specimens have been hardened or decalcified. If it be attempted to cut the specimens without further preparation, it will be found that the sections easily break directly they are made, if indeed it be possible to obtain any at all. In addition to these objections it is very difficult to hold the specimens firmly enough without crushing them and greatly interfering with their structure. In order to overcome these obstacles, it is necessary either to surround the material with some substance firm enough to afford a hold to the hand, or to saturate it with a medium which will impart to it a uniform consistency. This operation is known as "embedding." From the above prefatory remarks it will be seen that embedding is necessarily of two kinds, viz., "simple" embedding, which is fixing or placing the object to be cut in a medium firm enough to hold it, and " inter- stitial " or " infiltration " methods, which consist in permeating the specimens with some medium, which is at first fluid, but gradually becomes solid ; this being accomplished by the use of substances which are liquid when hot and solid when cool, such as paraffin, or by dissolving some substance in a volatile fluid, such as celloidin in ether and alcohol. A third method, and the 48 MEDICAL MICROSCOPY. one which is perhaps the most commonly adopted, con- sists in saturating the specimens with a solution of gum, which is afterwards frozen. The freezing media will be first described. The initial stage consists in abstracting the hardening re- agent. This is of course absolutely essential, as alcohol will not freeze, and the chrome salts would be deposited irregularly. The specimens therefore are placed in a basin con- taining ordinary tap water which should be frequently renewed. The process is shown to be complete by the specimens sinking to the bottom of the containing vessel if they have been hardened in alcohol ; or by the water remaining untinged if chromic acid or its salts have been used. They are now ready to be placed in gum. The solu- tion usually employed is gum and syrup, which is made of equal parts of gum solution (B.P. strength) and syrup, to which a little thymol should be added as a preservative. The following, however, recommended by Mr. T. L. Webb (" The Microscopic Journal," ix., 1889, page 344), will be found preferable. It freezes sufficiently to give a firm support without being too hard, it keeps better than gum, and is in addition cheaper. An aqueous solution of carbolic acid (i in 40) is taken and sufficient dextrin is dissolved in it to make a thick syrup. Dextrin dissolves slowly in cold water, so that a gentle heat must be used when making the mucilage. It solidifies if kept for some time, but may be liquefied again by gently warming the bottle. The specimens should be allowed to remain in these solutions for twenty-four hours, so as to become tho- roughly permeated. EMBEDDING. 49 SIMPLE EMBEDDING. Paraffin is usually employed for this method. A little tray is first made out of light cardboard in the following manner (fig. 19). The card should be oblong in shape as in the diagram. It is first folded along the lines A A' and BB ', next along the lines CC' and DD', keeping the folds of the paper on the same side. The lines aa, bb', cc and dd' are then marked with a pencil. The corners are now pinched up between the finger and of C DC' a/ ~b d \ />/ /nf/ /)/ f]/ i 15 parts Water 85 parts. Sections are allowed to remain in either of the above stains, undiluted, for at least twelve hours, a period of twenty-four hours being better. A dilute solution of ace- tic acid is then made by adding about three drops of the strong acid to a medium-sized capsule of water, or one drop to a watch-glass full. In this mixture the sections are washed after removal from the stain until they are 92 MEDICAL MICROSCOPY. light blue in colour ; they are then removed to absolute alcohol when still more colour is removed, and after- wards they are cleared in cedar oil and mounted . in balsam. It requires some practice to conduct the process of washing out the blue or " differentiating " the specimens properly ; usually too much colour is removed both from the tissues and the bacilli, or too much of the dye is left in the tissues so that the bacilli can hardly be distin- guished. Under such circumstances the margins of cells or other constituents of the tissues especially " mast-zellen " (see below), are very apt to be mistaken for micro-organisms. Kuhne's method. There are several points in which Kuhne's method of staining micro-organisms with methylene blue differ from those previously introduced, notably in the addition of carbolic acid to the stain, and the use of aniline oil for the purpose of dehydration in- stead of absolute alcohol. In his general remarks on this stain, Kiihne states that he has succeeded in staining in the tissues almost all forms of pathogenic bacteria, the only exceptions being the bacilli of leprosy and mouse septicaemia. The bacillus of glanders also stained in far fewer num- bers than in cover-glass preparations. He suggests that as leprosy bacilli will not colour by this method, it might be made use of as a differential test in doubtful cases of tuberculosis and leprosy. For this method the following solutions are required : Solution i. " Carbol. methylene blue." Absolute alcohol 10 parts Methylene blue 1-5 parts. Carbolic acid (5 per cent, aqueous sol.) 100 parts. The dye is placed in a mortar and the alcohol poured SELECTIVE STAINS. 93 over it, the carbolic acid solution being gradually added during stirring. Solution 2. " Weak acidulated water." Hydrochloric acid . . . . 10 drops Water 500 c.c. Solution 3. " Lithia water." Water 10 c.c. Concentrated aqueous solution of) 5 to g ^ rQ lithium carbonate 1 Solution 4. " Aniline oil solution of methylene blue." About as much methylene blue as will go upon the point of a pen-knife is rubbed up in a mortar with 10 c.c. of aniline oil. The whole of this mixture (the dye will not be wholly dissolved) is then poured into a small bottle. When required for use a few drops of the solution are added to a small capsule-full of pure aniline oil. Solution 5. " Aniline oil solution of safranin." This is made in the same way as solution 4, using safranin instead of methylene blue. Mode of procedure : Sections are removed directly from alcohol into the carbolic methylene blue (Solution i), where they re- main for about half an hour. The specimens are next washed in water, and then differentiated by rinsing in weak acidulated alcohol (Solution 2), being afterwards dipped for a few seconds in lithia water (Solution 3) and again immersed in ordinary water. The process of de- colourisation must be carefully watched, and stopped when the specimens have assumed a pale blue tint. The time for this varies from a few seconds up to a quarter of an hour or more. Experience alone can guide one, and many disappointments will probably be experienced before success is attained. After remaining gi MEDICAL MICROSCOPY. for about five minutes in water, the sections are dipped for a second or two into absolute alcohol, in which it is advisable to dissolve a pinch of methylene blue, and are then placed in methylene blue aniline oil (solution 4) ; owing to the alcohol they will spread, out well in the oil, and dehydration will be complete in a few minutes. If the sections have become very pale, by this stage of the proceedings, rather more of the aniline methylene blue should be added than is directed above. After dehydra- tion they are rinsed in pure aniline oil, which must then be extracted by thoroughly washing the specimens in xylol ; it is very important that this be effectually done, otherwise the preparations soon become brown and al- most worthless, two lots of xylol should be employed, the sections been transferred from one to the other. Finally they are placed on glass slides, and after draining off the superfluous fluid are mounted in balsam. The speci- mens may be counter-stained by immersing them in the safranin aniline oil (Solution 5) prepared in the same way as the aniline solution of methylene blue, the time required varies from two to ten minutes, according to the dilution of the stain and the material to be coloured. When removed from the stain they are immersed for a few seconds in pure aniline oil and then thoroughly washed in xylol. Although Kuhne states that all bacilli, with the ex- ceptions named above, are coloured by this method, the author has attempted in vain to stain the bacilli o pseudo-tuberculosis. " Mast-zellen." This seems to be the most suitable place to refer to these bodies. In sections of organs affected with diphtheria, glanders, and other processes, which have been stained by methylene blue or other aniline dyes, small round or spindle-shaped bodies are SELECTIVE STAINS. 95 often met with ; they are about twice the size of lymph cells, and consist of coarsely granular protoplasm with a well-marked nucleus. The granules stain with the aniline dyes, but the nucleus remains uncoloured, ap- pearing as a bright spot in the midst of the deeply- stained protoplasm. These bodies occur in large numbers in the connective tissue, especially in the mucous membrane, and are generally situated in the neighbourhood of the, vessels. The jr physiological and pathological significations are not known, they are very numerous around rapidly growing tumours and also in elephantiasis. '* Mast-zellen " have hitherto been chiefly described by foreign observers, and in England they are generally known under the term " plasma cells." They are very liable to be mistaken for colonies of micrococci by those unfamiliar with them, but may be distinguished from the latter by the presence of the nucleus and by the irregularity in size of the component granules. "Gram's method of staining." This process is the one most generally adopted for staining micro- organisms, it will stain all the most important patho- genic bacteria, with the exception of those associated with cholera, typhoid fever, and glanders. The following solutions are required : Solution i. Methyl" aniline violet. This should al- ways be freshly prepared. A few drops of pure aniline oil are placed in a medium-sized test-tube, which is then nearly filled with water and thoroughly shaken, the thumb being placed over the mouth of the tube. The emulsion is filtered through a double-folded paper, and poured through a second time if the filtrate be not quite clear. A little of this aniline water is placed in a cap- sule, and to it is added, drop by drop, a concentrated 96 MEDICAL MICROSCOPY. alcoholic solution of methyl violet, until the mixture just becomes opaque. This stage is most easily recog- nised by placing the capsule on the edge of a piece of filter paper, and adding the dye until the margin is al- most, but not quite, obscured. Solution 2. Gram's solution. Iodine i part Iodide of potassium .... 2 parts Water 300 parts. The sections are transferred from dilute spirit to me- thyl-aniline violet, where they are allowed to remain for five or ten minutes ; after staining they are transferred to Gram's solution until they become brown and opaque ; one minute is usually long enough. They are next re- moved to 60 per cent, alcohol, and gently moved about by means of a needle until no more colour can be re- moved. Professor Crookshank's procedure may also be adopted which is to transfer the specimens two or three times backwards and forwards between oil of cloves and the alcohol. When completely decolourised, the sec- tions may be counter-stained by placing them in abso- lute alcohol to which two or three drops of a saturated alcoholic solution of eosin have been added ; they are at the same time dehydrated, and after clearing in cedar oil may be mounted in balsam. One caution is here necessary : if the specimens have been embedded in celloidin previous to cutting, oil of cloves must not be used for decolourising, and it is better to dehydrate in 95 per cent, rather than in abso- lute alcohol. Weigert's modification of Gram's method. Weigert and Kiihne were the first to observe that after staining bacteria in methyl violet and treating with Gram's solution and decolourising with alcohol, SELECTIVE STAINS. 97 much of the colour was removed from the micro-organ- isms as well as from the tissues. These observers then proposed to use aniline oil for decolourising, and Weigert consequently introduced the following modification of the method just described. The sections are first stained in lithium carmine as described on page 75, decolourised with acidulated alco- hol and dehydrated with absolute alcohol. The sec- tions are then transferred one by one to glass slides, and each is treated in the following way : A couple of drops of methyl aniline violet freshly prepared (see above) are placed on the section and allowed to remain for about five minutes, the excess is poured off, and a small quantity of Gram's solution poured on ; after the lapse of one minute this is likewise poured off and all moisture removed by gently pressing a folded filter paper on the specimen. Pure aniline oil is now added, and made to flow gently too and fro by tilting the slide ; the colour will be removed at first rapidly and then more slowly, and fresh portions of oil must be used until no more colour comes away. It occasionally happens that the section adheres to the glass all round, but not in the centre, so that some of the stain may be thus retained, if such be the case, a corner of the section must be gently raised with a needle so as to permit some of the oil to flow beneath it, care being taken to prevent the whole section from being floated away. Decolourisation being complete, two or three lots of xylol are poured over the section, a light cloud will be observed at first, and the xylol must be used until this has quite disappeared ; unless this part of the process be thoroughly carried out, the section will speedily turn yellow. A few seconds are allowed to elapse for the 98 MEDICAL MICROSCOPY. xylol to evaporate, and finally a drop of balsam is placed on the specimen and a cover-glass applied. The lithium carmine solution is very apt to develop micro-organisms, and errors often arise on this account, the observer imagining that he has an extremely fine preparation. Some sections, such as of cirrhosis of the liver, known to be free from germs should therefore be passed through the above process so as to ascertain if the carmine solution be untainted. The above process is particularly suitable for staining anthrax bacilli and sections of ulcerative endocarditis. In the latter not only are the masses of micrococci beautifully demonstrated, but a delicate purple tint is left in the layers of fibrin. Neelsen-Ziehl method for staining tubercle bacilli. In order that this process may be carried out as successfully as possible, the specimens should be hardened in alcohol, or if any of the chrome solutions have been used, the hardening material must be tho- roughly extracted by water and the specimens soaked in alcohol for two or three days. Sections which have been cut in celloidin or paraffin will also be found more satisfactory to manipulate than those which have been frozen, as these last are very apt to curl up in the acid, and it is difficult to spread them out again. As stated in the Chapter on the " Examination of Sputum," this method depends upon the property which the tubercle bacilli possess of resisting for some time the action of dilute acids, which distinguishes them from all other bacilli, except those of leprosy which act in the same manner ; but as in this country leprosy is fortu- nately a very rare disease, this method of staining may be said to be an absolutely distinctive one. Tubercle bacilli also stain by Gram's and Kuhne's methods, but SELECTIVE STAINS. 99 these are never used for the purpose. I have used the double name for this process, for although introduced independently by the two observers, the processes are essentially the same. The solutions required are : Solution i. Carbol. fuchsine (Neelsen-Ziehl solution). Fuchsine - i part dissolved in a 5 per cent, watery solution of Carbolic acid 100 parts Alcohol 10 parts. Solution 2. Sulphuric acid, 25 per cent, watery solu- tion. Solution 3. Methylene blue : Methylene blue .... 2 parts Alcohol 15 parts Water 85 parts. The sections having been soaked in dilute alcohol, are placed in the fuchsine solution contained in a covered capsule, where they should remain for at least an hour, or preferably for twelve hours. They are then decolourized in the sulphuric acid. This process is carried out thus : Sections are allowed to remain in the acid for about thirty seconds, and are next washed in a large capsule full of 60 per cent, alco- hol, being gently moved about with a glass rod. Part of the colour will be probably restored. If so, they must be returned to the acid for about ten to fifteen seconds, then again washed in the alco- hol, this process being repeated until only a faint lilac tint remains. They are next counter-stained in the methylene blue solution. An immersion of half a minute will be sufficient, after which they must be again washed in water. The blue stain is rapidly re- moved by the water, and in about half a minute the H2 ioo MEDICAL MICROSCOPY. sections should be transferred to absolute alcohol. Still more colour will then be abstracted, and the specimens must therefore be watched, or they will be- come completely decolourised. As soon as they assume a light blue tint they should be removed to cedar oil for clearing, and finally mounted in balsam. The bacilli will be found to be stained a brilliant red, whilst the nuclei of the tissues are coloured blue. This stain is very permanent lasting for years. A more simple but not so reliable a method consists in immersing the sections for several hours in Gifobes' double stain, and subsequently washing out the excess of colour in methylated spirit, dehydrating in absolute alcohol, clearing in cedar oil, and mounting in balsam. Gibbes' double stain may be procured at any of the microscope makers. Its composition is rather compli- cated : Rosaniline hydrochlorate .... 2 parts Methylene blue i part. The mixture must be triturated, and 3 parts of ani- line oil mixed with 15 parts of rectified spirit slowly added, followed by 15 parts of distilled water. Methods of staining actinomyces. This fun- gus has been more frequently met with during the last few years than it was formerly, not because cases have been any more numerous, but on account of discharges from the chest and other parts being more carefully examined in doubtful cases. For demonstrating the fungus in sections I have found the following method most satisfactory. The sections are first stained in rubin (see p. 82) for ten minutes, are then washed in water and placed in abso- lute alcohol for about three minutes. If the sections are seen to become very pale they must be taken out SELECTIVE STAINS. IOI sooner. They are next transferred one by one to glass slides, and stained according to Weigert's modification of the Gram method (see p. 96). On examination the clubs of the actinomyces will be found to be stained red, whilst the mycelium appears violet. Plants' method, which is the one usually adopted, is as follows : The sections are placed for two hours in Neelsen's solution (see p. 99). The time of staining may be shortened to twenty minutes if the fluid is maintained at a temperature of 40 C. in an incubator. The sections are next washed in water and immersed for ten minutes in a saturated alcoholic solution of picric acid. After washing for twenty minutes in water they are transferred for about fifteen minutes to 50 per cent, alcohol. In about a quarter of an hour they will be properly decolourised, and may then be dehydrated in absolute alcohol, cleared in cedar oil and mounted in balsam. By this method the clubs only are stained, the mycelium remaining uncoloured. I now come to consider a small class of selective stains which are dependent upon micro-chemical reac- tions. These are practically confined to two, namely, the methods for demonstrating waxy degeneration (amyloid reaction), and for the exhibition of iron in the tissues. The amyloid reaction. There are several methods for showing this reaction, by means of which portions of tissue which have undergone waxy degenera- tion are clearly distinguished by change of colour from the normal portions. The chief processes are as fol- lows : i. A thin section of the tissue is placed on a slide, and a drop of an aqueous solution of iodine upon it. IO2 MEDICAL MICROSCOPY. The specimen may then be mounted in the iodine- mounting fluid (see p. 109). The edge of the cover- glass being fixed by a ring of Dammar varnish. On examination the affected portions appear as dark yellow or brown, the remainder of the section being light yellow. 2. This was Virchow's original method. The sec- tions as before are dipped into a watery solution of iodine, and are then conveyed to a 2 per cent, solution of sulphuric acid. As soon as the blue colourisation appears, the sections are swilled in water, and mounted in Farrants' solution. 3. Methyl aniline violet. The stain is made as described on p. 95. The sections are placed in it for about five minutes, are then washed in water for twenty-four hours, and mounted in Farrants' solution. The amyloid substance will be found to be coloured red, the rest of the tissue being a dull blue. The preparations will not keep well in the mounting medium unless they have been previously fixed for half an hour in a one per cent, solution of perchloride of mercury. . 4. Iodine green. The sections are soaked in this stain (see p. 80) for ten minutes, then washed in water and mounted in Farrants' solution. Very beautiful re- sults are thus obtained, the affected portions being coloured a rose pink, whilst the remainder assume a bluish green colour. Iron reaction. Iron is occasionally found free in the liver and other organs in diseases in which excessive destruction of red blood corpuscles is brought about. Dr. Mott in 1889, before the Pathological Society, showed some beautiful sections of liver from a case of pernicious anaemia. The iron was seen in the form of blue granules (converted by ferrocyanide of potassium SELECTIVE STAINS. IO3 into Prussian blue) in the cells and capillaries of the portal zone. The method for obtaining such specimens is as fol- lows : The organs must be hardened in alcohol, and after sections have been cut, they are placed in a solu- tion of ferrocyanide of potassium acidulated with hydro- chloric acid. In a very few minutes the sections will turn blue, and may then be removed and mounted in glycerine. An objection to this method lies in the partial solubility of the prussian blue, which escapes and tinges other parts of the tissue a similar colour. More exact definition of the deposit may be obtained by exposing the sections to a watery solution of sul- phide of ammonium, which precipitates the iron as dark greenish granules, or by placing them in a solution of sulphocyanic acid, when the granules will be of a blood- red colour. Before concluding the chapter, this seems to be the most suitable opportunity for referring briefly to the principle method which is employed for demonstrating the karyokinetic figures in nuclei, it is known as Flemming's method. The organ to be investigated must be removed from the body immediately after death ; the larynx, after an operation for excision for instance, is very suitable for this purpose. The tissues must then be " fixed," so that the various components are made to retain their living structural appearance. Flemming's fixing mixture has the following formula : Chromic acid (i per cent, solution) . . 15 parts Osmicacid (2 ) . . 4 Acetic acid i part. Small pieces of the tissue are placed in this solution for two days, and are then washed in a stream of run- 104 MEDICAL MICROSCOPY. ning water for at least an hour. They are afterwards hardened in alcohol, for which purpose three days are required. They may then be embedded in celloidin or paraffin (the former being preferable) and sections cut. The best staining fluid is undoubtedly safranin (see p. 80), after which they are washed in acidulated alcohol (see p. 76), then dehydrated in absolute alcohol, cleared in oil of cloves and mounted in balsam. Most of the nuclei will by this method be almost decolourised, whilst the karyokinetic figures will be stained a brilliant red. Another process for demonstrating these figures which produces very pretty results, but not quite so well de- fined as with Flemming's method, is after hardening the tissues in alcohol to stain them by Gram's method (see p. 95), when those nuclei which are undergoing division will be coloured a deep purple, whilst the others will be almost decolourised. CLEARING AND MOUNTING. 105 CHAPTER VII. CLEARING AND MOUNTING. THE final stage in the preparation of microscopic speci- mens is perhaps the most difficult, and requires more practice than any of those previously described. It not unfrequently happens that after a section has been successfully cut and stained, it is irretrievably damaged in spreading it out on the slide. Skill in manipulation can only be obtained after much practice, and each worker will probably arrange methods of his own, but a few general hints may be of service in indicating the more common errors and modes of correcting them. In order that a section may be satisfactorily examined its interstices must be filled by some material having a refractive index higher than that of air, in order to prevent the irregular reflection resulting from the want of homogeneity. Before these reagents can permeate the tissues, the sections have to be cleared. The liquids employed for this purpose have also a high index of re- fraction, by which the tissues are rendered transparent, and at the same time the alcohol which has been used for dehydration is removed, and the balsam or other resinous media can easily flow in. Two varieties of mounting media are employed, namely, fluid and solid, and the methods for employing these differ very much from one another. We will con- sider first the use of fluid media. After the completion of the staining process the 106 MEDICAL MICROSCOPY. sections are placed in water ; each one is then removed to a glass slide by means of the section lifter. The specimen must be carefully spread out on the lifter by means of needles, and then very cautiously pulled up out of the water. Unless this is slowly done the section is nearly sure to curl up, or float off from the lifter. Having successfully removed the latter, with the former adhering to it, the lifter is laid on the centre of the glass slide, and one corner of the section having been fixed by means of a needle, the lifter is cautiously pulled from under it and not the section from off the lifter. Sometimes in spite of all care, especially if the section had been cut by freezing, the specimen will obstinately refuse to lie properly on the lifter, whilst this instrument is being removed from the water. A very good ex- pedient is to place a clean slide in the dish containing the water, and then to spead a section on it by means of a needle, by gently steadying it thus, the slide may be raised, and so the specimen lifted out lying perfectly flat. The slide is now tilted, so as to allow superfluous fluid to strain off, and as much water as possible is removed by placing a piece of folded filter paper near the section, or if the object be not too delicate the paper may be gently pressed on it. A drop of the mounting fluid is then added, and a cover glass laid on it in the following manner. The cover glass is taken between the finger and thumb of the left hand and placed with one edge downwards, just beyond the drop of fluid. The upper edge of the glass is supported by a needle which is gradually lowered until the glass comes in contact with the liquid. If this be carefully done no air bubbles will be included, but if this should happen CLEARING AND MOUNTING. IOJ the glass must be lifted up, and a fresh one applied. The amount of fluid should be sufficient so as just to occupy the space beneath the cover-glass, as any excess interferes with the subsequent application of the fixing material. This quantity can only be gauged by practice. The cover-glass having been placed in position, it must be fixed by one of the cementing materials pre- sently to be mentioned. For this purpose the glass slide should be held in the left hand. Any excess of mounting fluid which has escaped must be removed with a soft cloth. A glass rod is then dipped in the cementing fluid and gently passed round the edge of the cover-glass. This should be done by holding the rod a fraction of an inch above the glass so as to avoid touch- ing it, but at the same time be near enough to guide the fluid. The rod will probably have to be dipped in the liquid two or three times before the circuit of the glass is complete. It is needless to add that only a very narrow portion of the cover-glass must be included in the ring ; but the same precaution need not be taken with the glass slide. As the , cement hardens a second or third coating should be applied, so as to form a raised border. The most common fluid media employed must now be considered. Glycerine. This is an excellent medium for sec- tions which have been previously hardened and are stained in dyes which are easily extracted by means of alcohol. It is also suited for specimens such as those containing fat which would be rendered useless by de- hydration, and further, for those sections which have been treated with metallic salts. It has the great disadvantage of causing fibrous tis- sue to swell up, and so lose its characteristic structure. I08 MEDICAL MICROSCOPY. In mounting fresh specimens this difficulty may be over- come by covering them the first day with equal parts of glycerine and water, and on the following day remov- ing the cover-glass and adding pure glycerine. This reagent is much used for preserving parasites. For this purpose such specimens should be immersed in equal parts of glycerine and water, contained in a small covered capsule for twenty-four hours. On the second day a small portion of the liquid must be removed with a pipette, and a few drops of glycerine added. This process being repeated day by day until the parasite is surrounded by the pure reagent. Another excellent way of treating delicate objects is to employ a mixture of glycerine, alcohol and water. By the evaporation of the alcohol the liquid gradually increases in density, and after some time a cover-glass may be applied and fixed, or the object brought into pure glycerine. Lee recommends the following mixture : Glycerine i part Alcohol i ,, Water 2 parts. Glycerine jelly. This should be procured already prepared. The bottle containing the reagent is placed in hot water until the contents are fluid. The object having been placed on the slide and dried, the latter is slightly warmed and a drop of the jelly added. The cover-glass is then applied and gently pressed down, any air bubbles being expelled by pressure with a glass rod. The jelly soon solidifies and fixes the glass, but it is more secure to protect the preparation with a ring of cement. This reagent is useful for vegetable substances and for parasites. CLEARING AND MOUNTING. ICX) Glycerine mounting fluid : Camphor water 2 parts Glycerine i part Pure gum Arabic . . . . nf parts. This preparation is chiefly used for sections which have been injected with Prussian blue. The method of using it does not differ from those of the other fluid media. Farrants' solution. Farrants' solution being very difficult to prepare is best procured ready made. It is an extremely useful reagent combining most of the ad- vantages of glycerine, with but few of its disadvantages. Sections preserved in it, however, are apt to become cloudy after some years. Sections are mounted in the usual way, and after some days the fluid dries at the edge of the cover-glass, so fixing it ; but it is better to add a ring of Hollis' glue, or zinc-white cement. It is especially useful for mounting fresh specimens or sections which have been stained to demonstrate the amyloid reaction (see p. 101). Iodine mounting fluid : Glycerine 6 parts. Liquor iodi (B.P.) . . . . 3^ ,, Water 6 The ingredients are mixed and six parts of gum Arabic added. This preparation is only employed for sections stained in iodine. The method of using it is the same as for glycerine. Two other fluid media may be mentioned, namely, acetate of potassium in a saturated solution for mounting vegetable tissues or those exhibiting fatty change ; and castor oil for mounting crystals which are soluble in Canada balsam. 110 MEDICAL MICROSCOPY. CEMENTING MATERIALS. A section having been mounted in one of the fluids just mentioned, a ring of cement is applied to fix the cover-glass. If a square one has been used the cement is applied in the manner already described (see p. 107) ; but if the glasses are circular a Shadbolt's turn-table may be employed. The most usual materials employed are Canada balsam which must be liquid enough to run easily, or Dammar varnish (see p. 115). Other cementing substances are : Brunswick black, Hollis's marine glue and gold size. Any of the above may be supplemented by a ring of zinc white. All these materials are best secured ready prepared, as their composition is complicated and not readily car- ried out. For sections mounted in glycerine, Dr. Woodhead (" Pathological Histology," p. 80) strongly recommends the method suggested by Dr. Marsh, namely, a solution of gelatine ; the rationale of the process being that gelatine readily mixes with the glycerine in its imme- diate neighbourhood. The solution is prepared by placing a small quantity of gelatine in a narrow glass beaker, covering it with water, and allowing the gela- tine to take up as much of the water as it will. Any superfluous water is poured off and the mixture is heated and three or four drops of creosote are added to each ounce of the fluid. The mixture will then solidify, and each time it is needed, the bottle contain- ing it is immersed in a cup of warm water to render the contents fluid. All superfluous glycerine must be re- moved by the aid of a camel-hair brush and a damp CLEARING AND MOUNTING. Ill cloth. A ring of the gelatine fluid is painted round the edge of the cover-glass ; as soon as this is set it is painted over with a solution of bichromate of potash, made by dissolving ten grains of that salt in one ounce of distilled water. Dr. Marsh recommends that " this application of bichromate of potash, should be made in the daytime, as the action of daylight upon it, in con- junction with the gelatine, is to render the latter insolu- ble in water." The preparation is finished by washing it over with methylated spirit to remove all the glycerine, and then a ring of zinc-white is applied. Mounting in solidifying media. This is ac- complished by preserving the sections in a transparent refracting material which solidifies on drying into a vitreous mass. Various resins are employed, dissolved in a fluid which easily evaporates. As these substances are insoluble in water this must be removed. This process is known as " dehydration." After staining and washing, the sections are placed in absolute alcohol, by which means all the water is rapidly extracted. The time usually required is five minutes, but if the specimens are thick a longer period may be necessary. Great care is needed in the dehydration of sections embedded in celloidin. As this material is soluble in absolute alcohol it is advisable to employ 95 per cent, alcohol to abstract the water, allowing about ten min- utes for the process. Very delicate specimens may be treated thus : A clean cover-glass is placed at the bottom of a shallow dish containing water, and the sections floated on to it. By means of needles one specimen is spread out on the cover-glass, which is removed with a pair of forceps, 112 MEDICAL MICROSCOPY. and two or three lots of absolute alcohol poured over it. The spirit is allowed to evaporate, and a clearing solu- tion applied in the same way. Finally the section is mounted in balsam. In the ordinary course of events each specimen is removed from the alcohol on a section lifter and im- mersed in one of the clearing agents. Owing to the rapid escape of the alcohol the section flattens out and floats on the surface of the fluid, but it should be gently forced below the surface with a needle. The specimen will soon become perfectly transparent. If any cloud appears, or if, white spots are seen (this being best tested by viewing it against a black surface, such as the sleeve of a coat), dehydration has been inefficiently per- formed, and the section must be again placed in the absolute alcohol. When perfectly transparent each section is removed to a clean glass slide by means of a lifter which is laid flat on the slide. One corner of the specimen is then fixed with a needle, and the lifter cautiously withdrawn. If this has been done with skill the section will be almost flat. If any part of it has become rolled up, .or wrinkled, this defect must be remedied by means of two clean needles, the one being used to fix the section, and the other to remove the creases. This is rendered easier by having an excess of the clearing reagent on the slide. The slide is now tilted to allow the clearing fluid to run off. This must be done gently, and if necessary the section steadied by a needle to prevent its slipping off. After draining, the superfluous oil is removed with a cloth or a folded filter paper may be laid on the section, and -gently pressed. . A drop of Canada balsam or Dammar varnish is then CLEARING AND MOUNTING. 113 placed on the section and a clean cover-glass applied in the same manner as described on p. 106. By this means the intrusion of air bubbles is prevented as far as possible, but should any occur an attempt may be made to remove them by gentle pressure on the cover- glass. Very often this is unavailing, and it is better to leave them alone, as they are generally expelled by the contraction of the balsam, and undue pressure may either damage the section, or break the cover-glass. Occasionally after two or three days gaps are noticed beneath the cover-glass which may even trespass upon the section. The balsam must then be liquefied by gently warming the slide, and a drop of balsam placed on the edge of the cover-glass nearest the gap. A little pressure will drive out the air, the resin taking its place. For some days after mounting, the slides should be allowed to lie flat, protected from dust, and when the balsam has thoroughly set they may be stored edge- ways. Slides should be labelled as soon as finished, the description including the name of the organ, or nature of the tissue, the disease, method of staining and date. We now have to consider the various clearing re- agents in use. Any of the essential oils will answer the purpose, but those most commonly employed are, oil of cloves, creo- sote, cedar wood oil, xylol, and oil of bergamot. Oil of cloves. Although more generally employed than any of the others it has several disadvantages ; it certainly produces excellent results, but has a clinging and penetrating odour, rapidly dissolves celloidin, and removes the aniline dyes, picric acid, and eosin. If sections be exposed too long to its action, they become i - MEDICAL MICROSCOPY. rigid and brittle, and should therefore be removed di- rectly they are transparent. Creosote. I prefer this to any of the others for ordinary microscopic work, except for use with speci- mens stained in the aniline dyes. Its odour is not dis- agreeable, sections may remain in it for any length of time without becoming brittle, and it does not dissolve celloidin. Cedar wood oil. For bacteriological purposes this is undoubtedly the best clearing reagent. It does not dissolve celloidin, or abstract the aniline dyes. It has, however, rather a tendency to stiffen the sections, espe- cially if they are left in it for some time. Xylol. This is not very often used, except in special methods, such as Weigert's modification of the Gram method, and Kiihne's processes for the demonstration of bacteria in the tissues. Oil of bergamot. This is a good clearing agent, and has the advantage of not dissolving celloidin or affecting the aniline dyes. It has, however, a very dis- agreeable smell. Turpentine, oil of cajeput, and oil of origa- num, are sometimes employed, but possess no advan- tage over the others. Practically the only two resins employed for mount- ing specimens are Canada balsam and Dammar varnish. Canada balsam. This is suitable for all speci- mens which will bear dehydration in alcohol, and clear- ing with an essential oil. It may be procured dissolved in various fluids. The most common being turpentine or chloroform, or a mixture of the two. Benzol also is sometimes employed. Any of these solutions are suitable for ordinary sec- CLEARING AND MOUNTING. 115 tions, but for bacteriological work, or for sections stained in the aniline dyes, xylol is decidedly the best solvent. Balsam dissolved in xylol may be procured from most of the firms that supply microscopical apparatus, or any of the ordinary solutions may be evaporated to dryness over a water bath, taking care that the balsam does not get over-heated and become brown. The dried resin is dissolved in xylol. The consistency of the solution should be such that it flows easily, that is to say, it should be nearly as thin as glycerine. Taking everything into consideration this is the best kind of balsam to use. It should be kept in a special "balsam bottle," which consists of a wide-mouthed bottle furnished with a ground glass cap and a small glass rod lying in it. Dammar varnish. It is also well to procure this ready made, but its composition is as follows : Gum Dammar 2 parts Gum mastic i part Turpentine 4 parts. Chloroform 2 ,, The gums are mixed with the solvents until they are dissolved, and the mixture filtered through cotton-wool. This preparation has the advantage of being quite colourless, and is therefore useful for photo-micrography. Sections preserved in it do not keep well, becoming after a time cloudy and granular. It must not be used for sections stained in the aniline dyes, or for those containing micro-organisms. Breaking down old specimens. In the course of preparation of a number of specimens a certain pro- portion will from one reason or another be spoilt. The glass slides and cover-glass need not be wasted, but a jar should be kept into which waste alcohol should be I 2 Il6 MEDICAL MICROSCOPY. thrown, such as that used for dehydration. Into this rejected slides may be put from time to time, and after soaking for a week or two may be removed, and will then be quite suitable for another occasion. At other times valuable specimens may be damaged in some way ; the cover-glass may be broken, or the sections may have faded. A section which has been mounted in glycerine is easily released by cutting through the cementing material with a knife, and then placing the slide in water, when the section will float off. It may then be stained, and mounted again in the usual way. One which has been preserved in balsam must be immersed for some time in chloroform. After some hours the section will be freed, and should then be allowed to lie in absolute alcohol for a day. It may subsequently be restained and remounted. PREPARATION OF SECTIONS. CHAPTER VIII. COMPLETE PROCESSES FOR THE PREPARATION OF SECTIONS. IN this chapter it is proposed to give as briefly as possible two methods of preparing sections. In the preceding chapters all the processes have been con- sidered in detail, and should be carefully studied before this chapter is consulted. Whilst working I have found it convenient to have a summary of the various stages through which the specimens have to pass before they are complete, and give two of them here ; others may be compiled by referring to what has already been de- scribed. Method I. Harden in Muller's fluid, cut by freezing, stain in haematoxylin and eosin, mount in Canada balsam. 1. Place the specimen removed as soon after death as possible, in a bottle containing several times its bulk of Miiller's fluid. Change in twenty-four hours, merely pouring the fluid off without removing the specimen from the bottle. Change again on the third and seventh days, and at the end of each week until the sixth. Examine to see if the specimen is sufficiently hardened, if this be the case, transfer to dilute spirit (fifty per cent.) until wanted, if not, put back in Miiller's for another week. 2. Place the specimen in water until it sinks, and the washings are no longer coloured. Transfer to solution Il8 MEDICAL MICROSCOPY. of dextrin (see p. 48) for twenty-four hours. Remove the specimen by means of forceps to the plate of an ether freezing microtome. Work the spray cautiously so as not to overharden. Float the specimens off the knife into water. 3. Take a dilute solution of hsematoxylin and allow sections to remain in it for several hours, or in a strong solution for about three minutes (p. 73). Wash the sections in water and allow them to float exposed to the light for two or three hours. 4. Transfer to absolute alcohol, containing a few drops of alcoholic solution of eosin, and allow the section to remain for five minutes ; or soak it in a watery solution of eosin for ten minutes and then place in absolute alcohol for three minutes. 5. Clear in creasote. 6. Mount in Canada balsam. Method II. Harden in alcohol, embed in celloidin, stain in carmine and picric acid, mount in Canada balsam. 1. Cut small portions of tissue and place them in absolute alcohol. Change this solution in twenty-four hours. The specimens will be hardened in from two to four days, depending upon their size, and may be left in the alcohol for an indefinite period. 2. Place the specimens in equal parts of absolute alcohol and ether for twelve hours ; transfer to celloidin of the consistence of mucilage for twelve hours ; place each specimen on a dry cork, and allow celloidin to set ; then immerse in a large jar containing eighty per cent, alcohol, for at least twelve hours. Select one specimen and place in the clamp of the microtome. Be careful to have the knife and specimen constantly covered with an excess of dilute alcohol (fifty per cent.). Transfer PREPARATION OF SECTIONS. 119 sections when cut by means of a camel's hair brush to water, where they will spread out, then keep them in dilute spirit. 3. Place the sections in lithium carmine (p. 75) for ten minutes. Wash in acidulated alcohol (p. 76) until the sections are a light pink, or until no more colour comes out. 4. Transfer the sections to absolute alcohol containing a few drops of a saturated alcoholic solution of picric acid for five minutes. 5. Clear in cedar oil. If after the preparation of one specimen the contrast-stain is found not to be strong enough, more picric acid must be added to the alcohol. If white patches are noticed in the sections, dehydration has not been properly accomplished, and the sections must be put back into the absolute alcohol. 6. Mount in Canada balsam. I2O MEDICAL MICROSCOPY. CHAPTER IX. METHODS OF PREPARING SECTIONS OF THE CENTRAL NERVOUS SYSTEM. IN order to prepare sections of the brain or spinal cord, very complicated and rather difficult methods have to be adopted. It has, therefore, been thought advisable to devote a separate chapter to their consideration, and to describe in detail the various processes from the time that the cord or brain are removed from the body, until the sections are complete. As the examination of the spinal cord is more frequently required than that of the brain, the following description will be understood to apply to the cord unless otherwise stated : Hardening. The spinal cord should be removed from the body as soon after death as possible, and there will be less chance of injury if it be removed from the front instead of from the back as is usual. The method of thus doing not being generally known, a brief outline of the procedure is here given. It is as follows : The viscera having all been removed, the inter-vertebral sub- stance above and below the second lumbar vertebra is cut through, the pedicles of the vertebra divided with bone- forceps, and the body then wrenched out with lion forceps. A specially devised chisel having a point projecting from its cutting edge is inserted with this point in the verte- bral canal, and is driven with a mallet through the entire line of pedicles on the right side to the base of the skull ; a like manoeuvre is performed with a similar CENTRAL NERVOUS SYSTEM. 121 chisel on the left side. The bodies of the vertebrae having been removed, the theca is fully exposed and is slit up with probe-pointed scissors. The cord is re- moved by dividing the nerves on either side, and those of the cauda equina and gently lifting it with the mem- branes out of the canal. Transverse cuts are made at intervals of half an inch if the whole cord is intended for preservation or the diseased portion is separated from the healthy. The best fluid for hardening purposes is a solution of bichromate of ammonium (two per cent.) or Miiller's fluid. The former is more rapid in its action, but the latter more regular, and is therefore to be preferred. The cord should be suspended in a tall jar, which should be large enough to contain sufficient fluid to measure several times the bulk of the specimen. The hardening reagent must be changed at the end of twenty-four hours, on the third and the seventh days, and at the end of each week until the process is completed, the jar in the meanwhile with its contents being kept in a cool place and in the dark. Five or six weeks will probably be required, and the cord should be tested from time to time to see if it is properly hardened. To do this, a small section of the specimen should be taken, and placed in water, it ought to remain flat, if it curls up it is not yet ready. Exposure to Miiller's fluid generally renders the tissue brown in colour. When sufficiently hardened, the cord is washed in water and steeped in eight per cent, alcohol, where it may remain until arrangements are made for cutting. For the sake of convenience the specimen may be cut into segments, which can be placed in small bottles carefully labelled. Cutting. Sections may be cut by freezing, but this 122 MEDICAL MICROSCOPY. will be found by no means satisfactory, and it is far better to embed the cord in celloidin or paraffin. The choice of these two will depend upon many circum- stances, in a great measure upon the microtome it is intended to employ. If a series of sections are to be cut by the Cambridge Rocking microtome, paraffin is un- doubtedly to be preferred, but if any other microtome be selected celloidin should be chosen. The method of embedding in paraffin does not differ from that described on p. 52. For celloidin, after re- moval from the hardening reagent the specimens are placed in absolute alcohol for two days, and are then transferred to equal parts of ether and alcohol, for at least twelve hours. They are next immersed in a thin solution of celloidin for some hours, and afterwards in another solution about the consistence of mucilage. After another twelve hours they are deposited upon pieces of cork a little larger than the specimens, and when the celloidin has commenced to set, the corks are thrown into a large jar containing eighty per cent, alcohol, care being taken that the celloidin is below the surface of the liquid. In twelve hours, sections may be cut by Richert's or Schanze's microtome. Hamilton has introduced a method by which sections, which have been embedded in celloidin, may be cut on a freezing microtome. The specimens must be removed from the corks and placed for forty-eight hours in Erlicki's fluid which has the following composition : Potassium bichromate . . . . 2-5 parts Sulphate of copper '5 part Water 100 parts. This is done in order to free the preparations from the spirit, and they are then placed in the following mixture : CENTRAL NERVOUS SYSTEM. 123 Sulphate of copper *5 part Potassium bichromate .... 2-5 parts Mucilage of syrup and gum . . 100 parts. This solution with the specimens should be kept for two or three days in a warm chamber at 38 C. Sections are then cut in the freezing microtome, re- ceived in Erlicki's fluid, washed in methylated spirit, and stained according to Weigert's method presently to be described. If the specimens are cut with a Richert's microtome, sections will, as a rule, be easily obtained, but if the at- tempts prove unsuccessful, the precautions and measures suggested on p. 62 should be carefully followed. For series-cutting the Cambridge Rocking microtome is undoubtedly the best, or series may be obtained by the process described on p. 64. It may so happen that after embedding in celloidin it is desired to cut series by the Rocking microtome. This may be done by remov- ing the specimens from the corks, and immersing them in benzol, and then transferring to the liquid wax as described on p. 49. They may next be fixed to the Rocking microtome in the usual manner. Staining. Nearly all specimens of the central nerv- ous system are now stained by one of three methods, namely, those of Weigert or Pal, or in aniline blue black. For the demonstration of certain lesions the ordinary methods are preferable. For instance, when it is de- sired to examine miliary aneurisms in the brain, ammo- nium carmine should be used (see p. 78), or haema- toxylin and eosin. A simple method which stains the ganglion cells very well, is to dilute Stephens' blue-black ink one-half to two-thirds (Harris and Power). 124 MEDICAL MICROSCOPY. As a rule sections are made for the purpose of in- vestigating tracts of degeneration, and for this purpose one of the processes named above, and which we now proceed to describe, must be employed. Weigert's method. The solutions required are : Solution i : Saturated solution of acetate of copper . i part Distilled water i Solution 2 : Haematoxylin i part Alcohol (90 per cent.) 10 parts Concentrated sol. of lithium carbonate i part Distilled water 90 parts. The haematoxylin must be dissolved in the absolute alcohol, the water added, and the mixture boiled. After it is cool the lithium carbonate is added. This solution will not be ready for use for a couple of days and will not keep for more than a month. Solution 3 : Borax 2 parts Ferricyanide of potassium . . 2-5 parts Water 200 parts. It is essential that the specimens should have been hardened in Miiller's fluid. The first step consists in placing the sections, whe- ther single or in series, in the solution of acetate of copper for twenty-four hours, and then thoroughly washing them for about half that time in water, which should be frequently changed. This part of the pro- cess may be performed in mass before sections are cut, by placing the specimens after embedding in the copper solution for two or three days, or if a warm chamber at 38 C. can be employed, forty-eight hours will be suffi- cient, sections being then cut. When the washing is CENTRAL NERVOUS SYSTEM. 125 complete the sections are placed in the haematoxylin solu- tion for one or two days, and are then removed to water. They should now exhibit a deep blue-black colour, and be perfectly opaque. If this be not the case a drop or two of a saturated solution of lithium carbonate may be added to the water, and if the desired appearance is not then produced the specimens must be returned to the haema- toxylin solution for another twenty-four hours. They must next be thoroughly washed in water which should be changed every few hours, until the washings are no longer coloured. Unless this process be thoroughly carried out the sections will ultimately develop a num- ber of dark spots which will render them practically useless. The next step consists in differentiating them. For this purpose the sections are placed in the borax and potassium ferricyanide solution (Sol. 3). They will at once be seen to undergo a change, becoming lighter in colour, and the grey matter gradually becoming separ- ated from the white. This part of the process must be carefully watched, and as soon as the two parts of the cord are clearly denned they must be removed to water. The time required varies from five minutes to half an hour. If left too long the haematoxylin will be removed and the specimens appear uniformly brown. After washing in water the sections must be dehy- drated, cleared in creosote, and mounted in balsam. If the specimens were previously embedded in celloidin, it will be better to use 95 per cent, alcohol for dehy- drating, otherwise owing to the solution of the embed- ding material in the absolute alcohol, the sections will probably fly in pieces, and thus all the labour and trouble bestowed upon them will be lost. Stained in this manner, the white substance of 126 MEDICAL MICROSCOPY. Schwann assumes a purple-black colour, whilst the connective tissues, ganglion cells, and axis cylinders are dyed an orange-brown. If there are tracts of de- generation (sclerosis) these also assume the latter colour. Pal's method. This is a modification of the pre- ceding. The solutions required are : Solution i : Haematoxylin -75 part Distilled water 90 parts. Alcohol 10 ,, to which is added immediately before use a small quan- tity of a saturated solution of lithium carbonate in the proportion of three drops of the solution to ten c.c. of the haematoxylin stain. Solution 2 : Permanganate of potash '25 per cent, solution. Solution 3 : " Pal's solution." Oxalic acid ......... i part Potassium sulphite i ,, Distilled water 200 parts. The specimens are hardened in Miiller's fluid, as de- scribed above and embedded in celloidin, the sections being cut either singly or in series. They are at once transferred to the hsematoxylin solution, where they remain for five or six hours, and are then transferred to water, containing a drop or two of a saturated solu- tion of lithium carbonate. They should then be bluish-black in colour and opaque. The sections must remain in water, which should be frequently changed, until no more colour will come out. The method of differentiating is the characteristic part of this process. The sections are first placed in the CENTRAL NERVOUS SYSTEM. 127 permanganate of potash solution for from fifteen to twenty seconds ; after which they are immersed in Pal's solu- tion. Here they must be carefully watched until the white and grey matters are distinctly defined. This will probably be brought about in from one to two minutes. If any black spots appear the sections must be again placed in the permanganate of potash for a few seconds, and then again washed in Pal's solution. After this they must be washed in water for a quarter of an hour. If they are now dehydrated (95 per cent, alcohol) cleared, and mounted in balsam, the medullary sheaths will be found stained a bluish -black colour, the remainder of the tissue being white. Counter-staining is therefore advisable, and for this purpose borax carmine (see p. 79) is the best solution to use. Pal-Exner method. This process is more rapid than the preceding (although in some respects more troublesome), and produces very good results. The nervous material cut into small segments is placed whilst fresh in about ten times its bulk of one half per cent, solution of osmic acid, where it remains two or three days, the solution being changed daily. The specimens are then removed, washed in water, and dipped for a few seconds into absolute alcohol, and are then embedded in celloidin or paraffin. Sections are cut in glycerine and removed to water. They are differentiated in the same manner as in Pal's solution, that is to say in permanganate of potash, and in the liquid known as Pal's solution (vide supra). Aniline blue-black. By staining sections of the central nervous system by this method, the nerve-cells are especially brought into prominence, being coloured a deep slaty blue. Two or three methods have been 128 MEDICAL MICROSCOPY. introduced, but the best is undoubtedly the one sug- gested by Lewis (" Human Brain," p. 125). Sections of the fresh brain or spinal cord are cut as thin as possible by means of an ether freezing microtome, are then floated on water, and afterwards immersed for the purpose of hardening in a two per cent, solution of osmic acid. After a few minutes (about eight to ten) they are washed in water and placed in -25 per cent, aqueous solution of aniline blue-black for two hours. The sec- tions are once more washed in water, and each is spread out on a slide, and allowed to become quite dry, after which it is mounted by the addition of a drop of Canada balsam and a cover-glass. In all the above processes a number of changes are required, and if the sections are moved from solution to solution by means of needles, they are very apt to be damaged, and much time is taken up in carefully man- ipulating them. To avoid unnecessary risk, a small perforated zinc sieve should be employed, in which the sections may be placed, and so transferred altogether from one solution to another. If it be desired to prepare specimens of the cortex of the brain, very good results may be obtained by adopt- ing Jolgi's "sublimate method." It consists of two processes : i. Hardening in bichromate of potash. 2. Treatment with bichloride of mercury. Small pieces of the tissue are placed in Miiller's for about four weeks, and are then passed directly into a watery solution of corrosive sublimate. The best strength to use is a -25 per cent, solution. The volume of fluid must be several times that of the specimens, and it must be renewed as often as it becomes yellow. An immersion of at least three weeks must be allowed, but longer than this im- proves the result. Sections are then cut by the freezing CENTRAL NERVOUS SYSTEM. 129 microtome, and afterwards thoroughly washed in water, otherwise they will be spoilt by the formation of a black precipitate. They are best preserved by mounting in glycerine. Prepared in this way the nerve cells and sometimes the blood-vessels are opaque and black. Oc- casionally the body of the cell and its finest processes are outlined with the utmost clearness. This is not a true stain, but Jolgi thinks that there is formed in the tissue elements a precipitate of some sub- stance that renders them opaque. I3O MEDICAL MICROSCOPY. CHAPTER X. INJECTION OF TISSUES. THE operation of injecting the blood-vessels is not often required for pathological purposes ; but very instructive specimens of emphysematous lung may be prepared in this way. In the physiological laboratory it is more often performed. The process is rather a difficult one, and for successful results requires more complicated apparatus than a private individual generally cares to set up. For the sake of completeness two simple methods will be described here, which with a little practice and pati- ence will answer very well. Two kinds of "injection masses" are in use, namely, one which is fluid at ordinary temperatures, and the other which is solid at the temperature of the air, but melts by heat ; the latter is more generally used. Several varieties of each have been introduced, but only two are of importance : Cold injection. Soluble Prussian blue ... 2 parts. Distilled water 100 ,, The Prussian blue is dissolved in the water, and a few drops of hydrochloric or acetic acid are added be- fore injecting. Carmine gelatine injection mass. Carmine 3 parts. Strong ammonia .... 6 ,, Glacial acetic acid ... 6 ,, Coignet's French gelatine . 7 ,, Water 80 The gelatine is cut up into small pieces, placed in 50 INJECTION OF TISSUES. 131 parts of the water, and allowed to swell up for four or five hours. The carmine is ground up in a mortar with a little water, and the ammonia added. This mixture is allowed to stand for two hours, and then poured into a bottle, the mortar being rinsed with the remainder of the water. The swollen gelatine, with any remaining water unabsorbed by it, is placed on a water-bath until it melts. A dark purple fluid results from the carmine mixture, to which the acetic acid is added, a drop at a time, the whole being thoroughly mixed ; as soon as the colour changes to a crimson the addition of the acid must be stopped. The crimson carmine is added little by little to the melted gelatine, the mass being continually stirred. Prof. Stirling states that this mass may be kept in a cool place for a long time if its surface be covered with methylated spirit. Before it is employed it must be melted over a water-bath, and filtered through fine flannel, rinsed out in hot water. The organ to be injected should be removed imme- diately after death, the main artery being carefully dissected out and cut as long as possible. The injec- tions may be made with a brass syringe, which should be short, and wide in the barrel to allow of its being readily cleaned, and it should have several nozzles of varying calibre, each provided with a shoulder and groove by which to secure it. Instead of fixing a nozzle directly on to the syringe, it should be fastened into the vessel by means of thread, and a stout piece of elastic tubing should be fixed over it, connected by the other end to the syringe. When in use the nozzle is filled with the injection fluid, as is also the syringe with the tubing attached, the latter being closed at its extremity by a brass clip, so that all air may be ex- cluded ; the tubing is then applied to the nozzle, the clip K 2 132 MEDICAL MICROSCOPY. removed and the piston slowly pushed in, the nozzle being steadied by an assistant. A pause must occasionally be made to allow the fluid to slowly force its way through the vessels. If the blue solution be used no further pre- cautions are necessary, but if the carmine injection-mass be employed, the organ to be injected must be placed in warm water (40 C.), and before use the syringe must like- wise be heated by drawing hot water up into it several times, the injection-mass itself being liquified over a water-bath. More frequently injections are made by a " constant- pressure " apparatus. That described by Prof. Stirling (" Practical Histology," p. 75) is the simplest and best. This apparatus consists of a tin trough large enough to contain the organ to be injected, and suffici- ent water is poured in to cover it. The water is kept at 40 C. by means of a gas burner or spirit lamp placed beneath it. In the same trough is placed the injection- mass in a Wolffs bottle. The bottle is connected with a large air chamber (a large wide-mouthed jar answers very well) into which water can flow from the tap, and thus compress the air. The pressure within the cham- ber can, if desired, be registered by means of a mano- meter. The compressed air acts on the surface of the injection-mass in the Wolft's bottle, and forces it through a tube which is attached to a cannula fixed in the main vessel of the organ. After the operation is over, the further treatment de- pends upon which material was used. If the Prussian blue solution has been employed, the organ must be at once placed in equal parts of methylated spirit and water to which a few drops of hydrochloric acid have been added ; it is left in this for twenty-four hours, after which it may be cut up and hardened in alcohol. INJECTION OF TISSUES. 133 When the warm carmine mass has been employed, the organ is rapidly cooled by being placed in running water until the mass is thoroughly set, when it may be cut into small pieces and hardened in alcohol. 134 - MEDICAL MICROSCOPY. CHAPTER XI. EXAMINATION OF FRESH TISSUES. IT may be often necessary, either in the post-mortem room, or especially in the operating theatre, to ex- amine a tissue as soon as it is removed from the body for purposes of diagnosis. Although the specimens so prepared are not as satisfactory as when they have been submitted to the processes of hardening, and the other stages necessary for mounting permanently, yet by means of certain precautions very fair results may be obtained. Tissues cannot be examined in a dry state on account of their opacity and the irregularity with which they transmit light. They must, therefore, be bathed in some medium which will change their appearances and vital properties as little as possible. The examination of blood, pus, effusions, &c., will be referred to in future chapters, and we shall consider here the treatment of tumours, and the method of examining such tissues as nerve fibres, splenic pulp, membranes, &c. The fluids usually employed for the purpose of moistening such specimens are, normal saline solution, serous fluid, aqueous humour, and oxidised serum. Normal saline solution. The preparation of this liquid was given on p. 33. It can be prepared in bulk and kept continually at hand. It alters the tissue but slightly, and is the most convenient of all these liquids. Serous fluid. This is most commonly taken ad- FRESH TISSUES. 135 vantage of in the post-mortem room where it may always be procured from the pericardial sac. Aqueous humour. This is obtained by punctur- ing the anterior chamber of the eye of a recently killed ox. Iodised serum. The usual formula for this is Tincture of iodine ... i part Serous fluid 100 parts Carbolic acid .5 part. Stirling (" Practical Histology," p. 24) recommends that a strong solution should be kept which may be diluted as required. The serous fluid obtained by allowing blood to coagulate, and pouring off the serum from the clot is placed in a bottle with a few crystals of iodine. This mixture must be frequently shaken. At first very little iodine is dissolved, but in a fortnight or three weeks the solutions become of a deep brown tint. When required, a little of the strong fluid is added to fresh serum until the latter has a light brown colour. In whatever manner prepared, this fluid alters the tissues slightly, and colours them a light yellow. Thin membranes may be placed at once on the slide, spread out as far as possible by means of scissors and forceps in a few drops of normal saline solution, and a cover-glass applied. A better result is obtained if a drop of a dilute watery solution of methylene blue is added. The stain must be very weak, or it adds to rather than detracts from the obscurity of the specimen. In the examination of fresh tissues, as a rule, the operation of teasing has to be adopted. A small por- tion of the muscle or other fibrous material is cut off with a pair of curved scissors and placed on a clean glass slide with a small quantity of one of the solutions previously named. The elementary parts of the tissue 136 MEDICAL MICROSCOPY. are then separated by means of needles. To effect this the particle under examination is fixed with one needle, whilst small fragments are torn off with the other. These are treated in the same manner until they are as minute as possible. The most suitable fragment is then placed beneath a cover-glass, the bulk of the tissue being removed. Another method which may sometimes be adopted, is to scrape the surface of the object to be examined, hav- ing previously moistened it with saline solution, and the scrapings are collected on the slide and examined under a cover-glass. Great assistance in either of the above processes is obtained by " softening " or " dissociation." Seve- ral reagents are used for this purpose, of which the following are the best : Iodised serum prepared as above may be used for isolating nerve fibres. Specimens must be allowed to remain in it for thirty-six hours, and the strong iodised serum should be diluted until the solution is of a deep cherry colour. A ten per cent, solution of common salt may be employed to soften connective tissue, being especially useful in the examination of tumours. Dilute alcohol, according to Ranvier, is very serviceable for epithelial tissues ; twenty-four to thirty- six hours is required for its full action. The best strength to use is one part of 90 per cent, alcohol with two parts of water. Caustic potash in 40 per cent, solution is recom- mended by Woodhead for isolating muscle cells. This is very rapidly accomplished, seldom requiring longer than half an hour. Potassium bichromate in two per cent, solu- FRESH TISSUES. 137 tion is useful for dissolving the cementing material be- tween the fibres of tendon, or for dissociating epitheliun. and the nerve cells of the spinal cord. It requires seven to fourteen days to complete the process. For specimens of the central , nervous system, Landois' fluid is perhaps as good as any. It has the following composition : Sodium sulphate '.., 5 parts. Neutral ammonium chromate . 5 ,, Potassium phosphate ... 5 ,, Distilled water 100 ,, Small pieces of tissue must remain in it from one to five days; In the investigation of tumours or any solid tissue for diagnostic purposes, it is advisable to attempt to pro- cure sections. After a considerable amount of prac- tice small sections may be cut with a sharp razor. The specimen is held in the left hand and short quick cuts are made across it, the blade of the razor then being dipped in a glass capsule containing water. The vessel should be placed over a black surface, and the cut particles gently moved about with a needle, two or three sections will then generally be found thin enough for mounting. A very useful little piece of apparatus for similar pur- poses is Valentin's knife (see fig. 18) which consists of two parallel blades fixed in a handle which can be ap- proached to or separated from one another by means of a screw. The tissue to be cut is held in the left hand. The blades of the knife are then set almost close to one another, moistened with water and drawn with a clean cut through the specimen, the resulting section being separated by a sharp turn of the instrument, and the blades afterwards separated under water. Although 138 MEDICAL MICROSCOPY. this operation appears very simple it requires consider- able practice before satisfactory sections will be ob- tained. The chief difficulty lies in judging the distance at which the blades should be set from one another for various substances. Experience alone can teach this accurately. The most satisfactory method of obtaining sections of fresh tissues is to dip them in gum (see p. 48) and to cut the sections by means of an ether freezing microtome. The manner of carrying this out has already been de- scribed on pp. 56 and 58. The sections may afterwards be stained, watery solutions of the dyes being preferable. Bismarck brown, magenta and methylene blue may be used, and the specimens afterwards mounted in gly- cerine. TISSUES AND ORGANS. 139 CHAPTER XII. PREPARATION OF INDIVIDUAL TISSUES AND ORGANS. IN commencing the study of the microscope in medicine some difficulty will be probably experienced as to which of the foregoing methods is applicable to particular tis- sues and organs. Some hints in regard to this matter will be found distributed through the previous chapters, but for the sake of easy reference directions will be given in this chapter to assist beginners in their choice of hardening and staining reagents gathered from the per- sonal experience of the author. But after some prac- tice the student will be competent to select his own favourite and successful processes. It should be borne in mind that in order to fully understand diseased organs and tissues the normal structure of these parts must be thoroughly known. The student will do well therefore, to examine a num- ber of healthy organs obtained from the post-mortem room, or from small animals such as cats, guinea pigs, &c., and in the preparation of these considerable pro- ficiency in hardening and staining will be acquired. We shall begin the list with normal histological struc- tures, such as epithelium and then pass on to the various organs. Squamous epithelium. This occurs either in a single layer, or in several layers ; in the latter case it is said to be stratified. As a single layer it lines serous membranes : blood and lymphatic vessels, &c., and is known as endothelium. A characteristic specimen may be obtained by killing some small animal such as a 14.0 MEDICAL MICROSCOPY. guinea pig or cat, and immediately opening the ab- domen, removing portions of the omentum, or the mesentery, and spreading them out on cork. They should then be placed at once in a solution of silver nitrate (p. 87), so as to demonstrate the outlines of the cells. Other specimens should be stained in haema- toxylin or alum carmine. Stratified squamous epithe- lium may be obtained from sections of skin, or better from the tongue of any small animal. They may be hardened in absolute alcohol, embedded in celloidin, and stained in picro-carmine or hardened in Miiller's fluid, cut by freezing and stained in carmine. For the purposes of demonstrating karyokinesis, the specimens should be stained in safranin or better, sub- mitted to the method described on p. 103. Columnar epithelium. This form of epithelium lines the mucous membrane of the alimentary canal from the cardiac orifice of the stomach, downwards, and the greater part of the ducts of the glands opening into it. Isolated cells may be obtained by scraping the mucous membrane of the intestine of some animal, or hardening a portion of the intestine in Miiller's fluid, embedding in celloidin and staining in haematoxylin and eosin. Secretory epithelium. Typical examples of this variety of epithelium may be obtained by scraping the cut surface of a liver and examining in glycerine, or by cutting sections of a liver, and staining in hasmatoxylin and eosin. Ciliated epithelium. This form is best obtained from the trachea of any small animal, hardening the specimen in Miiller's fluid, embedding in paraffin and staining in haematoxylin. Very satisfactory specimens may be procured from an ordinary nasal polypus by TISSUES AND ORGANS. 14! hardening immediately after removal in a saturated watery solution of corrosive sublimate for two or three hours, then thoroughly washing it in alcohol, and stain- ing in hsematoxylin. Transitional epithelium. This type of epithe- lium is best seen in the bladder ; to obtain specimens the bladder should be spread out on cork, and placed in dilute alcohol for twenty-four hours, and stained en masse in picro-carmine. A little of the mucous surface is scraped off and examined in glycerine. Areolar tissue. This is rather difficult to obtain. The best method is that described by Harris and Power (" Physiological Laboratory", p. 92), " By the injection into the subcutaneous tissue of a rat which has just been killed, of a 0-2 per cent, solution of nitrate of silver or osmic acid, a small artificial bulla is formed. This is allowed to remain for from ten to thirty minutes and is then opened with a pair of fine curved scissors, and the delicate subcutaneous tissue is rapidly removed and spread out on a glass slide. It is immediately covered with a cover-glass, and the preparation is stained for 24 hours with picro-carmine. Glycerine is passed through until all the superfluous staining material is removed after which the preparation is sealed up." White fibrous tissue. Typical examples of this tissue are found in tendons. Samples may be obtained either from the tail of a mouse or rat, or from an ampu- tated limb. Preparations may be made by teasing in normal salt solution, and staining with magenta, or por- tions may be hardened in Miiller's fluid, cut by freezing and stained in haematoxylin and eosin. Elastic tissue. This is most readily demonstrated by teasing out a small piece of the ligamentum nuchae of an ox in glycerine, or by mounting a piece of the 142 MEDICAL MICROSCOPY. omentum of some small animal, washing it freely with dilute acetic acid, staining with hgematoxylin and mounting in glycerine. Adenoid tissue (retiform).*-This is best exempli- fied by hardening a lymphatic gland in Miiller's fluid, cutting by freezing, and staining in haematoxylin. Some of the sections should then be mounted directly, and others placed in a test-tube half filled with water, and thoroughly shaken. This dislodges the lymph cor- puscles, and leaves the fine reticulum visible. The sections may then be dehydrated, cleared, and mounted in balsam. Adipose tissue. For this purpose specimens of skin hardened in Miiller's fluid, cut by freezing and stained in haematoxylin should be prepared, whilst other portions should be treated at once with osmic acid, so that the action of this reagent on fat cells may be ob- served. Gelatinous or embryonal tissue. This form of tissue occurs typically in the umbilical cord, and in the skin of the embryo. A portion of the cord may be hardened in Miiller's fluid and then in alcohol. Sec- tions are cut by freezing, and stained in picro-carmine. Very beautiful specimens may also be obtained by form- ing a bulla by the injection of a dilute solution of gold chloride into the subcutaneous tissue of an embryo, in a stronger solution of which it is subsequently stained (Harris and Power). Cartilage. There are three chief varieties, hyaline, elastic, and fibro-cartilage. Hyaline cartilage. Specimens may be obtained from the ribbed cartilages of young animals. They should be hardened in chromic acid or spirit, and sections cut by freezing, and stained in haematoxylin and eosin. TISSUES AND ORGANS. 143 Elastic cartilage obtained from the epiglottis or cartilages of the ear, should be hardened in spirit and stained in picro-carmine. Fibro- cartilage. This variety occurs in the inter- vertebral substance. Preparations may be hardened in spirit, and stained in haematoxylin and eosin. Other sections should be stained in chloride of gold. Ossifying cartilage. A long bone of a foetus, such as the femur, is decalcified in Perenzi's solution (see p. 45). Transverse and longitudinal sections are cut in celloidin, and stained in haematoxylin and eosin. Bone. Specimens are decalcified in Perenzi's fluid, cut in celloidin and stained in picro-carmine. . Teeth. Specimens are placed in a saturated solu- tion of picric acid until quite soft, then hardened in alcohol, cut in celloidin, and stained in picro-carmine or haematoxylin and eosin. In order to show the development of teeth, the jaw of a newly born kitten or puppy should be obtained, and first placed in Von Ebner's solution, then hardened in alcohol, and stained in lithium carmine and picric acid. Striped muscle. Any voluntary muscle may be taken, hardened in chromic acid and spirit, cut in cel- loidin and stained in picro-carmine. Fresh sections should also be examined by teasing in glycerine so as to make oneself familiar with unstained muscular fibres such as are often met with in sputum and vomited matters. Unstriped muscle. The most satisfactory method of demonstrating this tissue is by hardening portions of intestine or stomach in Miiller's fluid, cutting in cel- loidin, and staining in haematoxylin and eosin. Central nervous system (see special chapter). 144 MEDICAL MICROSCOPY. Brain. Specimens must be hardened in Miiller's fluid or in ammonium bichromate, and cut in celloidin. Sec- tions should be stained in various ways, in carmine, ani- line blue-black, and by the methods of Weigert and Pal. Spinal cord. The same methods as for the brain may be employed. If there is reason to suspect de- generation, Weigert or Pal's method should certainly be chosen. Nerve fibres. Portions of nerves should be taken from recently killed animals ; some of them may be hardened in Miiller's fluid, cut in celloidin, and stained in osmic acid, picrocarmine, or by Weigert's method. Others should be placed at once in osmic acid, then teased out, and mounted in Farrants' solution. Nerve cells. In order to demonstrate the different variety of nerve cells, specimens of the following should be hardened in Miiller's fluid, cut in celloidin and stained in aniline blue-black. 1. Spinal ganglia. 2. Sympathetic ganglia. 3. Spinal cord to exhibit multipolar nerve cells. 4. Cerebrum. 5. Cerebellum. Nerve endings. Simple tactile cells. These can be well demonstrated in a section of skin, placed immediately after removal (e.g., after amputation) in a solution of chloride of gold. Sections are then cut by freezing, and mounted in Farrants' solution. Pacinian corpuscles. To display these bodies, sections of skin should be cut, preferable from the foatus, hardened in alcohol, and stained in haematoxylin. (For further information as regards nerve-endings, see " Out- lines of Practical Histology," Lesson xxxiv., by Prof. Stirling). TISSUES AND ORGANS. 145 Blood-vessels. Arteries. The structure of an artery is best illustrated by hardening a piece of the aorta or carotid artery of a cat in Muller's fluid, cutting in celloidin and staining in picro-carmine. Veins. These may be prepared in a similar manner. Capillaries. These are best obtained from the pia- mater. The brain of an animal is placed for two days in a 2 per cent, solution of bichromate of potassium. The pia-mater is then stripped off, stained in haema- toxylin and eosin, and mounted in Farrants' solution. Lymphatic glands. The lymphatic gland of any animal may be hardened in Muller's fluid, cut in celloi- din, and stained in lithium carmine and picric acid. Specimens of sweat and sebaceous glands may be prepared in the same way ; the former being taken from the sole of the foot, and the latter from the skin of some animal. The eye. The structure of this organ may be demonstrated on the eye of a bullock which has been recently killed. It should be hardened in Muller's fluid for three weeks, embedded in celloidin, and the sections stained in haematoxylin and eosin. (For a full account of the pathological examination of the eyeball see a " Manual of Clinical and Practical Pathology," by Drs. Wynter and Wethered). Internal ear. The temporal bone of a cat or dog is removed and decalcified, and afterwards hardened in methylated spirit. It is then embedded in celloidin, and sections cut in the direction of the longitudinal axis of the cochlea. Tongue. Specimens of this organ should be hard- ened in Muller's fluid, cut in celloidin, or by freezing, and stained in safranin and in picro-carmine. Specimens of oesophagus, stomach, and intes- 146 MEDICAL MICROSCOPY. tine should be hardened first in Miiller's fluid, and then in absolute alcohol. After embedding in celloidin, the preparations should be so arranged that sections are cut transversely and longitudinally to the axis of the intestines. Haematoxylin and eosin is the best staining method. For the purposes of instruction, sections of the stomach should be made through the cardiac and pyloric ends, and from a portion of the greater curva- ture. From the intestine, portions should be selected from the upper part of the duodenum, the ileum, and colon. Lungs. Portions should be hardened in absolute alcohol, cut in celloidin, and stained in lithium carmine and picric acid. The student should make himself familiar witl) the structure of the bronchi and trachea. The process of embedding in celloidin is especially use- ful in examining specimens of lungs, more particularly in cases of pneumonia and phthisis. It is almost essen- tial when micro-organisms (e.g., tubercle bacilli) are to be sought for. Thyroid gland. Specimens are to be prepared by hardening the gland for twenty-four hours in a mixture of spirit and water, and then in absolute alcohol. Sec- tions should be cut in celloidin and stained in picro- carmine. Thymus gland removed from a foetus should be treated in the same way. Liver. Hardened in absolute alcohol, cut by freez- ing, and stained in haematoxylin and eosin. Sections of kidney are prepared in the same manner. Pancreas and supra-renal bodies. Hardened in Miiller's fluid, cut in paraffin or celloidin, and stained in haematoxylin and eosin. TISSUES AND ORGANS. 147 Spleen. Harden in Miiller's fluid. Place a few sections in a test-tube with water and thoroughly shake ; then mount the sections unstained so as to show the structure of the pulp. Other sections should be cut in celloidin, and stained in carmine and picric acid. Bladder. The organ should be distended with chromic acid and spirit, and immersed in the same mixture for twenty-four hours. Portions are then cut off and hardened in absolute alcohol, and treated in the same way as the intestine. Portions of the uterus, ovaries, and Fallopian tubes should be hardened in absolute alcohol, cut ii celloidin, and stained in haematoxylin and eosin. Specimens of testicle may also be treated in like manner. Embryological specimens should be hardened in Miiller's fluid, cut in celloidin, and stained in haema- toxylin and eosin. L 2 148 MEDICAL MICROSCOPY. CHAPTER XIII. THE EXAMINATION OF TUMOURS. ONE of the most frequent tasks which a pathologist is requested to undertake is the microscopic examination of tumours, and as this subject, therefore, comes strictly under the head of Medical Microscopy, it has been considered advisable to devote a chapter to a brief description of the most important growths of this kind. In the operating theatre, sections may be made, directly the tumour is removed, by means of a Valentin's knife, and examined either unstained in salt solution (see p. 33), or preferably stained by dipping them into a solution of haematoxylin. Their nature can then be generally easily recognized. Better specimens can be obtained in a very short time by cutting off a small piece of the tumour, placing it for a few minutes in gum solution, freezing it on a Cathcart's or Swift's microtome, and so obtaining thin and complete sections. If permanent preparations are required, the tissue must be hardened, stained, and mounted by the processes already described. The following classifications and descriptions are largely taken from Mr. Frederic S. Eve's chapter on tumours, in Mr. Mansell Moullin's " Surgery." Although cysts are usually described with tumours, an examination of their contents will be considered in a future chapter. Fibromata. These are tumours composed of adult or well-developed fibrous tissue. In consistence they EXAMINATION OF TUMOURS. 149 vary from a density approaching that of cartilage, to a succulent, yielding, but still not friable tissue. A section of the firmer fibromata displays a number of fibrous bands standing out on a grey, yellowish, or white ground ; or the section is firmly uniform and dull white. The fibres either interlace, or are arranged concentrically or in parallel lamellae. A scraping yields no juice. Microscopically, the firmer tumours are com- posed of looser or more compact bundles of fibres, often wavy and interlacing or disposed chiefly parallel to each other. Situated on the bundles in small numbers, are flattened nuclei, belonging to connective tissue cells, the protoplasm of which may be shown to anasto- mose by processes around the bundles. The softer forms are made up of interlacing fibrillae, more or less thickly studded with large round or oval connective tissue cells, whose protoplasm is continuous with the fibrillae. The chief diagnostic characteristics are then, fibrous tissue with scanty cells, and at growing points young cells surrounding adult and well developed fibrous tissue. Myxomata (fig. 20). Myxomata are new formations of loose, fibrillar, connective tissue, permeated with fluid which is rich in mucin. Common examples are polypi of the nose, rectum and uterus. A section exhibits a glistening, pale, jelly-like tissue, the surface of which is raised in the centre. Micro- scopically the tumour is composed of stellate and round cells, of which the protoplasm is prolonged in delicate filaments which meet one another and form a felting of fibrils. The stellate cells are chiefly charac- teristic of mucous connective tissue ; in some growths they exist exclusively, while in others the round cells preponderate. The vessels are usually abundant and 150 MEDICAL MICROSCOPY. clearly seen, owing to the transparency of the tissue ; they often form a wide meshwork. Enchondromata (fig. 21). These are tumours composed of cartilage chiefly of the hyaline variety. The section is glistening, smooth and translucent, and almost invariably shows a number of separate masses FIG. 20. Cells from a periosteal myxoma of the thigh (Ziegler).* or lobes divided by bands of connective tissue contain- ing blood-vessels. In some cases the section is often greatly modified by secondary changes. Microscopically, the commoner forms have practically the same structure as ordinary hyaline cartilage. In many the cartilage cells are more irregularly distributed * Figures 20, 21, 22, 23, 25, 26, from Ziegler's " Pathological Ana- tomy" are inserted with the kind permission of Messrs. Macmillan and Co. EXAMINATION OF TUMOURS. and more numerous in proportion to the matrix ; the capsules are larger, and in growing parts of tumours a single capsule often contains two or more cells. The . FIG. 21. Section from an osteoid chondroma of the humerus (Ziegler). a, cortical layer ; b, medullary spaces or cancelli ; c, periosteal growth ; rf, normal Haversian canals ; e, Haversian canals distended with cartilage, which at/ contains a core of new bone; g, cartilage developed from periosteum which at h contains bony tra- beculae ; i, cartilage developed from medullary tissue, which at k contains bony trabecula ; /, original trabecula ; m t remnants of medullary tissue. cells at the periphery beneath the perichondrium are flattened. Not rarely stellate ones are observed, es- pecially in the parotid tumours. 152 MEDICAL MICROSCOPY. Osteomata. Of these tumours there are three varieties ; the eburnated, compact and cancellous. The eburnated occur as small flat elevations on the surface of the skull. Similar growths also spring from the bones of the face. Compact osteomata are observed on the shafts of the long bones, with which their structure is identical. The name cancellous sufficiently describes the struc- FIG. 22. Section through the margin of a sarcoma affecting the intermuscular connective tissue (Ziegler). , normal muscle fibre ; ai, atrophied muscle fibre ; b, round cells intruded between the mus- cle fibres ; c, fully developed tumour tissue ; d, round cells resem- bling white blood corpuscles. ture of the third variety, the most common and widely distributed of all. With rare exceptions they are situ- ated on the diaphyses of the long bones, near to but not necessarily over the epiphysial disc ; they are covered with a layer of cartilage, the deeper surface of which grows continually and is gradually converted into bone. It is of course impossible to examine these tumours EXAMINATION OF TUMOURS. 153 by the methods described at the beginning of this chapter. They are, however, more easily recognised by their grosser characteristics, their position, &c., than by microscopical examination. In order to procure sections, it is necessary to place the portions which have been removed in one of the de- calcifying solutions previously described until all lime salts have been removed, when sections may be cut in the ordinary way. Myo-fibromata. As the name implies these tu- mours are composed of muscle and fibrous tissue in varying proportions. The muscle presents the ordinary characters of the unstriped, or involuntary variety, and is distributed in inter-lacing fasciculi. The outline of individual fibres is often not easily distinguishable in sections, but the fibres may be readily isolated by the process of teasing as described on p. 135, chromic acid being the best medium in which to perform the opera- tion. The naked eye characters resemble those of a coarse fibroma, the sections being marked by distinct interlacing fibrous bundles. Sarcomata. This large class of tumours consists of growths derived from the connective tissue constitu- ents of the body, but their elements never develope into fully formed or adult structures. The sarcomata are divided into three chief varieties, the round-celled, spindle-celled and myeloid. Between these many inter- mediate forms are observed ; thus, round and spindle- cells may be mingled in nearly equal proportions mixed sarcoma ; and myeloid cells are associated either with round or with spindle-cells. Again the spindle-celled sarcomata shade off indefinitely into the fibromata. Round-celled sarcomata (fig. 22). These again .are divided into two sub-varieties, the small and the 154 MEDICAL MICROSCOPY. large. The former is much the commoner, and its component cells are about the size of leucocytes or granulation cells. In the latter they are as large as those of squamous epithelium. A section of a round- celled sarcoma under the microscope shows an uniform surface formed of closely crowded cells, intersected here and there by a band of connective tissue. In this re- spect and in the character of the cells it differs essentially from cancer. The nuclei are large and the protoplasm FIG. 23. Section from a giant-celled sarcoma showing also spindle-shaped cells (Ziegler). around them scanty, in fact many of the small round- celled tumours appear to be composed entirely of nuclei and if the processes described on p. 103 are applied to the sections karyokinesis will be demonstrated. The inter-cellular substance varies ; it is usually scanty and homogeneous, sometimes formed of delicate interlacing nbrillae prolonged from the protoplasm of the cells, and sometimes of homogeneous bands. The blood-vessels EXAMINATION OF TUMOURS. 155 are usually abundant, and in many instances have no proper wall. Spindle-celled sarcomata (fig. 23), are com- posed of either small or large spindle cells analogous to the fibroblasts of young granulation tissue ; they pos- sess an oval nucleus, and their extremities are prolonged into a more or less delicate fibre. The cells are closely approximated, and are arranged in parallel or inter- secting fasciculi or bundles. The protoplasm of the cells unites to form a coarse stroma. Myeloid sarcomata (fig. 23) occur almost ex- clusively as tumours in the centre of bones, and in the gum as epulis. They are characterised by the presence of " giant cells" which consist of irregular masses of protoplasm containing many nuclei resembling those met with in tubercle. These cells are associated with others, either round or spindle shaped, or both. A sec- tion has, in parts, or over the whole surface, a peculiar maroon-red tint, by which the nature of the tumour may be recognised. Sarcomata connected with bone often undergo partial transformation into cartilaginous or osseous material. In the former (chondro-sarcoma) the intercellular sub- stance is abundant and hyaline. The osteo-sarcomata are composed either of round or spindle cells. The lime salts are deposited in a delicate fibrous reticulum in which the round cells are enclosed. Some other varieties of sarcomata have to be men- tioned : Myxo-sarcomata. These occur especially in the breast, parotid gland and testicle. In microscopical sections they exhibit, in varying proportions, branched and round cells, which stand out distinctly in an abun- dant homogeneous stroma. 156 MEDICAL MICROSCOPY. Alveolar sarcomata. This variety is much more uncommon. It occurs in bone muscle, and subcuta- neous tissue. The cells are large, round, and not un- like epithelial tissue. They are collected into alveolar masses bounded by delicate bands of fibrous tissue. Melanotic sarcomata. These originate in moles of the skin and in the choroid of the eye. They have a blackish colour, but the pigment is often distributed in irregular masses. In sections they exhibit an alveolar structure with a scanty stroma. The cells are usually round and large in size ; occasionally spindle cells are present. The pigment is chiefly deposited within the cells in the form of small granules, but some of it will be seen to lie free in the stroma. Lympho- sarcomata. This is a variety of round- celled sarcoma originating in a lymphatic gland, but never involving simultaneously the whole lymphatic system. Microscopically round cells are seen arranged in irregular cylinders lying in the meshes of a coarse fibrous stroma, but no definite reticulum can be dis- covered. Adeno-sarcomata. This can scarcely be called a distinct form of tumour. It consists of sarcomatous tissue generally of the round-celled variety, enclosing portions of the gland tissue in which it is situated ; thus it is frequently seen in the breast, parotid glands and testicle, but the gland tissue must not be mistaken for new growth. EPITHELIAL TISSUE TUMOURS. This is a large and important group, and consists of tumours derived from the epithelium of the surfaces and EXAMINATION OF TUMOURS. 157 glands of the body. There are two main divisions, the innocent, comprising papilloma and adenoma, and the malignant, i.e., carcinoma. Papillomata. In the simplest form these are merely overgrowths of the normal papillae of the skin or mucous membrane. Others are composed of a series of mesentery outgrowths, and some occur as branched FIG. 24. Fibro-adenoma of mamma (Boyce). masses which are seen under the microscope to be covered with columnar epithelium (villous growths). The external form varies greatly, but in microscopic sections they are seen to consist of layers of epithelial cells mainly of the pavement variety, more or less altered in shape from pressure. Adenomata (fig. 24). These are glandular tumours 158 MEDICAL MICROSCOPY. and differ in structure according to the gland which they attack. The most common variety is perhaps the mucous polypi which are composed of hypertrophied mucous glands. In microscopic sections branching tu- bules are seen which are lined with a single layer of columnar epithelium. The stroma is soft and made up of fibrillar tissue with stellate and round cells. Mr. F. S. Eve (/. c., p. 135) says that the statement that adenomata do not secrete is erroneous, for the tubules of rectal polypi pour out quantities of mucus ; in one specimen of nbro-adenoma of the breast he found a quantity of milk which exuded from the section. Carcinomata. Cancers are tumours whose struc- ture follows the type of the epithelial tissues. Anatomically they are divided into groups in accord- ance with the nature of their epithelium. Squamous-celled epitheliomata (fig. 2:5). These occur in situations where there is normally a flat epithelium, namely, the skin, mouth, pharynx, oeso- phagus, larynx, vagina, &c. In some of the cases the epithelial processes tend to penetrate into the under- lying structures so as to produce a more or less diffused infiltration and thickening of the part. As they grow, these processes soon become compressed, producing concentric whorls or small epidermic globes which are known as cell-nests or bird's-nests. The cells as a rule are large, and generally irregular in shape owing to pressure. Rodent ulcer or cancroid of the skin is also a slowly growing form of epithelioma affecting chiefly the face of people advanced in years. Under the microscope it is seen to be made up of round or lobulated masses of very small, round, or slightly flattened cells. Cell-nests are absent as a rule. EXAMINATION OF TUMOURS. 159 Cylindrical-celled epitheliomata. This affects parts where the surface is normally covered with cylin- drical epithelium, hence it is chiefly found in the stomach and intestine. Microscopically, the tumour FIG. 25. Section from an epithelioma of skin (Ziegler). a. epi- dermis ; b, corium ; c , subcutaneous areolar tissue ; d, sebaceous gland ; , hair follicle ; /, cancerous ingrowths from the epidermis ; g, deep set cancerous cell groups ; h, proliferating fibrous tissue ; *, (above) cell nest ; , (below) sweat gland. will be seen to be composed of a number of cylinders and alveoli lined with cylindrical epithelium. The stroma is well marked. Spheroidal-celled cancer. This is the most common form of carcinoma. It used to be divided into two varieties, hard or scirrhus, and soft or medullary, but these terms have lost their pathological significance, i6o MEDICAL MICROSCOPY. the difference depending only on the relative amount of stroma and epithelial cells respectively. In the hard variety (scirrhus, fig. 26), the cells are small and arranged in elongated groups ; they vary greatly in shape and often contain two nuclei. Fibrous tissue is abundant and dense so that the walls of the alveoli are broad and well marked. The cut surface of FIG. 26. Carcinoma of the mamma (Ziegler). a, stroma; b, nests; c, cancer cells; d, blood-vessel ; e, fibrous stroma infiltrated with small cells. the tumour is greyish and transparent, with opaque yellow markings indicating the existence of fatty de- generation in the cells. But little juice can be ob- tained from the cut surface. When placed under the microscope it is seen to contain irregularly-shaped cells. In the softer tumours (medullary cancers) the stroma EXAMINATION OF TUMOURS. l6l is scanty and delicate. The cells are large, often coalescent and arranged in long columns or alveoli. The cut surface of the tumour is grey in colour, and juice can be freely scraped from it. In this juice, cells and free nuclei may be found, the latter being large, and mainly oval in shape. Carcinomata undergo fatty mucoid and colloid metamorphosis. The fatty change is found in most cancers, especially those of the breast. When the mucoid degeneration has advanced (mucous cancer) the tumour as a whole is gelatinous in appear- ance. The change is chiefly found in the stroma, the alveoli being bounded by broad transparent hyaline bands of connective tissue. A section presents a uniform, moderately firm gelatinous aspect. Colloid cancer is found in growths of the stomach and intestines. The epithelium is chiefly affected by the metamorphosis, and the stroma only to a lesser degree. Small drops of colloid material make their appearance in the protoplasm of the epithelium, pressing the nucleus to one side, and distending the cell in the form of a vesicle. The cell wall gives way, and the cells merge together in a gelatinous mass ; to the naked eye such tumours have a markedly gelatinous appearance, and on section exhibit large alveoli bounded by thin bands of stroma and containing soft white jelly-like material. These tumours frequently occur as infiltra- tions penetrating among the constituents of the tissues. If cancer attacks the skin or eye-ball it may become pigmented and is then known as melanotic car- cinoma, but it is a rare form of tumour compared with melanotic sarcoma. l62 MEDICAL MICROSCOPY. CHAPTER XIV. EXAMINATION OF URINARY DEPOSITS. IN this chapter will be described the microscopic ex- amination of urinary deposits. The chemical examina- tion, which is even of greater importance, cannot be considered here. The reader is referred to larger works, notably " Clinical Diagnosis" by Prof. v. Jaksch (trans- lated by Dr. Cagney) and " Urinary and Renal Dis- eases " by Sir William Roberts. Many morbid conditions of the urinary tract, how- ever, are characterised by a particular deposit, and it is desirable that whenever disease of the kidney, bladder, urethra or genital tract is suspected, that a microscopic examination of the urine should be made as well as a chemical ; this is especially to be insisted on whenever there is a trace, no matter how small, of albumen in the urine, and also when there is any suspicion of the for- mation of a calculus. The urine should be collected in a conical glass and allowed to stand carefully protected from dust for about twenty-four hours, by which time the deposit will have fairly settled. It is advisable to add some disinfectant (not carbolic acid, which precipitates albumen) to the specimen to prevent decomposition. Salkouski's fluid is an excellent one for the purpose (" Deutsche Medi- cinische Wochenschrift," xiii., No. 16, 1888). It con- sists of a fluid containing 5 to 7-5 c.c. of chloroform in URINARY DEPOSITS. 163 a litre of water ; about 2 oz. of this preparation should be used. A few drops of thymol or oil of turpentine also answer satisfactorily. When the deposit has finally settled the supernatant fluid is carefully poured off. A pipette closed by the finger at the upper end is then introduced and some of the deposit allowed to enter by momentarily removing the finger. On the removal of the pipette the excess of fluid around the pipette is removed with a cloth, and the tube then allowed to rest for some seconds, standing perpendicularly on a glass slide; the heavier particles, such as crystals, will thus sink, and in this way a satis- factory sample of the deposit is obtained ; a drop or two of fluid is then permitted to escape, and a cover-glass applied. The specimen should then be examined first with a half-inch (to detect the larger crystals) and then with a quarter-inch lens. If the deposit be small, considerable assistance is afforded by adding a drop of a solution of iodine dis- solved in iodide of potassium (see p. 33) to the fluid on the glass slide, or a few drops (three are sufficient) of magenta solution to the deposit in the specimen glass. The most convenient formula for the magenta is Woodhead's : Magenta I part Rectified spirit .... 20 parts. Distilled water .... 180 ,, By either of the above means, the epithelial cells are brought more into prominence, and by their distribution in lines, or otherwise, may draw attention to other bodies, such as casts of the renal tubes, in which they are lying. In addition to this, should amyloid casts be present they will be stained a deep brown by the iodine solution. M 2 164 MEDICAL MICROSCOPY. Another advantage of using a stain is that the objects are more easy to focus when some colouring matter is employed. Unless this be done, the transparent cor- puscles, especially if the deposit is small, will often escape notice as the lens is gradually approached to the glass, with the result that the cover-glass is pushed hard on to the slide, and the fluid, carrying crystals, cells or other bodies with it, is forced from beneath the edges of the glass and that specimen rendered useless. Directions were given above that the urine should be allowed to stand for 24 hours before the deposit is exa- mined. In certain cases it is also advisable to look at a specimen about six hours after it is passed. Some urines undergo decomposition very rapidly, or the urine may be drawn from the bladder actually decomposed. If " cayenne-pepper " grains be deposited these should also be examined as soon after they crystallise out as possible, in order to recognise the uric acid formation ; if the urine becomes alkaline from decomposition these crystals are transformed to those of urate of ammonia, and thus false impressions may be derived. There is generally a slight deposit even with healthy urine, and if this be examined will be found to consist of epithelial debris, various crystals and leucocytes. Very often also there is a copious deposit of urates especially in the urine passed in the early morning. The various objects met with in urinary deposits will now be described seriatim, the pathological significance of each being duly considered. The sediments naturally divide themselves into or- ganised and non-organised ; the former including epithelial cells, the corpuscles of blood, pus and mucus, renal casts, parasites and portions of new growth ; whilst under the latter division will be described the URINARY DEPOSITS. 165 numerous forms of crystals which separate from the secretion under different conditions. ORGANISED DEPOSITS. i. Epithelium. As already stated some epithelial cells are found in the very slight deposit which falls in healthy urine ; these arise from the urethra and bladder, and in females from the vagina, and it is only when present in large numbers that their presence is of any note ; but a different construction must be put on the occurrence of renal cells. FIG. 27. Vaginal epithelium. The cells for the most part are of the squamous variety, are polygonal in shape and contain only one nucleus ; distributed amongst them will be found their earlier form, circular in appearance. These arise from the meatus and prepuce. Those from the vagina are of the same shape, but much larger (fig. 27). Bizzozero describes a form of epithelium which has for its origin the surface of the male urethra ; the cells are cylindrical, have well-marked outlines, and gradu- ally diminish in size towards their attached extremities, and thus appear sometimes tailed, at other times rounded, according to the surface which is presented to the eye. 1 66 MEDICAL MICROSCOPY. If any of the above cells are present in large numbers, some affection, most probably inflammatory, may be assumed, of the parts from which they come, thus, the cells described in the last paragraph are especially numerous in men suffering from an old gonorrhoea, and Sir William Roberts (loc. cit., p. 122) points out that the deposits found in the urine of persons subject to noc- turnal emissions have much the same appearance to the naked eye, a collection of whitish flakes and strings being found at the apex of the collecting glass. FIG. 28. Epithelium from the bladder, ureter and pelvis of the kidney. In women afflicted with leucorrhosa, large pavement epithelial cells are found in large numbers in the deposit, often united by their borders into patches of rude Mosaic. The cells derived from the epithelium of the blad- der, ureter and pelvis of the kidney (fig. 28) are difficult to distinguish from one another. A great variety of forms are found. Bizzozero and Eichorst maintain that the type of cells is the same for all these parts ; the cells may be spindle-shaped, cylindrical, tailed, spheroidal oval or irregular in shape. They URINARY DEPOSITS. 167 are smaller than those described above. Those that come from the superficial layers are spheroidal or oval ; they possess one nucleus and are generally coarsely granular. The cells arising from the deeper layers are more irregular, or spindle-shaped, and often present processes projecting from their sides. The single nucleus is larger, and the protoplasm is more finely granular. In cases of vesical or renal calculi these cells are often found in large numbers ; they also occur in the urine of patients suffering from pyelitis. The recognition of venal epithelium (fig. 29) is of much more importance. The cells are of two chief FIG. 29. Renal epithelium, healthy and fatty. varieties, according as to whether they are derived from the straight tubes or from the cortex, but the two kinds are often very difficult to distinguish from one another. The typical cells from the cortical portion consist of a circular nucleus about the size of a red blood corpuscle surrounded by a finely granular protoplasm, the outline of which is generally indistinct. The cells from the straight tubes are natter and more cubical in shape ; the cell wall is generally plainly seen. The cells, therefore, are rather smaller than those de- rived from the other parts of the genito-urinary tract. They are often aggregated together into small groups, or form complete casts of the renal tubules (see l68 MEDICAL MICROSCOPY. below). A few cells are frequently seen cohering to hyaline and other casts. The presence of these cells indicate some lesion of the kidney substance, most probably inflammatory. If the cells show fatty degeneiation, this is indicative of a similar process going on in the kidney, and thus is of grave import. Prof. v. Jaksch (" Clinical Diagnosis," p. 179) de- scribes another variety of cells which occurs in the con- valescent stage of acute nephritis (of scarlatina and ery- sipelas). These are small round cells with an eccentric nucleus. He considers that they are doubtless the. t o 00 FIG. 30. Blood corpuscles in urine. young kidney cells formed within the tubules in the process of repair. 2. Red blood corpuscles (fig. 30). The occur- rence of blood in the urine must always give rise to some anxiety, and a careful clinical examination should be made to try and ascertain its source. Much assist- ance may be gained by a diligent microscopical search. The presence of blood may be determined by certain chemical tests, of which the most satisfactory is the guaiacum test. One or two drops of tincture of guaiacum are added to a small quantity of urine in a test-tube. and about half a drachm of ozonic ether is floated on the surface. If blood be present, a blue colouration URINARY DEPOSITS. 169 appears at the junction of the two fluids, which gradu- ally spreads through the ether. The most reliable test for blood in the urine is, how- ever, the discovery of the red blood corpuscles by the microscope. The corpuscles do not always retain their form and colour, in fact they often appear much changed. If the urine be of very low specific gravity, especially if it become ammoniacal, the corpuscles gradually lose their colour, swell by inhibition and then appear as colourless rings and finally may disappear altogether. If, however, the urine be of specific gravity 1020-1025, they will retain their form for some days, or appear smaller and of a deeper colour. Occasionally they shrink and become crumpled and misshapen. They occur apart from one another and do not run into rouleaux. The corpuscles, then, are characterised by their thin, clear outline, the absence of a nucleus or cell contents and their feeble refracting power. As a rule there is not much difficulty in recognising them, but occasionally other objects occur which by the inexperienced may be easily mistaken for blood cor- puscles. The most likely bodies are the sporules of some fungi ; they may be distinguished by having a nucleus, and also double forms caused by gemmation may be noticed. These sporules also are not always circular, but are often somewhat oval. The nuclei of renal epithelial cells may easily be taken for red blood cells ; as already stated they are of the same size and shape and not infrequently the rest of the cells to which they belong cannot be made out. This error may easily be rectified by the addition of a drop of magenta solution (see p. 163) which will stain the nuclei but not the corpuscles. 170 MEDICAL MICROSCOPY. The most important point, having proved the presence of blood, is to attempt to ascertain its source. It may arise from the kidneys, renal pelvis, ureter, bladder or urethra. As regards the causes no better classification can be found than that given by Sir William Roberts (loc. cit., p. 141). 1. Local lesions. External injury, violent exercise, calculous concretions, ulcers, abscesses, cancer, tuber- cle, parasites, active or passive congestion, Bright's disease. 2. Symptomatic, in purpura, scurvy, eruptive and con- tinued fevers, intermittent fever, cholera, &c., mental emotion. 3. Supplementary or vicarious, to menstruation, haemor- rhoids and asthma. That microscopic examination can be of great use in determining some of these causes is obvious, thus, small crystals of uric acid suggest calculous con- cretions ; numerous pus cells denote a possible ab- scess ; tubercle bacilli are conclusive proof of a tuber- cular process somewhere in the genito-urinary tract ; the discovery of parasites ; and finally the concomitance of renal casts, indicating Bright's disease. Some of these points will be again referred to. Blood having its source in the kidneys produces certain characteristic appearances. The blood is intimately mixed with the urine, and generally does not form a de- posit even after standing several hours, but occasionally a chocolate-coloured grumous sediment subsides. The secretion has a peculiar reddish or smoky appearance. The cells may sometimes be seen to have partially lost their colour, and present themselves as pale yellow rings, and the lesion then is probably an acute nephritis, URINARY DEPOSITS. IJI either of fresh origin or an exacerbation of a chronic inflammation. If the cells are few in number, and have quite lost their colour, a suspicion should arise either of miliary tuberculosis of the kidneys, or congestion of those organs. In cases of abscess, embolism and tuberculosis, or from the presence of parasites, the haemorrhage is usually slight, whilst if malignant disease is the cause, it may be very frequent and profuse. Blood from the pelvis of the kidney and ureter has less marked characteristics, and very little help is afforded by the microscope. The occurrence of certain crys- tals, such as uric acid or oxalate of lime, may yield suspicion of a calculus, but the main evidence must be derived from the clinical aspects of the case. The same may be said if the blood comes from the bladder or urethra. When the bladder is the source, portions of villous growth may be found, or there may be other evidences of cystitis. If the bleeding is from the urethra, it will probably occur independently of micturition. Under either of these circumstances the blood will appear of a bright red colour, will not be intimately mixed with the urine, and clots will very likely be found in the deposit ; if the clots are long and of about the diameter of a goose- quill, the ureter is the probable source, as such clots are occasionally formed there. In the condition known as " hsemoglobinuria," a chocolate-coloured sediment is deposited. No blood corpuscles are found in it, but it consists mainly of amorphous granular matter, which Sir William Roberts considers to be disintegrated corpuscles. Tube-casts are also present, mostly granular in appearance, accom- panied by a few transparent nbrinous cylinders. Crys- 172 MEDICAL MICROSCOPY. tals of oxalate of lime occur, and the late Sir William Gull found large numbers of minute crystals of haematin. 4. Leucocytes (pus cells), fig. 31. A few iso- lated white blood cells will be found in any urinary deposit, but their presence only assumes importance when there is a sufficient number to justify the term " pus." The line to be drawn between the two con- ditions is naturally an arbitrary one, and no fixed rule can be stated. Generally speaking, no conclusions can be drawn from the occurrence of the leucocytes, unless FIG. 31. Pus cells in urine affected by acetic acid and unaltered. there is a distinct deposit at the bottom of the collecting glass. Urine which contains pus is turbid when first voided, but a sediment soon subsides. When the reaction of the urine is alkaline, as is usually the case, the deposit appears as a semi-gelatinous mass, which is not clearly separated from the general mass of the urine, and can be drawn out into long strings. Should the speci- men be acid, the pus forms a uniform white layer, clearly defined, but easily miscible with the supernatant liquid. The nature of the sediment is generally unmistakable, the only deposit likely to be confused with it is mucus. Pus may be distinguished from mucus by the addition of a little caustic soda, which converts the former into a viscid mass, which can be drawn out into long strings URINARY DEPOSITS. 173 when the liquid is poured from one test-tube into another. Under the microscope the pus cells retain their characteristic form spherical in shape, and about one- third larger than a red blood cell. They are granular in appearance, and almost colourless, having only a slight yellowish tinge when crowded together. If the urine is very concentrated they appear small and crumpled. The addition of acetic acid causes the granules to disappear, and the nuclei to become pro- minent. They are often observed to contain fatty mat- ter, which indicates that an abscess, of slow formation, has burst into the urinary tract from some neighbouring part, and not, as when occurring in epithelial cells, fatty degeneration of the kidneys. If there be any doubt as to the nature of the cells, this may be cleared up by the addition of a drop or two of iodine in iodide of potassium (page 33) to a little of the deposit on a glass slide ; when a cover-glass is applied, and the specimen placed under the microscope, the pus cells will assume a deep mahogany-brown colour, whilst epithelial cells and detritus will only be stained a light brownish-yellow. The possible source of the pus is the same as in the case of blood, namely, the kidneys, pelvis of the kidney, ureter, bladder, ure- thra or genital tract, or from the rupture of an abscess into the genito-urinary tract from some other part. Pus from the kidneys is rather rare, according to Sir William Roberts (loc. cit., p. 499, et seq.), it may arise from (i) phlegmonoid inflammations ending in circum- scribed abscess ; (2) multiple abscesses from purulent infection, and (3) occasionally from embolism, apart from pyaemia. The differential diagnosis of these con- ditions can only be arrived at from the clinical side. 174 MEDICAL MICROSCOPY. With the aid of the microscope pus may be assumed t come from the kidneys when the pus cells are accon' panied by renal epithelium, and more certainly when they are accompanied by casts of the renal tubes. Occasionally the cells themselves are moulded into this form, and are known as "pus casts" (see below). Pyelitis (suppuration in the pelvis of the kidney) is only productive of a few cells in the urine, and the same may be said when the pus comes from the ureter. The forms of epithelium accompanying the pus must rx carefully observed, and from these some conclusions may be drawn. With pyelitis the discharge of pus may be intermittent, owing to some obstruction blocking back the discharge. A tumour, appearing and disap- pearing, will then probably be made out in the loin. An absence of mucus points to the mischief being located above the bladder. In the above conditions the urine is acid. The most common cause of pus in the urine is cystitis. The local symptoms will most likely be quite sufficient for the diagnosis without the aid of the microscope. The urine is strongly alkaline, and often ammoniacal, except in old cases, when it may retain its acidity. The quantity of pus is often very considerable. Mixed with the pus cells, the numerous forms of bladder epi- thelium will be seen. If the urethra be the source of the pus (as in gonor- rhrea) the discharge will take place independently of micturition, and a small quantity can generally be squeezed from the orifice. If pus be found in the urine of a female patient, inquiries should be made as regards the existence of leucorrhcea, before any further conclu sions are drawn. The bursting of a neighbouring abscess is indicated URINARY DEPOSITS. 175 by the sudden discharge of a considerable quantity of pus, and as before stated, under these circumstances the cells often contain fatty matter. 5. Mucus. A translucent, flocculent cloud of mucus, separates from nearly all urines ; when the secretion is acid it remains thin and diffluent, but when alkaline, it becomes tenacious and ropy. There is nothing characteristic to be observed micro- scopically, the mucous cells being indistinguishable from leucocytes. On account of its common occurrence, it has little clinical value ; when present in large quantities it may indicate a catarrh of the urinary tract, the exact localisation of the trouble being indicated by the accom- panying forms of epithelium. In order to distinguish a deposit of mucus in acid urine, from one of pus, the caustic potash test should be applied. In the case of mucus, the deposit disappears, instead of being transformed into a stringy mass, as with pus. The mucin in which the corpuscles lie, is precipitated by acids and alcohol. 6. Fat. Fat globules are but rarely found in urine. They may occur as accidental ingredients after the passage of a catheter. As a pathological curiosity they have been stated to appear after a too excessive or pro- longed use of cod-liver oil. Small globules of fat occasionally accompany casts, owing to the fatty degeneration which they have under- gone, they also occur in the interior of epithelial cells (see page 168), and under similar circumstances, small globules may be found free in the deposit. In " chylous urine," large quantities of free fat are discharged. Globules of fat may then be distinguished under the microscope, but more commonly, only granular parti- cles are visible. 176 MEDICAL MICROSCOPY. In phosphorus poisoning also, free fat has been found in the urine, which may easily be explained by the intense degree of fatty degeneration which takes place in this condition. 7. Casts. If a specimen of urine is found to contain albumen, it has become almost a matter of routine to examine the deposit for " casts." This operation is a most important one, for all casts (with the exception of hyaline casts) are a sure indication of renal disease. Although in nearly all cases casts accompany albu- minuria, cases have been recorded in which these bodies were found in non-albuminous urine. Thus, Nothnagel (" Deutscher Archiv fur Klin. Med.," xii. 326, 1874) has found them in cases of jaundice in which there was no albuminuria, and Drs. Finlayson (" Brit, and For. Med. Chir. Review," January, 1876), Burkart and Fischl, have recorded similar cases. The form of these bodies indicate that they have been moulded in the renal tubules ; their size naturally varies greatly, ac- cording to the calibre of the tubule in which they were formed, their diameter varying from ^^ inch (small) to yj^ inch (large), the largest number being known as "medium sized," varying between these limits. Their nature is also very different, according to the circumstances under which they arise. According to their consistence they are known as blood, epithelial and pus casts ; cylinders composed of micrococci ; granular, waxy, fatty and hyaline casts, the last three often being studded with crystals and epithelial cells ; to these must be added cylinders formed of crystals and urates, which may be known as " false casts," and finally those strange bodies " cylindroids." There seems to be some doubt as to the precise URINARY DEPOSITS. 177 structure of these cylinders. The sub-stratum of most of them is apparently an albuminous material exuded from the capillaries, whilst in some cases the casts are formed by degeneration and disintegration of epithelial cells and blood corpuscles, white and red. This seems to be the origin of the granular casts. Blood fibrin is pro- bably the basis of the blood casts. Cornil has described hyaline globules in the epithelial cells, which are forced into the lumen of the tubules, and thus the hyaline variety may be formed. The above objects will be described in the order named together with their clinical significance. Blood casts (fig. 32^). There can be no difficulty in recognising these bodies. The uniform size of the red blood corpuscles which usually retain their charac- teristic shape renders the casts conspicuous objects in the sediment, more especially as they are generally of the large variety. The discs may, however, become crumpled or otherwise deformed, which will render their nature somewhat obscure. They often appear thick and opaque owing to the presence of a large quantity of dark pigment. These casts are not usually perfect, but generally occur as fibrinous casts with the corpuscles scattered more or less irregularly over them. The discovery of these bodies in a deposit has natu- rally much clinical importance. The most usual con- dition under which they appear is acute nephritis ; if sections of a kidney affected in this way are examined mi- croscopically many of the tubes will be seen to be filled with blood and the formation of the casts can easily be imagined. Intense congestion, such as occurs after the administration of cantharides or turpentine will also produce them. Whether the congestion due to ob- 178 MEDICAL MICROSCOPY. structive heart or lung disease will cause them to form is open to question. These casts are occasionally seen after injuries to the kidney in which hsematuria results. Other rarer conditions accompanied by blood casts are : renal calculus, infarct, new growth and tubercle. Pus casts. These are but rarely met with even when the amount of pus deposited is considerable. They appear as cylinders composed of pus cells, closely packed together ; they are very rarely complete, and many cells will be found lying near them, having become detached. It is most likely owing to this latter condition that they are so rarely found, as they very easily break up, and the majority of them are probably destroyed and the cells dispersed before the deposit settles. The nature of the cells can easily be demonstrated by the use of reagents (see p. 173). They are most constantly seen in connection with the discharge of multiple abscesses into the tubules ; conse- quently only very few occasions are presented for exa- mining them. Epithelial casts (fig. 326). When perfect, these cylinders consist of the characteristic cubical cells of the renal epithelium (see p. 167) closely pressed together. Care must be taken not to confuse them with blood casts, more especially as they occur under similar con- ditions ; careful focussing will exhibit the granular pro- toplasm surrounding the nuclei, and in cases of doubt a drop or two of magenta solution should be added to the deposit. As with the blood casts, however, this perfect formation is not often seen. More commonly the cells are scattered irregularly over a hyaline basis. The cells too, are generally broken or shapeless, or only URINARY DEPOSITS. 179 the nucleus is visible. Immature forms of epithelium, oval or circular in shape are particularly common. The presence of these casts indicate acute changes going on in the renal parenchyma, probably nephritis or congestion. This condition may either be primary or an acute attack upon old mischief; in the latter in- stance they will be accompanied by granular casts. FIG. 32. a. Blood casts, b. Epithelial casts, c. Granular casts. d. Fatty casts, e. Hyaline casts. As just hinted, cylinders composed of red blood cells and of epithelial cells are frequently found together. Casts composed of micrococci. These bodies are not often met with, and are very likely to be con- fused with finely granular casts. They are markedly opaque and have rather a greyish colour. Under a high power (% immersion) they may be resolved into N 2 l8o MEDICAL MICROSCOPY. their individual elements and the micro-organisms ,can be distinctly made out. Apart from this they may be distinguished by their resistance to caustic potash and nitric acid. They imply a very serious condition in the kidney ; most probably septic embolism ; but they may also arise from the extension upwards of septic pyelitis (v. Jaksch). Granular casts (fig. 32^). These are more fre- quently met with than any other form. They are opaque, usually have a sharp outline, and are grey or brown in colour. Fragments only of these bodies are usually found, and by their uneven extremities show further signs of having been broken. There are two distinct varieties ; in one the granules are so fine as only to be differentiated by a very high power, and in the other the granules are much coarser, so that they can easily be made out with a half-inch lens. When present they indicate the later stages of an acute .renal affection or a more chronic process ; and may be said to be diagnostic of Bright's disease. When the process is acute they are accompanied by epithelial and perhaps blood casts ; but when occurring alone, contracted granular kidneys may be assumed. No dif- ference can be drawn as to the anatomical conditions, by observing whether the granules be fine or coarse. Most authors now agree that they are formed by the degeneration of the blood and epithelial casts. Amyloid casts These are clear, homogeneous cylinders, highly refracting and possessing a clear outline with rounded extremities. They are often of a great length, but being brittle, are easily broken, so that fragments are more commonly seen than perfect casts. They are found of varying diameter, the largest URINARY DEPOSITS. l8l being almost an inch in breadth, whilst the smallest are not more than the breadth of a red corpuscle, and be- tween these limits all sizes occur. Owing to their transparency, these casts are difficult to see, and unless some staining solution, such as magenta or iodine in iodide of potassium be employed they are easily missed. Attention is sometimes drawn to them by the linear arrangement of crystals, leucocytes or epi- thelial cells clinging to them. They occasionally exhibit the amyloid reaction (see p. 101) with iodine in iodide of potassium, or methyl- aniline violet, but this is by no means constant. Their exact composition is not known but v. Jaksch concludes that they are very complex and may result from the destruction of epithelium, or from the exuda- tion products (amyloid material, fibrin) into the renal tubules. They sometimes occur in large numbers. Their diagnostic significance is not great, as they are found in all forms of chronic renal disease ; conse- quently amyloid disease of the kidney must not be dia- gnosed from their presence alone. Fatty casts (fig. 32^). Cylinders composed entirely of fatty globules are but rarely met with, although they do occur ; more commonly the globules are found scat- tered more or less numerously over a hyaline matrix. Fatty crystals frequently accompany them, being composed of crystals of the fatty acids or the combina- tion of these with the earthy metals. These casts are easily recognised by the sharply defined outline and high refracting power of the globules, but their nature may be proved by running a drop or two of ether under the cover-glass, when the globules will be immediately dissolved. l82 MEDICAL MICROSCOPY. Fatty casts occur in connection with sub-acute and chronic nephritis, when the disease has lasted for some time. They indicate, especially when occurring as pure casts formed entirely of fat, an advanced degree of fatty degeneration of the kidneys, and consequently necessi- tate a grave prognosis. Hyaline casts (fig. 320). These are very similar in appearance to the amyloid casts already described, but are not so highly refractive, and their extremities are somewhat more square. They are homogeneous, pale bodies, sometimes described as "ground-glass like," and are of varying length and thickness. They are even more difficult to see than the amyloid casts, and require to be stained with some medium, magenta being the best ; a few drops of the stain should be added to the deposit (p. 163). Again, as with the amyloid cylinders, attention is often drawn to them by the arrangement of the epithelial cells, &c., adhering to them, and this is more marked after the use of the staining material. Their diagnostic value is small when occurring alone. Henle found them when the kidneys were quite healthy. Huppert discovered them in the urine of epileptic pa- tients after an attack, no renal lesions being present. When, however, epithelial or blood cells are found upon them, some conclusions as regards renal disease may be drawn. In such cases nephritis may be as- sumed, more or less acute. In fatty degeneration of the kidneys, fat cells are often seen thickly crowded together upon them. Cylinders composed of urates, or crystals ("false casts.") When examining a urinary deposit rich in amorphous urates, collections closely resembling granular casts are frequently observed ; they are caused by the accidental accumulation of the urates, and are URINARY DEPOSITS. 183 consequently not in any way connected with true casts. They may often be caused to form by gently moving the cover-glass once to and fro with the finger. Unless the observer is aware of their nature, they are not at all unlikely to be mistaken for casts ; slight pressure on the cover-glass will immediately disturb the formation and cause them to disappear. Casts composed of haema- toidin crystals have been described, but I have never seen them ; they have been stated to occur in the urine of infants. Cylindroids. These bodies were first described by Thomas in 1870 (" Archiv. fur Heilkunde," xi., 130) and are not uncommonly seen in the urine under various conditions. They are ribbon-like bodies, much longer than casts, and are often twisted upon themselves, and studded with small crystals and epithelial cells. In general appearance they are not unlike threads of cot- ton, but are easily distinguished from them by a more irregular outline, the absence of fraying at the ends, and the cells and crystals adhering to them in a way which they never do with the extraneous ingredients of urinary deposits. Thomas first found these bodies in the urine of scarlet fever, but they often occur in Bright's disease, more particularly in the chronic forms ; in the urine of children, too, they are not uncommonly to be seen, and Bizzozero has discovered them when there was no renal disease. Their origin is doubtful, but they appear to be added to the urine after its escape from the kidney. Casts of the seminal tubes. These bodies are only seen in very rare instances. In general appearance they are similar to hyaline casts, but are much broader and usually have spermatozoa embedded in them 184 MEDICAL MICROSCOPY. They are occasionally found in the deposit of the early morning urine of those patients subject to nocturnal emissions. 8. Spermatozoa (fig. 33). Spermatic filaments when found in urine are seen as pear-shaped bodies possessing a minute oval head, not more than youoo- inch in breadth, and a long tapering tail, slightly thickened at its attachment to the head, the entire FIG. 33. Spermatozoa (Ultzmann). length being about ^ inch. They are thus easily recognised by a quarter-inch lens. In urine they are always motionless, but when examined fresh they ex- hibit active movements. In some instances semen escapes with the urine in such quantities as to form a mucous-like deposit, and whitish flakes are then often found, which may be re- moved with a pipette and the characteristic spermato- zoa demonstrated. In medico-legal cases the presence URINARY DEPOSITS. 185 of these bodies may be of the utmost importance, especi- ally if found in the vaginal mucus. In other respects they have very little diagnostic value, for many cases are on record in which they were constantly found in the urine for years, without any detriment to the general health of the patient. This is a matter of some importance, as quacks frequently im- pose upon their victims by exaggerating the danger of the occurrence of these bodies in the urine, and so reap a bountiful harvest whilst administering drugs for the ostensible purpose of averting the terrible fate which they predict for those who are unfortunate enough to consult them. 9. Fragments of morbid growths. In the early days of the use of the microscope " cancer cells " were supposed to be so characteristic as to be easily recognised, and consequently great stress was laid upon their detection in cases of doubtful diagnosis. Now, however, the term is but rarely used, as the cells from a morbid growth differ so little from the ordinary transi- tional epithelial cells, that it is impossible to distinguish the former from the latter. If a great number of large irregularly-shaped cells be found together with a blood-stained sediment, the diagnosis of a malignant growth may be suggested, but this is of no value unless the symptoms and physical signs also point towards the same conclusion. In some rare cases small fragments of the growth may be discharged (especially in females) which are large enough to be submitted to a more complete microscopical examination, and then the existence of a growth may be determined. Those most likely to be met with are portions of villi from a superficial growth of the bladder, or fragments from the surface of a can- l86 MEDICAL MICROSCOPY. cerous ulcer of the uterus,. rectum, or some other portion of the genito-urinary tract. 10. Parasites. The parasites found in the urine naturally divide themselves into pathogenic and non- pathogenic. Every urine when allowed to stand will develop large quantities of bacteria, their form depend- ing upon whether acid or ammoniacal fermentation is set up, but these do not necessarily interfere with the search for other more important organisms, such as tubercle bacilli and the ova of Bilharzia haematobia. In collecting the urine intended for the search of micro-organisms, the parts about the orifice of the urethra should be carefully cleansed and the secretion be received into a perfectly clean vessel ; the importance of these precautions is obvious. The deposit should be examined after the urine has stood for twelve hours. The parasites having a pathogenic value will be first considered, and then those which are non-pathogenic. PATHOGENIC MICRO-ORGANISMS. Tubercle bacilli. The discovery of tubercle bacilli in the urine is of course of great importance, for it in- dicates that a tubercular process is proceeding either in the genito-urinary tract or its immediate neighbourhood. After standing for several hours, the supernatant fluid is poured off from the deposit. A little of the deposit is removed by means of a pipette, and the outer side of the tube is wiped with a cloth ; the open end is then allowed to rest for a few seconds on a cover-glass, one drop allowed to escape, and distributed over the glass, either by means of the pipette or more satisfactorily by placing another glass over the first, and gently sliding them URINARY DEPOSITS. 187 apart. Owing to the dilution which the bacilli must undergo in the fluid, a considerable number of glasses should be prepared before a negative answer is given. After the glasses are dry they are stained in precisely the same manner as will be detailed under the ''exam- ination of sputum." The various steps, however, should be carried out as rapidly, yet as gently, as possible, as the film does not become very firmly fixed to the glass, and the salts being easily dissolved may carry some of the other matters away with them. If no bacilli be found, and yet the other features of the case point strongly to a suspicion of tubercle, the following method may be adopted : About 100 c.c. of the urine are mixed with 10 drops of caustic soda and boiled for five minutes. The mix- ture is then poured into a conical glass and allowed to stand for twenty-four hours. Portions of the sediment are then removed to cover-glasses and examined for tubercle bacilli in the ordinary way (Biechert). If the result is still negative, it by no means follows that the disease from which the patient is suffering is not tubercular, but granted that bacilli are found, what conclusions may be drawn ? In the first place, as already stated, it is quite certain that there is a tubercular lesion somewhere in the genito-urinary tract, or in some tissue immediately ad- jacent to it, which is indirectly affecting that tract. In every probability there is some ulceration, although bacilli have been found in miliary tuberculosis where no ulcers could be found. The character of the predominating variety of epi- thelial cells will lend considerable aid in localising the lesion. If, together with renal epithelium, casts are associated, the kidney may be safely assumed to be the l88 MEDICAL MICROSCOPY. organ affected, although it is very important to con- sider most carefully the history and clinical facts of the case, and take the microscopic examination as an aid to diagnosis, rather than an infallible test. As a rule the bacilli are very few in number, but occasionally large colonies are found, but no deductions as to the severity and extent of the disease must be made from whether few or many rods are seen. Erysipelas. Various authors, notably, v. Jaksch and Fehleison (" Die ^Etologie des Erysipels ") have described cases of erysipelas in which nephritis has supervened, and in which streptococci have been found in the urine when first passed, which are indistinguish- able morphologically from the streptococcus erysipelatos. This condition ceased when the erysipelas disappeared. Too much stress must not be laid upon this form of bacteriuria, as micrococci have been found in the urine in other septic diseases, such as ulcerative endocar- ditis and glanders so that they have no diagnostic value. The spirillum of recurrent feYer has occasionally been met with, but only when its association with blood pointed to the probability of haemorrhage into the kidney and the consequent escape of the organisms from the capillaries. The fungus of actinomycosis has also been stated to occur when the urinary organs are attached. Septic micro-organisms. These occur in all forms, rods and micrococci. They must be distinguished from those due to the decomposition of the urine after it has been passed. In septic bacteriuria the urine is turbid when passed. The most frequent cause of this condition is the use of unclean catheters. URINARY DEPOSITS. l8g This subject has been most exhaustively studied by Sir W. Roberts, and the results of his observations are given in his work " On Urinary and Renal Diseases." He divides bacteriuria into four groups : Group I. Bacteriuria associated with incipient putrefactive changes in the urine. The urine is feebly acid, neutral or feebly alkaline, and passes on quickly to decomposi- tion. Rods are most frequently seen under the micro- scope. This condition is not infrequent in women suffering from leucorrhcea, in men who have suffered from stricture and who have frequently used catheters or bougies. Group II. Bacteriuria with ammoniacal fermentation of the urine. This indicates a serious condition. The urea is transformed into carbonate of ammonia, the fermenta- tion being due to the action of the " micrococcus ureae " (Cohn). The organisms occur singly or in chains of two, three or four cocci. This form is apt to arise in old stricture cases, in the presence of calculi, after operations, in cases of enlarged prostate and in all other conditions in which the bladder is unable to empty itself completely. Group III. Bacteriuria without decomposition of the urine. Rods and cocci are found, and the urine though cloudy on passing becomes clear on standing. The super- natant liquid continues transparent and acid for many days, and the organisms in the deposit show no signs of multiplying. All this leads to the inference that the seat of growth of the organisms is not the urine itself, but some portion of the surface of the urinary mucous membrane. The condition is associated with painful and frequent micturition and pains about the neck of the bladder. Group IV. Micrococci in the urine without decomposition. I9O MEDICAL MICROSCOPY. This condition is extremely rare, Sir William Roberts having only met with one example. Sarcinae. This seems to be the most suitable place in which to mention the occurrence of sarcinae in the urine, although they cannot be strictly stated to be pathognomonic. The organism is discharged with the urine, sometimes forming a greyish-white deposit. In appearance the groups of elements are similar to those found in vomit, but are somewhat smaller. The seat of their production is probably the bladder. The symptoms accompanying its growth are said to be lumbar pains and painful micturition. Bilharzia haematobia (distoma haemato- bium). The ova of this parasite are discharged with the urine ; the worm itself will be described in a later chapter. The urine containing the ova is mixed with blood (endemic haematuria) and frequently contains much fat. This disease only occurs in hot countries such as Egypt and the Cape, and those cases which have been watched in this country have contracted the disease whilst abroad. If the urinary deposit be examined, amongst the pus cells and debris will be found minute oval bodies, about jfa inch long, furnished at the anterior end with a projecting spine, whilst the other is comparatively blunt. In the mature ova the embryo may be seen as a granular ciliated body. Occasionally the free embryo may be seen either rapidly moving about, or slowly stretching the body out to its full length and then again retracting it. In such cases the empty shell of the ovum will be seen lying near the organism. Filaria sanguinis hominis. This parasite is more easily discovered in the blood than in the urine, URINARY DEPOSITS. IQI and will be therefore described in the chapter on " The Examination of the Blood." In chylous urine this worm may, however, be found by examining the deposit in the usual way, and will be seen surrounded by blood and pus cells. As the animal is viviparous no ova are dis- charged. Eustrongylus gigas. This is the largest of the nematoid worms, but is so exceedingly rare in the human subject as to merit no more than passing notice. A specimen preserved in the museum of the Royal College of Surgeons of London is recorded as having been found in the human kidney after death. It is more common in animals. In general appearance it much resembles the Ascarus lumbricoides, but is considerably larger, reddish in colour and possesses six oval papillae instead of three. Ascarus lumbricoides. This worm is described in the chapter on the examination of faeces ; it is only found in the urine when a communication exists be- tween the lower bowel and the renal tract. Echinococci. For a description of the micro- scopical characters of the hydatids and their cysts the reader is referred to the chapter on "The Examination of Discharges." The occurrence of a hydatid cyst in the kidney is very rare, but the discovery of the hooklets, &c., in the urinary deposit may clear up an otherwise obscure dia- gnosis. The condition most likely to be mistaken for this disease is hydronephrosis. Complete scolices are very seldom seen, as the vesicles are broken in the passage down the ureters, and only hooklets and portions of the laminated structure of the cysts can as a rule be made out. The discharge of a cyst is usually attended with blood or pus in the urine. IQ2 MEDICAL MICROSCOPY. With the use of the microscope alone the site of the disease cannot be ascertained, and a careful clinical examination must be made. The kidney is the most usual organ attacked but the cyst may be situated in or near some other part of the genito-urinary tract. NON-PATHOGENIC MICRO-ORGANISMS. When any urine is allowed to stand for some time it soon becomes crowded with micro-organisms. Micrococci are usually most numerous, but rods are also seen (vibriones) frequently in active movement. The micrococcus ureae is often found as almost a pure culture on the surface of the fluid. Moulds are not so common, except on urines which contain sugar when they develop to a huge extent. If the yeast fungus (Saccharomyces cerevisiae) is found, it is a sure sign of sugar in the urine. This fungus occurs as oval budding cells, entangled in a mycelium of branching threads. Another mould which is fairly common is ordinary mildew (Penicillium glaucum) ; this appears in acid urine which has been allowed to stand. It consists of oval cells and mycelia, but aerial hyphae are soon sent off terminated by brush-like filaments arranged in groups, in which the spores are contained. Infusoria. Such organisms are never found in fresh urine, but only when fermentation has commenced. They are similar to those described in the chapter on the * Examination of Faeces." URINARY DEPOSITS. UNORGANISED DEPOSITS. Under this head will be described the numerous crystals and amorphous collections which occur in urinary deposits. The majority of the substances are naturally dissolved in the urine, but are thrown out of solution either on account of being developed in excess, or by reason of a change in the reaction of the secretion or even of its temperature (urates). In addition there are a few additional constituents, such as leucin or tyrosin, which depend for their production upon some abnormal chemi- cal process brought about by disease. A conclusion as to the chief nature of the deposit may sometimes be drawn from its naked-eye characters. Amorphous urates have a characteristic appearance. The sediment is loose and powdery, and varies in colour from a deep red to nearly white, according to the amount of urinary pigment carried down with it. A corroborative test for urates is the facility with which they dissolve with even slight heat ; should any deposit remain after warming, the sediment was a mixed one ; too much heat must not be applied, or albumen may be thrown down. Another deposit usually coloured is uric acid. The well-known "cayenne-pepper-like" grains are seen collected together at the bottom of the glass or adhering to its sides. If the urine be alkaline, and phosphates be deposited, a brilliant white crystalline layer falls to the bottom of the glass, and crystals will also be noticed in wavy horizontal lines on the sides of the containing vessel. If the sediment be shaken up and some of the cloudy urine placed in a test-tube and boiled, heat tends rather o 194 MEDICAL MICROSCOPY. to increase the cloudiness than to diminish it, but the fluid immediately becomes clear upon the addition of a drop of nitric acid. The above remarks chiefly apply to a deposit of triple phosphates. If the urine be alkaline when passed, a peculiar pellicle of amorphous phosphate of lime not uncommonly collects on the surface of the liquid. When shaken, this layer breaks up into small plates, which exhibit a play of prismatic colours. The only other deposit which has marked character- istics is oxalate of lime. This forms a double layer at the bottom of the .specimen-glass, the upper being well- defined, dense, and white in colour, whilst the lower is more like a deposit of mucus, loose, and of a grey colour. The crystals also form lines on the sides of the vessel, as if the glass had been finely scratched. In examining a deposit for crystals, the remarks made at the commencement of the chapter particularly apply. The outside of the pipette should be carefully wiped and the tube allowed to rest on the glass slide for a short time in order to allow the crystals time to fall to the point, and then the drop for examination may be allowed to escape. These sediments are most easily classified as those occurring in acid urine, and those occurring when the secretion is alkaline. SEDIMENTS OCCURRING IN ACID URINE. Uric acid (lithic acid), fig. 34. This is usually de- posited in crystals, but occasionally in a granular form. Crystals so minute as to appear as a flocculent cloud are occasionally seen after the addition of cold nitric acid to urine, and may be mistaken for a cloud of albumen. URINARY DEPOSITS. IQ5 The most common variety is the " cayenne-pepper-like ' grains already alluded to. The primary form of uric acid is a rhombic prism or lozenge, but this is so modified as to be hardly recog- nisable, and the number of varieties met with are almost countless. If the angles of the crystals become equal, quadrangular tables or cubes are produced. Sometimes six-sided crystals are seen, much resembling cystin, but differing from that substance in having two of their sides larger than the others. If the angles are rounded off ovoids or barrel shapes are obtained. At FIG. 34. Uric acid. other times rods are seen, generally aggregated together into stellate or fan-shaped masses. One ray is often larger than the others, and forms a well-marked spike. Other forms are seen too numerous to mention, but whatever shape the crystals may assume they may nearly always be recognised by their colour. The natural colour of uric acid is white, but it gener- ally carries down with it some of the urinary colouring matter, and therefore the crystals are usually reddish- yellow in colour, varying from a deep orange to a light fawn according to the tint of the urine. The urine is generally strongly acid, but if allowed to o 2 196 MEDICAL MICROSCOPY. stand until it is ammoniacal, the crystals seem to be gradually transformed into urate of ammonia, assuming the characteristic globular form ; on the addition of drop of hydrochloric acid they are re-dissolved, and uric acid again crystallises out. It is important, therefore to examine the original crystals soon after they an formed. If there be any doubt as to the nature of the deposit the " murexide " test should be applied. A little oi the sediment with a drop of nitric acid is evaporated to dry. ness on a glass slide over a spirit lamp. To the residue two or three drops of ammonia are added, if uric acid be present, a fine crimson colour makes its appearance, bul sometimes not for two or three minutes. The clinical significance of a deposit of uric acid i< greatly dependent upon what time elapses between the passing of the urine and the appearance of the crystals, In perfectly healthy conditons, uric acid separates out during the acid fermentation which occurs twelve oi twenty-four hours after emission. If the sediment occurs within four or five hours, it indicates an excess, but has still no special pathological importance. It is frequently thus met with in gout and chronic rheu- matic complaints ; in convalescence from febrile affec- tions ; in chronic diseases of the heart and lungs which interfere with oxidation of the tissues ; in certain dis- eases of the liver ; in strumous and tubercular subjects ; in rickets, scurvy, and leucaemia. In connection with gout, Sir A. Garrod has shown that the blood of a gouty patient contains an excess oJ uric acid, that it is deposited in combination with soda in the cartilaginous and fibrous tissues of the joints, and that in such a condition the kidneys themselves often suffer. URINARY DEPOSITS. I 97 It is, however, when the uric acid crystals are de- posited before the urine cools, or immediately after, that this sediment assumes great importance. It may then be feared that a similar process may take place in some part of the urinary tract, laying the foundation of calculi or gravel. Active treatment for the prevention of such a calamity is then immediately called for. Amorphous urates (fig. 35). This deposit was shortly referred to in the general remarks on unorganised deposits. Urates are naturally colourless, but as deposited in urine they are nearly always coloured, the tint varying from orange or purplish to the lightest pink or fawn, but FIG. 35. Amorphous urates. the sediment is always deeper in colour than the urine from which it separates. The deposit is loose, forming a uniform layer at the bottom of the glass, and is often accompanied by a peculiar film floating on the surface of the fluid. The corroborative test is the application of moderate heat, which quickly causes the urates to dissolve. The chemical nature of the sediment is variable, but it usually consists of the mixed urates of potash, soda and lime (rarely of ammonia), the bases occurring in varying proportions. Under the microscope minute granules are seen, but these have no special characteristics. The pathological significance of such a deposit is very ig8 MEDICAL MICROSCOPY. small ; its formation depends upon an acid state of the urine, and a certain degree of concentration. It is thus often observed in a state of perfect health, especially during the winter months and after profuse perspiration. In disease it is very frequently met with, especially in febrile conditions, when it need give rise to no apprehension. If urates are constantly deposited, without rise of temperature, there is probably organic disease some- where in the body, either in the heart, lungs, liver or elsewhere. FIG. 36. Urate of soda. Dyspeptic patients frequently complain that their urine is thick, especially that passed in the early morning. Urate of soda (fig. 36). This is a very rare de- posit. The specimen from which the accompanying sketch was taken, was obtained from the urine of a patient suffering from gout. The sediment was yel- lowish in colour, and at first glance was mistaken for one of uric acid. Under the microscope the charac- teristic appearances were seen, consisting of circular opaque masses, from which spiny crystals projected. Some of the globes presented several such spines, URINARY DEPOSITS. 199 others only one, variously curved, whilst still others were destitute of such projections. Some of the crystals seemed to be embedded in phosphatic plates. This sediment is chiefly found in the urine of children, and is deposited before the urine is passed. The spines, therefore, are very apt to cause irritation of the mucous membrane, and even to collect in masses and block the urethra, or form the nucleus of a calculus. In rare cases, of which the above is an example, these crystals are found in the urine of gouty patients. Oxalate of lime (fig. 37). The general character of this deposit has already been given on page 194. FIG. 37. Oxalate of lime. Under the microscope the crystals are seen to be trans- parent regular octahedra, or, especially in albuminous urines, they appear as dumb-bell-shaped bodies. The octahedra are generally known as " envelope crystals," this shape being assumed owing to one axis of the crystal being shorter than the other two. They are seen of various sizes, but not unfrequently the crystals are exceedingly minute, and may easily be passed over without recognition. Occasionally they crystallise as pointed octahedra. The " dumb-bell " forms are in reality flattened, rounded discs, with a central depression on each surface, and according to 2OO MEDICAL MICROSCOPY. the way in which they lie in the field, appear as dumb- bells, ovoids or circles, and may be made to apparently change their shape by a gentle movement of the cover- glass. These crystals are soluble in hydrochloric, but insoluble in acetic acid. Oxalate of lime occasionally falls as an amorphous deposit, and can then only be recognised by its solu- bility or more elaborate chemical tests. This sediment is frequently found in healthy urines, especially after a vegetable diet, and consequently no importance can be attached to its appearance, unless it occurs persistently in large quantities, in spite of alteration in diet. The chief danger is its connection with the formation of calculi. Much stress, however, has been laid on the condition known as " oxaluria " and " oxalic diathesis," by Drs. Prout, Bird and Begbie, but Sir William Roberts, Beneke, and others, have opposed these views, on the grounds, firstly, that intense oxaluria may exist per- sistently without any of the symptoms attributed to the oxalic diathesis, and secondly, that this group of sym- ptoms may exist typically without the occurrence of deposits of oxalate of lime in the urine. Beneke further shows that oxalic acid has almost its sole origin in the azotised constituents of the blood and food ; everything, therefore, which retards the metamor- phosis of these constituents occasions oxaluria. Very little value, again, attaches to the amount of deposit which occurs, for the urine may contain a large proportion of oxalic acid, without it or its salts being thrown out of solution. To ascertain the exact amount present, a troublesome quantitative analysis would have to be undertaken. Sulphate of lime. This substance is but rarely URINARY DEPOSITS. 2OI found in urinary sediments. The author has never met with it. Fiirbruger and Valentiner have described its occurrence in large needle-shaped crystals, and also in elongated tablets with abrupt extremities, they are sometimes arranged in sheaths and rosettes. Amongst them are seen masses of indeterminate crystalline struc- ture. Sulphate of lime is also met with in urinary sediments in the amorphous form. It is insoluble in ammonia and acids, and its detection can only be as- sured by a rather involved chemical analysis, for which the reader is referred to textbooks on that subject. The pathological conditions under which this deposit occurs have not been ascertained. Phosphates. The various forms of phosphatic de- posit are met with in feebly acid urines, especially the " stellar phosphates " or neutral phosphate of lime, but as they are more frequently found in urines having an alkaline reaction, their description will be postponed until such sediments are considered. Cystin (fig. 38). Crystals of cystin are in rare cases found in urinary deposits. The urine from which it falls has generally a sweet-briar-like odour, and is turbid when passed. It is particularly liable to decompose, evolving sulphuretted hydrogen, which blackens white glass vessels, and the colour of the liquid sometimes changes from yellow to green. A great part of the cystin dissolved in the urine does not crystallise spon- taneously, but may be brought down by the addition of a few drops of acetic acid. To the naked eye the deposit is fawn-coloured, very much resembling urates. It is not dissolved by heat, and is insoluble in acetic acid, but soluble in ammonia (difference from uric acid). When the ammonia solu- tion is allowed to evaporate, the cystin. separates out as 2O2 MEDICAL MICROSCOPY. beautiful six-sided crystals, taking the form either of prisms or plates, the former being single or arranged in stellate groups. To corroborate the presence of cystin in the deposit, the urine should be filtered, the sediment on the filter paper washed with a little water, and then transferred to a piece of platinum foil and heated in the flame of a Bunsen burner. Cystin burns with a bluish -green flame, emitting white acrid fumes, having an offensive odour resembling garlic, but does not melt. A spontaneous deposit of cystin, when placed under the microscope, exhibits six-sided plates, showing lines FIG. 38. Cystin. of secondary crystallisation ; they are not often seen singly, but are placed on one another, or joined together in long strings. They closely resemble one form of uric acid, but differ in having all their sides equal. Its occurrence in the urine is chiefly of importance as indicating the possible formation of gravel or a calculus. It has a peculiar tendency to run in families, especially affecting children and young male adults. Cystin may persist in the urine for many years, perhaps only ap- pearing at intervals, without any injury to the general health, but merely causing physical irritation to the bladder and urethra by the formation and passage of small concretions. URINARY DEPOSITS. 203 Xanthin. Bence Jones, Douglas Maclagan and Budd, have described this substance in urinary deposits. Bence Jones discovered it in the urine of a lad who three years previously had exhibited the symptoms of renal colic. The crystals resemble one form of uric acid, being pointed ovals, very much like whetstones. They are soluble in ammonia and insoluble in acetic acid (difference from uric acid). When the deposit is heated with nitric acid, a yellow mass is deposited on evaporation, which turns a violet-red colour when caustic potash is added. The clinical significance is not known, beyond indicating the possible formation of a calculus composed of this substance, of which there are now several on record. Tyrosin (fig. 39). Together with leucin, the crys- FIG. 39. T>rosin. tals of which will be immediately described, this sub- stance has been found in urinary deposits. It occurs as sheaves of very fine needles, sometimes in yellowish-green crystalline globules. If the nature of the sediment is doubtful, it should be dissolved in hot ammonia, and the following tests applied : i. If a little of the solution be allowed to evaporate, 204 MEDICAL MICROSCOPY. tyrosin will crystallise out in fan-like groups of colour- less needles. 2. Some of these crystals are dissolved in hot water, and to the warm solution some nitrate of potash and mercuric nitrate are added. A red precipitate, together with a red colouration of the fluid, indicates the presence of tyrosin. 3. Some more of the crystals are placed in a small capsule and a drop or two of sulphuric acid added. After standing for half-an-hour water is added. The solution is now boiled and saturated with carbonate of lime. The mixture is filtered and to the filtrate a little perchloride of iron solution (which must be free from acid) is added. In the presence of tyrosin a deep purple tint is produced. A spontaneous deposit of tyrosin is rare, but it may be obtained from urine which contains it by the addition of acetate of lead, until a precipitate is no longer pro- duced, and then passing sulphuretted hydrogen through the mixture. The precipitate is separated by nitration, and the clear solution which passes through concen- trated by evaporation, when the tyrosin will crystallise out. This substance is principally found in the urine of patients suffering from acute yellow atrophy of the liver, but has also been met with in phosphorus poisoning, typhus and other infectious fevers, also in leucocythaemia and certain nervous states. Leucin (fig. 40). As an urinary deposit this sub- stance is still more rare than tyrosin, although it usually occurs in solution with that substance in the above named conditions. The sediment, when it occurs, consists of yellowish oily looking drops, which have a concentric appearance. URINARY DEPOSITS. 205 The urine should be evaporated to dryness, and the residue heated with boiling alcohol. On cooling, the solution deposits shining white delicate plates of leucin, which are greasy to the touch ; they are insoluble in ether and chloroform (difference from cholesterin). As a confirmatory test, some of the crystals may be dissolved in boiling water, and the solution heated with proto-nitrate of mercury, when, if leucin be present, metallic mercury will be deposited. Hippuric acid. Crystals of hippuric acid appear as colourless, four-sided, rhombic prisms or needles, the FIG. 40. Leucin. former forming when it separates from a dilute, and the latter when from a strong solution. They are soluble in alcohol and insoluble in acetic acid, thus differing from uric acid and phosphates respectively. They have no clinical significance. They occur in large numbers after a patient has been taking benzoic acids or after eating certain fruits. Haematoidin and bilirubin. These substances are both found in urine, but it is extremely difficult, if not impossible, to distinguish them one from another, their crystals have the same form, and no accurate chemical tests have been discovered which one will re- act to and the other not. The crystals are of two kinds, clusters of needles or minute rhombic tablets. Their colour varies from a light yellow to a deep ruby-red. 206 MEDICAL MICROSCOPY. They are soluble in caustic soda. On treating the crystals with a little nitric acid, a green rim forms round them, and their red colour gradually changes to blue (Hoppe-Seyler). These two substances occasionally occur in the amorphous form. Haematoidin crystals have been found in severe acute nephritis, in carcinoma of the liver, in some of the acute specifics, such as scarlet fever and typhoid, and in jaun- dice. If they occur in large numbers, a previous hae- morrhage is indicated, or an abscess may have burst into the urinary passages. Bilirubin has chiefly been discovered in the urine of jaundice. Soaps of lime and magnesia. In the urine of various diseases, v. Jaksch (loc. cit., p. 201) has found crystals similar in form to those of tyrosin, forming rosettes of fine needles. Their nature is uncertain, but this observer considers that they are probably composed of the lime and magnesia salts of the higher fatty acids. They usually occur in the urine of febrile conditions, such as severe puerperal septicaemia. SEDIMENTS OCCURRING IN ALKALINE URINE. Triple phosphates (fig. 41). The most typical form in which the phosphate of ammonia and magnesia (MgNH 4 PO 4 -f-6Aq.) occurs in urinary sediments, is a triangular prism with bevelled ends ; but this is often considerably modified by the removal of the angles or the hollowing-out of the sides. Some of the crystals are more quadrilateral in form, whilst others appear almost like octahedra in consequence of the central part of the URINARY DEPOSITS. 2OJ crystals not being developed. If the urine be highly ammoniacal, the crystals may have a stellate arrange- ment, consisting of feathery rays, like snow-flakes, or may form peculiar jagged figures like leaves. Like all phosphatic deposits these crystals dissolve readily in acetic acid. They cannot be said to possess any pathological value, their presence simply indicating an excess of alkalinity of the urine. Neutral phosphate of lime (stellar phosphates), fig. 42. The chemical formula of this substance is CaHPO 4 + 2Aq. The crystals are formed of rods or FIG. 41. Triple phosphates. needles varying in size, often showing lines of secondary crystallisation, and arranged singly or in sheaths and rosettes. They are generally surrounded by small crystals of oxalate of lime. The urine is, as a rule, feebly acid, or just becoming alkaline. The deposit is decomposed by ammonia and dissolved by acetic acid. This sediment is rarer than the other forms of phosphates and has a rather more grave significance. Sir William Roberts states that according to his experience " the presence of this deposit in quantity is an accompani- ment of some grave disorder." The author has twice met with it in cases of advanced phthisis. 208 MEDICAL MICROSCOPY. In scanty numbers the crystals may be met with in healthy urine, especially during the " alkaline tide " after meals, and if the secretion be rich in lime. Basic magnesium phosphate. These are like- wise but seldom met with, chiefly owing to its easy solubility. The crystals occur as strongly refracting plates, having the form of elongated rhomboidal tablets. Nothing is known as regards their pathological value, but they simply seem to indicate that the urine is con- centrated. Amorphous phosphate of lime. This is the most common deposit when the urine is alkaline from FIG. 42. Stellar phosphates. fixed alkali ; crystals of the triple phosphate often ac- company it. In general appearance the sediment is not unlike the ordinary urate deposit, except that it is not coloured by the urinary pigments, and is consequently whiter than the supernatant liquid. Under the microscope minute granules are seen, resembling the amorphous urates, collected together into irregular groups. Such a deposit is frequently seen in healthy urine after a meal, and is a sure sign that the secretion is alkaline, and thus is a ready test of the success of drugs which have been administered in order to secure an URINARY DEPOSITS. 2OQ alkaline reaction of that fluid. The urine is turbid when first passed, and the deposit rapidly subsides, an iridescent film collecting on the surface. Amorphous phosphate of lime (bone-earth) forms a large portion of phosphatic calculi, but requires some nucleus around which to accumulate. Carbonate of lime. This substance is always deposited in the amorphous form from ammoniacal urine. The granules are often collected together into dumb-bell-shaped masses or small concretions. The sediment dissolves in acetic acid with the evolution of gas, thus distinguishing it from the amorphous phos- phatic deposit which dissolves in acetic acid without the evolution of gas. When voided as gravel or small calculi, carbonate of lime is found in the crystalline form as small spheres exhibiting a radiating appearance. Urate of ammonia. Urate of ammonia is thrown down when the urine becomes strongly ammoniacal. The deposit may easily be obtained by allowing urine which contains the amorphous urates or uric acid to stand until ammoniacal fermentation commences. To the naked eye the deposit is white, but occasion- ally it possesses a beautiful violet hue (Roberts). Under the microscope dark, opaque, globular masses of various sizes are usually seen. More rarely this substance takes the form of minute dumb-bells, arranged singly or in groups. If the deposit be dissolved by the addition of a drop of acetic acid, and a few drops of the solution placed on a glass slide, rosettes of uric acid will crystallise out, the crystals being quite colourless. Cholesterin (fig. 43). The occurrence of choles- terin in urine was first pointed out by Dr. Lionel p 2IO MEDICAL MICROSCOPY. Beale (" Microscope in Medicine," p. 355). This author showed that the oily particles so frequently seen in the deposits from the urine in chronic Bright's disease contain cholesterin, which may readily be separated in a crystalline form. But as a spontaneous deposit, cholesterin is ex- tremely rare. It has been found associated with pyuria. The crystals take the form of rectangular plates with a notch in one corner, and commonly appear superimposed one on another. Indigo. This substance occurs either as the colour- ing matter of deposits, such as urates, or pure, in the FIG. 43. Cholesterin. amorphous or crystalline form. The crystals are usually needle-shaped and arranged in rosettes. It not infre- quently occurs in putrescent urine as blue shreds and films on the surface of the liquid or adhering to the sides of the glass. The presence of indigo is due to the decomposition of indoxyl. It has no decided clinical significance. Gravel and calculi. The composition of these bodies is more easily determined by chemical analysis than by the use of the microscope. Reference has already been made several times, whilst URINARY DEPOSITS. 211 describing the various deposits to the possible formation of calculi. Thus gravel is most usually composed of uric acid or of urates, or of the two combined, more rarely of cystin or xanthin. Calculi may be formed of the mixed phosphates, car- bonate of lime, phosphate of lime, uric acid, urates (chiefly found in the kidneys of children), oxalate of lime (" mulberry calculi "), cystin or xanthin. Various other concretions are occasionally met with formed of fibrinous material, blood, fatty matter or cholesterin. As regards the microscopical evidence which may be obtained from scrapings of these bodies, reference must be made to the different heads under which the crystals are described. But as such scrapings will, in most cases, merely yield an amorphous powder, but little aid is rendered in the recognition of the stones, and it is there- fore advisable to proceed at once to a chemical analysis. Extraneous matters. Matters entirely foreign to the urine, which occasionally find their way into the urine, are of two classes, the first of which is of great importance, whilst the second is purely accidental. In the first class are included such substances as are the result of disease, or of a morbid process in some adjacent organ. Thus, substances belonging to the faeces may be found under such circumstances that they could not have been passed by the bowel, thus indi- cating a communication between some point of the urinary tract (most probably the bladder) and the in- testine. In other cases hairs have been passed, which, by the malignant course of the case, have been shown to come from a dermoid cyst. Fragments of tumours also occur, which have their source, not in the urinary organs, but in some neighbouring tissue. P 2 212 MEDICAL MICROSCOPY. The second class of extraneous matters includes any substances which have accidentally found their way into the urine, or in some hysterical cases, have been purposely placed there for the purposes of deception. It is always advisable, therefore, to cover specimens which have been set aside for examination, for other- wise, in hospitals especially, many small particles derived from the bedding, floors, food, &c., may fall in and be a source of much perplexity, before their true nature is ascertained. One of the most puzzling of these objects is caused by the use of dusting powders, especially in female patients. Starch grains are then seen, more or less altered by inhibition of fluid, and may be rather difficult to recognise ; if a drop of iodine solution is added, how- ever, their change in colour to a deep purple will at once explain their nature. Fragments of pine wood from the floor, becoming soft and swollen by soaking, often give the appearance of casts, unless carefully examined. Other substances 'encountered, and with which the observer should make himself acquainted, are hairs, both human and from animals (especially cats and dogs), fibres of cotton, worsted, silk, wool, &c., these being often coloured ; portions of feathers ; bits of tea- leaves, showing cells and spiral vessels ; particles of food, meat fibres, oil globules, &c. Young pediculi, and the larva of the blowfly (these last especially in diabetic urine) are also sometimes found, and hysterical people will add flour, sand and other powders, milk, or blood from the finger, in order to endeavour to deceive the physician and excite sympathy, generally of a practical form, for some pretended malady. Preservation of urinary deposits. A question URINARY DEPOSITS. 213 often asked is : " What is the best mode of making per- manent preparations of urinary deposits " ? This ap- plies more especially to the various forms of crystals. The ordinary mode of mounting with Canada balsam is not satisfactory, this medium being too transparent for the purpose. The best plan is to use some preservative fluid in which the crystals are not soluble. For uric acid, oxalate of lime, urates of soda and ammonia, as well as for casts, glycerine, diluted with water, answers as well as any, or a dilute solution (i in 50) of carbolic acid may be used. The method introduced by Dr. Lionel Beale is an excellent one and is carried out as follows (" Microscope in Medicine," p. 371) : A shallow " cell " has to be prepared. The most convenient form for the purpose is that which is made by painting upon a glass slide, with a fine brush, a narrow border of Brunswick black, enclosing either a square or circular space as may be most convenient. The urinary sediment is allowed to subside to the bottom of a conical glass, the supernatant liquid being poured off, and a small quantity of the preservative solution added. The deposit is again allowed to sub- side, and the solution poured off and replaced by a fresh quantity. This method is adopted in order that the deposit may become thoroughly saturated with the pre- servative fluid, otherwise there is danger of the prepara- tion deteriorating before many months have passed. Glycerine, if used, must be added to the deposit in the conical glass in very small quantities at a time, so that cells, casts, and other small bodies, may swell out again after they have been caused to shrink. After the final subsidence of the deposit, a small 214 MEDICAL MICROSCOPY. portion may be removed with a pipette, placed in one of the cells above described, and the glass cover applied to the surface of the liquid, care being taken that the whole surface of the glass is well wetted with the solu- tion, in order that no air bubbles may be included in the preparation. Any excess of fluid is now to be soaked up with a clean cloth or filter-paper, and the cover cemented to the cell by applying a little Brunswick black or Dammar varnish with a camel's hair brush. Crystals of the triple phosphate or cystin have to be preserved in a different manner. The cell is made as before. The triple phosphate may be kept in water, to which a little ammonia and chloride of ammonium have been added. In this solution the crystals preserve their beautifully smooth character, while in pure water the surface becomes roughened. Crystals of cystin are best preserved in organic acids, a very weak solution of acetic acid keeping them quite unchanged. EXAMINATION OF FAECES. 215 CHAPTER XV. EXAMINATION OF THE F^CES. UNDER the term " faeces " will be understood all those substances passed by the bowel, and consisting of the residue from the processes of digestion, mixed with the products of the secretions of the alimentary canal. In this chapter will be considered, not only the matters which are expelled as the result of alterations in the dejecta produced by disease, but also objects such as parasites, which are added to or passed independently of the stools. Before passing to the microscopical examination, a short epitome will be given of the chief alterations in the macroscopical characters of the faeces. The character of the stools in health is greatly de- pendent upon the nature of the food which is taken, as will be seen by the following remarks. No definite conclusions can be drawn from the quantity. The average in health is about five ounces daily, any considerable increase being principally due to the addition of fluid, which, however, bears no rela- tion to the quantity taken by the mouth during the day. Naturally, the quantity varies considerably with the solid food that has been eaten. The colour changes more than the other charac- teristics. In this respect the nature of the food has a marked influence. The usual brown colour be- 2l6 MEDICAL MICROSCOPY. comes lighter with milk diet, and darker when much meat has been taken ; and the same effect is produced when the faeces have been retained for some time in the intestines. An excess of green vegetables also com- municates their colour to the dejecta. Cocoa-nibs or chocolate often cause the stools to assume a greyish tinge. Coffee, claret and porter, especially when these liquids are taken in excess give their particular hues to the contents of the intestine. The colour is further considerably altered when various drugs have been administered. Iron, mercury, manganese and bismuth cause a black colouration owing to the formation of the sulphides of the metals. Calomel yields a green, rhubarb, santonin or senna a yellow, and logwood a red colour. Various diseases produce such changes in colour as to almost render them characteristic. The stools are almost colourless in obstructive jaundice, when they have a pale greyish, putty-like tint. In obstruction of the pancreatic duct, too, the stools are almost white : this being due to the fact that the action of the pan- creatic secretion is essential to the production of the ordinary faecal colouring matter (Walker, "Trans. Roy. Med. and Chirurg. Soc.," vol. Ixxii., 1890, p. 257). In the disease known as psilosis or sprue, the mo- tions are pure white, with occasionally a layer of yellowish matter over them ; they are partially or wholly formed according to the stage of the disease. The "rice-water" stools of cholera are also almost colourless, but are more characterised by the consistence. In typhoid fever the motions are the colour of pea-soup. The occurrence of blood in the evacuations, on the other hand, produces a black colour (melaena), if it is retained for some time in the bowel, but if due to the EXAMINATION OF F^CES. 217 bursting of a vessel through ulceration it retains its red colour. In dysentery the stools vary considerably with the different stages of the disease, being sometimes merely streaked with blood, at others consisting of almost pure blood, whilst sometimes they resemble meat-washings. Bile pigment not unfrequently makes its appearance, and is always pathological, depending upon some change in the liver or gall-bladder. Green stools are principally seen in intestinal affec- tions of children. As already stated this may be owing to calomel having been administered, but apart from this they may occur under two other conditions. Firstly, in infants diarrhoea is easily caused by excess of acidity, and the green colour which is then observed is caused by the presence of a bacillus (Lesage), which can be culti- vated on artificial media, and causes blood poisoning when injected into animals. Secondly, the condition of green stools in children is sometimes brought about by the presence of biliverdin. The consistence of the faeces depends partly upon the food which has been taken, but chiefly upon the quantity and nature of the secretions of the large intes- tine. The reaction is normally alkaline, but an acid re- action does not necessarily imply disease. In young infants errors in feeding frequently produce acid stools, with diarrhoea and excoriation of the buttocks, whilst the " clayey " stools are usually alkaline, owing to the presence of carbonate of ammonia, the result of decom- position. The odour of the motions yields little information. In children, a fcetid or acid smell often indicates that they are not being fed properly. The absence of bile, 2l8 MEDICAL MICROSCOPY. by withdrawing its deodorising action, causes a very bad odour. In sprue, the smell is almost characteristic being particularly pungent and penetrating. In stools of dysentery and melaena, and in any con- dition in which decomposition sets in rapidly, the odour becomes excessively foetid. Passing next to objects visible to the naked eye and not normal constituents of the faeces, we may mention first foreign bodies. These are of all kinds, and the case-books of lunatic asylums record many extraor- dinary things which have been swallowed and after- wards passed by the bowel. Among the most common of such bodies are portions of indigestible food, as seeds and stones of fruits, false teeth, masses of hair, small stones and portions of metal, &c. When there is overaction of the small intestine (lientery) undigested food is nearly always found in con- siderable quantities. In order to separate these and the following objects the faeces should be placed in muslin over which a stream of water should be directed ; by these means most of the solid matter will be broken up and the larger bodies easily detected and separated. Mucous and fibrinous shreds. Nothnagel (" Beitrage zur Physiologic und Pathologic des Darmes," p. 185) has described an affection of the bowel which he has termed "enteritis mucosa or mem- branacea." In this disease cylindrical shreds of mucous membrane, sometimes forming complete casts, are passed by the rectum, accompanied with violent straining. After retention in the lower part of the bowel, they may be consolidated into u hard, white, rounded masses, about the size of nutmegs " (Fagge). Dr. Lionel Beale (" Microscope in Medicine," p. 291) has described EXAMINATION OF F^CES. 2IQ several such cases. Some of the shreds seemed to con- sist of firm mucus, in which the epithelial cells from the large bowel and mucus corpuscles were embedded. In some of the cases the substance passed was firm enough to form a tough leathery membrane, and under the microscope exhibited fibres, mucus corpuscles and im- perfectly formed epithelial particles. Occasionally the casts are more distinctly fibrinous and are tougher and ribbon-like ; their form and character can be demonstrated by removing them to a large dish of water and gently moving them to and fro with a needle, when they will float out and exhibit their whole structure. On careful examination pits may be made out corresponding to the mouths of glands. Portions of bowel separated from intus- susceptions. These are more interesting from a purely pathological than from a clinical point of view, as the diagnosis of intussusception will in all probability have been made before the cast off portions are passed by the rectum. When floated in water they will be seen to involve the complete circumference of the intes- tine; they are generally gangrenous and emit a most offensive odour. Under the microscope the more or less altered structure of the part of the bowel from which they are derived may be demonstrated ; the examination will be made more easy if a drop or two of methylene blue be added to the water in which the specimens are teased out. Intestinal concretions. These objects are usu- ally the result of accident, and are mere pathological curiosities possessing no clinical value. Hard masses are often passed with the motions by lunatics ; thus Dr. Langdon Down records a case in which such a mass when examined proved to be cocoa-nut fibre. In other 220 MEDICAL MICROSCOPY. eases oat-hairs have been found matted together into a firm solid ball. In rare instances, drugs which have been administered for too long a period, or incautiously, produce similar results, and masses of carbonate of magnesia or insoluble salts of iron may be found. Mention may here be made of the occurrence of "scybala" in the rectum, which often give much trouble, especially in elderly people. These are caused by the retention of faeces and removal of moisture, and the combination of the constituents of the excrement with inspissated mucus. Gall-stones. -The passage of gall-stones is of great clinical interest, inasmuch as they often clear up a doubtful diagnosis. They are easily detected with the naked eye, and for closer examination may be obtained by shaking up the faeces with water, when they will immediately sink and are thus easily removed. Stones consisting of bile-pigment are the most com- mon. They often occur in large numbers. They are black in colour, irregular in shape, and sometimes present facets where they have rubbed against each other. They are friable and are easily crumbled. They vary much in size, but are usually about the size of hemp seeds. They are generally deposited round a nucleus, which consists of inspissated mucus. Gall-stones are more rarely light in colour, and then most probably composed of cholesterin. Their structure can easily be exhibited by scraping off a little with a knife and adding a drop of water. The characteristic crystals (see fig. 43) are then seen, consisting of rect- angular plates, with a notch out of one corner, super- imposed one on another. Their nature may be further corroborated by adding to some of the scrapings a drop of sulphuric acid, when a brilliant red colour will make EXAMINATION OF PIECES. 221 its appearance. These calculi are smooth, and some- times attain such a large size as to occupy the entire gall bladder. They are then of course only discovered after death. When several are present, they are facetted, and are much harder than those formed of bile-pigment. More rarely bile-stones are composed of carbonate or phosphate of lime. Before concluding the account of the macroscopic ex- amination of faeces, mention must be made of the bodies described by Virchow and Nothnagel (loc. cit., p. 96) resembling cooked sago grains or frog-spawn. Virchow thought that they came from an excess of starchy food. Finally the bodies known as " sable intestinal," and described by Dr. Sheridan Delapine in the "Pathologi- cal Transactions," xii., 1890, p. in, deserve brief notice. Labouline,in 1873, described small irregular granules, not unlike cork-dust, which he had found in the stools, and gave to them the name " sable intestinal." Dr. Delapine has investigated several cases of this kind. The granules or small masses vary considerably in size, some being very minute, whilst others measure -| inch in their largest diameter. In one case they were brown in colour, and were shown to be composed of vegetable cells, such as are found in sclerenchymatous tissue, a small amount of carbonate of lime, phosphates and oxalates, and a few silicious particles. In another instance they resembled coarse bran in appearance, and in yet another, small gall-stones. In the last case, however, it was shown that their nuclei consisted of undigested food matter, and that therefore they must have been produced in the intestine, and also that they were not composed of cholesterin, or of any of the pro- ducts which may accumulate within the gall bladder. 222 MEDICAL MICROSCOPY. After careful investigation Dr. Delapine came to the conclusion that all these masses were practically nothing else than residues oi articles ot food (fruit pips, &c.) which had not been digested, either because they were undigestible, or because the stomach and intestine failed to digest them. Yet they could not be said to be simply undigested matters such as those found regularly in the fasces, since by their accumulation they gave rise to suspicious appearances and symptoms. MICROSCOPICAL EXAMINATION. The stools should be examined in two ways. First, by placing a small particle on a glass-slide, covering it with a cover-slip and distributing it into a uniform layer by gentle pressure. Secondly, by shaking up a portion with many times its bulk of water, allowing the sediment to settle, then removing portions by means of a pipette as in the examination of urinary deposits. In the stools in health various particles of undi- gested food, animal and vegetable, are met with, and the student should make himself well acquainted with these, although it is true that mistakes are not so liable to occur in the examination of the faeces as in that of the sputum or urine. They are much the same as those which will be described as occurring in vomit (q. v.). When the individual from whom the specimen is obtained is on a meat diet, muscular fibres will invariably be found, but are sometimes so altered as to be hardly recognisable, being swollen and stained yellow by the bile ; with a little care, however, their characteristic striation can usually be made out. With these fragments elastic fibres will also be seen ; they EXAMINATION OF F^CES. 223 may be recognised by their clear outline, tendency to branch dichotomously, and the bold curves which are nearly always apparent. They are straighter and coarser than those described as occurring in human sputum, are very elastic, and are usually seen in masses. Bundles of areolar tissue will also be observed after meat has been taken, especially if the digestion is at all faulty ; the fibres are irregularly arranged, and generally appear in a shapeless mass the exact structure of which is difficult to make out. Fat is usually found in the form of crystals, the needles being arranged in rosettes; these are especially numerous when the pan- creas is diseased ; indeed their presence in combination with almost colourless stools is strong corroborative evi- dence of blocking of the pancreatic duct, but the exact nature of the lesion can, of course, only be made out clinically. Fat also occurs in globules. In chronic alcoholism it is found in abundance in the stools. When vegetables have been mixed with the diet, vegetable cells of various kinds are seen, spirals, hexagonal cells, single or in groups, &c. ; the forms being too numerous to describe and depending upon the particular vegetable which has been eaten. Starch granules. Owing to inhibition these bodies are at first sight difficult to recognise, but their nature can easily be demonstrated by the addition to the specimen under the microscope of a drop of iodine in iodide of potassium (p. 33), the development of a deep purple colour, turning to black when a drop of sulphuric acid is also added, proves the presence of starch. If only a milk diet has been given, as in infants, the 224 MEDICAL MICROSCOPY. appearance is different. The stools are loose instead of formed ; the constituents described above are absent, with the exception of fat globules and crystals, these being present in large numbers. Coagulated albumen (casein) is also found, usually as irregular yellow particles. Crystals. In the microscopic examination of faeces several varieties of crystals will be met with but very few of them have any diagnostic value. Fat crystals (see above). They occur very abun- dantly in the stools of patients suffering with jaundice. The investigations of Gerhardt, v. Jaksch and Oester- lein, show that they are formed of a combination of the al- kaline earths with the higher fatty acids. The crystals take the form of minute, sharp-pointed crystals, scat- tered through the field or arranged in clusters like rosettes. Haematoidin crystals. The author has never found these crystals in the alvine discharges, but von Jaksch, states (/. c., p. 156) that he has not infrequently done so, especially in chronic intestinal catarrh from over-eating, and in many instances in which blood had been discharged into the intestine some time before the stools were passed. The form of the crystals is usu- ally illdefined ; they are sometimes free and sometimes enclosed in masses of a shining substance resembling mucin. Charcot-Leyden crystals (for an illustration of these bodies see fig. 54). As these crystals are the result of decomposition of albuminous substances, it is only to be expected that they are some- times found in the stools. They have, however, no dia- gnostic significance and have been found in various diseases. EXAMINATION OF F^CES. 225 Oxalate of lime crystals (fig. 37, p. 199) are fre- quently met with and are probably derived from the food, being particularly abundant after a vegetable diet. Other crystals occasionally found, but having no clinical value are : triple phosphate, phosphate of lime, car- bonate of lime and sulphate of calcium. The formed elements which are found in the motions are blood corpuscles, white and red, and epi- thelial cells, but the most important from a practical point of view are the various parasites with their ova, and some pathogenic micro-organisms. The same plan will be adopted in their description as in the chapters on the examination of sputum, urine, vomit, &c. Red blood corpuscles. The occurrence of blood in the stools has already been briefly alluded to (p. 216), but although the colouring matter (somewhat altered by the action of sulphuretted hydrogen) is frequently pre- sent, yet the red blood cells are very seldom seen, even when the blood is apparently abundant, as in typhoid fever. Small irregular particles of haematoidin, reddish- brown in colour are met with, and more rarely, crys- tals of this substance. The presence of blood may be demonstrated thus : A small portion of the excrement is dried, and the powder placed on a glass slide ; a crystal of common salt is then added, together with a few drops of glacial acetic acid. The specimen is covered with a cover-glass, and heated until steam just begins to rise; after cooling, minute crystals of haematin will be visible by aid of the microscope. Leucocytes. White blood cells are but seldom met with, even when there is considerable inflammation of the intestine. If ulceration takes place, pus cells may be found in considerable quantity. The discharge Q 226 MEDICAL MICROSCOPY. of an abscess into the bowel is indicated by the appear- ance of pure pus. Epithelium. Epithelial cells in greater or less abundance are always found in the faeces, and their presence therefore is of very little clinical importance. Two chief varieties are met with, squamous cells from the lower end of the rectum, and cells which are fusiform or columnar in shape, from the remainder of the intestinal canal. These latter are sometimes diffi- cult to define, their outlines being faint, but their nuclei are always seen, and are especially distinct if some dilute staining fluid be added, such as methylene blue or magenta. Nothnagel has drawn especial attention to the fusi- form cells, which he considers a degenerative form, caused by the abstraction of fluid. In addition to the above constituents of the evacua- tions, a certain amount of amorphous material is always present, derived chiefly from the waste products of di- gestion ; it is of little importance medically, either with a microscopic or on chemical examination. The subject of parasites discharged from the bowel is a very large one. It will be most convenient to follow the usual division into vegetable and animal parasites, subdividing them into those which are patho- genic and those which are not. The former being the most important will be considered first in each division. VEGETABLE PARASITES. Pathogenic : Bacillus of tubercle. Tubercle bacilli are fre- quently found in the stools of phthisical patients af- EXAMINATION OF FAECES. 227 flicted with diarrhoea, and in such cases ulcera- tion of the intestines is almost invariably found after death. It is of course conceivable that the bacilli may be derived from the sputum which has been swal- lowed, but if such were the case, they would be very few in number and it would be no proof that an intes- tinal lesion did not also exist, as ulcers are frequently found after death, in which diarrhoea was not a pro- minent symptom during life. It is not, however, with obviously consumptive pa- tients that an examination of the faeces for tubercle bacilli is of value, but in those cases in which there is obstinate diarrhoea, with raised temperature, and in which the diagnosis is doubtful. If the organisms are then found the uncertainty is cleared up, and if they are very plentiful so as to resemble almost pure cultures, ulceration is nearly certain to be present. In order to search for tubercle bacilli, a small portion of the faeces is placed between cover-glasses, which are dried, passed three times through the flame and stained in the same way as will be described in the chapter dealing with the examination of sputum. Bacillus of typhoid fever. The pathogenic value of the bacillus first described by Eberth in 1880 in causal relationship with enteric fever is now accepted by most bacteriologists. The bacilli, however, unlike those of tubercle, possess no characteristic staining pro- perty ; it is therefore impossible to demonstrate them satisfactorily in typhoid stools, although they may readily be demonstrated in the tissues after death by the process described on p. 91 (LofHer's method). To prove their presence in the faeces a troublesome and prolonged process of separation of the colonies by plate cultivations has to be adopted, and this can only 2 228 MEDICAL MICROSCOPY. be accomplished in a properly appointed bacteriological laboratory. Bacillus of cholera. The discovery by Koch of the " comma bacillus," which has now been con- clusively proved to be the cause of Asiatic cholera, was the chief means of placing bacteriology in the position which it now holds in modern science. Fortunately in England at the present day, we have very rare oppor- tunities of studying the cholera bacillus except in laboratories. Koch found the organism in the intestine and alvine discharges of patients who had died or were suffering from Asiatic cholera. It was rarely found in the vomit, and never in the other secretions, urine, saliva, &c., nor in the blood or breath. The bacillus is a short thick rod, not so long as the tu- bercle bacillus, and generally more or less curved. The organisms are often united together into wavy threads (spirilla), especially in artificial cultures, owing to the rapidity of reproduction. As with the bacillus of enteric fever, the cholera organism is very difficult to demonstrate in the stools owing to the myriads of other bacilli which are present, but as in the typical rice- water stools it often occurs in huge numbers, an at- tempt may be made to do so in the following manner : A small portion of the flaky sediment of the stools is placed on a cover-glass, which is then covered by a second, so as to obtain two uniform layers by gently sliding the glasses apart. They are dried and the films fixed by passing them three times through a flame. They are then placed, prepared side downwards, in a watch-glass containing an alcoholic solution of fuchsine or methylene blue. After five minutes they are re- moved and thoroughly washed in water, after which they are dried and mounted in balsam. EXAMINATION OF F^CES. 22Q More satisfactory results are obtained by adopting Koch's method of plate cultivation, but this requires considerable experience in bacteriology. It may, how- ever, be briefly stated here that cultivated on plates of nutrient gelatine, the colonies commence to form in about twenty-four hours ; they are white in colour, roughly circular in shape, but have irregular outlines. Gradually a yellowish tint appears, which grows darker, especially towards the centre and the gelatine begins to liquefy, emitting a most unpleasant odour. For further descriptions of this bacillus and its growth in other media, the reader is referred to the text-books on bacteriology. Bacillus of cholera nostras. This organism is often known as the "bacillus of Finkler and Prior," these observers having first described it. It is found, in the stools of patients afflicted with " cholera nostras," and may be demonstrated in the same way as the bacillus of Asiatic cholera ; it differs from the last named in being broader and larger, but the most marked differences are seen in the cultures on nutrient gelatine. The colonies are very much larger than those of the comma bacillus of the same age. They have a faint yellowish-brown tinge, the borders are well-defined, and they exhibit a distinctly granular appearance. They liquefy nutrient gelatine very rapidly, so that the first plate of a series may be completely liquefied on the day following inoculation, emitting a very foul odour. NON-PATHOGENIC ORGANISMS. Micro-organisms of various kinds abound in the faeces, both in health and disease. So numerous are they that 230 MEDICAL MICROSCOPY. they often form a considerable proportion of the whole bulk of the stools. It is on account of this that patho- genic organisms such as those of typhoid or dysentery are almost impossible to recognise, not possessing any characteristic selective powers for particular stains. All the fission fungi stain readily with the ordinary stains, fuchsine and methylene blue being the best. A small portion of the discharge is spread out into a uni- form layer between cover-glasses, which are then separ- ated by a sliding movement, as in the examination of sputum, dried and passed three times through the flame. The glasses are then placed face downwards in a watch-glass containing the undiluted stain, or a drop or two of the dye is placed on the glasses. After three minutes, the preparations are washed thoroughly in water ; if fuchsine be used, a few drops of methylated spirits may be added. Very excellent specimens may be obtained by means of the Gram method. After the glasses are prepared in the ordinary way, a few drops of methyl-aniline-violet are placed on them and allowed to remain for three minutes, they are then washed in Gram's solution (p. 33) for one minute, and are finally dipped alternately in aniline oil and methylated spirits until no more colour comes out, after which they are dried and mounted in balsam. Another method by which good results are obtained, and which, as will be presently again mentioned, is strongly recommended by v. Jaksch, is to immerse the glasses for about a minute into a solu- tion of iodine in iodide of potassium. This reagent has the advantage of staining the yeast fungi as well as bacilli and micrococci, and for some of the latter it has a peculiar power of differentiation, colouring them blue or violet. EXAMINATION OF F^CES. 23! Moulds. Members of this group are very seldom found in the faeces when first passed. Practically the only one ever met with is the thrush fungus Oi'dium albicans, which consists of small round spores and mycelial threads. It is generally found in the stools of children suffering from a similar condition in the mouth. It is of very slight diagnostic importance. Saccharomycetes. These are very commonly found in the alvine discharges, both in health and disease, occurring as round or oval cells, exhibiting the characteristic gemmation ; they are especially abundant in the acid stools of children. They are also found in large numbers in acute catarrh of the small intestine in adults. When a cover-glass preparation is stained with iodine solution, the cells assume a mahogany-brown colour. Schyzomycetes. The fission fungi often occur in such large numbers as to constitute the main bulk of the discharge. In sprue, for instance, the peculiar whitish scum found in large quantities in the stools in the exacerbations of the disease, consists entirely of bacilli of various sizes. Dr. Thin has isolated thirteen varieties, probably one of these is pathogenic, but neither Dr. Thin nor the author has been able to verify this assumption. The organism most frequently met with is the one associated with ordinary putrefaction, namely, the Bacterium termo. Nothnagel was the first to describe the Bacillus sub- lilis in the stools. It has no pathological value. It occurs as long mycelial threads, the spores being situated at the end of the rods, or attached to the threads. These micro-organisms stain brownish-yellow with the iodine solution. 232 MEDICAL MICROSCOPY. Von Jaksch in a most exhaustive investigation on this subject, has described a large number of fungi which stain blue with the iodine solution, none of which, how- ever, have any clinical value (" Clinical Diagnosis," p. 132). Some of them are micrococci, which occur in large numbers, and are coloured reddish-violet with the solution. Next in order are small bacilli resembling those of mouse septicaemia, these stain in a similar manner. Larger rods are sometimes seen, which resemble the Leptothrix buccalis. Finally, large round cells are met with, strung together like beads, appearing very much like yeast cells. It may be here remarked that the colouring produced by the iodine solution is only transitory and has usually completely disappeared in forty-eight hours. ANIMAL PARASITES. Protozoa. Various forms of monads and infusoria are met with in the fasces, but are comparatively un- common, and merit only passing notice. Nothnagel found monads in the stools of consumptive and typhoid patients ; active movements had generally ceased. The Amoeba coli was first described by Losch (" Virchow's Archiv.," 1875, p. 196). These bodies are circular in form, contractile, and consist of coarsely granular protoplasm, and possess a round nucleus. Some valuable researches on amoebic dysentery have recently been made by Drs. Councilman and Lafleur ("Johns Hopkins Hospital Reports," December, 1891, EXAMINATION OF F^CES. 233 vol. ii., p. 395, et seq.). They found in certain cases of dysentery amoebae in the stools which did not differ materially from those described by Losch. In searching for the organisms some choice had to be exercised in the portions of stools to be examined. They were more numerous in the small gelatinous masses which were often contained in the faeces than elsewhere. The numbers which were found in different cases and even in the same case at different times, varied greatly. At times no single portion of the faeces which could be examined was free from them. In other cases single individuals were only found after long and careful search. Their numbers in general were proportional to the severity of the lesions. Drs. Councilman and Lafleur state that the amoebae differ somewhat in appearance according as they are active or inactive. In the latter condition they are round or slightly oblong and are more highly refractive than the other cells which may be found in the stools. In this resting condition there can frequently be dis- tinguished no division into an ecto- and an endo-sarc, but there is simply a body, enclosing vacuoles of greater or less size. They are difficult to recognise in this condi- tion unless active ones are found at the same time and so attention be directed to their presence, but when in motion their appearance is characteristic. Every de- gree of activity of movement can be seen. At times the change is so slow that one must look carefully for some time to be sure that any motion is taking place and at others the movements are so rapid that certain of their phases can scarcely be followed. The movements con- sist essentially of two kinds : one progressive, and the other limited to the thrusting out and retraction of pseudopodia. In some instances, there seems to be a 234 MEDICAL MICROSCOPY. certain rhythm in these changes of form and position, which occur at regular intervals. Various bodies were frequently enclosed in the amoebae. The most common of these were red blood corpuscles, often in considerable numbers. Pus cells, well preserved, or in various stages of disorganisation were also seen. Both bacilli and micrococci were oc- casionally enclosed. In some cases, in addition to red blood cells, blood pigment was found, either in the form of black granules or as irregular brown masses. Of infusoria the most common is the Cercomonas intestinalis. It occurs in the mucous discharges of young children. It is pear-like in shape, furnished with tentacles, and has a clear nucleus. An organism, closely resembling the one first de- scribed, is the Trichomonas intestinalis, but dis- tinguished from the former by a ciliated disk at one end. Another entozoon occasionally found, especially in diarrhoea, is the Paramaecium coli, it is oval in shape and covered with cilia, and the anterior extremity is narrower than the posterior. VERMES. The recognition of the various worms which infect the human intestinal canal is of great importance, not only with a view to undertake treatment, which may expel them, but also as a means of clearing up vague symptoms, which have previously evaded explanation. It is not intended here to enter into the full life history of each parasite, and to trace it from the ovum through the " intermediate host " to the human intestine; but EXAMINATION OF F/ECES. 235 only those forms will be described which are likely to come under the notice of the medical man in his clinical work. In addition to the worm, the ova are sometimes found in the stools ; these are never discharged through the genital canals of the parasite, but remain in situ until the proglottis is ruptured. CESTODA. The tape-worm as a whole is known as a " strobilus," and is divided into a " head," " neck," and a number of segments or proglottides. Taenia mediocanellata (saginata). This is the most common tape-worm in this country. The inter- mediate host is the ox. The head, which is the most easily recognised part of the parasite (fig. 44), is square in shape, and measures in the transverse diameter 1-5 mm. It is provided with four suckers, which usually have much pigment de- posited round them. As the head is generally difficult to find, or may not come away when the rest of the strobilus is discharged, it is important to be able to recognise the proglottides. The whole worm may attain the length of four yards, and acquires its full growth in about three months, so that if the head has not been discovered, although pro- glottides have been passed, we may conclude that the parasite has been destroyed, if no fresh segments are discharged for thirteen weeks. There is no distinct neck, but the first segment appears immediately below the head. The upper segments are broad and short ; 236 MEDICAL MICROSCOPY. about the centre of the animal they become square, and in the lower half the length exceeds the breadth. The sexual organs attain their full development about the 45oth joint from the head, the ova appearing about 400 joints lower down. The ripe proglottides measure f inch in length, and about -J inch in breadth. They usually rupture and discharge their ova while in the intestine, so that those FIG. 44. Head of Taenia mediocanellata (Boyce). which are passed per anum are shrivelled and empty (Fagge). The segments may be recognised and distinguished from those of the other tape worms by the form of the uterus. This organ has numerous branches, hav- ing twenty to thirty on each side of a median chan- nel ; the branching is dichotomous, and the termina- tions are round and club-shaped. The genital pore EXAMINATION OF FAECES. 237 does not alternate regularly, but may be situated on the same side in two or three successive segments, they are, however, always situated on the narrow edge. Malformations are particularly liable to occur, and sometimes there are two or three genital pores in a single proglottis, each corresponding with a separate double sexual apparatus ; so'metimes the segments are not regularly developed, so that imperfect triangular joints disturb the symmetry of the series ; or a super- numerary proglottis projects by the side of the con- tinuous line of joints ; very rarely there are two distinct chains united in their whole length by one edge at an acute angle. The eggs are oval, and exhibit the primordial yolk membrane ; no booklets are seen in the embryo as is the case in Taenia solium. Taenia solium. This worm was so named be- cause, according to an old idea, it was thought to exist alone in the intestine. This is now known not to be the case ; sometimes they occur in large numbers, as many as five-and-twenty having been passed by one indi- vidual. It was also formerly supposed to be the most common of the tapeworms in this country, but this, too, has been shown to be without foundation, the Taenia mediocanellata occurring far more frequently. The head of the Taenia solium (fig. 45) is of a generally rounded form, about the size of a pin's head. In front it is prolonged so as to form a proboscis, or rostellum, which is surrounded by a circle of twenty-six hooklets arranged with their points outwards. They are of two sizes, large and small alternately. The wide part of the head is provided with four large sucking discs, and is generally black from the presence of pigment. Owing to its small size it is difficult to find, and is MEDICAL MICROSCOPY. frequently overlooked by the patient, who should be directed by the physician to bring for examination every particle of white matter passed. The loss of the head is rendered more probable by the very slender neck which it surmounts. This neck is about \ inch in length, and it gradually merges in the anterior part of the body, in which fine transverse lines begin to appear as the first indication of the formation FIG. 45. Head of Taenia solium (Boyce). of segments. On passing down, the segments elongate and become more completely divided. The whole length of the strobilus is usually about nine feet. The joints are at first very small, and broader than they are long. They gradually increase in breadth and length until about the middle they appear square, and in the lower half they are longer than they are broad. The proglottides may be distinguished from those of EXAMINATION OF F^CES. 239 the mediocanellata by the shape of the uterus. This is more simple in the solium, consisting of a central passage running parallel to the length of the proglottis, and having about ten branches on each side, which ramify instead of dividing dichotomously. The genital pore is placed in a little papilla, situated on the lateral edge of the segment. It alternates regularly on the two sides of the animal throughout its whole length. Proglottides are best demonstrated by placing two of them on a glass slide and covering them with a large cover-glass, when they may be examined with a good lens. The specimens will be rendered more transparent by the addition of a little glycerine. The total number of segments is about 800. The sexual apparatus begins to appear about the 2Ooth segment from the front, and is mature about the 45oth. Its time of growth is the same as for the mediocanellata, and the same deductions may be drawn from the ap- pearance of the segments. The ova (fig. 46, g) are globular, and measure -036 mm. in their longest diameter. They have a shell which appears to be marked with a number of minute radi- ating lines, this being due to rod-shaped projections, which closely cover its surface ; when mature, embryos furnished with booklets are seen within the eggs. The intermediate host of this parasite is the hog, hence its greater frequency on the continent, where uncooked pork is a frequent article of diet. Taenia nana is the name given to a form of tape- worm prevalent in Sicily, Italy, and Egypt. Ranson mentions it as having occasionally been found in England. Its average length is nearly -| inch, and its breadth about -J^ inch. It has a circular head provided with four suckers, and a rostellum surrounded by about 240 MEDICAL MICROSCOPY. twenty booklets. The uterus is oblong. This parasite occurs in huge numbers in a single patient. Bothriocephalus latus. This worm is limited to the inhabitants of certain countries of Europe, being most commonly found in Switzerland and Sweden. It is the largest of all the tapeworms, attaining a length of from 1 6 to 20 feet, and possessing from 3000 to 4000 segments, which are broader than they are long. In the middle of the strobilus they are about ^ an inch broad by ^ inch in length, but the last ones gradually become longer and narrower so as to be almost square. This worm has a longitudinal projecting ridge traversing its whole length. The uterus occupies the middle of each segment. It is composed of a convoluted tube bent several times upon itself so as to give a rosette-like appearance. The genital pore lies in the centre of each proglottis, opening upon its ventral surface. When portions of the worm are passed by a patient, the segments do not come away separately, but portions two and three feet in length are expelled. The head of this parasite is oval, and about -^ inch in breadth. It is blunt at the anterior extremity, and has two deeply grooved longitudinal suckers, one on each side. The eggs (fig. 46, d) are .oval, and are about -07 mm. in length. They are covered with a brown shell, and open in a peculiar manner at one extremity by a kind of small lid, so allowing the embryo to escape. The intermediate host of this tapeworm is not known, but is supposed to be some fish. The frequency of fresh water lakes in Switzerland may explain its pre- valence in that country. Other tapeworms which are occasionally found in the human subject, but are so rare as only to need mention here, are the Tcsnia cucumevina which is about 8 inches EXAMINATION OF KECES. 24! long, having a small head with a rostellum surrounded with a quadruple circle of booklets ; the Tania flavo- puncta ; the Tania madagascariensis , and the Tania lepto- cephala. The ova of the above Cestodes are occasionally found in the faeces without any segments accompanying them. Should the presence of a tapeworm be suspected, the stools should be mixed with water and the sediment allowed to subside, and this process should be repeated several times until very little matter remains ; a small portion of this should be placed on a glass slide, a cover-glass applied, and the specimen examined by a % inch lens, when the eggs, if present, will be easily recognised. The same process should be adopted in searching for the smaller parasites now to be described. TREMATODA. These worms are commonly known as "Flukes," they are flat and more or less oval in form. On their ventral surfaces they have one or more sucking discs. They are rare in man, but have occasionally been found in the stools, or more often in the biliary passages or in- testines. Distoma hepaticum. This worm is very common amongst sheep, producing the disease called " Rot." As its name implies its chief habitat is the liver. It frequently dilates and obstructs the bile ducts. It is about an inch in length and half an inch in width. The body is flat and is elongated anteriorly, forming a kind of head which is furnished with a sucker. There is another sucker on the ventral aspect. Between the two lies the genital pore leading to the uterus which is R 242 MEDICAL MICROSCOPY. convoluted. The eggs (fig. 46, e) are small oval bodies, ' pj 03 C GO G hn 2 8 ? *&- 8 -S S^jr! ?llf - s .JS S ^ SQ d G c 22 '-^ o c -u *3 )r< i> 45 O s . u - N ^ o .a ^ E P S liST- o 5 S * 13 mm. long ; they are brown in colour, and one end which is broader than the other opens by a small lid. EXAMINATION OF FvECES. 243 Distoma lanceolatum. This species is about a J inch long and -^ inch in breadth. It is pointed at the extremities, but more so anteriorly than posteriorly. It has no alimentary canal, but possesses a complicated generative apparatus packed with numerous ova. These are oval, brown in colour, and about '04 mm. in length. This trematode is very rarely seen in man, and only occurs in comparatively small numbers in cattle. Distoma sinense. This parasite is more elongated than the two just described. It is about ^ inch in length and about -^ inch in breadth. It possesses a single suc- torial disc at one extremity, communicating with a bi- furcated intestine ; the genital pores open at the opposite end. When seen in the bile in the fresh state, a beau- tifully delicate green colour, tinged with yellow, may be observed round the edges, whilst the centre of the animal is a deep brown. The ova are extremely small and occur in very large numbers. This worm only oc- curs in man, and as many as five hundred may be found in the bile ducts of one person. Hitherto it has only been described amongst the Chinese. NEMATODA (Round Worms). Ascaris lumbricoides (common round worm). The Ascaris lumbricoides inhabits the small intestine, but may be passed either alone, or with the stools. In general appearance it closely resembles an ordinary earthworm. Whilst living it has a reddish-brown colour, fading after death to a duller grey. It measures six to sixteen inches in length, is marked by transverse striae, and tapers towards both extremities. The female is about twice as long as the male the head ; consists of R 2 244 MEDICAL MICROSCOPY. three conical projections which are furnished with fine teeth and touch corpuscles. The posterior extremity of the male is folded on the anterior surface like a hook. The ova (fig. 46,/)are circular or bluntly oval ; yellowish- brown in colour, and measure -07 mm. in length. They are covered with an albuminous sheath which is often tinged with bile. Beneath this covering is a dense shell. Several of these parasites (three to four hundred) may be passed by one individual ; but probably each worm only exists for a few months within the body of its host. Ascaris mistax. This parasite in general form much resembles the preceding, but is smaller, and the shape of its head is rather more pointed. Hitherto it has only been found in the cat. Oxyuris vermicularis (threadworm). As their common name indicates these worms are like small ends of white thread ; the male being about an eighth of an inch and the female about four-tenths of an inch in length. They have a comparatively blunt anterior and a tapering caudal extremity. The tail of the male is provided with six pairs of papillae. The male is much more frequently seen than the female. The eggs (fig. 46, b) are oval in form and flattened on one side. They measure '05 mm. in length. The con- tents are granular except when mature, when the em- bryo may be seen. The shell is membranous and pre- sents a treble contour. Trichocephalus dispar (whipworm), (fig. 47). This parasite inhabits the caecum. It is cylindrical in form, having a short thick body, and long whip-like neck. The male is about i-J- inches in length, and the female about 2 inches. The worm fixes its thin ex- tremity by means of four claw-like teeth into the wall EXAMINATION OF F^CES. 245 of the intestine, and is therefore only very rarely found in the faeces. The ova (fig. 46, a) are oval and about 05 mm. long, and are characterized by having a trans- lucent point at either extremity. Anchylostoma duodenale (Strongylus duodena- lis). This parasite does not occur in this country, but is frequently met with in Egypt and Brazil. It inhabits chiefly the jejunum. It is cylindrical in form. The male measuring about half an inch, and the female an inch in length. The head which is bent nearly at right angles to the body is armed with four conical teeth, by means of which the animal fixes itself on the mucous membrane. The posterior extremity is tapering, and in the male expands into a pouch with three flaps. The eggs (fig. 46, c) are oval, with a thin transparent shell, FIG. 47. Trichocephalus dispar. and have a long diameter of -05 mm. The division of the yolk (fig. 46, c) can be plainly seen. This nematode is only seen in the stools after some measures have been taken to expel it, but the eggs frequently appear. Strongylus gigas. This is a large worm chiefly met with in the kidney, bladder, lungs and liver of dogs, but a few cases are on record in which it has been met with in the pelvis of the kidney in man. It is a large worm, the male being about ten inches in length, and the female nearly a yard. 246 MEDICAL MICROSCOPY. Anguillula intestinalis. This is a small round worm, which has been discovered in the stools in cases of Cochin -China diarrhoea, but has not been met with in this country. Trichina spiralis. These parasites have in very rare cases been found in the stools. They closely re- semble the Trichina found in muscle, but are not en- capsuled. The male is 1-5 mm. in length, and the female 3 mm. The males are distinguished from the females by having two conical projections from the posterior extremity. EXAMINATION OF SPUTUM. 247 CHAPTER XVI. EXAMINATION OF SPUTUM. BY " sputum " is understood all the matter coughed or hawked up from the air passages. As this material includes the secretions from the buccal respiratory tract, it might naturally be expected that an examination of the contents of the spittoon would yield valuable results as regards the condition of the parts forming that tract. Since the discovery of the tubercle bacillus by Koch, in 1882, the microscopic examination of sputum has re- ceived a great impetus, and it has now become almost a matter of routine in any case of lung disease of a doubtful character. The naked eye features of the expectoration received much more attention from the older writers, before the physical examination of the chest by percussion and auscultation arrived at the present state of exactness, and before the clinical thermometer had been brought into routine use. Not only were the appearances of the secretion noticed, but attention was directed to its smell, taste, reaction, &c. With our modern methods of physical examination, much less stress is now laid on the macroscopic characters of sputum, but with the aid of the microscope a diagnosis, which may be doubtful, or extremely obscure (e.g., Actinomycosis), may be cor- roborated or established. Although this chapter will necessarily be directed chiefly to the microscopic examination of sputum, it will 248 MEDICAL MICROSCOPY. be necessary, or at any rate desirable, to briefly con- sider some of the more noticeable points in connection with its quantity, colour, odour, &c. Sputum may be described as mucous, muco-purulent, or purulent, according as it is transparent, semi-opaque, or resembles the matter drawn from an abscess. To attempt to describe its examination under these heads would be manifestly absurd, as instances of each are found in the course of most diseases of the respiratory organs. It is very important that a satisfactory sample should be obtained. In the first place then, the spittoon must be carefully washed out before use, and must never be made a receptacle for grape skins and various other odds and ends, which seem so frequently to find their way into it. It is well also to replace it with another during meal times, otherwise particles of food are cer- tain to become mixed with the expectoration, which gives much unnecessary trouble to the physician when searching for the minute white particles so characteristic of certain diseases. This is especially the case if the patient be on a milk or farinaceous diet. Thus, in the sputum of one case which I had sent me for examina- tion in several succeeding samples, minute white specks were seen, which combined with the history and sym- ptoms of the case had given rise to a suspicion that it was one of actinomycosis. By means of micro- chemical tests, however, they were shown to consist simply of collections of starch grains derived from food. Sputum coughed up early in the morning before the patient has partaken of any food is to be preferred, though for some purposes the whole quantity collected during twenty-four hours is necessary. In order to observe the stratification peculiar to some EXAMINATION OF SPUTUM. 249 diseases, the sputum should be collected in conical glasses. QUANTITY. The amount of sputum expectorated within twenty- four hours varies very greatly. In some conditions the spittoon may be filled several times daily, whilst in others there will scarcely be a drachm. The sputum is excessive in oedema of the lungs, and in some cases of haemoptysis ; also when an abscess bursts, derived either from the lung itself or breaking through into the lungs from neighbouring parts. Most commonly this is due to the rupture of an empyema, but the pus may be derived from the abdominal cavity. The quantity is also excessive in bronchiectasis, the patient may rest for several hours, suddenly bringing up many ounces of foetid purulent matter. In phthisis again, where large cavities exist, the expectoration is often very copious but this is by no means characteristic. CONSISTENCE. The consistence of the sputum stands usually in some relation to its quantity, for if this be very copious the expectoration is generally thin and watery, whilst if small in amount it is usually thicker ; but numerous exceptions to this statement are constantly met with. The consistence is dependent in a great measure upon the amount of mucoid material contained in it. As a type of a tough tenacious sputufm, that occurring in croupous pneumonia may be taken ; it is often so tena- 250 MEDICAL MICROSCOPY. cious that if the spittoon be turned upside down, it will not flow out. The reason for this is difficult to assign, for the proportion of water is not small, nor is the quantity of mucin contained nor its specific gravity unusually large. The expectoration in bronchial asthma is often very tough, as can be proved by the difficulty experienced in obtaining a thin layer for microscopic examination ; it contains a large amount of mucin. In the early stages of acute bronchitis also the secretion is very thick and tenacious. This condition was termed by the older writers Sputum crudum, in contra-distinction to the thin watery expectoration which they named Sputum coctum. COLOUR. In speaking of the colour of sputum we must con- sider briefly its transparency. Pure mucoid expectora- tion is colourless and almost transparent. The more cellular elements there are present, especially leuco- cytes, the more cloudy and opaque the secretion be- comes. Changes in colour are almost entirely due to the presence of blood or its derivatives. This is not the place to enter into a discussion on the different causes of haemoptysis. Roughly speaking, they may be divided into two classes, in the first of which a rupture of a blood vessel, either in the pul- monary or systemic circulation, gives rise to the haemor- rhage, whilst in the other there is a diapedesis of red blood corpuscles parenchymatous haemorrhage. The amount of blood may vary from fine streaks dis- tributed through the expectoration to almost pure EXAMINATION OF SPUTUM. 25! blood. The latter condition is only found when a vessel has completely ruptured. Investigations by Dr. Kidd and Mr. Taylor at the Brompton Hospital for Consumption showed that in a large proportion of the cases of profuse haemoptysis, a small aneurysm had burst, whilst erosion of the vessels was comparatively rare. When first ejected the blood is usually bright in colour, frothy in appearance and alkaline in reaction. Haemoptysis is by no means diagnostic of tubercle ; heart disease, especially mitral stenosis, bronchitis and pneumonia, croupous and catarrhal, congestion of the lungs, infarcts and the presence of parasites, new growths, &c., all may give rise to blood spitting, more or less profuse. Professor Albert Frankel in a most ex- haustive discussion (" Pathologic und Therapie der Krankheiten des Respirations Apparates," vol. i., p. no) on the occurrence of haemoptysis, comes to the conclu- sion that blood spitting without any previous symptoms, is most probably always the expression of commencing tubercular inflammation. If the blood has been retained in the lungs for any time it undergoes changes which produce considerable alteration in its colour and general appearance, this being due to decomposition, or metamorphosis of the haemoglobin. Thus in gangrene of the lungs, the ex- pectoration becomes of a dirty reddish-brown colour, owing to the formation of methaemoglobin and haematin. Very characteristic also is the peculiar chocolate colour which the expectoration assumes when a crou- pous pneumonia is passing into gangrene. This form was termed by Andral " prune juice expectoration." Under the microscope the blood corpuscles will be found to have undergone very considerable changes, which will again be referred to later on. The same 252 MEDICAL MICROSCOPY. appearance is sometimes noticed in the sputum of aged persons suffering from oedema of the lungs. A peculiar brownish colour of the expectoration is often found in gangrene of the lung more commonly when ulcera- tion of the lungs has occurred from some other cause. Leyden considered this to be due to the abundance of hsematoidin and bilirubin crystals which are present. Brown induration of the lungs produces a peculiar appearance in the sputum. In the mass of mucoid material rust coloured streaky deposits are seen, especially in the early morning, and these under the microscope are found to consist of pigmented epithelial cells. More common than the above is the well known rust- coloured sputum of croupous pneumonia. This colour, however, is not always constant, occasionally it assumes a lemon-coloured hue, or more commonly a grass- green colour, this being especially the case when the crisis has been deferred. This latter appearance must not be confused with those serious cases in which jaun- dice has supervened, for the green colour is then due to admixture with bile colouring matters. Traube also described this green colour in sub-acute cases of pneumonia, so that in any doubtful case when green expectoration is observed, micro-organisms should always be sought for. Various observers have described a yellow ochre coloured sputum ; this being associated with abscess of the liver, more especially when hydatid cysts are pre- sent. Under these circumstances large numbers of haematoidin and bilirubin crystals are seen under the microscope and the booklets of the parasite may be dis- covered on microscopic examination. The above colourations must not be confounded with EXAMINATION OF SPUTUM. 253 the yellow tint sometimes assumed by the expectora- tion in summer time. This occurs in various diseases and the tint is very similar to the yellow of an egg. The colour is confined to the superficial layers of the mucus, and often becomes more pronounced after the expectoration has remained some time in the spittoon. It is dependent upon the presence of bacteria, which in their growth produce the pigment. It is a fact worthy of remark that not infrequently several patients in the same ward exhibit the same colouration of the sputa, especially those in neighbouring beds. From similar causes a greenish tinge is sometimes observed. Black coloured sputa may be said to be normal, for they are almost always met with in towns or manufac- turing districts. The black hue is owing to the admix- ture of particles of carbon, dust, &c., which everybody inhales more or less. The particles are found partly free and partly embedded in cellular elements, hereafter to be described. In cases of malignant disease of the lungs the expec- toration is generally tinged a dark red, and has a peculiar gelatinous consistency, sometimes, however, both with carcinoma and sarcoma, various shades of green appear. Before quitting the consideration of the diagnostic value of the colour of the sputa, a few words must be said about what is known as " spurious haemoptysis." That is to say blood ejected from the mouth, but having its origin not from the lungs, but from the upper part of the air passages, the trachea, pharynx, mouth and nose. The diagnosis from lung disease, however, is more easily made clinically than by an examination of the contents of the spittoon. Hysterical patients often prick and suck their gums 254 MEDICAL MICROSCOPY. in order to elicit sympathy and obtain credit for being the subject of serious pulmonary disease. Dr. Douglas Powell (" Diseases of the Lungs and Pleurae," 3rd edit., p. 363, et seq.) divides false or spurious haemoptysis into seven divisions: i. In cases of epis- taxis, the blood commonly trickles down the back of the throat and excites cough, by which it is removed in clots, staining the saliva. 2. Ulceration of the throat, especially when malignant, may lead to copious haemorrhage. 3. Hysterical haemoptysis (already re- ferred to) . 4. A morbid state of the gums frequently arises from want of due attention to the teeth, or from the presence of decayed stumps in the alveoli ; blood may exude on the slightest friction. 5. An in- sufficient supply of vegetable food leads to a spongy congested state of the mucous membrane of the mouth and fauces, very similar to the lesions characteristic of scurvy, and this is one of the most common causes of spurious haemoptysis. 6. In certain cases of anaemia the mucous membrane of the mouth and fauces exudes a sanguineous fluid. The transudation is very slow, and in the daytime scarcely noticed, but during the night some accumulation takes place, and on waking the patient expels some bright red, unaerated fluid containing a few coagulated films, giving an appearance closely resembling that of currant jelly and water. 7. General haemorrhage from the whole mucous membrane of the mouth is sometimes seen in haemophilia. ODOUR. Inferences to be derived from the odour of the sputa are not of any material value. The chief exception is EXAMINATION OF SPUTUM. 255 that of gangrene of the lungs, in which complaint the expectoration has a peculiarly offensive and penetrating smell. In phthisis and other diseases in which the secretion is retained for some time in the air passages, it acquires a faint sweetish odour, due to decomposition. A few other naked-eye characters of the sputum remain to be considered. In the later stages of phthisis, when cavities have formed, the sputum becomes nummulated. It then consists of opaque greenish discs, about the size of a sixpenny piece. If ejected into water these discs sink and spread out into irregular and ragged masses. They are probably formed by the passage of a thick secretion, through the very fine bronchial tubes (Frankel). A similar condition is seen when an empyema has burst into the lungs (Traube). When some forms of sputa are allowed to stand they separate into three layers, the uppermost of which con- sists of semi-opaque, mucoid, and generally frothy liquid. The middle is a serous-like fluid, somewhat cloudy and whey-like ; whilst the undermost is made up of a mass of pus cells and debris, and is usually of a dirty yellow- ish colour. This formation occurs most commonly in bronchiectasis, and very typically also in putrid bron- chitis, and gangrene of the lungs. In one of the rarest diseases known to physicians, " plastic bronchitis," peculiar formations termed " bron- chial casts" or "stolons" (fig. 48) are expectorated. The sputum in this disease is either of a catarrhal type, or contains a large proportion of blood. In it are masses covered with mucus rolled up in the form of balls, and stained with blood. If these are thrown 256 MEDICAL MICROSCOPY. into water they spread out, and when freed from mucus are seen to consist of complete casts of parts of the bronchial tree. According to Biermer they may extend to its finest subdivisions, so that the minute terminal filaments may show bulbous ends, due to their having FIG. 48. Bronchial stolon (Bizzozero and Firket). been moulded in the infundibula. Their colour is a greyish-white, sometimes, however, having a reddish tinge. This depends either upon their being merely stained with blood externally, or owing to their central EXAMINATION OF SPUTUM. 257 cavity being filled with blood clot. In the great ma- jority of cases this central bore contains bubbles of air, producing an appearance as of a string of pearls within them (Frankel). When examined more minutely the casts are found to be composed of a number of concentric laminae, separ- ated at intervals by narrow spaces, and with a central cavity running their whole length, except in the finest branches. When submitted to microscopic examination they are found to consist of a fibrillar or hyaline base, in which are embedded large numbers of leucocytes. Charcot-Leyden crystals are often found in quantities within the meshes. The length of the casts is usually ! to 2 inches, but sometimes they are much longer, even attaining 7 inches. Their diameter is narrow, rarely exceeding that of a goose quill. They divide dichotomously, the branches gradually diminishing in length and thickness. A slight bulging is often seen at the points of division, dependent on a similar condition in the branches of the bronchi themselves. According to Biermer the site of formation of the casts may be inferred from the length of the intervals between succes- sive points of bifurcation. The short, rapidly branching stolons being derived from the tubes of the upper lobe of the lungs, and the comparatively longer tubes from the lower lobe. The masses expectorated at different times by the same patient are often so similar in size and arrangement as to give forcible corroboration to the suggestion that they have been produced in the same tubes. Similar bodies are found occasionally in pneumonic sputum, and in the rare cases in which diphtheritic membrane has penetrated to the finest branches of the bronchi. The casts in such a case are not so com- s 258 MEDICAL MICROSCOPY. plete as in plastic bronchitis, and are thicker and less elastic. Casts very similar to the above are occasionally seen after an haemoptysis, they are easily distinguished from the stolons, being homogeneous in structure, more trans- parent, and only consisting of a few branches, these being difficult to trace, and to separate from one another. We need not in this little work enter into fuller de- tails of the characters of sputa diagnostic of the different diseases of the respiratory organs, but will now pass to the MICROSCOPIC EXAMINATION. ' The same precautions must be taken in collecting the samples as were described in preparing for the macro- scopic examination, and especially is the sputum to be selected which is coughed up early in the morning, before any food has been taken. As a rule special portions have to be picked out and placed beneath the microscope, and this process is greatly aided by emptying the spittoon into a black vulcanite dish, similar to those employed by photo- graphers, or into dishes specially prepared for this work. By this means the opaque-white particles which yield the most useful information are rendered con- spicuous and more easily recognisable. These portions are most conveniently removed by forceps and scissors ; it is a good plan to slightly bend the tip of one of the blades of the forceps, so as to form a small hook. Another method very generally adopted is to employ two steel pens, these act admirably, both as tearers and lifters. EXAMINATION OF SPUTUM. 259 Some aid is rendered to the general microscopic examination of sputum by pouring it into a large conical glass containing water, the heavier portions, such as fragments of lung tissue, spirals, pieces of membrane, &c., will fall to the bottom after a few hours, and may be removed by means of a pipette. In the preliminary examination any small opaque portions are removed and placed in the centre of a glass slide, and a cover-glass laid on the specimen, and gentle pressure applied in order to obtain a uniform layer. If it be desired to preserve the specimen a drop of glycerine must be added, then any superfluous moisture removed from the edge of the cover-glass by means of filter paper and liquid balsam run round its edges. Certain objects, such as leucocytes, pus cells, salivary corpuscles, &c., will be found in all sputa, and in others objects of more diagnostic value. We now proceed to describe in detail the various formed elements which may be met with. i. Leucocytes and mucus corpuscles (fig. 49, /). In greater or less numbers these are constant ingredients of all sputa. The latter are less granular in appearance than the former, and are possessed of more nuclei ; they are larger too than the leucocytes, but owing to the effects of diffusion it is often almost im- possible to distinguish between them. In mucoid ex- pectoration the mucous corpuscles naturally predominate, whilst in purulent sputa the pus cells (leucocytes) may occupy the entire field. Both varieties of cells are seen in all stages of fatty degeneration, and may contain pigment granules, particles of carbon, &c., and after an haemoptysis, crystals of haematoidin. It will be obvious from these remarks that very few deductions can be with safety made from their presence. Closely s 2 260 MEDICAL MICROSCOPY. resembling these cells, but rather larger, are the "sali- vary corpuscles." 2. Compound granule cells (fig. 49, e). These bodies are found in most sputa, often in so great numbers as to conceal all the other elements. In general appearance they resemble leucocytes, but are three or four times as large. In shape they are round or oval. Their protoplasm is coarsely granular, and they contain several large oval nuclei which are ren- dered more conspicuous by the addition of a drop or two of dilute acetic acid. They are frequently the seat of particles of carbon and dust, and often appear in an extreme stage of fatty degeneration. Again, sometimes peculiar formations are found in them, first described by Virchow (" Archiv.," Bd. vi., page 562), and termed by him "myelin drops" (fig. 49, d) t owing to their similarity in appearance to coagu- lated nerve pulp. These objects are irregular in form, being sometimes circular, sometimes spindle- and some- times club-shaped, and having a tendency to flow to- gether and form unshapely masses. They are also found floating free in the general mass of mucus. The chemical nature of these bodies is uncertain, but most authors consider that they consist of lecithin. The origin of the compound granule cells has been much disputed. The majority of observers ascribe their source to the alveoli of the lungs. Against this view is the fact that they are not only found in conditions in which the alveoli are affected, but also in bronchitis and in the morning expectoration of perfectly healthy people. In favour of this view on the other hand, as before stated, is the fact that they are the principal seat of foreign par- ticles which have been inhaled into the lungs, and are especially numerous in miners, &c., suggesting the idea EXAMINATION OF SPUTUM. 26l that a slight irritation is set up, sufficient to loosen these cells, and so cause their presence in the pulmonary secretion. Other authors, of which Cohnheim is the chief, consider that they are simple lymph corpuscles which have become enlarged and swollen owing to the imbibition of cellular debris, &c. I most certainly in- cline to this last view. 3. Red blood cells (fig. 49, #). The subject of hae- moptysis has already been fully considered. The red corpuscles, contrary to what occurs in the urine, retain FIG. 49. Various cells in sputum, a. Squamous. b. Columnar. c. Cubical, d. Myeloid. e. Compound granule cells. /. Leuco- cytes, g. Red blood cells. as a rule their shape and colour. When they are very numerous they form rouleaux, and preserve their or- dinary histological behaviour. Under some circum- stances, as in pneumonia, the corpuscles yield up their colour to the general mass of sputum, and then appear as colourless rings, and the same process of destruction is also noticed in gangrene of the lungs. In pneumonia they may only be represented by crystals of haema- toidin. 4. Epithelium. Three varieties of epithelium are 262 MEDICAL MICROSCOPY. found in the sputa, namely, squamous cells derived from the buccal cavity, pharynx, and upper part of the larynx ; columnar cells from the greater portion of the respiratory passages ; and cubical cells from the terminal bronchi and alveolar walls. Squamous cells (fig. 49, a) are always found, and there- fore have no pathological interest. When heaped to- gether in big masses their edges are very likely to be mistaken for elastic fibres, but careful focussing will obviate this error. The columnar cells (fig. 49, b) are of more value in diagnosis, for when they occur in large numbers they denote an inflammatory state of the parts from which they are derived. They are usually more or less altered from their original form becoming swollen and pear- shaped, and their protoplasm more hyaline in appear- ance, and through partial loss of their contents form the well known " goblet cells." Although in their original state they are furnished with cilia, these appendages are very seldom preserved in the sputum. The cilia occur under only two conditions, namely, strong mechanical irritation of the bronchial mucous membrane, or ulcera- tive destruction of the same. From the first cause it is that they are sometimes seen in asthmatic sputum, for here the smaller bronchi are irritated by efforts to expel the thick and tenacious masses of secretion (Frankel). Cubical epithelium (fig. 49, c) is derived from the al- veoli ; the cells are not often met with in their original condition, but are more or less altered by fatty degenera- tion. In the expectoration of patients suffering from brown induration of the lungs, especially in that ejected in the early morning, small reddish-brown streaks and particles are found, which when examined microscopi- cally are found to consist of masses of pigmented epi- EXAMINATION OF SPUTUM. 263 thelial cells, the particles of pigment being circular or irregular, rarely crystalline. According to some authors the compound granule cells which have been just described are considered to be altered alveolar epi- thelium. 5. Elastic tissue (fig. 50). In the microscopic examination of sputum the search for elastic tissue stands second only in importance to that for tubercle bacilli. This tissue may have its source in the alveoli of the lungs, in the mucous membrane of the respira- tory tract, or in the vocal cords and epiglottis. In the large majority of cases, however, it is derived from the alveoli. When present it is essentially diagnostic of partial destruction of the lungs or other parts of the respiratory tract. A little experience will very soon enable the observer to recognize the fibres derived from the parenchyma of the lung from those of the bronchi or larynx. In order to search for elastic tissue, the sputum for twenty-four hours should be collected and thrown into a black dish. Sometimes the collections of elastic fibres are so large that they appear as opaque, white specks floating in the mucus. When one of these is removed and placed under the microscope it will be seen to consist entirely of large meshes of elastic tissue, showing quite plainly an alveolar arrangement, roughly tracing the forms of the alveoli. Such cases, however, are not common, and as a rule one must search in the general mass of expectoration for any particularly opaque particles, remove them by means of forceps and scissors, and place them on a slide. A cover-slip is next laid on, and gentle pressure used to obtain a uniform layer. The specimen must then be examined 264 MEDICAL MICROSCOPY. with a j- inch lens, and after a little practice the curled fibres will soon be recognized. By this method they retain exactly their original form, and much useful information may be gained from a study of them. II the sputum be very thick and tenacious, some aid in rendering it more transparent may be gained by running under the cover-glass a drop or two of a 30 per cent, solution of caustic potash. This dissolves the mucus and other matters, leaving the elastic fibres untouched. Several preparations should be examined before a nega- tive result is announced. Another method which is frequently practised is one which was first introduced by Dr. Samuel Fenwick. It has the advantage of being more certain of detecting elastic tissue if present, but the appearance of the fibres is considerably changed, and many characteristics pre- sently to be alluded to are lost. After considerable experience I have come to the conclusion that the former method, although perhaps more tedious, is to be preferred. Dr. Fenwick's method is carried out as follows : The sputum is collected for twenty-four hours, and poured into a beaker. An equal quantity of a solution of caustic soda, twenty grains to the ounce is then added, and the mixture boiled until it becomes perfectly liquid, being occasionally stirred with a glass rod. When this stage has been reached the heating must immediately cease, for over-boiling will either dissolve the elastic tissue or render it so transparent and irregular in ap- pearance that it can scarcely be recognized. When the boiled fluid has somewhat cooled it is thrown into a conical glass containing about four times its volume of water. In an hour or so a deposit will have formed in which the elastic fibres will be found, and portions of EXAMINATION OF SPUTUM. it may be removed with a pipette and examined under a microscope. Elastic tissue is composed of fasciculated fibres, highly refractive, but possessing dark contours. They branch dichotomously. They have a slightly yellowish tinge, and have a tendency to break across, leaving a sharply defined edge. They usually occur in wide hoops and curves. Their clearly cut outline is especi- ally characteristic, and enables them to be distinguished FIG. 50. Elastic tissue. from other bodies such as cotton and silk fibres, for which they might otherwise easily be mistaken. When derived from the parenchyma of the lung they will be seen to have a distinctly alveolar arrangement, as is well shown in the accompanying figure (fig. 50) which was procured from the expectoration of a patient about twelve hours after an injection of tuberculin had been administered. When coming from the larynx the arrangement of the 266, MEDICAL MICROSCOPY. fibres is more linear, the fibres being straighter, and devoid of the bold curves which are so characteristic of the last named variety. Fibres from the other parts of the mucous membrane of the respiratory tract are similar to those coming from the larynx in their general arrangement, but are finer, and the bundles are generally of smaller dimensions. Their diagnostic value is great. Roughly speaking, their presence indicates that a destructive process is occurring somewhere in the respiratory tract. If from the larynx or bronchi there is probably considerable ulceration. If derived from the former the laryngo- scope will give a far more satisfactory diagnosis than the contents of the spittoon ; but it is in those cases in which the arrangement of the fibres denotes that the parenchyma of the lung is affected that their recogni- tion proves of so much value. It was at one time thought that they were diagnostic of tubercular disease of the lung. Troup (" Sputum ; its Diagnostic and Prognostic Signification ") maintains that where the elastic tissue of the pulmonary alveoli is seen, the correct diagnosis in nine cases out of ten will be that of tubercular phthisis. If tubercle bacilli be also found of course the diagnosis is assured, but the same author considers that careful microscopy of the expectorated matters can frequently detect its presence in a very early stage of phthisis, even when percussion and auscultation practised by skilled observers give only doubtful or negative results. Dr. Troup even goes further than this, and states that he has frequently found elastic tissue months before bacilli made their appearance. I cannot say this has been my experience, and would most certainly give the palm to the tubercle bacilli for early diagnostic worth. EXAMINATION OF SPUTUM. 267 In addition to being found in phthisis, elastic tissue is occasionally seen in the sputum of pneumonia even when the disease has run a normal course (v. Jaksch). The fibres are also found in cases of abscess of the lung, and they are then seen on careful examination to be thickly set with pus corpuscles, and to be greyish- yellow in colour, especially at their edges. They are also found in the sputum from bronchiectatic cavities. In cases of gangrene of the lung these fibres are again occasionally found. They are generally of a dirty colour with jagged edges, and are surrounded by greyish-yellow masses. Their appearance in this dis- ease is not very common, in fact rather the exception, the reason probably being that though there is a con- siderable destruction of lung tissue the elastic tissue is dissolved by the ferments evolved during the process. Frankel gives a somewhat similar explanation, suggest- ing that the fibres are destroyed by the numerous bac- teria which are present. In addition to the information yielded by the pre- sence of elastic tissue of disintegration of lung tissue, more precise knowledge may be obtained by closer examination, as has been pointed out by Sir Andrew Clark. Thus if complete alveolar rings are present which are at the same time very elastic, we may assume that there is a rapid destruction of lung tissue taking place. If small "tailed" pieces only are seen which have lost their elasticity, a more chronic process is indicated, whilst occasionally fibres are found encrusted with lime salts (fig. 51) and these must have lain for a con- siderable period in the lung cavity. Care must be taken not to mistake other bodies which are often present in the sputum, for elastic 268 MEDICAL MICROSCOPY. tissue ; such objects are portions of fungi, as the Lepto- thrix buccalis and various forms of aspergillus. These are not so regular in outline as the elastic fibres and are generally surrounded by spores. A description of these fungi will be given later on. But the bodies which are most likely to mislead are fibres of cotton, silk or flax (see p. 278) ; a little prac- tice, however, will soon prevent the observer from making any such blunders. Fibrillation of the mucin of viscid sputa will sometimes give rise to an appear- ance, at first sight, closely resembling a bundle of fibres, FIG. 51. Fragments of elastic tissue partially encrusted with lime-cells. but careful focusing will easily discern between the two. 6. Fragments of connective tissue. These only occur in rare cases and are derived from the walls of the alveoli and bronchi. They occur as dark grey particles, and are easily recognised. They are chiefly found in cases of gangrene and abscess of the lung. In ulcerative processes affecting the larynx, small portions of cartilage may be loosened by the violence of the cough, and be ejected with the expectoration. 7. Curschmann's spirals (fig. 52). These pecu- liar bodies are chiefly found in the sputa of asthmatic patients, where they are sometimes exceedingly mime- EXAMINATION OF SPUTUM. 269 rous. They were at one time considered characteristic of asthma, but have since been shown to occur, al- though only in small numbers, in the expectoration of pneumonia and of acute and chronic bronchitis. They were first minutely described by Curschmann, and hence their name. FIG. 52. Curschmann's spirals (Troup). If the sputum from a case of bronchial asthma be poured out on a black plate small opaque white par- ticles will be seen which, even with a low power lens, may be seen to consist of spiral formations. When one of them is placed under the microscope, an intricate 270 MEDICAL MICROSCOPY. arrangement of spiral threads can be discerned. In the centre there is a highly refracting thread sometimes straight and sometimes twisted corkscrew fashion. Around this, in a complicated manner, is entwined an ensheathing layer of fine threads, in the meshes of which are seen leucocytes and epithelial cells. Some of the spirals will be noticed to possess a peculiar prominence, and if these be more closely examined a large collection of pointed octahedral crys- tals of various sizes will be found. The exact structure of these spirals has not yet been definitely settled. According to some observers they consist largely of collections of bacteria, but the majority of authors consider that they are formed of mucus which has been moulded into spiral shape in its progress through the small bronchioles. The central core is due partly to an optical effect and partly to the greater compression which it has apparently undergone. Three modifications of the spirals are generally met with. Firstly, the complete spiral, consisting of the central thiead surrounded by the mass of mucoid material ; secondly, the central thread alone is often seen, and thirdly, the spirally twisted portion of mucus may occur without the central thread being visible. The diagnostic value of these bodies has not been es- tablished with any certainty. They are probably due to a catarrh of the finest bronchioles, and as before stated they are most commonly associated with bron- chial asthma. 8. Tonsillar casts (fig. 53). Casts of the crypts of the tonsil, or more frequently fragments of such casts are occasionally found in the sputum. They chiefly occur in connection with acute inflam- matory diseases of the tonsils, but in comparison with EXAMINATION OF SPUTUM. 2 7 I the clinical aspects of the case have little diagnostic value. They bear considerable resemblance to masses of mucus, and are consequently easily overlooked, but they are more likely to be mistaken for small leashes of elastic tissue. Tonsillar casts appear as irregularly- shaped bodies composed of parallel fibres finely knit together into a close net-work. When teased out the FIG. 53. Cast of a tonsillar crypt (from a drawing kindly lent by Dr. Sheridan Delapine). appearance represented in the figure is produced. A complete cast assuming the exact form of a tonsillar crypt is rarely met with, but as already stated, more commonly only fragments are found. 9. Corpora amylacea. These bodies are very rarely met with ; they were first described by Friedreich in 1856. They appear as oval or -circular grains and 272 MEDICAL MICROSCOPY. are very similar in form to starch grains. They are composed of a vitreous looking substance, stratified in irregular layers around a central nucleus, which differs in appearance from the peripheral layers which are arranged so as to present a radiated formation. The central portion is sometimes scooped out so as to form a small hollow from which shallow fissures extend, and occasionally is filled up with a material of different composition to the rest of the grain. These amyloid bodies are characterised by their chemical reactions. If a solution of iodine in iodide of potassium be run in under the cover-glass a deep blue colour is produced, which becomes almost black on the addition of sulphuric acid ; the nucleus, how- ever, is stained yellow by the iodine. Methyl violet causes the same reaction as in amyloid disease gene- rally [ (see p. 101), colouring the grains red, whilst the surrounding tissues assume a blue tinge. Picro-car- mine has a peculiarly selective action, staining the bodies a deep red. With methylene blue the corpora amylacea assume an emerald-green colour. As regards the possible significance of these amyloid concretions many diverse theories have been suggested, the two principal being those of Friedreich and Lang- hans. The former considers that they are derived from remains of haemorrhoids in the lungs. In all his cases there was a history either of recent haemorrhagic in- filtration, or traces of past inflammation. His theory is that a minute clot forms locally, thus constitut- ing the nucleus, and fibrin is deposited in layers around it, and that this formation undergoes modi- fication (of what kind is at present unknown) which causes the bodies to assume their characteristic appear- ance and properties. EXAMINATION OF SPUTUM. 273 Langerhans on the other hand assigns to the corpora amylacea a cellular origin. In this view he has but few supporters, and moreover the bodies described by him do not agree in all particulars with those now under consideration. The observations of Jurgens and Zahn, and those more recently of Curtis, corroborate those of Friedreich. 10. Portions of membrane. a. Diphtheritic membrane. In cases of mem- branous laryngitis, whether occurring after scarlet fever or being undoubted instances of diphtheria, portions of the membrane are often coughed up and should be submitted to careful microscopic examination. The membrane consists of a soft, thin, whitish layer presenting fine striation and wavy lines, in the meshes of which are pus cells and epithelial debris. The fibril- lated substance and cells alternate so as to produce a laminated structure. To demonstrate its characters it should be placed in water when it can easily be spread out. Portions of it should be stained in methylene blue (see page 91) in order to ascertain whether any organ- isms be present. In the membranous laryngitis occur- ring after scarlet fever this is never the case. In diphtheria (it being assumed that "croup" and diphtheria are the same disease) the pathogenic or- ganisms are often found, though not invariably so. They consist of bacilli of various shapes, the organism being polymorphous. b. Echinococcus membrane. Hydatid disease of the lung is an extremely rare one ; there are, how- ever, several cases on record. The membranes are the same as those found in disease of the liver, and further description will be postponed until a future chapter. 274 MEDICAL MICROSCOPY. Occasionally the cysts themselves or loose booklets are expectorated, rendering the diagnosis easy. The source of these bodies, however, is not necessarily the lungs, as they may arise from a suppurating hydatid cyst of the liver perforating through the diaphragm into the lung. 1 1 . Crystals. Many forms of crystals are found in the expectoration, but few of them have any diagnostic importance. a. Hasmatoidin. These crystals occur in the form of ruby-red rhombic prisms or needles, often grouped together into bundles. Complete crystals are not often met with in the sputum, haematoidin more commonly being found as irregular red particles. Both crystals and fragments are frequently enclosed in white blood corpuscles. Their presence indicates that blood has lain for some time in the air passages, or that an abscess has per- forated into the lungs. They often occur in large quantities after an haemoptysis, or some time after a pulmonary infarct. If haematoidin is found chiefly enclosed in cells, an old haemorrhage of the lungs is the most probable cause, but if the crystals are chiefly free there has most likely been a rupture of an abscess from some neighbouring organ into the lung. b. Charcot-Leyden crystals (fig. 54). In the sputum of asthmatic patients these crystals are often found in large numbers. They are long and pointed octahedra, or sharp or truncated fusiform bodies ; they vary greatly in size. They are soluble in alkalies, mineral acids, and warm water, but are insoluble in ether, alcohol and cold water. Their exact chemical composition is not known. Schreiner supposes them to EXAMINATION OF SPUTUM. 275 consist of phosphoric acid combined with an organic base. Curschmann and Ungar are of opinion that these crystals are derived from the decomposition of organic matter in the bronchioles. In connection with this, v. Jaksch's remarks are worthy of note ; he says that in fresh cases of bronchial asthma, or at the commence- ment of a new series of attacks, Curschmann's spirals are chiefly found, but no crystals ; if such prepara- tions are prevented from evaporating for about forty- eight hours, Charcot-Leyden crystals will be found to FIG. 54. Charcot-Leyden crystals. have been formed. In the further course of the attack the sputum when first ejected will contain the crystals. This seems to suggest that the crystals are formed by the decomposition of the spirals. They are not absolutely diagnostic of asthma, being present in other conditions. c. Cholesterin crystals (fig. 43, p. 210). The well known rhombic, iridescent plates, with notches at one corner, are not often met with in sputum. T 2 276 MEDICAL MICROSCOPY. The expectoration of phthisis, or the pus from a pul- monary abscess or empyema, are the most frequent conditions under which they occur. They generally lie together in heaps, are easily soluble in ether, but in- soluble in acids, alkalies and water. When treated with dilute sulphuric acid and tincture of iodine, the crystals gradually change their colour, becoming violet- blue, green and red. With sulphuric acid alone they slowly turn from yellow to violet-red. d. Crystals of the fatty acids. These occur as groups of long, sharply-pointed needles, often arranged in small rosettes. They are characterised by their easy solubility in ether. They are most usually found in connection with putrid bronchitis and gangrene of the lungs, but also occur in the sputa of bronchiectasis and phthisis. They have therefore but little diagnostic value. As regards their chemical nature, they consist chiefly of palmitic and stearic acids, in combination with sodium, potassium, calcium and magnesium. e. Leucin and tyrosin (figs. 40 and 39). These two substances are but rare constituents of the sputum. When present they in no way differ in form from that when appearing in urinary sediments. They have been described by Leyden in a case of putrid bronchitis, and in another in which an empyema burst into the lung. /. Oxalate of lime (fig. 39, p. 199). These occur occasionally in their characteristic envelope-shaped crystals, or as an amorphous sediment. They have no diagnostic significance. g. Triple phosphates (fig. 41). In appearance these crystals are the same as those described on p. 206. Being soluble in acids, they are only found EXAMINATION OF SPUTUM. 277 when the sputum is alkaline. They sometimes occur in the sputa when a sanious exudation has broken into the lungs, and when decomposition of albuminous matters has taken place with evolution of free ammonia. 12. Concretions. It not uncommonly happens that small portions of cretaceous matter are coughed up by phthisical patients. When these particles are of a large size, they are known as "lung stones." Frankel also describes " bronchial stones." These latter show indications of branching, and are of the size and shape which would be expected if they had been formed in the bronchi. The origin of both varieties is uncertain. Andral describes a case in which after death in a patient forty years old, who died of phthisis, he found a copious deposit of chalk in the cartilaginous portions of the smaller bronchi ; the mucous lining was in some places ulcerated, and some of the calcareous matter had escaped. Another source suggested is calcification of caseous matter which has been retained in a pulmon- ary cavity. Finally, some observers consider that these cretaceous particles are derived from bronchial glands which have become calcined and ulcerated through into the bronchi. As regards their size, this varies from that of a small pea to that of a plum-stone, or even of a walnut. The number ejected by one patient may be very great. Andral records a case of a phthisical patient who, in eight months, expectorated no less than 200 stones, the largest of which was the size of a cherry. 13. Foreign bodies. For the most part these have little diagnostic worth. Some of them have evi- dently been inhaled, and again ejected, sometimes in so changed a condition as to be hardly recognisable. 2 7 8 MEDICAL MICROSCOPY. Such are particles of food which occasionally lie for a considerable period in the ventricles of Morgagni. Other instances of foreign bodies so expectorated are nut shells, teeth, threads, &c. The retention of these FIG. 55. Silk fibres. bodies in the air-passages often gives rise to seriou symptoms, the explanation of which is only afforded on the expulsion of the offending body. Fio. 56. Cotton fibres. Occasionally when malignant disease of the larynx is present, portions of tissue slough off and are ejected. Microscopic examination of such fragments may yield most important aid in diagnosis and prognosis. EXAMINATION OF SPUTUM. 279 Dr. Fagge records two cases in which hairs were expectorated from mediastinal dermoid cysts. FIG. 57. Linen fibres. FIG. 58. Wool fibres. 14. Accidental ingredients. In the. opening part of this chapter stress was laid on the necessity of the prevention of extraneous matters from entering the 280 MEDICAL MICROSCOPY. spittoon. But sometimes in spite of all care such acci- dental substances will find their way into, and become mixed with the sputum. Most commonly these are particles of food matter, such as animal and vegetable tissues, globules of fat, starch grains, &c. Bundles of elastic tissue derived from the food may be distin- guished from those coming from the patient by being coarser in texture, and arranged in a more parallel manner. Starch grains may be known from the cor- pora amylacea by their chemical reactions, the former assume a purple colour when treated with iodine dis- solved in iodide of potassium. Fibres of silk, cotton, linen, &c., are also often met with. Fibres of silk (fig. 55) are tapering, possess sharp outlines, and do not branch. Fibres of cotton (fig. 56) are twisted, and have curious central markings. Fibres derived from linen (fig. 57) are jointed, cylindrical, have ragged uneven ends, and fine branching filaments at intervals. The fibres of wool (fig. 58) are rounded, with fine cross-markings and reticulations in the border at the site of the cross-markings. The central longitu- dinal canal is usually invisible. Any of these fibres may be coloured. To the above list must be added hairs, particles of dust, chips of wood from the floor, &c. Many of the above objects, unless carefully examined, may give rise to much misapprehension. MICRO-ORGANISMS OF SPUTUM. 28l CHAPTER XVII. THE MICRO-ORGANISMS OF SPUTUM. PROBABLY no department of medicine has made greater advances during the last few years than bacteriology, and this has been largely made use of for clinical purposes in the examination of sputum. In this chapter will be described the most convenient and reliable methods employed for the detection of micro-organisms in the expectoration. i . Tubercle bacilli. A great number of processes have been from time to time introduced for the clinical detection of the bacillus tuberculosis in sputum. The method which long experience has proved to be speedy and reliable will be here described at length, short reference only being made to other processes. At the risk of some repetition of the directions given in the previous chapter, this method will be detailed throughout. In the first place the patient must be directed to reserve that portion of expectoration which has been ejected in the early morning before any food or drink has been taken. If nourishment has been partaken of during the night, it is well to request the patient to carefully rinse out his mouth with some water before using the clean spittoon. Nothing is more annoying than to have food matter mixed with the sputum. I have received samples so contaminated in this way that a satisfactory examination was out of the question. 282 MEDICAL MICROSCOPY, The collected sample is thrown out into a dish (prefer- ably a dark coloured one). Search is made for the small, yellowish, opaque, round particles so common in phthisical sputa, and if any are found they should be transferred to a cover-glass. Failing these, any other opaque portions must be selected. These having been placed on a cover-glass, another glass is laid on it, and the specimen of sputum gently spread out by means of pressure. A layer as thin as possible should be ob- tained, any excess that exudes from between the glasses being wiped away with a soft cloth. The cover-glasses are now separated by a sliding movement, that is, not "sprung" apart. Two films of sputum are thus obtained, which are allowed to dry in the air, this process being expedited, if desired, by holding the glasses between the finger and thumb over a spirit lamp, and as long as the heat does not burn the hand it will not injure the specimens. When dry the film must be fixed to the glass, this is accomplished by seizing the glasses one by one with forceps, and rapidly passing them three times through the flame of a spirit lamp or Bunsen burner, thus co- agulating the albumen. A pause must now be made in the description of the process, to give the composition of the various fluids required. They are as follows : Solution i. Neelsen's solution (Ziehl). Fuschine i part . Dissolved in a s per cent, watery so-) - .,. ., J h 100 parts, lution of carbolic acid ) Alcohol 10 ,, Solution 2. Dilute watery solution of sulphuric acid, 25 per cent. MICRO-ORGANISMS OF SPUTUM. 283 Solution 3. Methylene blue. Methylene blue 2 parts. Alcohol 15 ,, Water 85 To resume, a small quantity of Neelsen's solution is placed in a watch glass, and heated until steam begins to rise. Into this the cover-glasses, prepared as pre- viously described, are placed, film-side downwards. It is sometimes difficult to decide, especially in the later stages of the process, which is the prepared side of the cover-glass, but this can be determined either by lightly scratching each surface with a needle, and observing whether a mark is made, or more simply still by holding the cover-glass towards the light, when the side on which the film is appears dull, whilst the unused side yields a bright reflex. The specimens are allowed to remain in the heated stain for two minutes. Most books state that five minutes are necessary, but experience has proved that two minutes are amply sufficient. In order to prevent the glasses sticking together, which they will do when both are submerged, the first glass should be placed slantingly in the fluid, so that it sinks. The second glass, however, should be held be- tween the finger and thumb, and dropped on to the fluid. If this be done from a short distance, the glass will float. After a stay of two minutes in the stain the glasses are removed one by one by means of forceps, and thoroughly decolourised in the dilute sulphuric acid con- tained in a shallow capsule. A few seconds usually suffice for this, although contrary to what is generally believed, the bacilli retain the stain for at least thirty minutes in the acid ; they are in fact difficult to deco- lourise, and it is important that the red stain should be 284 MEDICAL MICROSCOPY. removed from other bodies as far as possible, so that any excess should be on the side of a longer stay in the acid rather than the reverse. The best mode of procedure is as follows. The glass is retained in the forceps, and gen- tly moved about in the acid, and after a few seconds is transferred to a dish of water. The colour will probably be partially restored, and the specimen must then be returned for a few seconds to the acid, again rinsed in water, this process being repeated until no more colour is visible. The specimen is next placed, prepared side down- wards, in the methylene blue solution contained in a watch glass. It is allowed to remain for about half a minute, and is again washed in water. It is then al- lowed to dry, either by exposing it to the heat of the flame of the spirit lamp, or by gently pressing it between two folds of filter paper. It is finally mounted in water if only required for diagnostic purposes, or in balsam if a permanent preparation is desired. Thus prepared the bacilli (Frontispiece, fig. i) appear as short red rods, whilst the mass of the sputum is stained blue. The tubercle bacilli are of variable size, but have an average length of half the diameter of a red blood cor- puscle. They are usually straight, but may be found slightly curved. Their grouping is very characteristic, being generally arranged in small numbers of two or three, lying cross-wise or at angles to one another. Sometimes, however, they are so numerous as to form large clumps, in which the individual members can only be recognised with difficulty. Their characteristic property is that of retaining the stain in spite of exposure to acid. By this they are distinguished from all other bacilli, except that of MICRO-ORGANISMS OF SPUTUM. 285 leprosy, which in this country is hardly likely to be met with, and certainly never in expectoration. Their presence in sputum, therefore, is absolutely di- agnostic of a tubercular process seated somewhere in the respiratory tract, the exact locality being soon recognis- able by the stethoscope or laryngoscope. A peculiar beaded appearance is often noted, supposed by some to be due to the presence of spores which will not retain the stain. As regards their number, opinions differ considerably whether any definite conclusion as to the extent and activity of the disease can be drawn from observing whether the bacilli are numerous or not. Dr. Theodore Williams considers that a large number of rods denotes rapid spread of the disease, but I am decidedly inclined to agree with Dr. Kidd and Mr. Taylor (" Transactions of the Royal Medico-Chirurgical Society," vol. 71, p. 173), who after an exhaustive ex- amination of the results at the Brompton Consumption Hospital, came to the conclusion that no importance, as regards prognosis, could be attached to the large or small number of rods seen in the specimens. A negative result is of little value and proves nothing, but if after repeated examinations of several satisfactory samples of sputum no bacilli be found, this may be taken as presumptive evidence that the case is not a tubercular one, although it is no absolute proof. A positive result on the other hand is everything, and cannot be ignored. Although no other bacilli stain like the tubercle, yet errors sometimes arise owing to foreign bodies retaining the colour. Such matters as hair and horn are difficult to decolourise, and if the specimen has not remained sufficiently long in the acid, the edges of cells often appear red, but this mistake may easily be remedied by careful focusing. 286 MEDICAL MICROSCOPY. In order to examine satisfactorily for bacilli, an Abbe's condenser should be used, and at the lowest a one- sixth inch lens, preferably a one-twelfth, although they may be seen with a quarter. The method given above for staining tubercle bacilli can be thoroughly relied upon, but there are several modifications of it and other methods, only a few of which will be considered here. A rather more rapid process may be conducted with the same stains. The cover-glasses are prepared in precisely the same way, but instead of being placed in a watch glass, a couple of drops of Neelsen's solution are placed on the prepared side of the cover-glass, which is seized in forceps. The glass is held over the flame of the spirit lamp until bubbles are observed to form ; the excess of stain is then poured off, and the glass swilled in the dilute sulphuric acid and washed in water. A drop or two of the methylene blue solution is then placed on the specimen, allowed to remain for half a minute, and washed off in water. The preparation is next dried, and examined in balsam- or water. According to Ehrlich it is very difficult to obtain the acid solution without free acid fumes, which tend to decolourise the bacilli as well as the mucus. He there- fore makes use of the following process : The staining solution must always be prepared fresh. A few drops of pure aniline oil are placed in a medium sized test-tube which is filled with water, the mixture being thoroughly shaken and filtered through a double fold of paper. A little of the clear fluid is placed in a watch glass, and a few drops of a saturated alcoholic solution of fuchsine are added until the solution just becomes opaque. This being most easily judged by placing the watch glass on the edge of a piece of filter MICRO-ORGANISMS OF SPUTUM. 287 paper, and observing when this edge becomes obscured. Cover-glasses are floated, prepared side downwards, on this fluid for several hours (six to twelve). They are decolourised in a mixture of one part of nitric acid to two parts of a saturated solution of sulphanilic acid. After being swilled in water and rendered quite colourless by re-immersion in the acid solution, if necessary, they are counter-stained with methylene blue, washed in water, dried, and mounted in balsam. The rationale of this process is that the sulphanilic acid absorbs the nitrous fumes, so allowing the pure acid to act on the bacilli. The process recently introduced by Dr. Gabbet seems to be an excellent one. It closely resembles those already described, but is more rapid ; on the other hand one has not the same control over the process of decolourisation. The first staining solution has the following compo- sition : Carbolic acid (5 per cent.) .... 100 c.c. Magenta i gramme Spirit 10 c.c. Some of this solution is placed in a watch glass, the cover-glasses immersed in it and heated until steam begins to arise. They are then placed for one or two minutes in a solution, which at once decolourises and counterstains them. This solution is made as follows: Sulphuric acid (25 per cent.) . . . 100 c.c. Methylene blue >.. - 2 grammes. On being taken out the specimens are washed in water, and mounted in the usual way. In some cases in which no bacilli are found by the above methods, but where the history and physical signs point to a tubercular process in the respiratory 288 MEDICAL MICROSCOPY. tract, Biedert's method (" Centralblatt fur Klinische Medicin," 1891, No. 6) should be adopted. To a tablespoonful of the sputum seven or eight drops of caustic soda are added, and two tablespoonfuls of water. This mixture is boiled until it becomes liquid. Four to six tablespoonfuls of water are now added, and the mixture again boiled until the whole is a thin uni- form liquid. It is next thrown into a conical glass and allowed to stand from twenty-four to forty-eight hours. Part of the sediment which results is removed with a pipette to cover-glasses, spread out in as uniform a layer as possible, dried, and then stained for tubercle bacilli by one of the methods already detailed. The only other process for the detection of tubercle bacilli that will be described here is the one introduced by Dr. Heneage Gibbes. It is very simple and rapid, but in the opinion of the author not so reliable as the foregoing. Dr. Gibbes has a special staining fluid pre- pared as follows : Rose aniline hydrochlorate 2 parts Methylene blue . . . . . . . . . i part. The mixture is triturated and three parts of aniline oil dissolved in fifteen parts of rectified spirit is slowly added, and finally fifteen parts of distilled water. Cover-glasses are prepared in the usual way, and im- mersed in the stain, \vhich has been heated until steam has arisen, for five minutes. They are next washed in methylated spirit until no more colour can be extracted, when they are dried and mounted. 2. Pneumococci (fig. 59). In 1883 C. Friedlander announced that he had found the pathogenic organism of pneumonia. In the rusty sputum of pneumonia cer- tain organisms are almost always found, sometimes in very large numbers. Unfortunately, however, for their MICRO-ORGANISMS OF SPUTUM. 209 diagnostic value, micro-organisms, morphologically pre- cisely similar to them, are found in many other diseases, and indeed in the saliva of healthy individuals. These micrococci take the form of spindle-shaped bodies united at their flat bases, so forming diplococci. Sometimes three or four are joined together. But what especially characterises them is the capsule with which they are surrounded ; they thus appear as oval bodies provided with a sheath of similar shape. FIG. 59. Bacterium pneumonia crouposa, from pleural cavity of a mouse X 1500. A, B. Thread-forms. C, D, E. Short rod-forms. G. Diplococci. H. Cocci. I. Streptococci. (After Zopf, from Crookshank). There are two principal methods by which they are stained. Cover-glass preparations of the expectoration are pre- pared in the same way as was described when treating of tubercle bacilli. A few drops of aniline oil are placed in a test-tube, which is filled up with water and thoroughly shaken, the emulsion being filtered through a double fold of filter paper. A few drops of a saturated alcoholic so- lution of gentian-violet are added until the mixture becomes opaque. The cover-glasses are immersed in u 290 MEDICAL MICROSCOPY. this solution for five minutes, and are then swilled for a few seconds in absolute alcohol, washed in water, and mounted in the usual way. Very satisfactory specimens may also be prepared as follows : The aniline water is made as above, but instead of the violet a few drops of a concentrated alcoholic so- lution of fuchsine are added. The cover-glasses are immersed in this stain for about a minute, and are then placed for two minutes in a watery solution of methylene blue. Finally, they are washed in water, dried, and mounted in balsam. Stained in this way the diplococci are coloured blue, the capsule assuming a rose tint, whilst the ground substance is a bluish-red. A. Frankel found a coccus precisely similar in ap- pearance to the above, not only in the sputa of pneu- monia, but also in other diseases, and even in normal saliva. I have found very characteristic specimens in the sputa of phthisical patients. It is conceivable that these organisms are normally present in saliva and only excite pneumonia when other causes combine to produce a favourable pabulum upon which they may grow, and evolve the poisons which are necessary to cause the conditions found in the disease. Frankel's coccus and its capsule stain readily in methylene blue. After cover- glasses have been prepared in the usual way they are immersed in methylene blue solution for five minutes and then washed in water acidulated with acetic acid. 3. Actinomyces. This disease has been very rarely diagnosed in this country during life. Dr. Douglas Powell read a paper before the Royal Medico- Chirurgical Society (see "Transactions," vol. 72, p. 175) on a case which occurred at the Brompton Consumption Hospital. MICRO-ORGANISMS OF SPUTUM. 2QI Very seldom, however, in any country has the charac- teristic fungus been found in the sputum. Baumgarten, Paltauf and others, have recorded such cases. Possibly if the sputum from obscure cases was more frequently examined, more cases could be added to the list. The expectoration in actinomycosis is usually small in amount, muco-purulent, often of putrid odour, occa- sionally stained with blood, and contains minute white or yellowish granules about the size of a small pin's head. When one of these granules is examined with a quarter-inch lens, a number of small oval bodies (clubs) are seen surrounding an indistinct mass, but to bring out clearly the appearance of the fungus, specimens must be stained in the following manner. A few of the granules are placed on a cover-glass, allowed to dry, and passed three times through the flame. It is stained by Gram's method (see p. 95), and afterwards counter- stained by immersion for twenty-four hours in a satu- rated watery solution of safranin, containing two per cent, of aniline oil, then cleared in xylol, dr.ed, and mounted in balsam. The clubs of the fungus are thus stained yellowish-red, and the threads blue. Instead of the safranin, alcohol containing a little picric acid may be added after the Gram's process is complete, a stay of five minutes being necessary ; the clubs are then stained a bright yellow colour. 4. Micrococcus tetragonus. This peculiar or- ganism, as its name suggests, consists of four cocci united sarcina-like, and surrounded by a hyaline cap- sule. The cocci are stained by all the aniline dyes, and are frequently seen in sputum when this has been stained by methylene blue. The capsule, however, does not stain. This microbe is not pathogenic, being found in the u 2 292 > MEDICAL MICROSCOPY. sputa of various diseases, or even in the expectoration of healthy people. It is most frequently seen in phthi- sical sputum. According to Koch, it assists in the destructive process taking place in the lungs. Gaffney showed that when injected into guinea pigs and white mice, the animals died of septicaemia. 5. Septic micro-organisms. Various forms of micro-organisms, both rods and cocci, are found in all sputa, being especially numerous in the expectoration of bronchiectasis and gangrene of the lungs. They vary in size, and take the forms of staphylococci, strepto- cocci, &c. Diplococci are especially common, and are usually surrounded by a thin colourless zone. Occasionally zoogkea masses occur, made up of large numbers of micrococci. These are especially common in the sputum of phthisis. 6. Fungi. Several forms of fungous growth occur in the mouth, and are consequently not unusually found in the sputum. More rarely they grow in the deeper respiratory passages or even in the lungs themselves. The most common is the Oidium albicans or thrush (fig. 60), it occurs in the form of branching threads, composed of elongated cells, amongst which numerous spores are seen. They stain with methylene blue. The Leptothrix buccalis (Frontispiece, fig. 2) often grows round the teeth, and portions being de- tached and appearing in the sputum may be a source of difficulty as regards recognition, unless the observer be familiar with their appearance. Its reaction with iodine is characteristic. If a little of the tincture of iodine, or of a solution of iodine in iodide of potassium be added to the specimen, the threads of the fungus assume a deep violet colour, which, however, only lasts a few hours. MICRO-ORGANISMS OF SPUTUM. 293 The Aspergillus fumigatus has also been de- scribed by Virchow as occasionally growing in the lung. In some cases of extensive destructive ulceration in the lung, especially in pulmonary gangrene, bodies known as " Sarcinae pulmonis," are found in the sputum. They were first described by Virchow and Friedreich (" Virchow' s Archiv.," ci., 401, 1856, and 390, 1864). In appearance they closely resemble the Sarcinae ventriculi, but are rather smaller. FIG. 60. Oidium albicans (v. Jaksch). The only case in which I have found these bodies, was one of phthisis running a slow course, the physical signs being obscure, but tubercle bacilli were present in the sputum in large numbers. 7. Infusoria. These have been described by Kan- nenberg (" Virchow's Archiv," Ixxv., 471, 1879) in the sputum of patients afflicted with pulmonary gangrene. They were chiefly found enclosed in small yellow droplets surrounded by fatty needles. They were of the monad 2Q4 MEDICAL MICROSCOPY. and cerco-monad varieties. They exhibited sluggish movements. According to v. Jaksch the best method of demon- strating them is as follows. A few of the droplets are picked out and placed upon a cover-glass. About three or four drops of a one per cent, solution of common salt are added. The preparation is dried in the air and stained in a watery solution of methyl violet, washed in water, and while still wet placed in a concentrated solution of acetate of potash. By these means the protoplasm of the monads is coloured a beautiful blue. 8. Entozoa. Members of this group are very seldom found in the sputum. In the rare instances of hydatids in the lungs, the hooklets or even vesicles may be dis- covered. The Ascaris lumbricoides has occasionally found its way into the oesophagus from the intestines, and has been ejected, and Manson has recorded cases in which the eggs of Bilharzia hsematobia have appeared, having escaped from the parent worm in the blood vessels. EXAMINATION OF VOMIT. 295 CHAPTER XVIII. EXAMINATION OF VOMIT. AN investigation by the microscope of matters which have been vomited, does not yield such useful informa- tion as a similar examination of sputum, or even of the faeces. But occasionally, considerable aid to diagnosis is rendered by such means and the student ought to make himself familiar with the appearances which are met with when food has been partially digested. For this purpose he should soak pieces of bread, meat, tendon, cartilage, and the more common vegetables (such as potatoes, cabbages, &c.) in slightly acidulated water, and then tease out particles of these substances on a glass slide, and examine them with the microscope. Much more valuable information is gained from a chemical examination especially as regards the detec- tion of poisons, but the nature of this work forbids me entering into this subject here. The vomited matter is placed in a conical glass and allowed to stand for a few hours, when portions of the sediment should be removed with a pipette, placed on glass slides, protected with cover-glasses, and investi- gated with an inch and quarter-inch lenses. Separate portions of the supernatant liquid should also be exa- mined. The following particles of food matter will probably be seen : - 1. Muscle fibres (fig. 61, b) recognised by the trans- verse striations. 2. Elastic fibres, characterised by their clear outlines, and tendency to form curves. 296 MEDICAL MICROSCOPY. 3. Fat globules and needles. The nature of these bodies may be proved by their solubility in ether. 4. Vegetable cells of various forms. 5. Starch granules (fig. 61, c) more or less altered by the process of digestion, but stained blue by the action of iodine dissolved in iodide of potassium, a few drops of which should be run under the cover-glass. Epithelium (fig. 61, a). Two varieties of epithelium may be noticed. Squamous cells occur in small num- FIG. 61. a. Epithelial cells, b. Muscle fibre, c. Starch granules. d. Sarcinae. e. Yeast cells, f. Non-pathogenic micro-organisms. bers only, and are derived from the mouth and other parts of the oesophagus, having been swallowed with the saliva. Columnar cells are sometimes very numerous and then are indicative of catarrh of the gastric mucous membrane. They are generally more or less altered in shape by the action of the secretions. In the vomit of cholera numerous small white flocculi are found, which may be shown to consist of collections of epithelial cells, both squamous and columnar. The occurrence of so- called " cancer cells " in cases of carcinoma of the stomach is doubtful. No doubt cells derived from EXAMINATION OF VOMIT. 297 tumours do make their appearance in the vomit when such conditions exist, but they cannot with certainty be distinguished from squamous epithelial cells. If large irregularly-shaped cells are found in large numbers, together with red blood corpuscles and pigment, this would be presumptive evidence that cancer of the stomach did exist, although without accompanying clinical signs, which are far more constant and valuable, such a diagnosis could not be ventured on. Red blood cells. The appearance of red blood cells in the vomit depends upon how long they have been retained in the stomach. If a vessel has rup- tured and a considerable quantity of blood has been poured out, they will present their normal colour and biconcave form. As a rule, they are more or less altered by the action of the gastric juice, and are then seen as colourless rings. If the blood has been some time in the stomach the haemoglobin becomes reduced to haematin ; a dark brown fluid then results, in which masses of pigment may be seen, recognisable as blood only by means of the spectroscope. Leucocytes. A few scattered white blood cells are always present. If they occur in large numbers there is probably gastric catarrh, but the cells may be derived from the saliva, sputum, or nasal mucus which has been swallowed. These cells are seldom present in such quantity that the term pus may be applied, and practically this only happens when an abscess, situated in neighbouring parts, has burst into the stomach or resophagus. The cells themselves are generally so much altered by the secretions that only their nuclei are visible. In cases of diphtheria portions of membrane may become detached and ejected by the effort of vomiting. 298 MEDICAL MICROSCOPY. Their nature may be demonstrated by floating them in water, when they will easily spread out. Vegetable parasites. Various kinds of fungi are occasionally found in the vomit, some such as leptothrix have evidently been swallowed, and only two are of any importance. Sarcinse (fig. 61, d). These bodies appear as round cells grouped together in fours, and very much resemble wool-packs in form. They are of dark silver-grey colour, and are stained mahogany-brown with a solution of iodine in iodide of potassium. They are generally asso- ciated with obstructive disease of the pylorus, so that food is retained and undergoes fermentation. Yeasts (fig. 61, e). v. Jaksch (I.e., p. 108) describes three forms of yeasts as occurring in the vomit. (a). Saccharomyces cerevisise. They are about the size of leucocytes and cohere in small groups. If a solu- tion of iodine in iodide of potassium be run under the cover-glass, they stain a brownish-yellow. These bodies are occasionally elliptical in shape. (b). Very small yeast-like fungi in thick clusters. (c). Rod-like bodies, highly refracting, and of con- siderable length and thickness. They have rounded extremities, and occur separately or in strings. It has been stated that these are the agents in lactic acid fer- mentation of sugar. Micro-organisms (fig. 6i,/) are very frequently present in varying numbers. They take the form both of rods and cocci, but are non-pathogenic and therefore of no diagnostic worth. Animal parasites. These are purely accidental, and have entered the stomach from the intestines. The only ones which have been described are the Ascaris lumbricoides, Anchylostoma duodenale, and Oxyuris vermicularis. EXAMINATION OF DISCHARGES. 2QQ CHAPTER XIX. EXAMINATION OF DISCHARGES AND CONTENTS OF CAVITIES. THE vast proportion of specimens which medical men are called upon to examine with the microscope, are .derived either from the sputum or urine. But occa- sionally they have to investigate deposits of fluids drawn from the chest or abdomen, or the discharges from abscesses, fistulae, &c. These materials, though hitherto commonly neglected, may yield valuable as- sistance in diagnosis, and the student therefore should make himself acquainted with at least the most im- portant of such fluids, whenever occasion offers for their study. The fluids will either have been drawn off by puncture, or in some other way, or a discharge may have occurred spontaneously. If sufficient quantity of the material can be obtained, it should be placed in a conical glass, and allowed to settle for some hours, when the super- natant liquid can be poured off, and portions of the sediment removed by means of a pipette for investiga- tion. Unstained specimens should always be examined, and subsequently such staining processes, as experience may suggest, should be applied. SEROUS EXUDATIONS. These resemble in general appearance blood serum. They are almost clear, have a pale yellow colour, and coagulate on standing, yielding a clot which is rich in 300 MEDICAL MICROSCOPY. fibrin. As a rule there is but very little sediment, and when placed under the microscope in the usual way, it is often very difficult to make sure that one is focusing the layer of fluid between the glasses. A few red cor- puscles will usually be seen, and they generally preserve their form and colour. Leucocytes are usually more numerous. Some endothelial cells derived from the surface of the serous membranes will be seen, but these are also scarce. In addition to these, Bizzozero (" Microscopic Clinique," p. 115) describes some larger cells, varying in diameter from 7 to 30 p ; they contain small grey droplets, which are sometimes so abun- dant that the cells appear to be entirely made up of them. These bodies seem to undergo a kind of cystic degeneration, so that large vacuoles are formed, con- taining a clear fluid. Micro-organisms are frequently present, chiefly micrococci, and large numbers may be present although the fluid is quite clear. In one specimen which I examined, the liquid was yellowish in colour and perfectly clear, with the ex- ception of a few small white flakes. Yet tubercle bacilli were found in large numbers, especially in the flakes just mentioned. In order to search for the organisms, a little of the fluid is distributed over several cover- glasses, which are then examined in precisely the same manner as films of sputum (see p. 281). If the serous fluid is of old standing, cholesterin crystals are generally seen. H^EMORRHAGIC EXUDATIONS. Such liquids are of a brownish or red colour, due to the presence of blood. In the deposit red blood cor- puscles, more or less altered in colour and appearance, EXAMINATION OF DISCHARGES. 3OI are seen in large numbers, but occasionally the haemo- globin seems to have been dissolved out, and then only a few colourless rings are visible. Endothelial cells, often with advanced fatty degeneration, are likewise to be found. In regard to these bodies, Quincke (" Deutsches Archiv fur Klin. Med.," 1882, 569) as- serts that when treated with a solution of iodine in iodide of potassium, a marked glycogen reaction is often produced, and that the probable presence of carcinoma is then indicated. Should they be accompanied by large epithelial cells, the diagnosis of cancer may safely be made. CHYLOUS EXUDATIONS. Beyond the appearance of particles of fatty matter no characteristic bodies can be found in the deposit of the fluid in such conditions. SERO-PURULENT EXUDATIONS. These often occur in tubercular cases, and the patho- genic organisms can be found without difficulty. Pus cells are of course numerous, but otherwise no special characteristics can be mentioned. PUTRID EXUDATIONS. These are usually brownish in colour, and are at once recognised by their penetrating and disagreeable odour. The deposit is peculiar, exhibiting in addition to shrunken red and white blood cells, numerous crystals, chiefly fatty needles, cholesterin and hsematoidin. These fluids swarm with septic micro-organisms, I 302 MEDICAL MICROSCOPY. PURULENT EXUDATIONS. If pus be allowed to stand in a cool place it separates into two layers, the upper of which is almost trans- parent, whilst the lower is whitish and opaque. A few streaks of blood are almost always present. When fresh, pus is of a greyish colour, but when putrid it appears like a thin green fluid with an extremely strong and unpleasant odour. If much blood be present the colour depends upon the proportion of the two fluids. The discharge from hepatic abscesses has a peculiar chocolate colour. In the sediment of a purulent exudation pus cells of course almost fill the field ; if perfectly fresh, amoeboid movements may be seen, and if a solution of iodine in iodide of potassium is run under the cover-glass, the corpuscles assume a mahogany colour. If the discharge is old, white cells, more or less shrunken and granular in appearance, are seen. Cells containing fat will also be noticed. Red blood corpuscles are usually present, sometimes in very large numbers. Crystals are seldom absent, the most common being needles of the fatty acids, cholesterin, haematoidin and triple phosphates, but no special significance can be attached to their presence. The search for micro-organisms is of the greatest importance ; this is obvious, from the fact that the formation of pus has been shown to be almost entirely due to the action of bacteria for if these be excluded from entering the fluids of the body, as is now accom- plished by antiseptic surgery, a purulent process never occurs. Certain organisms therefore are peculiar to pus ; these are various forms of micrococci. They are EXAMINATION OF DISCHARGES. 303 more numerous in old than in fresh pus, although they can also generally be detected in the latter. No fewer than eight varieties of micrococci have been separated from pus, but the most common are the following. Staphylococcus pyogenes aureus. These are cocci occurring singly, in pairs, in very short chains, and in irregular masses. Their presence causes no odour in the pus. They are found in the discharge from boils, and in the abscesses of pyaemia and puerperal fever. Staphylococcus pyogenes albus. Morphologi- cally, these are indistinguishable from the above and occur under similar conditions, but differ somewhat in cultivations. Various pathogenic organisms are found in purulent discharges, and these are of the utmost im- portance, as their detection will be a most valuable guide to the surgeon or physician in the future treat- ment of the case. Tubercle bacilli. The method of detecting these organisms is the same as that described in the chapter on sputum. The layer of pus on the cover-glasses must be made as thin as possible, and to avoid errors it is important to allow the glasses to remain in the acid until the red fuchsine is entirely got rid of. Ten minutes or a quarter of an hour are not at all too long. The lesions in which the detection of tubercle bacilli is of great importance are many, amongst which may be mentioned tubercular nodules of the skin ; I have seen two or three cases in which so-called "post-mortem warts " were proved to contain bacilli. Discharge from the ear in cases of tubercular menin- gitis in children is another example. Actinomyces. This is a rare disease, although cases seem to have been more numerous during the past few years, probably owing to our improved means 304 - MEDICAL MICROSCOPY. of investigation, and partly, perhaps, because a sys- tematic examination of purulent discharges in doubtful cases is more frequently made, than was formerly the case. It should be laid down as a rule that when a collection of fluid is diagnosed, if the nature of the dis- ease is doubtful and especially if the discharge is puru- lent, a microscopic examination should always be made. In actinomycosis the pus is thin and viscous, and will be seen to contain small nodules of a greyish colour. If one of these be placed under the microscope, the characteristic club-shaped bodies will be seen. Cover-glass preparations should be made in the usual way and stained by the method described on p. 100. The central mycelium will then be stained purple and will be seen to consist of closely interwoven threads surrounded by the radiating mass of clubs. This ap- pearance is unique, and the " ray fungus" cannot be mistaken for any other. In a case under Dr. Douglas Powell at the Brompton Consumption Hospital Mr. Taylor recognised the fungus in the discharge dur- ing life ; and Prof. Crookshank undertook an ex- haustive enquiry into the nature of the parasite, the results being published in the Medico-Chirurgical Tran- sactions, vol. Ixxii., p. 193. He showed that the fungus appeared to be closely related to the Basidio-mycetes, and was able to demonstrate that the threads of the mycelium pass directly into the narrow central ends of the clubs. Bacillus of leprosy. The nodules which appear on various parts of the skin and mucous membrane in leprosy occasionally ulcerate, discharging a consider- able quantity of thin pus. If cover-glass preparations of this discharge are made in the usual way, and stained in the same manner as sputum is stained for EXAMINATION OF DISCHARGES. 305 tubercle bacilli, numerous fine slender rods will be seen, hardly distinguishable morphologically from the tubercle bacilli. They stain, however, more readily, and unlike the bacilli of tubercle are not coloured by Kuhne's method (see p. 92). Bacillus of anthrax. In this country two affec- tions are produced by this organism. " Malignant pustule," due to a direct inoculation, and consisting of a local inflammation followed by the development of a brawny swelling ; " wool-sorter's " disease resulting from inhalation, or swallowing of the spores so that the pulmonary or intestinal mucous membranes are first affected. In order to demonstrate the pathogenic micro-organ- ism, the local affection or carbuncle should be incised, and cover-glass preparations made of the discharge. When stained by Gram's method, the bacilli can be well seen as straight rods, sometimes curved. Their extremities are sharply rectangular, and the bacilli are usually joined together in chains, the segments being marked off by clear linear spaces. In sections the organisms should be stained by Weigert's modification of the Gram method as described on p. 96. Bacillus of syphilis. The pathogenic character of this organism has not met with general acceptance. It was first described by Lustgarten. In form it some- what resembles the tubercle bacillus, but it is always found in the interior of nucleated cells about twice the size of leucocytes. They have been found in the dis- charge of the primary lesion and in hereditary affections of tertiary gummata. Cover-glass preparations of the pus should be stained in the following way : A fresh solution of gentian aniline violet is made (see p. 95) in which the sections are placed for from x 306 MEDICAL MICROSCOPY. twelve to twenty-four hours. They are then re- moved, and washed for a few minutes in absolute alcohol, and afterwards immersed for ten minutes in a i -5 per cent, solution of permanganate of potash. Next they are rinsed in a watery solution of pure sulphurous acid, and finally washed in water. If by these means the preparations are not completely decolourized, they must again be washed for a few seconds in permanganate of potash, and then again in sulphurous acid and water, the process being repeated until no more colour is visible. This method is not very satisfactory as it stains other bacilli, as well as those of syphilis, and the whole matter requires further research before it can be determined that the characteristic organ of syphilis has been found. Another method has been introduced by De Giacomi which consists in staining cover-glass preparations in aniline fuchsine made in the same way as methyl aniline violet. The stain is heated, and the glasses allowed to remain in it for about 10 minutes, after which they are washed in water containing a few drops of perchloride of iron. They are next decolourized in a strong solu- tion of perchloride of iron and counter-stained in Bis- marck brown. The bacilli are then coloured a deep red. Gonococcus. The discharge obtained from pa- tients suffering from gonorrhoea may be shown to contain a distinctive micro-organism in the form of micrococci, occurring either singly, or more usually in groups of ten or twelve. Cover-glass preparations of the pus are stained in methylene blue, and then thoroughly washed in water. Bacillus of glanders. The bacillus mallei is morphologically very similar to the tubercle bacillus. EXAMINATION OF DISCHARGES. 307 It may be found in the pus derived from the ulcerated nasal passages of patients affected with this disease. The organisms are difficult to stain. The process intro- duced by Loftier is the best. The staining solution has the following composition : Concentrated alcoholic solution of methylene blue i part Potash solution (i in 10,000) . . . i part. The potash solution must be added immediately be- fore use. Cover-glasses are immersed in the mixture for five minutes, and afterwards washed in water containing two or three drops of acetic acid. Next the prepara- tions are transferred to another mixture containing two drops of concentrated sulphurous acid, and one of a 5 per cent, solution of oxalic acid in 10 c.c. of water. The alkaline staining fluid is thus decomposed, and the colour removed from everything except the bacilli. Although not occurring in discharges, two other parasites may be mentioned here, both being more common in the flesh of cattle than in man, but, never- theless, it is important to be able to recognise them. Trichina spiralis (fig. 62). This nematode has already been referred to on p. 246. In its characteristic form it is best seen in sections of muscle. It then appears coiled up in a lemon-shaped capsule, lying parallel to the muscular fibres, and surrounded at each end by a collection of fat globules. In some specimens the worm can hardly be distinguished, looking like a black mass, and the capsule will then be found to be calcified. Cysticercus celluloses. This has in rare cases been found in man. A case is recorded by Dr. Betham Robinson in the " Transactions of the Pathological x 2 308 MEDICAL MICROSCOPY. Society," 1889, p. 388, as occurring in the trapezius muscle of a child, aged four. Most cases that have been reported have been in the eye and brain, where its presence is most conspicuous, but in animals it is found chiefly in the muscles. It occurs as a small round body FIG. 62. Trichina spiralis in muscle (Parkes). visible to the naked eye, being sometimes as large as J- inch in diameter. Under the microscope the head (fig. 63) will be seen to have four suckers and a circle of hooklets. FIG. 63. Head of cysticercus celluloses (Parkes). CONTENTS OF CYSTS. For the purpose of diagnosis the contents of cysts are usually obtained by means of an exploring needle, but EXAMINATION OF DISCHARGES. 309 if possible it is better to procure a larger quantity by aspiration. The fluid is allowed to stand for some hours, and then some of the deposit is removed by a pipette, and examined by the microscope in the usual way. Hydatid cysts. The fluid obtained from these cysts is clear and limpid, with a specific gravity of about 1008. It contains very little albumen, and abun- dance of inorganic salts. The deposit is characteristic ; it contains the im- mature form of the Taenia echinococcus, a parasite FIG. 64. Hydatid cysts. which inhabits the intestine of the dog. The ova are discharged with the faeces of the host, and in some way, probably through drinking water, find there way into the human stomach, and thence into some other organ, generally the liver. There they develop and form minute round bodies known as scolices (fig. 64). In the sediment these can hardly be seen with the naked eye. They are provided with two circles of booklets and four suckers. Each scolex is oval in form, transparent, and often studded with granules, which are frequently 310 MEDICAL MICROSCOPY. stained yellow by the bile. In addition, free booklets will also be seen scattered through the specimen. In shape these resemble tiger's claws. In most specimens of this parasite, the original cyst formed from the ovum usually developes another within itself, known as the " daughter-cyst," and these again FIG. 65. Wall of hydatid cyst (Boyce). develop still more, " granddaughter-cysts." In this way the echinococcus becomes filled with a number of small vesicles of various sizes, which may amount to thousands. This illustrates the difference between a " cysticercus " and an "echinococcus," whereas the former gives rise to only one scolex, and can ultimately form only a single tapeworm, the latter may develop thousands of both. EXAMINATION OF DISCHARGES. 311 Occasionally the parasites cannot be found, but only portions of the cyst wall (fig. 65) or " cuticula." This is made up of a number of concentric layers, composed of a chitinous material which has a very characteristic appearance, and when once seen cannot be mistaken. These cysts occasionally suppurate, and the booklets then are generally the only recognisable feature that remain. Ovarian cysts. The fluid contents of ovarian cysts vary considerably in character. The fluid ob- tained may be either perfectly limpid or of a gelatinous consistence. The deposit as a rule is copious. The cells which are formed are but seldom well preserved, fatty degeneration being so far advanced that they are scarcely recognisable ; free nuclei are abundant. Epithelial cells may be either squamous, columnar, or ciliated. Colloid concretions which are probably de- rived from epithelium are an invariable component of " colloid cysts." Red and white blood corpuscles occur in varying numbers, and cholesterin crystals are also generally present. Dermoid cysts. The fluid in these cysts is thick and brownish in colour. The deposit is very copious, containing, in addition to epithelium, hairs and various forms of crystals, especially fat, cholesterin and haema- toidin. Hydronephrosis. The diagnosis of cystic kidney can be best assured by chemical tests, namely, those fcr the detection of uric acid and urea. The deposit, though usually slight, is of importance, and must be carefully collected in a conical glass. On microscopic examination epithelium from the renal tu- bules must be sought for, and if found, a diagnosis of cystic kidney may safely be made. 312 MEDICAL MICROSCOPY. Sperm at ocele. The nature of such cysts is a? once determined by the presence of spermatozoa (fig. 33, p. 184) and occasionally crystals closely resembling the Charcot-Leyden crystals. DISCHARGES FROM GENITAL ORGANS. 313 CHAPTER XX. i DISCHARGES FROM THE GENITAL ORGANS. A MICROSCOPICAL examination of discharges from the genital organs often yields important information, espe- cially from a medico-legal point of view. A medical man may be called upon to decide as to whether certain stains on linen are spermatic or not, or whether a piece of membrane is derived from the decidua of an early abortion. In this chapter a brief description will be given of the most important objects seen under the microscope in the examination of such discharges. , For full details and the conclusions to be derived from them, the reader is referred to treatises on Medical Jurisprudence and Obstetrics, although I have been unable to find any accurate descriptions of the various forms of membrane which may be discharged from the female genital organs. DISCHARGES FROM THE MALE GENITAL ORGANS. Seminal fluid. Semen, when fresh, is of a viscid, honey-like consistence, somewhat opaque, of slightly alkaline reaction, and possesses a faint characteristic odour. As it cools it becomes temporarily gelatinous, and afterwards changes into a thick liquid. Its normal colour is white, but under various conditions this may be altered. If mixed with blood it may appear red, brown, or 314 MEDICAL MICROSCOPY. yellow. The blood may be derived from the prostatic urethra, in which case spots of pure blood are usually noticed in addition to the brownish colour. If the haemorrhage come from the vesicular seminales, the spots are all equally coloured, denoting an intimate mixture of blood and semen (Ultzmann, " Sterility and Impotence," p. 15). A yellow colour is usually due to the admixture of pus, and the stains on linen are then greenish or yel- lowish. Occasionally indigo is present, and a violet or claret tinge is produced. Under the microscope the most prominent objects are the spermatozoa (fig. 66, a). In normal conditions these are present in large numbers. In fresh semen they are seen in active movement. They are about fifty //, in length. The "head "is pear-shaped, and 4-5 //, long. The tail is tapering, if a little water is added it will be seen to roll up and form a loop. If the organisms are dead, the tail will be seen to be spirally coiled or bent at an angle. Their characteristics are very well dis- played by the addition of a drop of iodine solution. A question of sterility may be raised, when the micro- scope may be of great value. Distinction must be drawn between Aspermatism and Azoospermism. By aspermatism is meant a condition in which there is inability to ejaculate semen. It may be per- manent or temporary, congenital or acquired. In azoospermism, although fluid may be ejected, no spermatozoa can be found, and the individual in question therefore is sterile. This condition also may be permanent or temporary, congenital or acquired. The spermatozoa may be greatly diminished in DISCHARGES FROM GENITAL ORGANS, 215 number without being quite absent, this is known as Olizoospermism. Such a condition is common in advanced age, or may be produced by disease, such as gonorrhoea. As long as the spermatozoa can be seen to exhibit active movements, the patient cannot be said to be absolutely sterile, although impregnation is improbable. A FIG. 66. Normal semen (Ultzmann). a. Spermatozoa, b. Seminal cells, c. Epithelium, d. Seminal granules. In addition to spermatozoa, certain other bodies are seen under the microscope in semen. Seminal cells (fig. 66, b). These are circular in shape, finely granular, and represent the spermatozoa in an embryonic state ; they usually exhibit one or more nuclei. Epithelium (fig. 66, c). These are of the squamous or columnar varieties. Masses of amyloid substance, which are finely 316 MEDICAL MICROSCOPY. granular within, stratified, and enclose a central nucleus. These are derived from the prostatic secretion (von Jaksch). Seminal granules (fig. 66, d). These form a fine molecular debris. They are often found attached to the seminal casts, which have been already described on page 183. Spermatic crystals. In general form these bodies closely resemble the Charcot-Leyden crystals (see page 275). Many authors consider them to be chemically the same. Fiirbinger asserts that they are derived entirely from the prostatic secretion. In normal semen they are not deposited for several hours and are seen to be most numerous and most perfectly formed in cases of azoospermism. Spermatic stains. When semen has dried on linen or cotton, the fibre of the stuff is stiffened, and the stain has a slightly translucent appearance. In order to demonstrate the nature of the stain small pieces of the linen should be carefully separated and placed in a watch glass with a few drops of water, just sufficient to soak the fibre thoroughly. The watch glass should be covered to keep out dust and prevent evaporation, and set aside for an hour. Afterwards some of the fibres are removed and gently pressed on clean glass slides. A slightly turbid fluid will exude, which can be rendered clearer, without in- jury to the specimens, by the addition of a little dilute acetic acid. On application of cover-glasses, and ex- amining the preparations under a quarter inch lens, the dead spermatozoa will be found if the stain was caused by semen. Observation should at the same time be made of accompanying hairs or fibres of dress stuff, &c. If too much fluid has been added the spermatozoa may be- DISCHARGES FROM GENITAL ORGANS. 317 come so transparent as to be almost invisible. One or two specimens should, therefore, be allowed to dry by evaporation, and a couple of drops of iodine solution added to the sediment, the organisms will then be well seen. Discharges from the genital organs in cases of gonorrhoea and syphilis may be examined for the characteristic organisms. The methods for so doing have been described on pages 305 and 306. DISCHARGES FROM THE FEMALE GENITAL ORGANS. The vaginal secretion under normal circum- stances exhibits under the microscope squamous epi- thelium cells and a few large leucocytes. When the leucocytes are very numerous, vaginal catarrh is probably present, and a few red blood cells will be seen mixed with the white corpuscles. Cancer cells. In cases of cancer affecting the vaginal portion of the os uteri, in which there is ulcera- tion, large epithelial cells, irregular in shape, and con- taining large nuclei, are stated to be found in large numbers, and to be true " cancer cells," but it is very doubtful, and whether they can be distinguished from the epithelial cells of the vagina, &c., I am much in- clined to agree with those who consider "cancer cells" to be a myth. Parasites : Trichomonas vaginalis. In chronic catarrh of the vagina, in addition to a large quantity of epithelial cells, and mucous and pus corpuscles, an infusorium is found, known as the " trichomonas vaginalis " (fig. 67). It is oval in shape, and has a long caudal appendage, 3 i8 MEDICAL MICROSCOPY. three flagella and a lateral row of cilia. In the specimen from which the accompanying sketch was taken, the cilia could not be seen. Fungi. Numerous micrococci are nearly always found in the vaginal secretion, they are greatly increased after childbirth. Tubercle bacilli have occasionally been found ; cover-glasses should be prepared and stained in the usual way (see p. 28). Gonococci (see p. 306) are found in patients suffer- ing from gonorrhea a. FIG. 67. Trichomonas vaginalis. In cases of suspected rape, the vaginal mucus should be examined for spermatozoa. Membranes. Various forms of membrane may be passed from the female genital organs, and the micro- scope may be of great aid in helping to decide the nature of the case. Mucus coagulated by astringents may simulate a membrane. By floating it out in water its true com- position will probably be recognised ; if placed under the microscope, a large number of cells about the size DISCHARGES FROM GENITAL ORGANS. 319 of leucocytes will be seen variously altered in shape, and irregularly distributed amongst ill-defined fibres. When an attempt is made to tease the material out, it breaks up into fragments, so that it will be found almost Impossible to obtain a thin layer, and the component parts do not adhere together as is the case with true membrane. Fibrinous clots. These may closely resemble the menstrual decidua of membranous dysmenorrhoea. On floating in water, however, the clot is easily broken up. Under the microscope red blood cells are seen in abun- dance together with some fibres, the former constituting the chief bulk of the specimen. Vaginal casts. These are easily recognised with the microscope. They consist almost entirely of squamous epithelial cells closely packed together, amongst which a few leucocytes and red blood cells may be seen. Their characteristics are best demon- strated by the addition of a couple of drops of a two per cent, solution of methylene blue. Membranous dysmenorrhosa. In this condi- tion, during the menstrual period, generally on the second or third day, membrane is expelled either in shreds or forming a more or less complete cast of the uterus. Under the microscope, layers of fibrin enclosing red blood corpuscles are seen ; one surface is rough and uneven, whilst on the other small round cells embedded in a reticular net-work may be made out, amongst which a few tubular channels are noticed ; these are the aper- tures of uterine glands, and are therefore of high dia- gnostic value. The epithelium lining the ducts is rarely complete but is usually more or less destroyed. In complete casts the orifices of the Fallopian tubes 320 MEDICAL MICROSCOPY. can be demonstrated, and the whole structure of the mucous membrane is seen including enlarged orifices of glands and an unusual amount of fibrillar tissue. Membranes of early abortion. The recogni- tion of an early abortion is often attended with diffi- culty ; when the decidua have fully formed it is of course easier, but if the event occurs within the first few months a microscopic examination may often be of great value. Portions of the amnion only may be found. It is a tough membrane, perfectly smooth and transparent. The internal surface is smooth and glistening and consists of a layer of flattened cells, each containing a large nucleus. This layer rests on a stratum of fibrous tissue, which gives the membrane its firmness, and is attached to the inner surface of the chorion. No vessels, nerves or lymphatics can be seen in it. As a rule, the amnion and chorion are found to- gether. One surface of the membrane is then smooth the amnion and the other rough owing to the presence of the chorionic villi. If the membrane be teased out and examined under the microscope the following structures can be distin- guished, although their exact distribution will probably have been disturbed. 1. A large number of small round cells. 2. A certain amount of fibrous tissue. 3. Irregular spaces filled with red blood corpuscles, many of them looking like large blood vessels. 4. Flattened cells with large nuclei, these belong to the amnion. 5. Whorl-like collections of young connective tissue cells. These formations are circumscribed, and consist of delicate branching fibres with nuclei. They are the chorionic villi. Their appearance is very characteristic DISCHARGES FROM GENITAL ORGANS. 321 and when once seen can hardly be mistaken. I have not found anything precisely similar to them in any other structure, and I think one would be safe in dia- gnosing foetal membranes when they are present. 6. A few cubical cells with large nuclei, the so-called " lentil-shaped " cells (Virchow). These are derived from the decidua. If the decidua are also seen the arrangement of the layers is usually more distinct and have the following arrangement : On one surface are the flattened cells of the amnion and beneath is the young connective tissue of the chorion. Next comes a thick layer of the "lentil-shaped" cells, those nearest the foetal membranes being closer together and smaller than those nearer the uterine surface. Then a certain amount of glandular tissue will probably be seen, the epithelium lining the ducts being often per- fectly intact and finally some muscular bundles detached from the uterus ; the characteristic portions being the " lentil-shaped " cells just alluded to. 322 MEDICAL MICROSCOPY. CHAPTER XXI. EXAMINATION OF THE BLOOD. A MICROSCOPICAL examination of the blood is usually undertaken for the purpose of ascertaining the relative proportion in number of the red and white corpuscles to the normal, and to one another. In addition, certain changes in form of the red corpuscles are of importance and the amount of colouring matter is also usually es- timated. More rarely, various micro-organisms are sought for. In normal blood the proportion of white to red cells is about i to 300, this varying considerably within physio- logical limits ; thus, the white cells are greatly increased in number after a meal when the proportion may rise to i to 150, afterwards falling to i to 600. The red corpuscles are -^-^ inch (7-5 ( a) in dia- meter, and x^oo inch (2 p) in thickness. When seen sideways they are bi-concave or dumb-bell-shaped and of a pale buff colour which appears of a reddish tint when a number of corpuscles are collected together. They have a tendency to run together, forming rolls or rouleaux. They are non-nucleated. When the circumference of a corpuscle is brought sharply into focus, a darker area is seen in its centre, but if the lens is brought nearer the corpuscle by means of the fine adjustment, the darker centre is replaced by a lighter area, while the ring becomes darker ; this being due to the fact that these bodies are bi-concave, circular discs. EXAMINATION OF BLOOD. 323 The white corpuscles are usually spherical in form and faintly granular. If the blood is examined fresh on a warm stage, amoeboid movements may be noticed. Their average size is about ^J^ inch, or 10 p in diame- ter. One or more nuclei are present, but are usually not visible unless a little weak acetic acid is run under the cover-glass. A third element will sometimes be observed, namely, blood-plates. These were first noticed by Bizzozero. To demonstrate them the blood must be " fixed " by the addition of some preservative fluid, such as Hayem's. This has the following composition : Chloride of sodium i part Sulphate of sodium .... 5 parts Corrosive sublimate .... *5 parts Distilled water 200 parts. A couple of drops of this fluid is added to a drop of the blood obtained from the finger, and placed on a glass slide. A cover-glass is applied, and the specimen examined by the highest obtainable power (preferably a T X 2- oil immersion) and a small diaphragm. The plates will then be seen as slightly oval or rounded granules about one-third the size of the ordinary red corpuscles. Methods of examination. For the histological examination of the blood it is sufficient to tie a handker- chief tightly around one finger so as to compress as much blood as possible into the tip, then prick it with a sharp needle, and remove the drop which exudes on a clean cover-glass. This should be placed on a glass slide and its edges surrounded with a rim of oil or Canada balsam, in order to prevent evaporation. Two processes of the utmost importance in the ex- amination of the blood will now be described, i.e., " counting the corpuscles," and the " estimation of Y 2 324 MEDICAL MICROSCOPY. haemoglobin." The latter does not strictly come under the head of Medical Microscopy, but a chapter on ex- amination of the blood for clinical purposes can hardly be complete without it. COUNTING THE CORPUSCLES. This is usually accomplished by means of Dr. Gowers' haemocytometer, fig. 68, (made by Hawksley, Oxford Street), which consists of (i) a pipette graduated to 995 cubic millimetres for measuring the diluting solution ; (2) a capillary tube for measuring the blood and capable of holding 5 cubic millimetres ; (3) a small glass jar and and stirrer for making the dilution ; and (4) a glass slide on which is arranged a cell -2 millimetres deep, and ruled at the bottom in squares, each ! millimetre in length and breadth. The slide is attached to a metal plate furnished with two springs for maintaining the cover-glass in position. The diluting solution recommended by Dr. Gowers is* as follows : Sulphate of soda 104 grains Acetic acid ...... i drachm Distilled water 6 ounces. The instrument is used in the following manner : 995 cubic millimetres of the saline solution are mea- sured off in the pipette and placed in the mixing glass. A drop of blood is obtained from the patient's finger in the way described above, and drawn up by the capillary tube to the level of the 5 cubic millimetre mark, and then thoroughly mixed with the saline solution in the glass. One caution must here be given, and that is that if the drop of blood does not readily exude, too much EXAMINATION OF BLOOD. 325 pressure must not be applied, or otherwise too great a proportion of serum will be obtained, and thus an incorrect calculation will result. The diluted blood is then thoroughly stirred with the small glass rod ; one drop is transferred to the floor of FIG. 68. Gowers' haemocytometer. A. Pipette for measuring the diluting solution. B. Capillar)' tube for measuring the blood. C. Cell with divisions on the floor. D. Vessel in which the dilution is made. E. Spud for mixing the blood and solution. F. Guarded spear-pointed needle. the cell, and another to a cover-glass. The two drops are then approximated, which ensures a regular layer extending throughout the depths of the cell, and the springs are arranged so as to keep the cover-glass in position. It is a good plan before placing the blood in 326 MEDICAL MICROSCOPY. the cell to rub the ruled portion of the cell over with a soft black-lead pencil, afterwards removing the loose dust. This renders the squares distinct, otherwise, especially in a new glass, they are often barely visible (Wynter). The slide is now examined with a quarter-inch lens, and the number of corpuscles counted in 10 squares. In order to explain the computation we quote Dr. Gowers' own words in his article in Quain's " Diction- ary of Medicine." " The dilution of 5 cmm. of blood in 995 cmm. of solution is i in 200 ; each square contains the corpuscles from a volume of dilution -2 mm. in one, and ! mm. in each of the other dimensions that is, 2 cubic ! mm., or the -002 part of a cubic mm. But the dilution being i in 200, this volume of dilution contains just 'ooooi cm. of blood. The number of corpuscles in a square multi- plied by 100,000 is thus the number in a cubic milli- metre of blood the common mode of statement. In order to limit error, the total number of corpuscles in 10 squares should be counted, and this number multiplied by 10,000 is the number per cubic millimetre." The average number in normal blood is about 5,000,000, which corresponds to about 50 in a square ; from this it is evident that normal blood will contain 100 corpuscles in 2 squares, and that, therefore, the number of corpuscles of any blood in 2 squares of the haemacytometer represents the percentage of corpuscles compared with normal blood. In counting the white corpuscles, if they are not in considerable excess, it is most convenient first to ascer- tain the number of red corpuscles per square, and note how many squares are contained in the field of the microscope. If then the focus is raised so that the cor- EXAMINATION OF BLOOD. 327 puscles gradually become indistinct, the white ones, from their higher re- fracting power, will appear like bright points, and the number in a series of fields can easily be counted. For ex- ample, the number of red corpuscles per square has been found to be 40, and the field contains 15 squares, that is, 600 corpuscles per field. 10 fields contain 15 white corpuscles; the pro- portion of white to red will, therefore, be i to 6ooxIQ = ito 4Q0 . i5 Another excellent instrument, though not so well-known in this country is the Thoma-Zeiss hamocytometev (may be procured from Baker, 243 High Hoi- born). Dr. Archibald Garrod has had considerable experience in the use of this apparatus, and I have to offer him my best thanks for kindly writing for me a description of the Kill 0*5 hsemocytometer, and the mode of using it. The dilution and mixing of the blood with saline solution is carried out in a pipette (fig. 69) made after the pattern of Potain's melangeur. The capillary bore of this pipette is at one point expanded into a bulb in which lies a small glass ball. The FlG 6 Q .- Pipette lower portion of the tube is graduated, (Thoma-Zeiss hasmo- and into it are drawn 5 or 10 cubic cy c millimetres of blood ; saline solution (of the composition 3 28 MEDICAL MICROSCOPY. given above) is then drawn in until the bulb, and a very small portion of the capillary tube beyond, are filled with the mixed blood and solution. According to the amount of blood taken, the dilution will be 200 or 100 times. A gentle rotary motion is then imparted to the pipette, which, by causing the glass ball to move within the bulb FIG. 70. Counting slide (Thoma-Zeiss apparatus). brings about a complete mixture of its contents. A small portion of the liquid is then expelled so as to clear out the unmixed saline solution from the capillary tube, and afterwards a drop of the diluted blood is placed upon the counting slide (fig. 70). The centre of this slide is formed into a circular cell by a rim of glass, and FIG. 71. Cross section of Thoma-Zeiss apparatus. in the middle of the cell so formed is a small platform not quite so thick as the surrounding rim (fig. 71). When the cover-glass is placed over the cell, a space of *oi millimetre is left between it and the central platform, whilst the small trough between the platform and the rim serves as a receptacle for any overflow of fluid. EXAMINATION OF BLOOD. 329 When the cover-glass is accurately applied, Newtonian colours will be produced where it is in contact with the rim of the cell. When these colours cannot be obtained, it must be concluded that some dust is lying between the two surfaces of glass which must then be separated and cleansed, and a fresh drop of liquid from the pipette placed in the cell. The preparation must also be re- jected if any of the liquid makes its way between the cover-glass and the external rim. Upon the centre of the platform is a square divided 'IL FIG. 72. Counting scale (Thoma-Zeiss apparatus). into 400 lesser squares, each n square millimetre in area and -^ mm. in diameter. Since the depth of cell is 'Oi c.mm. the space over each square is -$-5-5 c.mm. The contents of 16 such squares gives the per- centage of corpuscles. Sixteen sets of sixteen squares are marked off by triple lines (fig. 72), and if the whole of these are counted, or 256 squares in all, the area included in the counting equals that of 32 squares of Gowers' haemocytometer. In order to estimate the number of red corpuscles per cubic millimetre of undiluted blood, if after counting the 330 MEDICAL MICROSCOPY. corpuscles in a certain number of squares, m be found to be the average number of corpuscles per square of ^ n millimetre area, this number would have to be multiplied by 4000 x 100 or 200 (according to the dilution taken) to give the result sought for. Or the calculation may be performed thus :-4 * 2O ( or )_JL = n> where x = total number of corpuscles counted, y = the number of squares and the result () will be the number of corpuscles in a cubic millimetre of undiluted blood. Dr. Garrod says: "I always count the squares in sets of 1 6, counting successive rows of four squares, includ- ing in my counting those corpuscles which touch the upper and left hand borders of the square, either from the outer or inner side, and excluding those which touch the lower and right hand borders." The small size of the squares of this instrument very greatly facilitates the process of counting ; which may also be interrupted if necessary, and continued later on from the point at which it was stopped, as a note can be made of the number, and row of the square last counted. One of the drawbacks to the instrument is the trouble required for the cleaning of the pipette which should be washed out several times with water, and then dried. The drying is effected by means of alcohol, and after the expulsion of the spirit a current of dry air should be sent through the pipette until the little glass ball moves freely about in the bulb, without adhering to the sides. The pipette should also be washed out occasionally with ether, and at intervals with caustic potash. For the counting of the white corpuscles a second pipette is provided, of similar form, but giving a dilution of i o or 20 times. The liquid employed is a ^ per cent, solution glacial acetic acid, which destroys the red EXAMINATION OF BLOOD. 331 corpuscles and at the same time renders the white more clearly visible. A drop of the mixed blood and acid is placed in the cell and the corpuscles are counted by fields of the microscope, the area of which is determined in the following manner. The diameter of the field is ascertained by means of divisions on the floor of the cell, each of which equals -o millimetre. If the field has a chamber of ten divi- sions, the radius will be J- millimetre, and the area if (ff square millimetre, and since the depth of the cell is 01 millimetre, the cubic contents of the field will be o-i XTT (i) 2 cubic millimetre '(if = 3-1416). The number of white corpuscles may be then calculated by means of the following formula : D_x_N F^TC when D = the dilution i.e., 10 or 20 N number of corpuscles counted F = number of fields counted C = cubic contents of a field. In practice the calculation may be greatly simplified by always counting the same number of fields (e.g. 50), and by always using the same sized field, and the same dilution of the blood. ESTIMATION OF THE AMOUNT OF HEMOGLOBIN IN THE BLOOD. Various modes of estimating the richness of the blood in haemoglobin have been introduced. It may be ac- complished by the amount of dilution necessary to obscure a certain absorption band in the spectrum, or by the coloured discs invented by Hayem. The two 332 MEDICAL MICROSCOPY. most practical methods for clinical purposes are those of Dr. Gowers and von Fleischl. Gowers' haemoglobinometer (fig 73). This ap- paratus consists of two small tubes of exactly equal diameter, which when in use are placed in a stand made to receive them. One tube contains a preparation of FIG. 73. Gowers' hasmoglobinometer. A. Bottle for holding the diluting solution. B. Capillary pipette for measuring the blood. C. Graduated tube for measuring the amount of haemoglobin. D. Standard tint of normal blood. E. Support for D and C. F. Punc- turing needle (Havvksley). carmine and glycerine jelly coloured to represent a solu- tion of i part of healthy blood in 100 of water. The other tube is graduated, each division being equal to the volume of blood taken (20 cubic milli- metres). So that 100 divisions equal 100 times the volume of blood. A capillary pipette is also supplied capable of holding 20 cubic millimetres. This is filled EXAMINATION OF BLOOD. 333 with blood which is blown into the graduated tube in which a few drops of distilled water have previously been placed. The blood and the water are then tho- roughly mixed. Distilled water is now added drop by drop from the pipette until the tint of the mixture corre- sponds with that of the standard. FIG. 74. Von Fleischl's haemometer (for explanation of lettering see text). The height of the diluted fluid is then read off, the division which is reached representing the percentage amount of haemoglobin. For example, a specimen of blood is diluted until the mixed fluids reach the 55 mark on the tube, when the tint of the standard is found to be attained. The blood examined, therefore, contains 55 per cent, of the normal quantity of haemoglobin. Yon Fleischl's haemometer (fig. 74). For an 334 MEDICAL MICROSCOPY. account of this excellent piece of apparatus, I am once more indebted to Dr. Archibald Garrod, who describes it and the means of using it in the following words : " In this instrument the standard of comparison is an elongated wedge of tinted glass (K), which is so mounted that it can be moved backwards and forwards beneath a small stage, by turning a milled wheel (T). In the stage is a circular opening through which the light of a lamp is reflected upwards from a disc of plaster of Paris (S) mounted like the mirror of a microscope. The wedge of glass underlies one-half of this opening. The mixture of blood and water to be examined is placed in one division of a cylindrical vessel (G), which has a glass bot- tom, and is divided in half by a vertical septum (a). This vessel fits accurately over the circular opening in the stage, and must be so adjusted that the septum coin- cides with the edge of the tinted prism. On looking down upon the instrument, one-half of the illuminated circle will then receive its tint from the prism, the other half from the mixture contained in the vessel. " The blood is received from the ear or finger into a minute glass tube, into which it is drawn by capillary attraction. This tube is held by a small metal handle, which afterwards serves as a convenient stirring rod. The tube being accurately filled, and any blood which may have adhered to its outer surface having been care- fully removed, its contents are immediately washed out into the mixing vessel by means of a small pipette filled with water. Water is then added with careful stirring, until a plane surface is obtained, level with the top of the cylindrical vessel, and showing neither a con- cave nor a convex meniscus. If any clotting has oc- curred in the tube, a fresh specimen should be taken, but after a little practice this accident will seldom occur. EXAMINATION OF BLOOD. 335 "The other half of the vessel, which lies over the tinted prism, must then be filled in the same manner with pure water. The comparison of the tints is facili- tated by placing a cardboard tube, blackened internally, upon the stage of the instrument, so as to enclose the mixing vessel. " The prism is then swept backwards and forwards until an accurate match is obtained, when the percent- age of haemoglobin may be read off upon a scale (P) which is visible through a second small opening (M) in the stage. It is well to repeat the comparison several times, until the successive readings exhibit a close agreement. " The estimate should be arrived at as quickly as possible, because the eye soon becomes tired, rendering an accurate comparison almost impossible. In case of doubt the eye should be rested, and a fresh determina- tion made after an interval. " The comparison requires a darkened room and an artificial light, since by daylight the tint of the prism is entirely unlike that of the diluted blood. " The standard of haemoglobin is somewhat higher than that of Gowers' haemoglobinometer, and I have not yet found the blood of any Londoner to attain to the 100 per cent. " I have made repeated observations in quick succes- sion, taking a fresh specimen of the blood of the same individuals, and have obtained results not differing by more than i per cent., but it should be mentioned that the scale is only divided into tens and fives, although I believe that the delicacy of the instrument would admit of finer graduation. As it is the units of percentage must be estimated by the observer." Having described the methods of estimating the 336 MEDICAL MICROSCOPY. number of the corpuscles, we must now proceed to consider what results may be arrived at from this com- putation. The red corpuscles may be decreased, this condition being known as oligocythsemia. As before stated the normal number of red corpuscles in the blood is 5,000,000 in a man, and 4,500,000 in a woman to the cubic millimetre of blood. In various forms of disease this number may be diminished, the number falling to 2,000,000 or even as low as 360,000 per cubic millimetre (v. Jaksch). The lowest number I have ever found was 450,000 ; this occurred in a case of pernicious anaemia. Temporarily this decrease may be due to haemorrhage. As a permanent condition it is found in those diseases which are attended with deficient production of the cor- puscles. The quantity of haemoglobin is roughly proportionate to the number of corpuscles, but the individual corpus- cles often contain less haemoglobin than normal. The loss may be so great that the proportion of haemoglobin falls to 25 per cent, of the normal, as in some cases of chlorosis. Hayem drew attention to the fact that in pernicious anaemia the number of red cells in the blood is inversely proportional to the quantity of haemoglobin which they contain. In the case just referred to, the blood, tested by v. Fleischl's haemometer only yielded 10 per cent, of haemoglobin. Polycythsemia. Increase in number of the red corpuscles is never great, being very transitory, and within physiological limits, thus it is found after meals and in the newly born. In plethoric conditions also the number is above the average, and in the algid stage of cholera the red corpuscles are relatively in excess. Leucocy thaemia. By this is meant a condition in which there is a considerable and permanent increase in EXAMINATION OF BLOOD. 337 the number of the white red corpuscles ; it is also known as leucamia. Microscopical examination sometimes shows an enor- mous increase in the number of the white corpuscles. Virchow has found them as high as in the proportion of 2 : 3 of red corpuscles. In this condition the red cor- puscles are also diminished, being generally about two or three millions to the cubic millimetre of blood. Von Jaksch (I.e., page 20) has made some remarkable observations as regards the character of the leucocytes. It is usual to distinguish leucaemia as splenic, lymphatic, and myelogenic, according to the anatomical seat and clinical symptoms of the disease. When large and small leucocytes, the latter preponderating, occur, the leucaemia is of a lymphatico-splenic character. When the larger cells alone are found, v. Jaksch considers that the disease is almost entirely splenic. If the corpuscles are of a transitional form, nucleated red cells being also present, the bone-marrow is probably the seat of serious changes. Although the more pronounced forms of leucocy- thaemia are recognised without difficulty, it is not so easy to detect an early stage of this condition. Prof. Ehrlich has pointed out how this may be ac- complished, by taking advantage of the selective pro- perties of the " granules " present in the protoplasm of the leucocytes towards eosin. Preparations are made as follows : Two films of blood are procured by pressing a drop of blood between two cover-glasses, and then sliding them apart. They are next dried in an exsiccator and heated upon copper foil for a considerable time (ten to twelve hours) at 120* to 130 C. A more simple expedient may be adopted by placing the glasses as soon as the blood has partially z 338 MEDICAL MICROSCOPY. dried in absolute alcohol for a couple of hours, and then driving off the spirit by heat. A drop of concentrated eosin-glycerine solution is then added to each cover- glass, the excess of stain is washed off with water, and the glasses are finally dried and mounted in balsam. Ehrlich found that the granules presented consider- able differences in their staining properties. He dis- tinguished five several varieties, classifying them as a to s granules. When the disease is acute, and the number of leuco- cytes only slightly diminished, the mono- and poly- nuclear forms (g-granules) are alone increased, whilst the a-granules (" eosinophile ") are decreased. When the condition is a more chronic one, exactly the reverse condition of affairs results, the a-granules being increased and the -granules diminished. Various changes in the red corpuscles are noted in leucocythaemia, but these will be described separately. Leucocytosis. This term is applied to the condi- tion in which the white corpuscles are temporarily in- creased. This occurs physiologically after a meal, the proportion of white rising as high as i to 100 red cor- puscles. According to Virchow, leucocytosis accom- panies every case of lymphatic excitement, such as inflammation, and tubercular, scrofulous or cancerous enlargements, or swelling of the glands, and allied structures in Peyer's patches, solitary follicles, the spleen and the tonsils. I have found an increase of white cells in cases of cancer, when the cachexia has been very profound. This condition differs from leucocythaemia then in its transitory character, by the absence of deficiency of the red corpuscles, and by the degree in which the white corpuscles are diminished. EXAMINATION OF BLOOD. 339 Von Jaksch and Tumas maintain that leucocytosis occurs regularly in croupous pneumonia. Microcythaemia. Various observers have from time to time described bodies in the blood which appear like small red corpuscles. Manassein (" Centralblatt," 1871) produced changes in the diameter of the red blood cells by introducing chemical substances into the circu- lation. He noticed a diminution in size of the cor- puscles in cases of hyperpyrexia, in septic poisoning, and when the oxidation of the blood was interfered with, as during the inhalation of carbonic acid. He found the diameter of the corpuscles increased by the action of alcohol and quinine, also under the influence of cold, in cases where an excess of oxygen had pro- duced an acute anaemia. The bodies known as " microcytes " were first de- scribed by Vaulair and Masius ("Bulletin de 1' Academic Royale de Med. de Belgique," 1871, p. 515). They are spherical in form, rather redder than the ordinary cor- puscles, and have a diameter of 2 or 3 //,. They do not run into rouleaux. Some authors (Gram, Hayem) consider that they are caused by the fluids added to the blood for diluting purposes, but they are seen when no such agents have been used, and when the pure blood has been examined directly. Portions of colour- ing matter may be removed by the said agents, and simulate closely the microcytes, but they will be found not to possess such clear outlines, and as a rule only occur sparsely, whilst the microcytes are usually numerous. They are found in various conditions, such as in- fectious diseases, pronounced anaemia, and burns. Vaulair and Masius met with them in pathological states in which there was a diminished activity of the z 2 340 MEDICAL MICROSCOPY. liver, with an increased activity on the part of the spleen. I have also observed a similar case. In per- nicious anaemia they are very abundant ; Eichorst goes so far as to consider them pathognomonic of this disease. As regards their nature, some authorities believe that they are caused by the rapid and uniform abstraction of water from the corpuscles, others that they are the final stage of the changes which the red corpuscles undergo in the spleen, and that they are destroyed by the liver. Their true significance is not yet known, and the matter requires further elaboration before any diagnostic im- portance can be attached to them. Poikilocytosis (fig. 75). In certain affections many p p FIG. 75. Blood in poikilocytosis. of the red corpuscles are found to have undergone re- markable changes in shape and size. Some are seen to be much larger than usual (megaloblasts), but the majority show various departures from the normal spherical form. Some are oval, others pear-shaped, others kidney-shaped, whilst some exhibit minute pro- cesses, like small knobs projecting from them. Von Jaksch (loc. cit., p. 24) considers that in this condition the red corpuscles are endowed with an abnormal power of contractility. These appearances were at one time thought to be peculiar to pernicious anaemia, but have since been observed in many cases in which the blood is greatly EXAMINATION OF BLOOD. 34! affected, such as severe anaemia of whatever kind, and in chlorosis. They have also been recorded as occurring in the blood in cancerous cachexia, and in myeloid de- generation of the viscera. The most pronounced speci- men I have ever seen was obtained from a patient suffering from gout and profound anaemia with splenic enlargement. To observe these bodies the blood should be examined pure, without the addition of diluting fluids. Melanaemia. In the blood of patients who have suffered for some time from malarial disease, particles of pigment are observed in the blood. These are usually black, more rarely brown or yellow. They are some- times free, and sometimes enclosed in round or oval cells, probably leucocytes. This appearance is often associated with an enlarged and deeply pigmented condition of the spleen ; it is not improbable that the pigment particles are produced by the malarial fever, and find their way from the spleen into the blood. PARASITES IN THE BLOOD. Vegetable parasites. Several of the most im- portant pathogenic bacteria have been found in the blood of patients suffering from the particular dis- eases caused by the germs. The number of micro- organisms, however, falls far short of that found in animals under similar conditions. In order to search for the organisms, the finger of the patient should be well scrubbed with soap and water, and then washed over with corrosive sublimate of the strength of i in 1000. The tip of the finger is then compressed and pricked with a sterilised needle. The 34 2 MEDICAL MICROSCOPY. drop of blood which exudes is taken off with a clean cover-glass and immediately covered with another, the two being pressed together and then slid apart, so as to obtain two films ; when dry they are passed three times through the flame, and afterwards stained in an appro- priate manner, according to which bacillus is to be sought for. Bacillus of anthrax. Cover-glasses prepared as above should be stained by Gram's method (see p. 95). Their appearance does not differ from that described on p. 305, as obtained from the local lesion. Bacillus of tubercle. Cover-glass preparations must be stained by Neelsen's method (p. 98). Tubercle bacilli are found with difficulty in the blood, and a large numbers of specimens must be obtained. Meisels found them in the blood of a patient suffering from miliary tuberculosis and his discovery has since been confirmed by other observers. Although I have made several attempts, I have never been able to detect the bacilli in the blood of tuber- cular patients. Bacillus of typhoid fever. Some observers have succeeded in finding bacilli in the blood of patients afflicted with enteric fever, which morphologi- cally, in their manner of growth on artificial media, and in the effect they have when inoculated on animals, are similar to those which are found in typhoid stools and in the tissues after death (see p. 227). Cover-glass pre- parations should be stained in methylene blue, and washed in water acidulated with a drop or two of acetic .acid. Bacillus of glanders. The pathogenic bacilli of glanders, in addition to being found in the farcy buds, and discharges from ulcers in patients suffering from EXAMINATION OF BLOOD. 343 that complaint, have been found in the blood in small numbers. They are difficult to stain ; cover-glass pre- parations are best coloured by Kiihne's method (see p. 92). Bacillus of tetanus. Nicolaier and Kitasato were the first to describe this organism. The rods are long and slender and exhibit terminal spore formation. They have only very rarely been found in the blood. Spirillum of relapsing fever. The organism found in the blood of patients suffering from relapsing fever may be demonstrated in a drop of fresh blood placed under a cover-glass, or dried preparations may be made in the usual way, stained with fuchsine, and washed in water containing a few drops of alcohol. The spirillum is usually known as the " spirillum Obermeieri," having been originally described by Dr. Obermeier of Berlin in 1873. It takes the form of a delicate, homogeneous, spirally-twisted filament, hav- ing a length of from ^hm to ^ inch (15-40 //,). In fresh blood it exhibits active movements, rotating on its long axis, and with a lashing movement progressing backwards and forwards. The " spirochaetae," as the spirilla are sometimes termed, are only found in the blood during the actual attacks of relapsing fever, and cannot be found during the intervals. According to Heydenreich, they appear before the febrile attack begins, but cease to be dis- coverable before the commencement of the crisis. The same observer also finds that their numbers vary greatly from day to day. Sometimes, after the fila- ments have been present for two or three days, they suddenly disappear, but a few hours later may reappear in large numbers. They are very sensitive to reagents of all kinds. If 344 MEDICAL MICROSCOPY. the blood be examined in the intervals of the disease, peculiar refractive bodies resembling diplococci are found ; these are especially numerous when the par* oxysm commences. Von Jaksch (I.e., p. 31) considers that when the attack begins, these bodies grow out, as it were, into short thick rods, from which the spirilla are finally evolved. They are therefore probably spores. Haematozoa of malaria. Lavaran in 1880 was the first to notice the existence of peculiar structures in the blood of patients suffering from malaria, and his observations have been confirmed by several observers, notably by Marchiafava and Celli. The different forms met with may be divided into two groups ; those within the red blood corpuscles and those free in the serum. Intr a- corpuscular bodies. -There are three varieties of these : Firstly, irregular protoplasmic bodies much smaller than the corpuscles. They exhibit active amoeboid movements. Marchiafava and Celli who were the first to observe them suggested the name Plasmodium malariae. Secondly, minute masses of finely granular or hyaline protoplasm enclosing granules of pigment. They are usually circular in shape, and likewise exhibit amoeboid movements. The pigment granules also change their shape. Thirdly, forms which appear like isolated grains, and in addition large homogeneous bodies surrounded by clear spaces. Extra-corpuscular bodies. These are also of various forms. Firstly, what are known as the "semi-lunar bodies of Laveran." These objects are crescent-shaped, either rounded or pointed at the extremities ; occasion- ally they are almost spherical. They are motionless. Pigment granules are found in the centre of these bodies, which seem to be composed of homogeneous EXAMINATION OF BLOOD. 345 protoplasm. Secondly, protoplasmic masses, finely granular in appearance. The pigment is collected into the form of a rosette, and the protoplasm gradually undergoes segmentation, forming small spherical bodies, which are ultimately set free. Thirdly, circular pear- shaped, or ovoid bodies, a little smaller than red blood corpuscles, provided with one or more actively mobile, long lash-like filaments. Fourthly, small spherical pig- mented bodies about one quarter the size of the red blood corpuscles. These exhibit amoeboid movements. To demonstrate these bodies fresh blood is examined in the usual way. For permanent specimens, dried cover-glasses are prepared, and stained by the Gram method. Klebs and Tommasi-Crudeli found bacilli in the soil of the Campagna, which they regard as the specific organism of malaria. ANIMAL PARASITES. These are the Filaria sanguinis hominis and the Bilharzia haematobia. Filaria sanguinis hominis (fig. 76). This para- site has already been referred to (see p. 190). The dis- covery of the embryos of this nematode worm in the blood was first made by Dr. T. R. Lewis in 1872. Four years later the parent worm was found by Dr. Bancroft of Brisbane. It has been shown to inhabit the lymphatics of the inhabitants of tropical countries. It does not necessarily produce disease and many natives (i in 10 in South China) in perfect health were found to be affected. Usually the condition is brought to notice by the patients becoming afflicted with chy- 34$ MEDICAL MICROSCOPY. luria or elephantiasis. Dr. Stephen Mackenzie found them in the blood of a native of the Upper Congo suf- fering from " sleeping sickness." The parent worm is about 3 to 3^- inches long, and has a diameter of about T ^ n inch, and resembles " a delicate thread of catgut, animated and wriggling " (Manson). The mouth is circular, without papillae ; there is a narrow neck, and the tail is bluntly pointed. It is with the embryos, however, that we are immediately interested. Speci- mens are obtained by pricking two or three fingers of the patient, transferring the drops to a glass and covering with a long cover-glass. FIG. 76. Filaria sanguinis hominis (specimen in the urine, Boyce). The embryos are seen as active organisms about -^ inch in length, and having a diameter 3-^0 5 each is enclosed in a delicate sac or sheath which fits it accurately, except that a collapsed or unoccupied part is seen projecting at one end. Most observers agree that this sheath is the envelope or shell of the ovum, which, as the embryo developes becomes stretched out over the skin. This enveloping membrane is quite structureless, but the contained parasite is seen under a high power to be transversely striated and very EXAMINATION OF BLOOD. 347 granular. The embryos only occur in the peripheral vessels whilst the patient is asleep, generally, therefore, at night. They begin to make their appearance at about 6 or 8 p.m. By midnight their numbers reach the maximum. As morning approaches they become fewer and fewer, and finally disappear altogether ; but that this is not due simply to daylight, was shown by Dr. Stephen Mackenzie, who caused a patient to be up all night, and in bed all day, with the result that the filariae were only found during the day ; on the other hand, the most dense London fog, if the patient was up and about during the day, did not tempt the parasites to appear. Dr. Manson has twice obtained ova in a much earlier stage of development, consisting of oval bodies g-J n inch in length, and T J n in breadth. FIG. 77. Bilharzia haematobia, male and female; eggs (von Jaksch). Bilharzia haematobia (distoma haematobium). Patients who have dwelt in tropical countries, and be- come affected with endemic hsematuria, have been shown to be the hosts of a parasite, to which the name of Bilharzia haematobia was given by Dr. Cobbold. The adult parasites (fig. 77) occupy smooth walled spaces in communication with the veins of the pelvis. This fluke is white in colour, the male and female being distinct. The former is flattened, and about half an inch in length ; the hinder part is more cylindrical in form, from the edges being thinned, and folded inwards so as 34$ MEDICAL MICROSCOPY. to form a groove in which the female is received during sexual congress. The female is round and thin, and about f inch long. The parasite itself is but rarely seen in the blood, but the ova are discharged, and are often found in the urine (see p. 190). EXAMINATION OF BLOOD STAINS. A medical man is occasionally called upon to give a decision as to whether a stain on some portion of cloth- ing is caused by blood. Although in important cases such a question is generally referred to an expert, yet all doctors ought to be prepared to make such an ex- amination. The suspected stain, should first be exam- ined by a good lens ; if caused by blood, it will not be a mere colouring of the fibres, but will have a shiny glossy appearance, and each fibre will be invested with a por- tion of dried coagulum or clot. The most simple plan of procedure, when small par- ticles of coagulum can be removed, is to place them on a glass slide, and then breathe over them several times. A cover-glass is applied, and if blood be present, a red margin will soon appear, and by the aid of a quarter- inch lens the red blood corpuscles may be recognised. Should this be unsuccessful, a very small drop of water should be run in under the glass any excess must be avoided, or the corpuscles will swell and loose their characteristic appearances. Such a simple method, however, is not always practicable. The stained portion of the material must then be cut out and macerated in a small quantity of equal parts of glycerine and water, or better in a mix- ture proposed by Hofmann EXAMINATION OF BLOOD. 349 Water 300 parts Glycerine 100 Chloride of sodium .... 2 ,, Corrosive sublimate .... i part. If small portions of the fibres of the material be placed under the microscope, corpuscles will be seen either free in the fluid or entangled in the meshes of the fibres. A very important question often arises, granted that the stain is caused by blood, is it human ? At present our knowledge on this subject is very limited. In all animals with red blood the globules have a disc-like form. In the mammalia with the exception of the camel tribe the outline of the corpuscle is circular. In this tribe, and in birds, fishes and reptiles, they are oval in form. In the three last mentioned classes of animals, they possess a central nucleus. We are able to say with safety then that blood corpuscles are either mam- malian and may be human, or that being oval in shape, the corpuscles came from one of the other classes above mentioned. Dr. Lionel Beale in his work on " The Microscope in Medicine " figures a large number of corpuscles, which are worthy of reference. 35O MEDICAL MICROSCOPY. CHAPTER XXII. CUTANEOUS PARASITES. IN order to complete the account of the most important parasites found in disease, a short description must be given of those producing various affections of the skin. The clinical features are usually quite sufficient upon which to base a diagnosis, but the evidence afforded by the microscope will corroborate the opinion, and in a few atypical cases may yield the only means of arriving at a definite conclusion. Some of the bacteria producing cutaneous affections, such as those pathognomonic of tubercle, leprosy, &c., have already been considered. . Cutaneous parasites may be divided into animal and vegetable. The latter will be considered first as being the more important. VEGETABLE PARASITES. Trichophyton tonsurans. This is the charac- teristic fungus of "ringworm." The diagnosis is not difficult clinically, the presence of short, broken-off hairs, in small patches, sufficiently indicating the nature of the complaint. As corroborative evidence one or two of the hairs should be removed with forceps and soaked for a little time in dilute liquor potassae. The hair seems to be covered near its roots with an asbestos -like covering, CUTANEOUS PARASITES. 351 of a dull white colour. After soaking, they are placed on glass slides with a little of the potash solu- tion, a cover-glass applied, and light pressure made so as to separate the component fibres of the hairs. Attention should first be directed to the hair itself. The surface will be seen to be rough, the cortex and FIG. 78. Trichophyton tonsurans (Alder Smith). medulla being almost undistinguishable. The free end, instead of being pointed, is ragged and split. The fun- gus (fig. 78) consists almost entirely of spores ; these are circular in shape, and about '004 mm. in diameter; they 352 MEDICAL MICROSCOPY. possess nuclei, and are chiefly arranged in chains, dis- tributed between the disordered fragments of the hair, being particularly numerous about the root, the my- celial threads are few, jointed, somewhat curved in their course, and small granules are seen in their in- terior. When attacking the head this disease is known CUTANEOUS PARASITES. 353 as Tinea tonsurans ; when the body is affected it is termed Tinea circinata or Tinea marginata. Tinea marginata, formerly called " eczema margina- tum," occurs only in adult males ; the same fungus is found in all the forms. Microsporon furfur (Tinea versicolor). The af- fection caused by this fungus is also known as Pity- riasis versicolor. It occurs as yellowish-brown spots, scarcely rising above the level of the skin, and dis- tributed generally over the body, especially over the chest and back. To demonstrate the fungus some of the scales should be removed and examined in a drop of dilute potash. If an opportunity should occur of obtaining sections of skin thus affected, very satisfactory specimens may be obtained by staining them with Weigert's modification of the Gram method (see p. 95). The spores are found collected together into heaps, and are a little larger than those of Tricophyton tonsurans. The mycelial threads are very numerous and form a thick wavy mass, between the component parts of which the spores can be seen. Achorion Schonleinii. If a small piece of the crust from a case of favus (fig. 79) is examined in liquor potassae, dense masses of mycelia will be seen ; they are often so thick that the spores can scarcely be made out. These latter are oval or circular in shape and a little smaller than those of Tricophyton tonsurans. ANIMAL PARASITES. Pediculi. Three species of lice are parasitic on man; (i) Pediculus capitis ; (2) Pediculus vestimenti vel corporis ; (3) Pediculus pubis. AA 354 MEDICAL MICROSCOPY. 1. Pediculus capitis. In all cases of pustular inflammation of the scalp the hair should be carefully searched for pediculi, although the impetigo which re- sults is produced more by the scratching of the patient than by the irritation of the lice. Children are most frequently attacked, and women more than men. The louse is about a line in length, of a dirty- white colour, and covered with short scattered hairs. The abdomen is oval, and distinct from the head and thorax. The head has two short antennae, and large prominent black eyes. The parasite has six well-developed legs, furnished with strong claws. The male, which is rather smaller than the female, has a peculiar long projection on its back the penis. The animal itself cannot always be found, but equally diagnostic are the presence of "nits." These contain the ova, and are small, white, semi-transparent bodies, made of a hard material and triangular in form. They are firmly attached to the hairs, about three quarters of an inch from the roots, by short pedicles being glued to them by a material secreted from the lice. 2. Pediculus vestimenti. In general appearance this louse closely resembles the pediculus capitis ; it is, however, rather larger in size. Its ova are deposited in the clothing and not on the surface of the body. 3. Pediculus pubis. As its name indicates this parasite chiefly attacks the hair about the generative organs. It is different in shape to the other two varie- ties, resembling a small crab. It is also smaller, and the line of separation between the abdomen and thorax is not so well marked. The legs are short and curved, and terminated by strong claws. Sarcoptes hominis. This parasite, also known CUTANEOUS PARASITES. 355 as the Acarus scabiei, or itch mite, produces the well known disease scabies. The clinical features are too well known to need description here, and we need only consider the parasite itself. The male insects are but rarely seen, as they do not burrow but live upon the surface of the body. The female, after impregnation, makes her way under the skin, forming the characteristic cuniculus or "run." To demonstrate the mite, a burrow should be laid open with a stout needle, from the entrance to its blind ex- tremity ; the acarus will then be seen as a small white particle, and usually clings to the point of the needle. It should be transferred to a glass slide and covered with liquor potassae, or if it be desired to preserve it, the best medium for the purpose is glycerine jelly. The animal is in shape something like a tortoise, it has four pairs of legs, and is covered with a chitinous integument furnished with short spines and scattered hairs. The female is larger than the male and on the four anterior legs suckers will be noticed. The male has suckers on all the legs. Another method for more completely examining the mite and its burrow is to excise the burrow, parasite and all, by means of a sharp pair of scissors, although this is rather a difficult little operation. By means of a good lens, the passage can then be seen, often con- taining a row of oval eggs in chitinous cells, with the female acarus lying at one end. Acarus folliculorum. This is a small parasite occasionally found in the hair follicles and sebaceous glands. It cannot be said to be pathological as it is found in about ten per cent, of all healthy adults. The acarus may be demonstrated by squeezing out the contents of some of the follicles and placing them on AA2 356 MEDICAL MICROSCOPY. a glass slide with dilute liquor potassae. The animal will then be seen to possess a pointed abdomen, dis- tinct from the head and thorax which are continuous. It has four pairs of legs each terminating in three strong claws. EXAMINATION OF FOOD AND WATER. 357 CHAPTER XXIII. EXAMINATION OF FOOD AND WATER. THE examination of drinking water is almost entirely conducted by chemical analysis, although an investiga- tion of the sediment by the microscope, and especially by bacteriological processes, may yield important infor- mation. The adulterations of food are detected partly by chemical and partly by microscopical methods. We shall naturally consider here only the points that can be determined by the use of the microscope; these are so many that only the most important can be touched upon. If the student is desirous of going more deeply into this subject, he cannot do better than consult the work by Dr. A. H. Hassall, " Food : its Adulterations, and the Methods for their Detection." EXAMINATION OF FOOD. In looking down the list of substances commonly used for the adulteration of food, the various cereals occupy a prominent place, and an intimate knowledge of their structure and the appearances of their grains is abso- lutely necessary for the purposes under consideration, and we shall therefore begin with a description of these bodies, afterwards indicating the food-matters with which they may be found mixed. It may be here stated, however, that food is adul- terated with three main objects, firstly, to increase its MEDICAL MICROSCOPY. bulk and weight ; secondly, to improve its appearance and colour ; thirdly, to add to its taste, smell or other properties. Wheat. Several structures enter into the formation of the grain of wheat, as well as that of the other cereals. The seed is surrounded by membranes, technically known as the "testa"; the surface of the seed con- sists of angular cells, filled with granular oily matter, whilst its substance is made up of cells filled with starch corpuscles, and it is with these latter that we are most closely concerned, as the testa is in great part re- o o'6 FIG. 80. Wheat-starch grains. moved in the process of grinding and dressing the flour, and the same may be said of the cells forming the sur- face of the grain. The general structure of a " starch grain " is as fol- lows : Each granule exhibits a peculiar spot, termed the "hilum," round which are seen a set of circular lines which are for the most part concentric with it. A characteristic effect is produced by polarised light, each grain shows a dark cross, the point of intersection being at the hilum. The starch grains of wheat (in fact of all cereals) are best viewed with a quarter-inch lens. They will be EXAMINATION OF FOOD AND WATER. 359 seen to consist of round or oval particles of various sizes (fig. 80), some being very small and others of consider- able size, whilst only a few are of intermediate dimen- sions. The small grains are chiefly circular in shape, with a central hilum ; the larger granules form rounded or flattened discs, with thin edges, in these the hilum and concentric lines are barely visible, if at all. Occasionally some of the larger granules are twisted or turned up at the edges, and when seen sideways, present the appearance of a longitudinal furrow, giving a very good representation of a hilum ; but if the cover- glass be slightly moved so as cause the grains to turn over, their real nature is at once apparent. FIG. 81. Barley-starch grains. Portions of the outer envelopes of the wheat grain may be detected in the coarser and more branny flours. The testa consists of three layers of cells, two of which are disposed longitudinally and the third transversely to the axis of the seeds. The cells forming the surface of the seeds are large and angular. Barley. Like that of wheat, barley starch (fig. 81) consists of small and large grains, in fact the two starches are almost indistinguishable from one another. The larger grains are as a rule more distinctly ringed, while a greater proportion of them present the longitudinal furrow. If portions of the testa can be secured the 360 MEDICAL MICROSCOPY. difference from wheat is seen to be more marked. The testa of the barley grain consists of four layers of cells, smaller than those of wheat. There are three layers of longitudinal cells which are not beaded as in the case of wheat. The cells of the surface of the grain are not nearly so large as those of wheat, and there are three layers instead of one. Rye. In general form the starch grains resemble those of wheat, but a few differences may be noticed. The lesser grains are decidedly smaller than the corre- sponding grains of wheat, and many of the large rye gra- nules have a peculiar rayed hilum. With the polariscope FIG. 82. Oatmeal-starch grains. they exhibit a very strongly marked cross, thus distin- guishing them from barley-starch. The testa of rye also closely resembles that of wheat. Oatmeal (fig. 82). The starch grains are smaller than those of wheat and more uniform in size ; they are polygonal in shape and no hilum or concentric rings are visible. They tend to cohere together forming rounded masses, presenting a reticulated surface. With polar- ised light, unlike the starches of other cereals, no crosses are produced. The longitudinal cells of the testa are large and well-defined, and long and pointed hairs arise from some of them. The transverse cells form a single layer, and are almost square in shape. EXAMINATION OF FOOD AND WATER. 361 Maize (Indian corn flour). The starch grains (fig. 83) are polygonal in outline, and exhibit well-marked central depressions, and occasionally a radiated hilum. They are larger in size than those of the oat, and do not tend to cohere into masses. With the polariscope a well-defined cross is visible. FIG. 83. Maize-starch grains. The testa is made up of two membranes, the outer consisting of seven or eight layers of cells which are much longer than they are broad, and the margins of the outermost layer are beaded. The inner membrane consists of a single layer of cells. The cells containing the starch granules are very angular and are sub-divided by numerous septa. * *Q(jy o Q o o FIG. 84. Rice-starch grains. Rice (fig. 84). In shape the starch grains resemble those of the oat, being polygonal ; they are, however, much smaller. They exhibit well-marked central de- pressions and raised edges. The testa is rather complex. The outer surface is 362 M-EDICAL MICROSCOPY. thrown up into ridges, arranged both longitudinally and transversely. The ridges contain silica in the form of granules. The substance of the husk is composed of short fibres, arranged like the ridges. Beneath this fibrous membrane is a thin layer of angular cells, which are longer than they are broad. Beans (fig. 85).* Bean starch cells are more or less oval and somewhat flattened ; they exhibit a longitu- dinal cleft in the centre of the grain, crossed by trans- verse fissures. Peas (fig. 86). Pea starch grains are smaller than FIG. 85. Bean-starch cells. FIG. 86. Pea-starch grains. those of the bean and less flattened, but otherwise similar in shape. The longitudinal cleft which runs the whole length of the grain does not show the trans- verse fissures. Sago (fig. 87). The starch is obtained from the pith of several kinds of palms. The starch grains are elongated in form and of considerable size ; they are larger at one end than at the other. The large end is rounded, and the other truncated. The hilum is usually circular, but sometimes cracked, when it appears as a * Figures 80-94 are borrowed with the kind permission of the author from Parkes' " Hygiene and Public Health." EXAMINATION OF FOOD AND WATER. 3 63 slit or star. In some of the granules a few concentric rings may be seen. Tapioca (fig. 88). The starch is procured from the root of the plant. The granules are small in size, some- times united into groups of three or four. They are roughly circular in form, although owing to mutual pressure they may become truncated at one extremity as with the sago grains. The hilum is round and well- marked. Arrowroot (fig. 89). There are several kinds of arrowroot. The starch grains of each variety differ slightly from each other, but it would be of no advan- ^ <^y (8) .S^ /""*""> \^^*