A 82S Broadway Xi \ ,oix>«» REESE LIBRARY UNIVERSITY OF CALIFORNIA. Received '.._ (2 EMBRYONIC TISSUE, . . • MUSCULAR TISSUE. 62 NON-STRIATED MUSCLE, . STRIATED MUSCLE, . .... 6j> CARDIAC MUSCLE, . ....•• BLOOD-VESSELS. /»/» ARTERIES, . . .... CAPILLARIES, . ... 67 VEINS, . PART THIRD. ORGANS. THE SKIN. LAYERS OR STRATA, HAIRS. . •'.'•'• 2 SUDORIFEROUS GLANDS, ..... SEBACEOUS GLANDS, ...••• PRACTICAL DEMONSTRATION, THE TEETH. THE PULP, DENTINE, .... • ENAMEL, CRUSTA PETROSA, PRACTICAL DEMONSTRATION, THE STOMACH AND INTESTINES. GENERAL HISTOLOGY, . THE STOMACH, . j*J PRACTICAL DEMONSTRATION, SMALL INTESTINE, PRACTICAL DEMONSTRATION, THE LUNG. BRONCHIAL TUBES, . . • COATS, . . PRACTICAL DEMONSTRATION, .... yt PULMONARY BLOOD-VESSELS, . . • • • • X CONTENTS. PAGE THE PLEURA, : . . . . . . . 100 PULMONARY ALVEOLI, . . . . . . .101 PRACTICAL DEMONSTRATION, LUNG OP PIG, . . . 103 PRACTICAL DEMONSTRATION, HUMAN LUNG, . . . .105 THE LIVER. •GENERAL SCHEME, . . ... . . 106 THE PORTAL CANALS, . . . . . . .108 THE LOBULAR PARENCHYMA, . . . . . 109 PRACTICAL DEMONSTRATION, LIVER OF PIG, . . . .110 PRACTICAL DEMONSTRATION, HUMAN LIVER, • « . . 113 PRACTICAL DEMONSTRATION, THE PORTAL CANALS, . > .. .114 PRACTICAL DEMONSTRATION, THE LOBULAR PARENCHYMA, . 116 PRACTICAL DEMONSTRATION, ORIGIN OF BILE DUCTS, . . .118 THE KIDNEY. •GENERAL DESCRIPTION, . . . . . . . 120 THE TUBULI URINIFERI, , . . . . . .122 BLOOD-VESSELS, ' . . . . . • . . 124 PRACTICAL DEMONSTRATION, with Low Power, . . . .128 PRACTICAL DEMONSTRATION, THE CORTICAL PORTION, . . 130 PRACTICAL DEMONSTRATION, THE MEDULLARY PORTION, . .133 THE GENITOURINARY TRACT. URETER, BLADDER, UTERUS, VAGINA, ETC. •GENERAL HISTOLOGY, . . ... . . . 185 PRACTICAL DEMONSTRATION, UTERUS AND VAGINA, . . .136 PRACTICAL DEMONSTRATION, PELVIS OF THE KIDNEY, . . 140 PRACTICAL DEMONSTRATION, THE URINARY BLADDER, . . 141 PRACTICAL DEMONSTRATION, URINARY DEPOSITS, ... 143 THE OVARY. PRACTICAL DEMONSTRATION, ADULT HUMAN OVARY, . . 144 DEVELOPMENT OF THE OVUM. PRACTICAL DEMONSTRATION, FOETAL OVARY, ... 147 THE SUPRA-RENAL CAPSULE. . . .150 PRACTICAL DEMONSTRATION, . . . . . . 151 SALIVARY GLANDS. PANCREAS. PLAN OF GLAND STRUCTURE. TYPICAL GLANDULAR HISTOLOGY, ..... 153 TCBULAR GLANDS, . . . . . . . .153 •COILED TUBULAR GLANDS, . . . . . . 154 BRANCHED TUBULAR GLANDS, . . . . . .154 ACINOUS GLANDS, ... .... 155 THE PAROTID GLAND, . ... 157 CONTENTS. XI PAGE SUB MAXILLARY GLAND. . . . . . 158 PRACTICAL DEMONSTRATION, PAROTID GLAND, SUBMAXILLARY GLAND, PANCREAS, ........ 157 THE LYMPHATIC SYSTEM. GENERAL DESCRIPTION, ....... 161 LYMPH CHANNELS, ........ 162 PRACTICAL DEMONSTRATION, LYMPH CHANNELS OF CENTRAL TENDON OF THE DIAPHRAGM, . . . . . . 163 LYMPHATIC NODES OR GLANDS, x . . . . . .167 PRACTICAL DEMONSTRATION, MESENTERIC LYMPH NODE, . . 169 THE SPLEEN. SCHEME OF ORGAN, . . . . . . .173 PRACTICAL DEMONSTRATION, . >. / . . . . 174 THE THYMUS BODY. •GENERAL DESCRIPTION, . . . . . . .177 PRACTICAL DEMONSTRATION, . . . . . . • 178 THE NERVOUS SYSTEM. STRUCTURAL ELEMENTS, . . . . . . .180 NERVE FIBRES, ........ 180 NERVE CELLS, ..... . . 181 •CONNECTIVE TISSUE OF NERVE TRUNKS, .... 182 NEUROGLIA, ......... 183 THE SPINAL CORD. •GENERAL DESCRIPTION, ....... 186 PRACTICAL DEMONSTRATION, CERVICAL SPINAL CORD, . . .187 THE BRAIN. MEMBRANES, . . . . . . . . 191 PRACTICAL DEMONSTRATION, CEREBRUM, . . . . .192 PRACTICAL DEMONSTRATION, CEREBELLUM, . . . 195 MISCELLANEOUS FORMULAE, ETC. DAMMAR MOUNTING VARNISH, ...... 197 XYLOL BALSAM, . . . . . . . . 197 VARNISHES AND CEMENTS FOR RINGING MOUNTS ; DAMMAR VARNISH ; ZINC CEMENT ; ANILINE COLORS ; OIL COLORS ; BLACK VARNISH ; SHELLAC VARNISH, ....... 197 PRESERVATIVE FLUID, ....... 199 NORMAL SALT SOLUTION, . . . . . . .199 RAZOR-STROP PASTE, . . . . . . 199 Xll CONTENTS. PAGE SILVER STAINING SOLUTION, ....... 200 OSMIC ACID SOLUTION, ....... 200 WEIGERT'S STAINING FOR MEDULLATED NERVE TISSUE, . . .201 BAYBERRY INFILTRATING METHOD, ..... 201 KARYOKINESIS, ......... 202 FIXING AND STAINING CORPUSCULAR ELEMENTS OF BLOOD, . . 20S LIST OF ILLUSTRATIONS. FIG. PAGE 1. LABORATORY MICROSCOPE, . . . . . .2 2. COURSE OF LIGHT THROUGH THE MICROSCOPE, ... 3 3. FREE-HAND SECTION CUTTING, . . . . . .10 4. STIRLING MICROTOME, . . . . . . .12 5. METHOD OP IMBEDDING WITH PITH, TURNIP, ETC., . . .13 6. SECTION CUTTING WITH STIRLING MICROTOME, ... 14 7. THE SCHRAUER MICROTOME, . . . . . .14 8. THE AUTHOR'S LABORATORY MICROTOME, .... 15 9. METHOD OF HONING RAZOR, . . . . . .17 10. TURNING THE RAZOR ON THE HONE, .... 17 11. PARAFFIN SOLDERING WIRE, . . . . . .18 12. CEMENTING HARDENED TISSUE TO CORK, .... 19 18. NEEDLE FOR HANDLING SECTIONS, COVERS, ETC., . . .20 14. DIAGRAM ILLUSTRATING STEPS IN STAINING WITH H^EMA., . 25 15. DIAGRAM ILLUSTRATING STEPS IN STAINING WITH H^EMA. AND Eosm, . . . . . . . . .26 16. DIAGRAM ILLUSTRATING STEPS IN STAINING WITH BORAX-CAR- MINE, ......... 26 17. SECTION LIFTER, . . . . . . . .32 18. APPEARANCE OF BALSAM MOUNTED SPECIMEN, ... 33 19. MODE OF HANDLING COVER-GLASS, . . . . .32 20. DIAGRAM SHOWING EFFECT OF OIL AND AIR GLOBULES, . 36 21. EXTRANEOUS SUBSTANCES— HAIRS, ETC., . . . . .37 22. EXTRANEOUS SUBSTANCES— STARCH, ETC., . . 38 23. ELEMENTS OF A TYPICAL CELL . . . . . .39 24. STRUCTURE OF A CELL-NUCLEUS, . . . . .40 25. SQUAMOUS CELLS FROM SALIVA, . . . . . .41 26. PAVEMENT EPITHELIUM, ...... 42 27. FROG'S MESENTERY— SILVER STAINING, . . . . .43 28. COLUMNAR CELLS FROM INTESTINE, . . ... 45 29. CILIATED CELLS FROM BRONCHUS, . . . . .46 30. DIAGRAM SHOWING ORGANS OF THE OYSTER, ... 46 31. CORPUSCULAR ELEMENTS OF HUMAN BLOOD, . . . .47 32. DIAGRAM. HUMAN RED-BLOOD CORPUSCLE IN PROFILE, . . 48 33. BLOOD PLAQUES . . . . . . . .49 34. GLANDULAR CELLS FROM LIVER, ..... 50 XIV LIST OF ILLUSTRATIONS. FIG. PAGE; 35. FlBRILLATED CONNECTIVE TISSUE, . . . . .52 36. YELLOW ELASTIC TISSUE, ...... 53 37. TRANSVERSE SECTION OF LIGAMENTUM NUCH^, . . . 53 38. CELLS CONTAINING FAT, ...... 55 39. ADIPOSE TISSUE FROM OMENTUM, . . ... .55 40. HYALINE CARTILAGE FROM BRONCHUS, 56- 41. FIBRO-CARTILAGE FROM INTERVERTEBRAL Disc, . . .57 42. ELASTIC CARTILAGE FROM PINNA OF Ox, . . . .57 43. BONE — SHOWING LAMINATED STRUCTURES, . . • . .58 44. BONE — SHOWING HAVERSIAN SYSTEMS, 59- 45. CONTENTS OF HAVERSIAN CANALS, . . . . .60 46. CONTENTS OF BONE LACUNA, ...... 60 47. BLADDER OF FROG — SHOWING NON-STRIATED MUSCLE, . . 62. 48. DIAGRAM— ILLUSTRATING STRUCTURE OF STRIATED MUSCULAR FIBRE, . . . . . . . . .63 49. STRIATED MUSCULAR FIBRE FROM TONGUE, . . . .64 50. CARDIAC MUSCULAR FIBRE, . . . . . .65 51. ARTERY IN TRANSVERSE SECTION, . . . . .66- 52. BLOOD CAPILLARIES, . . . . . . 67 53. LAYERS OF THE EPIDERMIS, . . . . . .69' 54. STRUCTURE OF THE DERMA, . . . . . .70' 55. HAIR IN TRANSVERSE SECTION, . . . . . .71 56. HAIR FOLLICLE, . . , . . . . .72 57. SUDORIFEROUS GLAND, , . . . . .73 58. SEBACEOUS GLAND, ....... 73 59. SKIN IN VERTICAL SECTION, . . . . . .75 60. HUMAN CANINE TOOTH IN VERTICAL SECTION, . . .79 61. FANG OF TOOTH IN TRANSVERSE SECTION, . . . .82 62. STOMACH. DIAGRAM, ....... 83 63. CARDIAC GASTRIC GLANDS, . . . . . .85 64. PYLORIC GASTRIC GLANDS, . . .. . . . 86 65. STOMACH OF DOG, . .. . . , . . .87 66. DIAGRAM ILLUSTRATING INTESTINAL SECRETION, . . . 89» 67. DIAGRAM OF INTESTINAL ABSORPTION, . . . . .90 68. SMALL INTESTINE WITH PEYER'S LYMPHATICS, . . .92 69. BRONCHIAL TUBE ARRANGEMENT, . . . . .94 70. BRONCHIAL TUBE — SMALL, ...... 96 71. BRONCHIAL TUBE— MEDIUM, . . . . . .98 72. PULMONARY LOBULE — PERSPECTIVE, . . . . .101 73. PULMONARY LOBULE— LONGITUDINAL SECTION, . . .101 74. PULMONARY ALVEOLUS— CAPILLARIES FILLED, . . . 102 75. LUNG OF PIG, ........ 103 76. PULMONARY ALVEOLUS SHOWING LINING, .... 104 77. LIVER. DIAGRAM ILLUSTRATING PLAN OF STRUCTURE, . . 107 78. GLANDULAR CELLS IN CONNECTION WITH BLOOD-VESSELS AND DUCTS, ........ 109- 79. LIVER OF PIG, . . . . . . . .111 80. HUMAN LIVER — Low POWER, . . . . . .114 81. PORTAL CANAL, . . 115. LIST OF ILLUSTRATIONS. XV FIG. PAGE 82. HEPATIC CELLS— DETACHED, . . . . .117 83. HEPATIC LOBULE IN TRANSVERSE SECTION, . . . .us 84. BILE CAPILLARIES — ORIGIN OF BILE DUCT, . . . 119 85. KIDNEY— DIAGRAM ILLUSTRATING PLAN OF STRUCTURE, . . 121 86. KIDNEY TUBULES — ISOLATED ...... 123 87. BLOOD-VESSEL ARRANGEMENT IN KIDNEY, . . . .135 88. KIDNEY— Low POWER, ....... 128 89. KIDNEY — CORTEX IN VERTICAL SECTION, . . . .130 90. KIDNEY— MEDULLA IN LONGITUDINAL SECTION, . . .132 01. KIDNEY— MEDULLA IN TRANSVERSE SECTION, . . . .133 92. UTERUS WITH VAGINAL eul-de-sac, ..... 136 93. EXTERNAL UTERINE Os, . . . . . . .13$ 94. VAGINAL EPITHELIUM, . . . . . . . 139 95. EPITHELIUM OF URETER, . . . . . . .140 96. EPITHELIUM OF URINARY BLADDER, ..... 142 97. EPITHELIUM FROM URINARY DEPOSIT, ..... 143 98. OVARY — ADULT, . . . . ... . . 145 99. OVARY— FCETAL, . . . . . . .148 100. SUPRARENAL CAPSULE — Low POWER, . . . .151 101. SUPRARENAL CAPSULE — HIGH POWER, ..... 152 102. SIMPLE TUBULAR GLAND, ...... 153 103. COILED TUBULAR GLAND, . . . . . . .154 104. BRANCHED TUBULAR GLAND, ...... 155 105. DILATED TUBULAR GLAND, ...... 156 106. PAROTID GLAND, . . ' . . . . 156 107. SUBMAXILLARY GLAND, ....... 157 108. PANCREAS, . . . . .' . .158 109. PERIVASCULAR LYMPH SPACES, ...... 162 110. LYMPHATICS OF CENTRAL TENDON OF THE DIAPHRAGM— Low POWER, ........ 165 111. LYMPHATICS OF CENTRAL TENDON OF THE DIAPHRAGM— HIGH POWER, . . . . . . . . .166 112. LYMPH NODE— DIAGRAMMATIC, . . 168 113. MESENTERIC LYMPH NODE — Low POWER, . . . 170 114. MESENTERIC LYMPH NODE — HIGH POWER, . . . .171 115. BLOOD-VESSEL ARRANGEMENT IN THE SPLEEN, . . . 173 116. SPLEEN, ......... 175 117. THYMUS BODY, . . . . . . . .178 118. NERVE FIBRE, . . . . . . .180 119. CONNECTIVE TISSUE OF NERVE TRUNK, ..... 182 120. CONNECTIVE TISSUE OF BRAIN, ..... 183 121. SPINAL CORD— DIAGRAM, . . . . . . .185 122. CERVICAL SPINAL CORD— TRANSVERSE SECTION, . . . 188 123. ANTERIOR CORNU OF GRAY MATTER— CERVICAL SPINAL CORD, . 189 124. CEREBRUM, . . . . . . . .193 125. CEREBELLUM— Low POWER, ...... 194 126. CEREBELLUM — HIGH POWER, ...... 195 PRACTICAL MICROSCOPY, PAKT FIRST. TECHNOLOGY. THE LABORATORY MICROSCOPE. THE histologist should be provided with a microscope, in which the principal features of the laboratory instrument, Fig. 1, are embraced. The body A, which carries the optical parts, is made of two pieces of brass tubing, one sliding within the other and providing for alter- ations in length. The objectives, C, D, are attached to the body by means of the angular carrier E. The carrier is so pivoted that either objective may be turned into the optical axis, at will. The eye-piece, F, slips into the upper part of the body, with but little friction, so that it may be quickly and easily removed. The coarse or quick adjustment for focussing consists of a rack Gr, which is attached to the body, and into this gears a small (concealed) pinion turned by the milled-head H. The fine steel screw 1, by means of which the more delicate adjust- ments for focussing are accomplished, terminates below in a hardened point, which impinges upon one end of a lever (concealed in the arm) the fulcrum of the same being indicated at the point J. The oppo- site end of the lever is inserted in a notch in the split arm K. By turning the milled-head L, the lever is moved, and the optical body raised or lowered with extreme delicacy. The sfage, upon which objects are placed for examination, is per- forated at M, and a rotating disc — not indicated in the drawing — 1 PRACTICAL MICROSCOPY. enables one to alter the size of the opening at will. Below the stage an arm may be seen which carries a fork supporting the mirror N. The whole is supported on a short, stout pillar rising from the foot 0. FIG. 1. — THE LABORATORY MICROSCOPE. This instrument was designed and constructed for the laboratories of the New York University Medical College. It is strongly built; the mechanism is simple; and the height— 10^ inches— not too great for use in the vertical position. LENSES OF THE MICROSCOPE. Fig. 2 shows the arrangement of lenses, including a high-power objective of the Wenhain construction. The objective A is provided with one simple and two compound lenses. The lens B, nearest the object, and the one upon which the magnifying power mainly depends, is an hemisphere of crown glass. Such a figured glass possesses both chromatic and spherical aberration in high degree. These faults are corrected by the compound flint and crown lenses, C and D, placed above the hemispherical glass. The eye-piece consists of two crown glass, plano-convex lenses, with LENSES OF THE MICROSCOPE. their plane surface upward. The lower, E, is known as the field-lens, the upper, F, as the eye-lens. Eye-pieces add very materially to the magnifying power of the instrument, and are constructed of various strengths depending upon the curvature of the lenses. They ar& FIG. 2.— DIAGRAM SHOWING THE RELATION OF THE OBJECTIVE TO THE EYE-PIECE. named according to power, A, B, C, etc. The medium, B, is more commonly employed. The microscope previously described stands, with the draw-tube in place, about ten and one-half inches high; and represents the in- 4 PRACTICAL MICROSCOPY. struments used in the New York University Laboratory of Biology and Pathology. They were constructed by Schrauer, of this city, costing about fifty-five dollars each. They are provided with a single eye-piece, and Hartnack objectives Nos. 2 and 7, giving from. 30 to 400 diameters. Such an instrument is well adapted to the work of normal and pathological histology, though a condenser * should be attached below the stage and in the optical axis for high-power work with immersion lenses, and especially for bacteriological research. The stand is a rigid one, and if the height of the table upon which it is placed, and the chair of the observer be in a proper relation, no discomfort need be experienced in using the microscope in the verti- cal position. ADJUSTMENT OF THE MICROSCOPE. The microscope should be placed in front of the observer, on a table of such height that, when seated, he may, by slightly inclining the head, and without bending the body, bring the eye easily over the eye-piece. The slightest straining of the body or neck should be avoided. The light should always be taken from the side, and it matters little which side. Clouds or clear sky serve as the best source of light for our present work. Always avoid direct sunlight. If artificial illumination be employed — though it is not advised for prolonged investigation — a small coal-oil flame may be tempered by blue glass. ADJUSTMENT FOR ILLUMINATION". It will be observed that there are two mirrors in the circular frame below the stage — one plane and the other concave. The latter will be employed almost exclusively in the work of this volume ; and its curvature is such that parallel rays, impinging upon its surface, are focussed about two inches from the mirror. It will also be noticed * A non-achromatic condenser, after the formula of Abbe, of Jena, is in quite general use in this country. Its value has been very markedly increased here by the addition of a rack-and-pinion motion. In use for high power work with tissues, it is first placed so that the plane surface of the upper lens is in con- tact with the under surface of the glass slip holding the object to be exam- ined. Light is then reflected into the condenser as usual, excepting that the plane mirror is employed. This will give a strong illumination, but too diffuse for tissues. The light is then modified by diaphragms, or by racking the con- denser downward until the best effect is secured. For bacterial search the strong illumination is employed. This gives prominence to the stained mi- crobes, as the other elements in the field are lost in the excess of diffuse light. ADJUSTMENT FOE FOCUS. 5 that the bar, carrying the mirror-fork, may be made to swing the mirror from side to side. The work which we are about to under- take is of such a character as to require the avoidance of oblique il- lumination. We must, therefore, keep our mirror-bar strictly in the vertical position. If — the mirror-bar being vertical — a line be drawn from the centre of the face of the mirror, through the opening (dia- phragm) in the stage, passing on through the objective, and so con- tinued upward through the body and the eye-piece, such a line would pass through the optical axis. The centre of the face of the mirror must be in this axis. If, then, having gotten the mirror-bar properly fixed once for all, the light from the adjacent right or left hand window impinges upon the concave surface of the mirror, and the latter be properly inclined, the rays will pass through the diaphragm in the stage, and become focussed a little above the same. The light rays will afterward diverge, enter the objective, and finally reach the eye of the observer. The field of view (as the area seen in the microscope is termed) we will suppose to have been properly illuminated — and by this I mean that it presents as a clear, evenly lighted area. Turn all the factors spoken of out of adjustment, and proceed to readjust. Observe that, if the mirror be turned — not swung — slightly out of proper position, one side of the field will appear dim or cloudy : this must be corrected, and the student must practise until this adjustment becomes easy of accomplishment. Then proceed to the ADJUSTMENT FOE FOCUS. Observe that the largest opening in the stage diaphragm is in the optical axis. Swing the low-power objective into use, and rack the tube up or down until it is about one inch from the stage. Place a mounted object upon the stage (a stained section of some organ — say kidney — will be preferable). Examine the field through the eye-piece, and it will be found obscured by the stained object, and perhaps a dim notion of figure may be made out. Eack the body down carefully, watching the effect. The image becomes more and more distinct until, at a certain point, the best effect is secured. The object is in focus. Note carefully the distance between the object and the objective (with the Hartnack No. 2 this will be about seven-eighths of an inch), and hereafter you will be able to focus more quickly. Having observed the details of structure as shown with the inch objective, swing the high power into use. Rack the tube down, until the objective is about one-eighth of an inch from the glass covering 6 PRACTICAL MICROSCOPY. the object. The field is much obscured. Watching the effect through the eye-piece, rack the tube down with great care until the image appears sharp. Note the distance with this objective as before with the low power, probably about one thirty-second of an inch. Then endeavor, by slight alterations in the inclination of the mirror, to increase the illumination. Turn the diaphragm so that the light passes through a small opening, and note the improvement in defini- tion. The rule is : The higher the poiver, the smaller the diaphragm. You have doubtless observed, before this, that you cannot control the focussing as easily as when the low power was in use. Slight movements of the rack-work produce marked changes in definition ; and it is difficult, with the coarse adjustment alone, to make as slight movements as you may desire. Recourse must be had to the fine adjustment. Place the tip of the forefinger (either) upon the milled-head of the fine focussing-screw, and the ball of the thumb against its side, so that the hand is in an easy position. By a little lateral pressure the milled-head may be turned slightly either way. Note the effect on the image. You thus have the focussing under the most perfect con- trol. Remember that the fine adjustment is only necessary with high powers, and then only after the image has been found with the coarse adjustment. METHOD IN OBSERVATION. The study of objects under the microscope should be conducted with order or method. The body being in the position before advised, so that the sitting may be prolonged without fatigue, let one hand be occupied in the maintenance of the focal adjustment. It will be found, however flat an object may seem to the unaided eye, that as it is moved so as to present different areas for examination (and with the higher powers only a small area can be seen at once) constant manipula- tion with the fine adjustment will be required. It will also be found that even the various parts of a simple histological element — like a cell — cannot be seen sharply with a single focal adjustment. The forefinger and thumb of one hand must be kept constantly on the milled-head of the fine focussing screw. Supposing the light to be on our right, we devote the right hand to the focussing. The left hand will be engaged with the glass slip upon which the object has been mounted. The forearm resting upon the table, let the thumb and forefinger rest on the left upper side of the stage, just MAGNIFYING POWER AND MEASUREMENT OF OBJECTS. 7 touching the edges of the glass slip. The slightest pressure will then enable you to move the slip smoothly, steadily, and delicately. Proceed' to examine the object with method. Suppose a section of some tissue to be under examination — say one-fourth of an inch square. With the high power you will be able to see only a small fraction of the area at once. Commence at one corner to observe ; and with the left hand move the object slowly in successive parallel lines, preserving the focus with the right hand, until the whole area of the section has been traversed. Practice will soon establish perfect co-ordination of the movements involved, and will result in the ability to work with ease, celerity, and profit. CONSERVATION OF THE EYESIGHT. I The beginner should not become accustomed to the use of one eye alone, or of closing either, in microscopical work. It will require but little practice to use the eyes alternately, and the retinal image of the unemployed eye will soon be ignored and unnoticed. MAGNIFYING POWER AND MEASUREMENT OF OBJECTS. The microscope is not, as the beginner usually supposes, to be valued according to its power of magnification, but rather according to the clearness and sharpness of the image afforded. Magnifying power is generally expressed in diameters. A certain area is by the instrument made to appear, say, ten times as large as it appears to the naked eye. This object has, then, its apparent area increased one hundred times ; but reference is made in describing such phenomena only to amplification in a single direction. The dia- meter has been increased ten times and would be expressed by pre- fixing the sign of multiplication, e. g., X 10. A convenient unit of approximate measurement for the histologist is the apparent size of a human red blood-corpuscle with a given objec- tive. Thirty-two hundred corpuscles, placed side by side, would measure one inch ; or, we say, the diameter of a single corpuscle is the thirty-two hundredth of an inch. After considerable practice, you will become accustomed to the apparent size of this object with a cer- tain objective and eye-piece. This will aid in an approximate mea- surement of objects by comparison, and will further give the magni- fying power of the microscope. If a corpuscle appears magnified to one inch in diameter, it is evident that the instrument magnifies thirty-two hundred times. Should the diameter appear one-quarter of an inch, the power is eight hundred; one-eighth of an inch, four hun- 8 PRACTICAL MICBOSCOPY. dred, etc. The instrument which I have heretofore described, with the high power in use and the tube withdrawn, will present the corpuscle as averaging very nearly one-eighth of an inch in diameter — x 400. Whil ethis gives a gross idea of amplification, the method will often prove inaccurate because of individual errors in the estimation of proportions. Use of the Stage- Micrometer. — From a dealer^in optical goods pur- chase a Rogers'' * glass stage-micrometer, ruled in hundredths, thou- santdhs, and five-thousandths of an inch. Also procure from the dealer in drawing instruments a two-inch boxwood rule divided deci- mally to fiftieths. Place the micrometer on the stage of the microscope and focus the lines. Then place the rule also on the stage, but Justin front of and parallel with the micrometer. By a little practice, using both eyes, the two rulings may be seen simultaneously, and by adjusting the position of the rule, the lines may be made to appear superposed. Let us suppose that, with a given eye-piece and objective, the thousandth divisions on the micrometer correspond exactly with one of the tenths of the rule. Keeping this in mind, remove the micro- meter scale and substitute an object, say a blood slide. Let us again suppose that the image of a given red corpuscle appears to cover three of the one-tenth inch rulings, the latter scale having been left in position. It is evident that, as we found the value of one of the rule tenths to be, by the micrometer, the one-thousandth of an inch, the globule measures one three-thousandth of an inch in diameter. The value of the rule divisions must be determined for each objec- tive ; and a memorandum will then provide the means of quickly ob- taining a very close approximation of the size of objects as viewed in the microscope, and at the same time indicate the degree of ampli- fication of the instrument itself. SKETCHING FROM THE MICEOSCOPE. Let me most emphatically urge the practice of sketching in con- nection with microscopy. " I am no artist," or " I have no skill in drawing," is often the reply to my advice in this matter. I then sug- gest that no special skill is needed to begin with, only patience and a dogged determination to succeed. The pictures in the microscopic field have no perspective, and may be reproduced in outline merely. * The micrometer rulings of Professor Rogers, of Cambridge University, are without doubt of surpassing excellence. They are the result of many years of unwearying experimentation and are recognized standards through- out the scientific world. SECTION CUTTING. Begin with simple tissues, reserving intricate detail until a short period of practice gives the technique needed. I do not recommend the camera lucida, as my experience strongly impresses me with this as a fact, that he who cannot sketch without a camera will never sketch with one. Pencil drawings may be very effectively colored with our staining fluids, diluted if necessary. PREPARATIONS OF TISSUES EOR MICROSCOPICAL PURPOSES. TISSUES ARE STUDIED BY TRANSMITTED LIGHT. The microscopical study of both normal and pathological tissues is invariably conducted by the aid of transmitted light. Tissues, if not naturally of sufficient delicacy to transmit light, must in some way be made translucent. Delicate tissues like omenta, desquamating epithelia, fluids con- taining morphological elements, certain fibres, etc., are sufficiently diaphanous, and require no preparation. Such objects are simply placed upon the glass slip, a drop of som'e liquid added, and, when protected by a thin covering glass, are ready for the stage of the microscope. PREPARATION BY TEASING. The elements of structures mainly fibrous, e. g., muscle, nerve, ligament, etc., are well studied after a process of separation, by means of needles, known as teasing. A minute fragment of the organ or part having been isolated by the knife or scissors is placed upon a glass slip, and a drop of some fluid which will not alter the tissue added. Stout sewing needles, stuck in slender wood handles, are commonly employed in the teasing process. The separation of tissues is frequently facilitated by means of dissociating fluids which remove the cement substance. fi SECTION CUTTING. After having become familiar with the various elementary struc- tures of animal tissues, we proceed to the study of their relation to organs. As the teasing process is not available with such complicated structures as lung, liver, kidney, brain, etc., we resort to methods of slicing, i. e., section cutting. Sections must be made of extreme tenuity, in order that the natu- 10 PRACTICAL MICROSCOPY. rally opaque structures may be illuminated by transmitted light. This becomes an easy matter with such tissues as cartilage; but some, like bone, are much too hard to admit of cutting, and others are as much too soft; so that while certain tissues must be softened, the majority must be hardened. Fortunately, both of these conditions may be secured without in any way altering the appearance or rela- tions of the structures. Hardening processes, from necessity, become a prominent feature in histological work; but I propose here to indi- cate some of the more useful methods of section-cutting, reserving the hardening processes for another place. FREE-HAND SECTION CUTTING. My students, when ready for this work, are provided with some tissue which has been previously hardened. We will take, for ex- ample, a piece of liver which has been rendered sufficiently firm for our work by immersion in alcohol, and proceed to direct the steps in obtaining suitable sections by the simple free-hand method. FIG. 3.— FREE-HAND SECTION CUTTING. I wish to strongly emphasize the importance of this mode of cut- ting. A moderate amount of practice will render the microscopist independent of all appliances, save those of the most simple character and which are always obtainable. An ordinary razor, with keen edge, and a shallow dish, preferably a saucer, partly filled with alcohol are required. The razor best FKEE-HAND SECTION CUTTING. ll adapted to the work is concave on one side (the upper side as seen in Fig. 3) and nearly flat on the other, although this is largely a mat- ter of personal preference. Fig. 3 indicates the proper position of the hands in commencing the cut. I have made the sketch from a photograph of my esteemed colleague, Dr. Wesley M. Carpenter. The student should be seated at a table of such height as to afford a convenient rest for the forearms. A small piece of tissue is held between the thumb and forefinger of the left hand, so that it projects slightly above both. (In the cut, a cube of tissue, too small to handle in this way, has been cemented to a cork with paraffin in the manner hereafter described, and the cork held as just mentioned.) The hand carrying the tissue is held over the saucer of alcohol. The razor, held lightly in the right hand, as seen in the figure, is, previous to making every cut, dipped flatwise into the alcohol, so as to wet it thoroughly; and is then lifted horizontally, carrying several drops, perhaps half a drachm of the fluid on the concave, upper surface. The alcohol serves to prevent the section from adhering to the knife, and to moisten the tissue. If allowed to become dry, the latter would be ruined by alterations of structure. Now as to the manner of moving the knife. Resting the under surface upon the forefinger for steadiness, bring the edge of the blade nearest the heel to the margin of the tissue furthest from you. Then, entering the edge just below the upper surface of the tissue, with a light but steady hold draw the knife toward the right, at the same time advancing the edge toward the body. This passes the knife through the tissue diagonally, and leaves the upper surface of the latter perfectly flat or level. Remove the piece which has been cut and repeat the operation. Do not attempt to cut large or very thin sections at first. A minute fragment, if thin, is valuable. As the razor is drawn through the tissue, the section floats in the alcohol; depress the point of the knife and the section will slide into the saucer of spirit, and thus prevent its injury. If it does not leave the knife readily, brush it along with a camel's hair pencil which has been well wetted with the alcohol. Proceed in the above manner until the tissue is exhausted, when you will have a great number of sections, large and small, thick and thin. Selecting the thinnest, lift them c tref ully with a needle, one at a time, into a small, wide-mouthed bottle of alcohol ; cork and label for future use. When the work is finished, and before the spirit has evaporated from your fingers— it is impossible to avoid wetting the skin more or 12 PRACTICAL MICROSCOPY. less — wash them thoroughly, and wipe dry. This saves the roughen- ing of the hands which is apt to result when alcohol has been allowed to dry upon them repeatedly. SECTION-CUTTING WITH THE STIRLING MICROTOME. Of the numerous mechanical aids to section-cutting, I shall men- tion only two or three. One of the earlier and better known instru- ments is seen in Fig. 4. The Stirling microtome consists essentially of a short brass tube, into which the tissue is fixed, either by pressure FIG. 4.— STIRLING'S MICROTOME. or by imbedding in wax. A screw enters below which, acting on a plug, raises the contents of the tube. As the material to be cut is raised from time to time by the screw, it appears above the plate which surrounds the top of the tube. This plate steadies and guides the razor ; and it is evident that more uniform sections may be cut with this little apparatus than would be possible with nothing to sup- port the knife, or to regulate thickness, beyond the unaided skill of the operator. Much depends upon the manner in which the material is fixed in the tube or well of the microtome. If the tissue be of a solid character, like liver, kidney, spleen, many tumors, etc., it may be SECTION CUTTING WITH THE STIRLING MICROTOME. 13 surrounded with some carefully fitted pieces of elder-pith,* carrot, etc., and the whole pressed evenly and quite firmly into the well. A small piece of tissue which, by cutting, can be made somewhat cubical in shape, may be surrounded by slabs of pith, carrot, or turnip, shaped as in Fig. 5. Indeed, the fragments of imbedding material can be shaped so as to fit tissue of almost any form. Before the whole is pressed into the well of the microtome, the bottom, against which the brass plug fits, should be cut off square. The wax method of imbedding is employed with tissues such as brain, lung, soft tumors, etc., which might be injured by the previous treatment. To three parts of paraffin wax (a paraffin candle answers perfectly) add one part of vaseline, and heat until thoroughly mixed. The microtome having been previously warmed — standing upright, is filled with the imbedding mixture. The piece of tissue is then FIG. 5.— MANNER OF CUTTING AND ARRANGING PIECES OP PITH, TURNIP, ETC., FOR SUPPORTING HARDENED TISSUE IN THE WELL OF A MICROTOME. carefully wiped dry with the blotting paper and, just as the imbedding begins to congeal around the edges, is pressed below the surface with a needle and held until the cool mixture fixes it in position. The whole is now allowed to become thoroughly cold. By turning the screw, the plug of wax is raised ; and it must be gradually cut away, by sliding the knife across the plate, until the upper part of the tissue appears. Before commencing to cut sections — however the tissue may have been imbedded — provide yourself with a saucer of alcohol and a camel's-hair pencil. Having wetted the knife, turn the screw so that the tissue, with its imbedding, appears slightly above the plate of the microtome ; and then, resting the blade of the razor on the plate *The pith from the young shoots of Ailantus glandulosus (improperly called " Alanthus "), gathered in early autumn, is the best material for this method of imbedding with which I am acquainted. The wood is easily cut from the pith, and the latter is very large and firm. PRACTICAL MICROSCOPF. (vide Fig. 6), make the cut precisely as in free-hand cutting. The- section is then brushed off into the saucer, the screw turned up slightly, the razor wetted, and a second cut made. These steps are repeated until the required number of sections has been obtained. FIG. 6.— METHOD OP CUTTING SECTIONS WITH THE STIRLING MICROTOME. The imbedding will leave the cuts as they are floated in the alcohoL They may now be selected, lifted with the needle into clean spirit^ and preserved as before indicated, for future operations. THE SCHRAUEE MICROTOME. Fig. 7 represents an improvement on the Stirling instrument, and a most convenient, practical and inexpensive microtome for the phy- sician. FIG. 7.— SCHRAUER'S IMPROVED STIRLING MICROTOME, With clamp for holding the tissue. The tissue, if sufficiently hard, is held in a clamp or vice in the well of the instrument, the pressure being regulated by the side screw. By this means the necessity of imbedding is avoided. If the THE AUTHOR S LABORATORY MICROTOME. 15 tissue be too soft to withstand such treatment, it is best cemented to a cork, and the cork then fastened in the clamp. A screw-thread is cut upon a short cylinder, which works in a corresponding thread chased on the inside of the well-tube. The short cylinder carries the knife-plate, and, as the latter is turned to the right, the whole de- scends and the tissue projects, ready to be sliced off. THE AUTHOR'S LABORATORY MICROTOME. For certain work, some form of microtome becomes necessary in which the operator is relieved from supporting the knife. Fig. 8 is a sketch from such an instrument which I have contrived and which has been in daily use in my laboratory for over three years. The carriage A, supporting the knife B, is of solid cast-iron ; and has, upon the under side, a V guide, which fits into the longitudinal groove 0 of the base D. Parallel with this groove is a smooth flat FIG. 8.— THE AUTHOR'S LABORATORY MICROTOME. The instrument consists of a very heavy cast-iron bed upon which a carriage supporting a knife is made to slide. The tissue is cemented with paraffin (or held in an adjustable clamp not shown in the cut) to a table, which can be raised by a fine steel screw. The thickness of the section to be cut is controlled by turning the milled head actuating the finely threaded screw. surface, upon which also travels the rib E of the knife-carriage. A second Y has been avoided, in order to diminish friction. The knife is clamped rigidly to the upper surface of the carriage, by means of a Willis' tool-holder, consisting of a steel plate F, a nut G-, and washer H. The mechanism for supporting and positioning the tissue — not shown in the sketch— is built upon a plate I, which can be quickly fixed to the body of the microtome at the height and lateral incline required by the large set-screws J, J'. The mechanism for raising the tissue to the knife, between the cuts, consists of a screw K, of fifty threads to the inch which, working in the nut L, elevates the bevelled slide 16 PRACTICAL MICROSCOPY. M, to which the tissue N is affixed. An ether-freezing attachment may be substituted for the plate I. The milled-head 0 is divided into one hundred parts, so that each fraction of a turn raises the tissue -g- oW °f an inch. The knife should possess an edge of the most exquisite keenness ; and this holder admits the employment of almost any cutting instru- ment. In order to the production of the best results, the knife should be set at the most acute angle compatible with the use of the •entire length of the cutting edge, from heel to point. Both knife and tissue are to be flooded with alcohol, in ordinary work, as in free- hand cutting. A drip pan is provided, and is placed below the tissue- carrier. A groove in the front upper edge of the base prevents the pirit from flowing over the track, which., mixing with the lubricating oil covering the latter, would interfere with the delicacy and ease of the sliding motion. The value of this instrument is largely consequent upon its great solidity — the base weighing from eighteen to twenty-five pounds, with the knife carriage correspondingly heavy. Just why such weight and solidity are necessar}7, and contribute so largely to our success in cutting sections, is not at once apparent. The microtome is now made by Mr. L. Schrauer, of New York, in two sizes ; the smaller carrying a knife fourteen, and the larger, eighteen inches. A smaller pattern would present no special advantages over microtomes already in use. SHARPENING KNIVES. In the majority of instances of failure to produce suitable sections for microscopical work, the cause can be set down to dull knives; and I would urge the student to practice honing, until able to put cutting instruments in good condition. If he will but start properly, success is sure. Nine-tenths of the microtomes are purchased because of failure in free-hand work with a dull knife; but no advantage will be gained by a machine, if 1 he student be incapable of keeping the knife up to a proper degree of keen ness. A knife is a wedge, and for our purposes the edge must be of more than microscopical tenuity — it being impossible, with the microscope, to discover notches and nicks if properly sharpened. It is impossible to secure the best results with indifferent tools. The knife should be hard enough to support an edge, but not so hard as to be brittle. The proper temper is about that given a good razor. We need at least two hones — one comparatively coarse, for removing slight nicks; and another, for finishing. The first part of the work is best done by means of a sort of artificial hone made with SHARPENING KNIVES. 17 ground corundum. These are kept in stock by dealers in mechanics' supplies of great variety in size and fineness. For razors a "00" corundum slip will best answer. This will very rapidly remove the inequalities from an exceedingly dull razor. A Turkish hone will be best for finishing. For my large knives I use a third, very soft and fine stone, known as water-of-Ayr. Let the corundum slip be placed on a level support (mine are fitted into blocks like the carpenter's oil-stone), and cover the surface liber- ally with water.* The hones should always be worked wet. Place the knife flat on the stone near the right hand as at A, Fig. 9. Draw steadily in the direction of the curved dotted line, i. e., from right to left — holding the blade firmly on the stone B with slight pressure FIG. 9. FIG. 10. FIGS. 9 AND 10. — METHOD OF HONING. The knife is first brought with its heel in the position shown at A, Fig. 9. It is then drawn forward as indicated by the curved dotted line until, at the end of the stroke, the position C is attained. Fig. 10 indicates the method of turning the blade before reversing and between each stroke. until the position C is attained. Eotate the razor on its back — vide Fig. 10 — so as to bring the other side on the stone; and draw from left to right. Observe that as the knife is drawn from side to side (the edge invariably looking to ward the draw) it is always worked from heel to point. The amount of pressure may be proportioned to the condition of the edge. If it be badly nicked, considerable pres- sure may be employed; while, as it approaches keenness, the pressure * A few drops of glycerin added to the water retards evaporation and appears to keep the surface of the hone in good condition. 18 PRACTICAL MICROSCOPY. is to be lessened, until the weight of the blade alone gives sufficient friction. Kepeat the process fifteen or twenty times, and examine the blade. If the nicks are yet visible, continue honing until they can no longer be seen. Then draw the edge across the thumb nail. Do this lightly and the sense of touch will reveal indentations which the eye failed to recognize. Continue the use of the coarse stone until the edge is perfect, as far as the thumb-nail test indicates. The knife is then to be carefully wiped, so as to remove any coarse particles of corundum, and applied to the wetted Turkish hone with precisely the same motions as were employed in the first process. Af- ter a dozen or two strokes, examine the edge, by applying the palmar aspect of the thumb, with repeated light touches, from heel to point. This looks slightly dangerous to the novice, but it is an excellent method of determining the condition. Of course actual trial with a piece of hardened tissue is the best test. Some most skilful technologists prefer to finish by stropping. I have not used a strop in my laboratory for over two years, preferring to use the knife as it comes, highly finished, from the water-of-Ayr hone. If a strop be employed, the leather should be glued smoothly to a support of wood, otherwise the edge of the knife will become rounded. Stropping is conducted in the same manner as honing, only the edge of the knife follows the stroke instead of leading it. SUPPORTING TISSUES FOR CUTTING. Frequently small bits of tissue are required to be cut — pieces too small to be held with the fingers. I am in the habit of cementing such tissues into a hole in a bit of ailantus or elder pith, when the FIG. 11.— INSTRUMENT FOR SOLDERING TISSUE TO CORK SUPPORTS WITH PARAFFIN. It consists of an awl handle of wood into which a short piece of wire, preferably copper, is driven and bent as shown. whole may be cut as one mass. Tissue is frequently cemented to cork for convenience of holding in free-hand cutting; or the cork SUPPORTING TISSUES FOE CUTTING. 19 may be held in the vise of the microtome. The edge of the knife should not be allowed to touch the cork. Fig. 11 shows a simple little instrument, very convenient for using paraffin as a cement. A piece of stout copper or brass wire is bent as indicated, pointed, and driven into an ordinary awl handle. Paraffin wax possesses the very valuable property of remaining solid at ordinary temperatures, not cracking in the cold of winter or softening in summer. It is unaltered by most reagents, is easily rendered fluid, and quickly solidifies. As a cement, it is invaluable to the microscopical technologist. FIG. 12.— MODE OF CEMENTING TISSUE TO A CORK SUPPORT WITH PARAFFIN. Fig. 12 indicates the method of cementing a piece of tissue to a cork or other support. The tissue having been properly placed, the wire tool is heated for a moment in the alcohol flame, and then touched to a cake of paraffin. The paraffin is melted in the vicinity of the hot wire, a drop adheres to the latter and is carried to the edge of the tissue. In the cut the wire tool is seen in the position neces- sary for cementing one edge. The wire being removed, the wax im- mediately cools and becomes solid. The other sides are afterward cemented in like manner. The whole is done in less time than is necessary to the description of the process. 20 PRACTICAL MICROSCOPY. PREPARATION" OF TISSUES FOR CUTTING, ETC. We have already seen that most animal tissues are unsuitable for the production of thin sections until hardened. It is also a fact, paradoxical though it may seem, that fresh tissues do not present truthful appearances of structural elements. The old- school histologists insisted upon the presentation of structures unal- tered by chemical substances, while the modern worker has discarded such tissue with very few exceptions. Many descriptions of structure and growth, the result of study upon fresh material, have been proven by later methods grossly inaccurate. It is impossible to remove tissues from the living animal and to sub- ject them to microscopical observation without, at the same time, ex- posing them to such radical changes of environment as to produce structural alterations. Certain tissues, presenting in the living con- dition stellate cells with the most delicate, though well-defined branching processes, when removed from contact with the body, how- ever expeditiously, afford no hint of anything resembling such ele- ments, as they are quickly reduced to simple spherical outlines. In short, it is impossible to study fresh material, as such, without constant danger of erroneous conclusions, as retrograde alterations of structure commence with surprising rapidity the moment a part is severed from the influences which control the complete organism. From what has been said we appreciate the necessity of agents which, when applied to portions freshly removed from the animal, or even before removal, shall instantly stop all physiological processes and retain the elements in permanent fixity. Very much of the human structure which is available will be se- cured only after functional activities have long ceased, and the structure essentially altered. We are, therefore, compelled to resort to the use of material from the lower animals in very many instances. ALCOHOL HARDENING. The tissue, whatever process may be in contemplation, having been removed from the body as quickly after death as possible, ivithout washing or allowing contact with water in any way, should, with a sharp scalpel, be divided into small pieces. Of the more solid organs, pieces one-half inch square by one-fourth inch thick will be suf- ficiently small, and they will harden rapidly. The smaller the pieces and the larger the quantity of hardening fluid the more quickly will the process be completed. The volume of fluid should exceed that of CHROMIC ACID FIXING AND HARDENING. 21 the tissue at least twenty times. Wide mouth, well-stoppered bottles, from one ounce to a pint, or even larger, are best ; and they should be carefully labelled and kept in a cool place with occasional agita- tion. Quick Method. — A piece of any solid organ, say liver, spleen, pan- creas, kidney, uterus, lymph-node, etc., not larger than one half inch square by one-eighth thick, may be perfectly hardened in twelve hours by immersion in one ounce of ninety five percent, alcohol. No more should be thus prepared than is to be cut within twenty-four hours, on account of the shrinkage which results after the prolonged immersion of solid structures in strong spirit. After the tissue has been one hour in the above, it maybe hardened in one or two hours more, if transferred to absolute alcohol. This method is of frequent advantage in pathological histology. Ordinary Method. — The method quite general here, and intended to prevent shrinkage, is as follows : The organs, cut into pieces from one-half to three-fourths of an inch cube, are placed in a mixture of alcohol one part, water two parts (called in this laboratory " Alcohol A") for twelve hours. This re- moves the blood, and prepares the tissue for the next mixture — alco- hol one part, water one part, ("Alcohol B ") where it remains twenty- four hours. The pieces are afterward removed to ninety-five per- cent alcohol ("Alcohol C"). The strong alcohol completes the hard- ening, and serves as a preservative until such time as sections may be required. The process is complete in from two to four weeks, and the material will keep without deterioration for three or four years, especially if the spirit be changed occasionally. Ordinary anatomical specimens which have been preserved in dilute alcohol are of no value for our purpose. CHROMIC ACID FIXING AND HARDENING. Chromic acid is a very deliquescant salt, and is best preserved by making a strong solution at once, and then diluting it as may be needed. A stock solution may be made as follows : Chromic acid (crystals), . . . . 25 grammes. Water (distilled or rain), . . . .75 cubic cent. M. For general use, dilute 20 parts with 600 parts of water, which gives a strength of nearly one-sixth of one per cent. The tissue, as soon as secured and properly divided, is placed in the above, remembering the rule regarding quantity. Change in twenty- four hours to fresh solution, and again on the third day. In seven days, or thereabout, change the fluid again. The tissue must now be 22 PRACTICAL MICROSCOPY/. watched carefully and when, on cutting through a piece, the fluid is found to have stained the blocks completely, taking from two to three, or even four weeks, remove to a large jar of clear water and wash, changing the water frequently for twenty-four hours. The washing having removed the chromic acid, the tissue is further hard- ened in Alcohol A, B, 0. The special applications of this method, as well as of those which will follow, are indicated in Part Third. MULLER'S FLUID (MODIFIED).* Bichromate of potash, .... 25 grammes. Sulphate of copper, ... . 5 " Water, 1,000 c.c. M. This may be employed in precisely the same manner as the dilute chromic-acid solution. DECALCIFYING PROCESS. 6fc Chromic acid solution, . . ' .. 9 parts. Citric acid, C. P., . . . . . 1 part. Water, . .90 parts. The earthy salts may be removed from teeth and small pieces of bone with a liberal supply of the above in about twenty days. A frequent change of the solution will greatly facilitate the process; and an occa- sional addition of a few drops of the nitric acid may be made, with very dense bone. After the removal of the lime salts, the pieces may be preserved in alcohol until such time as sections are needed, when they may be cut with the microtome without injury to the knife. DISSOCIATING PROCESS (W. STIRLING). Artificial Gaslric Fluid. Pepsin, 1 gramme. Hydrochloric acid, . . . . 1 c.c. Water, . • 500 c.c. M. This process depends for its value upon the fact that certain con- nective tissues are more rapidly dissolved by the fluid than others. BAYBERRY TALLOW, HARDENING OR INFILTRATING PROCESS. Some three years since, I devised a method of infiltrating tissues with bayberry tallow. Tissues like lung, etc., which are delicately * The original Muller's fluid consists of the above (minus the copper salt) with an addition of 12.5 grammes of sulphate of soda. CELLOIDIN INFILTRATION. ^# cellular and hence very difficult to cut, when infiltrated with this material are supported in such a manner as to render the production of thin sections a very easy matter. Bayberry tallow is found in commerce in various grades. The best is white, clean, and of a consistency about equal to that of hard mut- ton tallow. It is instantly soluble in benzol, and dissolves rather slowly in alcohol. Having selected a piece of alcohol-hardened tissue for cutting, care- fully wipe it dry with blotting-paper and drop it into a capsule containing melted bay berry tallow. In order to render the tallow sufficiently fluid, and yet prevent the heat from becoming great enough to injure the tissue, the capsule should be set over a water- bath. Bubbles immediately arise as the spirit is vaporized and the tallow gradually fills the interstices of the tissue. If the latter be of a somewhat dense character it will be best, before placing it in the tal- low, to allow it to remain for an hour in pure benzol which, evaporating at a very low temperature, gives more ready admission to the infiltrat- ing medium. The length of time required for complete infiltration will depend upon the density and the degree of heat employed. Usually from ten to thirty minutes will suffice. The tissue, having become sufficiently infiltrated, is lifted out with the forceps, placed on a cork support, and allowed to cool. It is then cut, either free-handed or with the microtome, and ivithout alcohol. The dry sections, resembling tallow or wax shavings, are brushed into a saucer of pure benzol when in a moment the tallow will be dis- solved from the tissue. The sections are then lifted with a needle singly into a saucer of alcohol to remove the benzol. Afterward, they are transferred to a bottle of spirit, and labelled for future use. They will keep indefinitely. This process is peculiarly advantageous with such tissues as lung, pancreas, cerebellum, intestine, etc., where the structures require support only while they are being cut. The infiltrated blocks of tis- sue can be kept dry until such time as they may be wanted. CELLOIDIN INFILTRATION. Certain structures require permanent support, i. e., not only while being cut, but during the subsequent handling of the sections. The celloidin infiltrating process is best adapted to such material. Con- siderable time is needed for the successful employment of the process, but results can be secured that cannot be equalled with any other method. 24 PRACTICAL MICROSCOPY. Celloidin is the proprietary name of a sort of pyroxylin, very solu- ble in a mixture of ether and alcohol, producing a collodion. If thick collodion be exposed for a few moments to the air it becomes semi-solid — not unlike boiled egg-albumen; and to this property is due the value of a solution of celloidin in histology. It may be used as follows: To a mixture of equal parts of ether and alcohol add celloidin,* until the thickest possible solution has been obtained. A piece of alcohol-hardened tissue having been selected and kept for the preceeding twenty-four hours in a mixture of equal parts of alcohol and ether, is placed in about an ounce of the solution, and allowed to remain twenty-four hours. The bottle containing the whole should be well corked to prevent evaporation. The tissue after infiltration is to be placed on a cork support and allowed to remain in the open air for a few minutes, after which it should be plunged into a mixture of alcohol two parts, water one part. Here it may remain for twenty-four hours, or until wanted. Cut in the usual way using a mixture of alcohol two parts, water one part, for flooding the knife; the sections should be finally preserved in the. same instead of pure alcohol which would dissolve the celloidin. In infiltrating the tissue with the collodion it is best, especially if it be very dense in parts, to use, first, a thin and subsequently the thick solution. A more perfect infiltration is often obtained in this way. In some cases I have been obliged to continue the m acera tion for several days. The solution should be kept in well-stoppered bottles, as the ether is exceedingly volatile. Should the collodion at any time become solid from evaporation, it may be easily dissolved by adding the ether and alcohol mixture. The process is of inestimable value where delicate parts are weakly supported, and where it is important to preserve the normal relations. The gelatin-like collodion permeates every space and, as it is not to be removed in the future handling of the sections, it affords a support to portions that would otherwise be lost or distorted. It offers no ob- struction to the light, being perfectly translucent and nearly colorless. HARDENING BY FREEZING, ETC. I do not recommend the freezing process. Other fixing and hardening methods, which are of special applica- tion only, will be introduced in our future work as occasion may de- mand. * I find, after repeated trial, that the ordinary soluble gun-cotton, such as is employed by photographers, is in no way inferior to the celloidin. STAINING AGENTS AND METHODS. 25 STAINING AGENTS AND METHODS. STAINING FLUIDS. It is a very interesting fact (and one upon which our present knowledge of histology largely depends) that, on examination of tis- sues which have been dyed with special colored fluids, the dye will be found to have colored certain anatomical elements very deeply, others slightly, while others still remain unstained. Not only are different depths of color thus obtained, but different tints, even with a single dye, are often presented. If a section of some animal tissue be immersed in a mordanted solution of logwood, for example, besides the different depths of blue which are communicated to certain parts, other elements present pink and violet tints in various shades. The rule concerning the selection of dyes seems to be that those elements of a tissue which are the most highly endowed physio- ogically take the staining most readily. The minute granules of nuclei are so deeply stained in the logwood dye as to appear almost black. The nuclei are plainly stained, while the limiting membrane of cells is usually but slightly colored. Old, dense connective tis- sues stain feebly, or fail entirely to take color. The differentiation is, without doubt, due to chemical action between the elements of the dye and those of the tissue. A very great number and variety of materials have been used for histological differentiation, and a simple enumeration of them all would very nearly fill the remainder of our pages. It will be found, however, that leading histologists confine themselves to two or three standard formulae for general work. I shall notice only those methods which have been thoroughly demonstrated by years of em- ployment as best for the purpose suggested. Special cases will re- quire special treatment, which will be indicated in proper connection. H^MATOXYLIN * STAINING FLUID. To about eight fluid-ounces of a hot, saturated aqueous solution •of common alum, contained in a porcelain capsule, add, a few grains at a time, one drachm of hcematoxylin, with constant stirring. Boil over the spirit lamp very slowly for fifteen minutes. Add sufficient water to compensate for evaporation; and, when cold, pour into a wide-mouth bottle. Throw in a piece of camphor, say 30 grains, * The coloring principle of the Hsematoxylon Campechianum. Merck's pre- paration should be used. 26 PRACTICAL MICROSCOPY. allow the whole to remain exposed to the air for one week, and thea filter. The solution should always be filtered before using. Keep the filter paper in a funnel, and use it as a stopper for the bottle. The dye improves in strength of color for two or three weeks. Should the solution, which is of a beautiful purple or violet color, at any time turn red, a small piece of common chalk may be added. This will restore the color by neutralizing the acidity. A few crystals of alum should always be kept in the bottle to insure saturation. Prepared as above, the dye will keep perfectly for at least eight months, and gives a permanent stain. BOKAX-CARMINE STAINING FLUID. To eight ounces of a saturated aqueous solution of borax (borate of soda) add one drachm of the best No. 40 carmine (previously rubbed into a paste with a little water). In order to insure satura- tion, some borax crystals should always be left undissolved at the bottom of the bottle. Agitate frequently, and, after twenty-four hours, add fifteen drops of liquor potass®. Always filter or decant before using. It will keep indefinitely, im- proving, to a certain extent, with age. EOSIN SOLUTION. Alcohol, 2 ounces. Eosin, 1 drachm. This will give a saturated solution. PICRIC ACID SOLUTION. Picric acid, i ounce. Water, 4 ounces. The acid is in excess, insuring saturation. NITRATE OF SILVER SOLUTION. Nitrate of silver, o grains. Distilled water, .... 4 ounces. If the water be pure, light will have no effect upon the solution. STAINING METHODS. 27 STAINING METHODS. H^MATOXYLLtf STAINING PROCESS. You will require for future work a needle like Fig. 13, several saucers, preferably of white ware; a few watch-glasses—large, odd sizes are usually cheaply obtainable at the jewellers; half a dozen glass salt-cellars — small ones known as "individual salts;" and a two-ounce, shallow, covered porcelain box, such as druggists use for ointments, dentifrices, etc. Place on the work-table (best located so as to be lighted from your side and not from the front) in order, as in Fig.. 14. 1. A wa/ch-glass, containing say fl. 3 ij. of haematoxylin fluid. 2. Saucer, filled with water. 3. Salt-cellar, filled with alcohol. 4. The covered porcelain box, containing about an ounce of oil of cloves.* FIG. 13.— NEEDLE FOR^LIFTING SECTIONS, ETC. Select a section from some one of your stock bottles, lifting it out with the needle, and place it in the haema. solution. The section having been taken from alcohol and transferred to an aqueous staining fluid, will twirl about on the surface of the latter, inasmuch as cur- rents are formed by the union of the water and the spirit. "How long shall I let the section remain in the haema. ?" The only answer I can give is, " Until properly stained! " Nothing but experience will give you any more definite information. Much de- pends upon some peculiar property in the tissues: some stain rapidly, others stain very slowly. The strength of the dye is another deter- mining factor. Usually with the haema. formula, as given, from six to ten minutes will suffice. Place the needle under the section (if the fluid be so opaque as to hide the tissue, place the watch-glass over a piece of white paper or a bit of mirror), and gently lift it out; drain off the adhering drop of dye on the edge of the glass, and drop into the saucer of water. Here we can judge as to the color, and we, perhaps, find it to be of a light * The oil of Bergamot must be used for clarifying sections which have been infiltrated with collodion, as the clove oil is a solvent of the pyroxyline. PRACTICAL MICROSCOPY. purple — too light; so you may return it to the haema. for another period of two or three minutes, which will probably give sufficient •depth. As the section floats on the washing water, you will notice that the latter will be colored by the dye, some of which leaves the tissue. Allow the water to act until no more color comes out. The tint of the section changes from purple to violet, and the water must be al- lowed to act until the change is complete. Again, you will remem- ber that this dye contains alum and, if you hurry the washing, you will undoubtedly find crystals covering your specimen after it has been mounted. From five to ten minutes will complete the washing. If you were to examine your section at this stage, you would find it opaque, and, as we are obliged to study our objects mainly by trans- mitted light, we must find some means of securing translucency. The essential oils are used for this purpose, oil of cloves being com- monly employed. Lift the section from the water with the needle; let it drain a moment, and then drop it into the alcohol with which the salt-cellar was filled. The object of this bath is the removal of the water from the tissue, and this will be accomplished in from five to ten minutes. Again lift the section, and place it in the oil of cloves. The tissue floats out flat, and in a few minutes sinks in the oil. We might proceed to the examination of the stained section; but I shall ask you to let it remain in the oil, covering the box carefully to exclude our great enemy, the dust, until we have learned more about staining. FIG. 14.— DIAGRAM INDICATING THE SUCCESSIVE STEPS IN STAINING WITH THE H.EMATOXYLIN SOLUTION. To recapitulate: The essential steps in the haema. process are: 1. Staining the tissue — haema. 2. Washing — water. 3. Dehydrating — alcohol. 4. Rendering translucent — oil of cloves. STAINING METHODS. 2if As the section is put in the dye, care should be taken to so float it out that it may not be curled. This is easily done with the needle. After the alcohol bath, however, this becomes difficult, as the tissue is rendered stiff by the removal of the water. This is the simplest and best of all methods for general work, and you are advised to master every detail of the process. After reading the directions which I have given, and having never seen the work actually done, you will not be singular if you conclude the staining of tissues to be a tedious and slow process; but after a month's work you will be able to stain fifty different sections in half an hour, and have them ready for mounting. H^MATOXYLIN AND EOSIN. DOUBLE STAINING. Very beautiful and valuable results in differentiation are obtained by staining first with hasma. and subsequently with eosin. Eosin is a salt of resorcin, staining most animal tissues pink, and it affords with the haema. a good contrasting color. The tissue is to be stained in haema. and washed in water as usual; then it is placed in the eosin solution, and afterwards washed again. The subsequent treatment is as with the plain haema. process, viz., dehydration with alcohol, after which the oil of cloves. FIG. 15.— DIAGRAM INDICATING THE SUCCESSIVE STEPS IN DOUBLE STAINING WITH . AND EOSIN. The diagram, Fig. 15, shows the process complete: 1. Watch-glass with haema. 2. Saucer with water. 3. Watch-glass two- thirds filled with water, witli five drops of eosin solution added. 4. Saucer containing water. 5. Salt-cellar filled with alcohol. 6. Covered oil-dish. The eosin stains very quickly, generally in about a minute. Care should be taken not to overstain with it, as it cannot be washed out. If the sections are found at any time to be overstained with haema. the color may be removed to any desired extent by floating them in a 30 PRACTICAL MICROSCOPY. saturated aqueous solution of alum. They must afterward be washed in clean water. BORAX-CARMIKE STAIKING PROCESS. Arrange your materials as in the diagram, Fig. 16. 1. Watch-glass two-thirds filled with the carmine fluid. 3. Saucer containing about an ounce of alcohol. 3. Salt-cellar filled with a saturated solution of oxalic acid in al- cohol. 4. Salt-cellar with alcohol. 5. Porcelain dish containing oil of cloves. The carmine solution will stain ordinarily in from three to ten minutes. After the section has been for a few minutes in the dye, FIG. 16.— DIAGRAM INDICATING THE SUCCESSIVE STEPS IN STAINING WITH BORAX-CARMINE. you will lift it with the needle, drain, and transfer to the saucer con- taining alcohol. You^will then be enabled to determine whether the section is sufficiently stained; it should be a deep, opaque red. The alcohol washes off the section, removing the adhering solution of carmine. The carmine must now be fixed in the tissue or mordanted; and this you proceed to do by transferring the section to the watch-glass of oxalic acid solution. Notice the change in color, from a dull red to a bright crimson, and when the change is complete, lift it into the salt-cellar containing clean alcohol. This dissolves out the acid, which, if left, would appear later on the specimen in crystals. Five minutes suffice for this washing, after which transfer to the oil of cloves. This process does not give as sharp contrasts as the haema. and eosin, but it is simpler and very permanent. It is best to select 3ome one process for general work, and adhere to it. The acid of the Carmine process must be guarded with extreme care, as the smallest particle is sufficient to spoil the hsema. solution. Look to it that the MOUNTING OBJECTS. 31 dishes are kept scrupulously clean, and the same care must be be- stowed upon the needles, forceps, etc. You may, of course, stain several sections at once, providing you take care to keep them from rolling up or sticking together. While the vessels which I have recommended will be found of con- venient, proportionate, and economical size for general work, larger ones are sometimes needed; and almost any glass or porcelain vessel may be impressed for duty. CARMINE AND PICRIC ACID STAINING. After having washed the tissue, subsequently to mordanting with oxalic acid in the borax- carmine process, a bright yellow may be communicated to certain anatomical elements by means of picric acid. This often gives a valuable differentiation. The sections are placed in the picric-acid solution and allowed to remain for ten minutes. Kemove to water one ounce, glacial acetic acid ten drops for a moment, to fix the yellow; after this dehydrate with alcohol, and clarify with oil of cloves as usual. The sections should be transferred to the picric and acetic acid solutions by means of a platinum wire or a minute glass rod. The ordinary needle would be corroded, and the sections thereby discolored. MOUNTING OBJECTS. CLEANING SLIDES AND COVERS. When purchasing "slides, let me urge you to get them of good quality. The regular size is one by three inches, and the edges should be smoothed. As furnished by the dealers they are usually quite clean, and only require rubbing with a piece of old linen to prepare them for use. The cover-glasses should be thin, not over TJ^th of an inch, called in the trade-lists " No 1." Circles or squares three-quarters of an inch in diameter are generally convenient. They must be thoroughly cleaned: Drop them singly into a saucer containing hydrochloric acid. Then pour off the acid, and let clean water run into the dish for several minutes. Drain off the water and pour an ounce of alco- hol on the covers. Kemove them one at a time with the forceps or needle, and wipe dry with old linen.* The glass may be held between * We are indebted to Professor Gage, of Cornell University, for suggesting the use of Japanese tissue paper for wiping cover glasses, lenses, etc. Or- dinary manilla toilet paper is also an excellent material for such work. 32 PRACTICAL MICROSCOPY. the thumb and forefinger, the linen being interposed. Very slight pressure and rubbing will complete the process. The surface of the glasses should be brilliant, and they are to be preserved for future use in a dust-tight box. TRANSFERRING THE SECTIONS TO THE SLIDE. Procure a piece of either very thin sheet copper or heavy tin foil, three inches long and one-half inch wide, and bend it about three- fourths of an inch from one end, making a section lifter as shown in Pig. 17. FIG. 17. — SECTION SPOON. Strip of copper or heavy tin foil, best for lifting sections from staining and other fluids. For use in fluids which would attack metals, the spoon should be constructed from horn. Place a clean slide on the table before you and with the section - lifter used like a spoon dip up one of the sections from the clove-oil. By inclining the lifter, the section may be made to float to the centre of the slide. A small sable brush is often convenient for coaxing the section off the lifter. If it were our present object to simply examine the section, we could drop a thin cover-glass on the specimen, and it would be ready for study. Such an object would afford every requirement for present observation, but would nofc be permanent. The oil of cloves TRANSFERRING SECTIONS TO THE SLIDE. 33 would evaporate after a few days and the section be ruined. We pro- ceed to make a permanent mounting of our object. The clove oil, surrounding the section on the slide, is first to be re- moved; and it can easily be done by means of blotting-paper. With a narrow slip of thin filter paper wipe up the oil, exercising care not to touch the section or it will become torn. Proceed carefully, taking fresh paper until the oil will no longer drain from the section FIG. 18 —METHOD OF LABELLING A MOUNTED SPECIMEN. when the slide is held vertically. With a glass rod remove a little of the dammar solution (vide formulae) from the bottle and allow a drop of this varnish to fall upon the section. Pick up a clean cover-glass with a needle, and place it on the drop of dammar. This operation is seen in Fig. 18. The point of the needle may be placed beneath the cover-glass, the tip of the forefinger FIG. 19. — MODE OF HANDLING THE COVER-GLASS IN MOUNTING TISSUES. pressing lightly over it, and you will be enabled to carry the thin glass wherever desired. As the cover settles down the air is pressed out, until finally the section appears imbedded in the varnish— the latter filling the space between the cover and the slide. The object is "mounted." You \\WQ2o permanent specimen. The slide must be kept flat, as the dammar is soft. After some weeks, 3 34 PRACTICAL MICROSCOPY. the varnish around the edge of the cover will stiffen, and eventually become solid. Do not paint colored rings around the specimen. Nothing can present a neater appearance than the simple mount, as I have described it, after having been properly labelled. Labels seven-eighths of an inch square may be put on one or both ends, with the name of the object, date, method of staining, or whatever par- ticulars you may prefer. Specimens should be kept in trays or boxes so as to always lie flat. CARE OP THE MICROSCOPE. The objectives constitute the most valuable part of the instrument. The lenses should never be touched with the fingers; indeed the same rule applies to all optical surfaces. When the glasses become soiled, they may ce cleaned, but it should be done with great care. While the effect of a single cleaning would probably not be to the slightest appreciable injury to the glass, repeated wiping with any material, however soft, will destroy the perfect polish, and result in obstruction of light and consequent dimness in the field. Never use a chamois leather on an optical surface, as these skins contain gritty particles. Old, well-worn linen and Japanese paper are by far the best mate- rials for wiping glasses. If a lens be covered with dust, brush it off ; breathe on the surface, and wipe gently with the linen or paper. Should you get clove oil on the front lens of the objective (as fre- quently happens when examining temporary mounts) wipe it dry and then clean with the linen moistened with a drop of alcohol. Dammar varnish can be very readily removed from any surface after having softened it with oil of cloves. The front lens of the objective, being the only one exposed, is the one usually soiled. Particles of dirt on the objective, as I have said, cause a dimness in the field — the image is blurred. Dust on the lenses of *t he eye-piece, however, appears in the field. These lenses are readily cleaned by dusting, and wiping with the linen, after having breathed on the surface. Never wipe a lens when dusting with a camel's hair brush will answer the purpose. The microscope should either be covered with a shade or cloth, or put away in its case, when not in use. The delicate mechanism of the fine adjustment becomes worn and shaky if not kept free from dirt. PART SECOND. STRUCTURAL ELEMENTS, PRELIMINARY STUDY. FOKM OF OBJECTS. From a single and hasty view of bodies under the microscope, we are liable to form erroneous ideas of form. Either a sphere, disc, ellipsoid, ovoid, or cone may be so viewed as to present a circular out- line. It therefore becomes important to view objects in more than, a single position. This can easily be accomplished with isolated particles by suspension in a liquid. In this way the true shape of a blood -corpuscle, e. g., may be determined. Again, much information concerning the actual form of bodies may be gained by a proper adjustment of the fine focussing screw. You may remember that the depth of the field of view in the microscope is exceedingly slight. Speaking accurately, only a single plane can be seen with a single focal adjustment; but by gradually raising or low- ering the tube of the microscope, the different parts of a body maybe focussed and studied and an accurate idea of form secured. With a glass rod place a drop of milk, which has previously been diluted with three parts of water, on a slide, and put a cover-glass thereon as in Fig. 19. Focus first with the low power (L). A mul- titude of minute dots are observed. Then switch on the high power (H), and the dots will resolve into circular figures. Select one of the smaller particles and, as you raise the focus, the centre of the figure retains its brilliancy, while the edges become dark or blurred, show- ing convexity. Reverse the focus, and the centre again retains its sharpness long after the edge has become blurred. The figure, then, '36 PRACTICAL MICROSCOPY. is a spheroid. These bodies are fat globules. Particles of free fat always assume the spheroidal form when suspended in a liquid. Note the larger globules; they have become flattened by pressure of the cover-glass. Clean the slide, and make a second preparation from the diluted milk— first, however, shaking it violently in a bottle. Note the flat- tened air bubbles among the oil globules. Observe that these air A| FIG. 20.— DIAGRAM SHOWING "THE EFFECT OF AIR BUBBLES AND OIL GLOBULES IN A MOUNTED SPECIMEN UPON THE RAYS OF LIGHT. The lines A, B, show the refraction of the rays (so as to produce a ring of color) by the action of two plano-concave water lenses which are formed by the air-bubble. The oil is seen to correct the refraction cf C D, thus giving but little color to the margin of this globule. bubbles have no intrinsic color, while the fat globules are faintly yellow. Observe the change in the ring of prismatic color about the edge of the air bubble, as the focus is altered. No such color will be .seen in connection with the oil globule. The bubbles assume various figures from pressure of the cover glass. MOVEMENT OF OBJECTS. Objects are frequently seen moving in the field of the microscope, the movement being magnified equally with their dimensions. Thermal Currents. — When with the previous specimen, or any other fluid mount, the warm hand is brought close to one side of the stage, the globules in the field will be seen swimming more or less rapidly. These currents are due to the unequal heating of the liquid under observation. The direction of the current is in the reverse of its apparent motion. Brownian Movement. — Place a fragment of dry carmine on a slide; add a drop of water, and with a needle stir until a paste is formed. Add another drop of water, and immediately put on the cover- glass. With H, note the most minute particles, and observe their peculiar, dancing motion. This occurs when almost any finely-divided and generally insoluble solid is mixed with water; it ceases after a short time. The movement has been attributed to several causes. EXTRANEOUS SUBSTANCES. 3T Vital Movements. — Place a drop of decomposing urine on a slide, cover and focus with H. The field contains innumerable minute spherules and rods (bacteria) which are in active motion, resembling somewhat the Brownian movement, although sufficiently distinctive after close observation. After having rubbed the tongue for a moment against the inner surface of the cheek, put a drop of saliva on a slide, cover and focus H. Among the numerous thin, nucleated scales and debris, small granular spherules — the salivary corpuscles — will be found. Select one of the last, centre, and focus H. with extreme care. The minute granules within the cells are in active motion, resembling the Brown- ian movement; but with proper conditions the motion may continue for many hours. EXTRANEOUS SUBSTANCES. Before we begin the study of animal tissues, I wish to have you be- come somewhat familiar with the appearance of certain objects which FIG. 21.— EXTRANEOUS SUBSTANCES. A. Cotton fibres, showing the characteristic twist. B. Linen fibres, with transverse markings indicating segments. C. Wool. The irregular markings are produced by the overlapping of flattened cells. Wool may be distinguished from other hairs by the swellings which appear at irregular intervals in the course of the former. D. Silk. Smooth and cylindrical. are frequently, through accident or carelessness, and often in spite of the utmost care, found mixed with our microscopical specimens. 00 PRACTICAL MICROSCOPY. Among the more common objects floating in the air and gaining access to reagents, to subsequently appear in our mounted specimens, are the following: Fibres. — Procure minute pieces of uncolored linen, cotton, wool, and silk. With a needle in either hand, tease out or separate a few fibres on slides, add a drop of water and cover.* Starch. — Procure samples of wheat, corn, potato and arrow-root starch, or scrape materials containing any one of these substances FIG. 22. — EXTRANEOUS SUBSTANCES. A. Granules of potato starch. B. Corn starch. C. Wood fibres. The circular dots are peculiar to the tissue of cone-bearing trees. D. Spiral thread from a tea leaf. E. Fragment of feather. with a sharp knife. To a minute portion on the slide add a drop of water, cover and examine L and H. Wood Shavings, Feathers, Minute Insects, Portions of Larger In- * These substances, as well as most of those which follow under the same heading, may be mounted permanently as follows: Put the dry material in clean turpentine for a day or two, to remove the contained air. Transfer to the slide, tease, separate, or arrange the elements, after which wipe away the turpentine with strips of blotting paper. Add a drop of dammar and place the cover-glass thereon. The weight of the cover will be sufficient to press the object flat, if it be properly teased or separated. Although I do not advise the making of colored rings around cover -glasses, they may be formed after first protecting the dammar with a ring of gelatin. — Vide formulae. CELLS. 39 sects, Pollen, etc., are easily mounted temporarily or permanently as above. They are very commonly found in urine after it has been ex- posed to the air. and their recognition is very important. Let me urge you to become familiar with the microscopical appear- ance of the commoner objects which surround us in every-day life. The most serious mistakes have resulted from ignorance of this sub- ject. Vegetable fibres have been mistaken for nerves (!) and urinary casts, starch granules for cells, vegetable spores for parasitic ova, etc. STRUCTURAL ELEMENTS. Certain anatomical structures, of a more or less elementary nature, are united in the composition of organs. These structural elements will with propriety first claim notice from us. CELLS. A typical cell is a microscopical sphere of protoplasm, constituted .as follows (vide Fig. 23): A. Limiting membrane. B. Cell-body. C. Nucleus. JX Nucleolus. 0 FIG. 23.— ELEMENTS OF A TYPICAL CELL. The wall consists of an apparently structureless membrane of ex- treme tenuity. The cell-body may be either clear (jelly-like), granular, or fibrillated. The nucleus is a minute spherical vesicle, with a limiting mem- 40 PRACTICAL MICROSCOPY. brane inclosing a clear gelatinous material, traversed by a reticulum of fibrillae. The nucleolus consists of a spherical granular enlargement upon the fibrillae of the nucleus. Deviations from the type are most frequent, and vary greatly as to form, number of elements, and chemical composition. FIG. 24.— A CELL NUCLEUS, WITH NETWORK AND NUCLEOLUS. DIAGRAMMATIC. The typically perfect cell is rarely seen in human tissue on account of the length of time which commonly elapses between death and observation of the structure, the delicate fibrillae of the nuclei usu- ally appearing as a mass of granules. CELL DISTRIBUTION. The complex mechanism of the body had its origin in a single cell. This preliminary structure, endowed with the power of proliferation, became two cells. Two having been produced, they became four ; the four, eight; and thus progression advanced until they became countless. Some of these ceils remained as such ; others altered in form and composition gave birth to muscle, bone, etc., etc. The study of these processes belongs to physiology. The adult body is composed largely of cells of various forms. The different physiological processes, as secretion, absorption, respiration, etc., are effected through the intervention of these anatomical elements. All free surfaces, within or without the body, are covered ivith cells. The entire skin, the outside of organs, as lung, liver, stomach, intestine, brain, etc., etc.; all cavities, as alimentary tract, heart, ventricles of the brain, blood-vessels, ducts, all present a superficial layer of cells. VARIATION" IK FORM OF CELLS. Alteration from the typical or spherical form is effected mainly through pressure consequent upon active proliferation of contiguous cells, or growth of surrounding fibrous tissues. SQUAMOUS, STRATIFIED AND TRANSTIONAL EPITHELIUM. 41 FLAT CELLS. If a cell be subjected to pressure on two opposite sides, a flattening ensues, and a scale-like element results. Flat cells are united to form a continuous structure in different ways. SQUAMOUS, STRATIFIED, AND TRANSITIONAL EPITHELIUM. The simplest method of tissue production by means of flat cells is that of superposition, constituting squamous epithelium. Cells are placed one over the other, generally without great regularity. If regular, and in several layers, the structure is called stratified epithe- lium ; if only in a few layers, it is termed transitional epithelium. The superficial layer of the skin aifords an example of squamous,. FIG. 25.— SQUAMOUS CELLS FROM BUCCAL EPITHELIUM. A. Typical cell. B. Its nucleus. C. Union by overlapping forming laminae. D. Salivary corpuscles. X 400. stratified epithelium. The bladder, pelvis of the kidney, and vagina are lined with transitional epithelium. The thin, flat scales from the mouth may be demonstrated by scraping a drop of saliva from the tongue with the handle of a scalpel, transferring it to the slide, and applying the cover. The size of the drop of saliva should be carefully adjusted so as to fill the space between the cover-glass and slide. Too little will cause the cover to adhere so tightly to the slide as to press the cells out of form; too much, and the saliva flows over the cover and soils the ob- jective. With a glass rod, place a drop of the dilute eosin solution 42 PRACTICAL MICROSCOPY. on the slide, and with a needle lead it to the edge of the saliva. The dye will pass under the cover slowly; and, gradually, whatever anatomical elements there may be present will be stained. Observe that the nuclei of the flat scales first take the dye, and appear of a deep pink; while the other portions are either colorless or very lightly stained. Find a typical field and sketch it with pencil, afterward tinting with dilute eosin. PAVEMENT EPITHELIUM. When thin flat cells are disposed in a single layer, like tiles, the epithelium is termed pavement or tessellated. These cells are often •quite regularly polygonal (although this obtains more frequently with tissue from the lower animals), and they are always connected by their «dges by means of an albuminous cement. FIG. 26.— PAVEMENT EPITHELIUM. DIAGRAMMATIC. This structure is very extensively distributed. Most serous sur- faces, e. g., the pleurae, omenta. mesenteries, and peritoneal surfaces generally, are so covered. The lining of the heart , of arteries and veins, and of lymph channels is constructed with these cemented cells. Blood capillaries are formed almost entirely of such elements. The best demonstration is made by coloring the cement which unites the cells. If a tissue, covered with this epithelium, be placed for a few minutes in a solution of nitrate of silver a chemical union ensues; an albuminate of silver is formed which blackens in the light, •thereby mapping out the cells with great precision and clearness. It is nearly impossible to procure human tissue for this purpose, as the cement substance decomposes soon after death. The mesentery of the frog affords a good example of pavement cell structure; and -differs but little from the arrangement on human serous surfaces. PAVEMENT EPITHELIUM. 4:6 Kill a large frog by decapitation, and open the abdomen freely by an incision along the median line. Pull out the intestines by grasping the stomach with the forceps. This will expose the small intestine, which you will remove, together with the attached mesentery, by means of quick snips of the scissors. Work as rapidly as possible and avoid soiling the tissue with blood. Throw the gut into a salt-cellar filled with silver solution, vide formulae, where it must remain for ten FIG. 27. PAVEMENT EPITHELIUM FROM FROG'S MESENTERY. SILVER STAINING. A. Area showing the- outlining of the pavement cells by the silver-stained cement sub- stance. The nuclei have been brought out by the carmine. Minute stomata may be seen be- tween certain cells. B. A blood capillary terminating below in an arteriole. The silver has outlined the en- dothelia of the vessels. C. An area showing both layers of the pavement. The deeper cells are faintly outlined, being out of focus. The silver has been deposited over the lower portion of the specimen, nearly obscuring the cement lines. X 250. minutes covered from the light. Lift the tissue from the solution by means of a strip of glass (or a platinum wire), and throw into a saucer of clean (preferably distilled) water, changing the latter repeatedly for ten minutes. After washing, and while yet in the water, expose to sunlight (perhaps fifteen minutes) until a brown tint is acquired which indicates the proper staining. 44 PRACTICAL MICROSCOPY. Proceed to stain the intestine, with mesentery attached, with borax- carmine as directed for sections, excepting that, as the mass is great, it must be washed twice in alcohol after the oxalic acid. We have allowed the mesentery to remain connected with the gut, that the former might not curl, as it would have done had it been sep- arate. The preparation having reached the oil of cloves, proceed with a pair of scissors to snip off a small, flat piece of mesentery. Remove it to a slide, clean off the oil, apply dammar, and cover. The mesentery, you have learned from descriptive anatomy, consti- tutes a'support for blood and lymph vessels which are in connection with the intestine. The vessels are held together with a little deli- cate fibrous (connective) tissue, and are covered above and below with a layer of pavement epithelium. You will observe prominently some dark lines (the larger vessels) traversing the specimen. Select a thin spot between the vessels and focus H, you have a picture like Fig. 27. The field is traversed by very delicate dark lines, indicating the position of the cement sub- stance ; while the nuclei of the cells are pink from the carmine stain- ing. With the fine adjustment-screw run the tube of the microscope down carefully. The cement lines will disappear, and before they are completely out of focus, another set of cells will appear below the first set. So you may alternately bring into view the upper and under layers of cells covering the respective sides of the mesentery. Observe the irregular shape of the cells. Note, also, that the cells on one side average larger than those on the other side. You may also notice in various parts of the specimen blood-vessels lined with cells which are outlined with the silver staining. Sketch a field showing the elements as in Fig. 27, £nd stain the nuclei with the carmine solution. COLUMN AE CKLLS. Columnar Epithelium. Columnar cells are found, generally, throughout the alimentary and respiratory tracts. They also line the cerebral ventricles, the urinary and Fallopian tubes, the uterus, etc. This epithelium is quickly de- stroyed after death and is difficult of perfect demonstration except in an animal recently killed. Procure from the abattoir a portion of the small intestine and bron- chus of a pig, and with the curved scissors snip out small pieces from the mucous surface of each. Macerate in one-sixth per cent of chromic acid for twenty-four hours. CILIATED COLUMNAR EPITELIUM. 45 Place a piece of the gut on a slide and, after having added a drop of the acid solution, scrape off the mucous surface with a knife and remove the remainder of the gut. Add a cover-glass and focus H. You will find cells in various conditions, from isolated examples to small groups like Fig. 28. FIG. 28.— COLUMNAR CELLS FROM SMALL INTESTINE OF RABBIT. A. Tapering attached extremity. B. A swollen goblet cell. C. Finely striated free border. D. Transparent line of union between the striated portion and the body of the cell. X 400. Observe that the attached ends of the cells are often small and pointed, and that spheroidal and ovoidal cells are frequently wedged in between them. Note the -free border : It consists of striae, and is separated from the body of the cell by a translucent line. This ap- pearance is also that of the epithelium in the human intestine. Ciliated Columnar Epithelium. Prepare, by scraping, a slide from the mucous surface of the pig's bronchus (which has been macerating in the chromic acid). Observe the cilia on the free border of the cells. Interspersed be- tween ciliated cells, much enlarged individuals may be found, the so- called beaker, goblet or mucous cells. The motion of the cilia may be demonstrated as follows : Carefully open an oyster so as to preserve the fluid. On examina- tion you will notice the leaflets, shown in Fig. 30, commonly called the beard. With the scissors snip off a fragment. of the free border of 46 PRACTICAL MICROSCOPY. this beard, add a drop of the liquid from the oyster, and tease with a pair of needles. Apply the cover and focus H. At first, the individual cilia cannot be demonstrated on account of Fio. 29.— CILIATED COLUMNAR CELLS FROM BRONCHUS OF PIG. X 400. their rapid vibration. After a few moments, however, the action be- comes less energetic, and the hair-like appendages of the cells are to be plainly seen. FIG. 30.— OYSTER, OPENED TO SHOW METHOD OF PROCURING LIVING CILIATED CELLS. A. The divided muscle. This must be sectioned before the shell can be opened. B. The Heart. C. Liver. D. D. The so-called " beard." These laminae are covered with cells provided with cilia ; and a fragment of the free border of one of the leaflets may be snipped with the scissors, and examined as described in the text. Of course none of the above objects are intended to be perma- nent. BED BLOOD-CORPUSCLES. 4T SPHEROIDAL CELLS. The only cells which have, in any very great number, retained their primitive spheroidal form are the corpuscles of the blood and of the- lymphatic system. In solid organs, the cells, primarily spheroids, often become poly- hedral from pressure. Cells, developed spheres, not unfrequently send out prolongations, forming either stellate or polar cells according to the size of the radi- ating processes. RED BLOOD-CORPUSCLES. The human red blood-corpuscle is a flattened, bi-concave disc, one- three thousand two hundredth of an inch in diameter. It presents a. FIG. 31.— CORPUSCULAR ELEMENTS OF HUMAN BLOOD. A. Colored corpuscles adhering by their sides— rouleaux. B. The same crenated. C. The same shrunken. D. The same having absorbed water. E. The same still more swollen. F. The same with the plane C D, Fig. 32, in focus. G. The same with the plane A B, Fig. 32, in focus. H. Colorless corpuscles, x 400. mass of protoplasm destitute, as far as the microscope shows, of nuclei,, cell- wall, or any structure whatsoever. 48 PRACTICAL MICROSCOPY. Certain changes in form result, after removal from the circulation, yiz. : 1. They may adhere by their broad surfaces forming columns. 2. From shrinkage they may become crenated. 3. Still further shrinkage produces the chestnut-burr appearance. 4. From absorp- tion of water they may swell irregularly, obliterating the concavity of one side. 5. From continuous absorption they swell, forming spheres which are finally dissolved. Wind a twisted handkerchief tightly around the left ring finger ; w FIG. 32.— DIAGRAM OF A COLORED BLOOD-CORPUSCLE, SIDE VIEW SHOWING THE BI-CONCAVITY. A, B, Upper plane; which, in focus, gives the appearance shown at G, Fig. 31. C, D, Plane giving the appearance F, Fig. 31. prick the end with a clean needle, and squeeze a minute drop of blood on a slide, add a drop of saliva, cover and focus M. Observe : — 1. That considerable variation in size of the red blood- corpuscles exists. 2. The color — a delicate straw tint. 3. That the concave centre of the corpuscles which lie flat can be made to appear alternately dark and light according to the focal adjustment. 4. That the concavity is also demonstrated as the corpuscles are turned over by the thermal currents.* BLOOD-PLATES. Minute corpuscular elements in the blood about one-fourth the size of the red discs exist in the proportion of about one of the former to twenty of the latter. They are colorless ovoid discs; and are regarded by Osier as an essential factor in the coagulation of the blood. Prick the thoroughly clean finger with a needle. Over the puncture place a drop of solution of osmic acid (vide formulae) and squeeze out a minute drop of blood so that, as it flows, it is covered by the acid solution. This fixes the anatomical elements, providing against further change. The blood, as soon as drawn, must, with the acid, be * The student is at this time advised to study the corpuscular elements of the blood of such animals as he may be able to command. The red corpuscles of mammals (excepting the camelidse) do not vary in appearance from those of man, excepting in size. Those of birds, fishes, and reptiles are elliptical with oval nuclei. Corpuscles of the blood of invertebrates are not colored. POLYHEDRAL CELLS. 49 immediately transferred to a slide and covered. To provide against evaporation, run a drop of sweet oil around the edge of the cover. FIG. 33.— HUMAN BLOOD PRESERVED WITH OSMIC ACID. A. Colored corpuscles. B. Colorless corpuscle. C. C, C. Groups of plaques. X 400 and reduced. The blood-plates may be found, after careful search, bearing the relation to the red corpuscles seen in Fig. 33. WHITE OE COLORLESS BLOOD-CORPUSCLES. The white blood-corpuscle is a typical cell, spherical in form, presenting generally a nucleus — often two or more — with nucleolL In diameter about the one-twenty-five hundredth of an inch, they are usually found in the blood in proportion of one to three hundred to one thousand red corpuscles. The nucleus of the white corpuscle possesses nearly the same refractive index as the body of the cell, and is there- fore difficult of demonstration without the use of reagents or staining. Procure a drop of pus from a healing wound, mix it on a slide with an equal quantity of dilute eosin solution, cover and examine H. Pus is colorless, containing spherical nucleated corpuscles, the perfect ones resembling exactly those found in healthy blood. Ob- serve that the nuclei, some cells containing three or even four, are stained with the eosin. Minute pigment granules and fat globules appear in many of the pus cells, and others are broken and distorted. POLYHEDRAL CELLS. With a scalpel scrape the cut surface of a piece of liver from a recently killed pig ; place a minute portion of the finer part on a 50 PRACTICAL MICROSCOPY. slide; add a drop of normal salt solution (vide formulae); mix with a needle, and put on the cover-glass. With H. observe, among the numerous blood-corpuscles, fat globules, etc., the polyhedral liver cells, about twice or three times the FIG. 34.— GLANDULAR EPITHELIA. A, A. Polyhedral cells from human liver. B. Double nuclei. O. Cells from same showing connection with a capillary. D. Same cells infiltrated with globules of fat. E. Cells from liver of pig showing intracellular net- work, x 400 , diameter of a white blood-corpuscle (Fig. 34). Notice the large spherical nuclei, with nucleoli. Note, also, the yellow pigment granules and the fat globules in the body of the cells. Masses of these cells resemble somewhat pavement epithelium; they are not flat but poly- hedral. STELLATE CELLS. When we arrive at the study of the skin, I shall show you some very beautiful examples of stellate cells. I prefer to leave their demonstration until you have become more familiar with tissue cut- ting. POLAR CELLS. . As I have stated, spheroids may send off processes. These pro- longations may be one, two, three, or more in number, constituting unipolar, bipolar, tripolar, etc., cells. The best demonstration is made from the nervous system, where these poles are continued as nerves, etc. WHITE FIBROUS TISSUE. 51 From a freshly slaughtered ox, sheep, or pig (the first being the best) obtain a piece of the spinal marrow from the region of the neck. Out it transversely into discs about one-eighth of an inch thick, and place them in the chromic-acid fluid diluted with an equal bulk of water. After thirty-six hours, place one of the pieces in water, and with a needle pick out minute fragments from the anterior horn of the gray matter (refer to the diagram of the spinal cord) and trans- fer them to a slide. Add a drop of water and break the tissue into minute fragments by teasing with a pair of needles. Examine from time to time with L. to note the progress of the teasing. When properly teased, put on the cover-glass and search for large nucleated cells from which the prolongations or horns are given off. Compare with Fig. 123. Cells may be classified as follows: Epithelial — covering-cells, as in skin. Endothelial — lining-cells, lining vessels or cavities, n' Glandular — constituting the parenchyma of organs. \ CONNECTIVE (FIBROUS) TISSUES. Certain elementary structures of similar origin and mode of de- velopment, and serving alike to unite the various parts of the body, have been termed connective tissues. Custom has restricted the term, in its every-day employment, so as to apply to white fibrous tissue or, at least, to tissue which always resembles this more closely than any other, and I shall so use the expression in this work. WHITE FIBROUS TISSUE. This, the connective tissue par excellence, is composed of exceed- ingly fine fibrillae (one-fifty thousandth of an inch), which are aggre- gated in irregularly sized and variously disposed bundles. It forms long and exceedingly strong tendons connecting muscle and bone; its fibres interlace, forming the delicate network of areolar tissue ; it forms thin sheets of protecting and connecting aponeuroses; or, sup- porting vessels, it permeates organs, and sustains the parenchyma of glands. The fibres are held together by means of a transparent cement, which may be softened or dissolved in acetic acid. They may exist, as in dense tendons, without admixture. Cells are found between the bundles of fibres, known as connective- tissue corpuscles or fibro-blasts. The older and more dense the struc- ture, the less frequent are these cells ; while in young (recent) con- 52 PRACTICAL MICROSCOPY. nective tissue, stained, the nuclei of the corpuscles constitute a prominent feature of the specimen under the microscope. Having obtained a piece of tendon from a recently killed bullock, tease a fragment on a slide in a few drops of water. Select a portion which splits easily and separate the fibrils as much as possible. Cover and examine H. FIG, 35.— CONNECTIVE TISSUE. A. Teased fibres from a tendon. C. Fibrillfie. B. New connective tissue from a cirrhotic liver. D. Elongate cells in last showing mode of formation of fibrillae from cell elements, x 400. Fine, wavy fibres are seen composing the fasciculae. If the dissec- tion has been sufficiently minute, you may succeed in demonstrating ultimate fibrillae. These are best made out, as at 0 in Fig. 35, where the parts of a bundle have been separated for some distance, leaving the finer elements stretching across the interval. B in Fig. 35 shows recently formed connective tissue from the liver, where this structure had so increased as to largely obliterate the parenchyma of the organ. YELLOW ELASTIC TISSUE. This tissue consists of coarse shining fibres (averaging one-three thousandth of an in.) which frequently branch and anastomose. They are highly elastic. Under the microscope the fibres are colorless ; but when aggregated, as in a ligament, the mass is yellow. YELLOW ELASTIC TISSUE. 53 FIG. 36.— TEASED YELLOW ELASTIC TISSUE FROM THE LIQAMENTUM NUCHJE. X 250. FIG. 37.— TRANSVERSE Sscn >N OF PART OF THE LIGAMENTUM NUCH.E. S. Sheath of the ligament, sanding prolongations within -as at T T— dividing the structure into irregular bundles or fasciculae. L. Lymph spaces in the connective tissue. A. Adipose tissue in the sheath. V. Blood-vessels in transverse section. E5 E. Primitive fasciculae of yellow elastic tissue fibres. X 250. 54 PRACTICAL MICROSCOPY. Procure a small piece of the ligamentum nuchce of the ox, and tease- it on the slide after its having been macerated in acetic acid for a few moments. The acid softens the fibrous connective tissue and facili- tates the teasing process. The individual fibres having been isolated, they appear as in Fig. 36. When broken, they curl upon themselves like threads of India rubber. This tissue is variously disposed throughout the body where great strength with elasticity becomes necessary. The large arteries are abundantly supplied with elastic fibre, arranged in plates, in alterna- tion with muscle. As a network, it is mixed with connective ti>sue in the skin, and in membranes generally. It contributes elasticity to cartilage where the fibres form an intricate network. Ligaments are composed largely of yellow elastic tissue. Fig. 37 is drawn from a portion of a stained, transverse section of the ligamen- tum siifrflava. A strong sheath of fibrous tissue is thrown around the whole liga- ment, a portion of which is seen at S. This sheath sends prolonga- tions, T, T. into the structure, dividing it into irregular bundles, which support nutrient vessels. The elastic fibres seen in transverse section, as at E, E, are observed strongly bound together with fibrous tissue, which penetrates the smaller fasciculae, dividing them into the ultimate fibr illce. ADIPOSE TISSUE. Adipose or fat tissue is a modification of, and development from ordinary connective tissue. It originates in certain contiguous connective-tissue corpuscles, be- coming filled with minute fat globules. These ultimately coalesce and form single, large globules, which bulge out the cell-bodies until they become spheroids ; the nuclei at the same time are displaced to the periphery. An aggregation of such cells forms a lobule of adi- pose tissue. The cells are often so closely packed as to assume a polyhedral form. From malnutrition, this fat may be absorbed, ordinary connective tissue remaining. You will bear in mind the fact that whenever fat exists in a condi- tion of minute subdivision, the particles always assume the globular form; and that while adipose tissue contains fat, fat alone is not adipose tissue. ADIPOSE TISSUE. FIG. 38.— CONNECTIVE-TISSUE CELLS CONTAINING FAT— INDICATING THE MODE OF FORMATION, or ADIPOSK TISSUE. A. Ordinary elongate connective-tissue cells. B. Same containing minute globules of fat. C. Coalescence of the fat globules and displacement of the nucleus. D. Still greater increase of the fat. x 400. FIG. 39. — ADIPOSK TISSUE FROM TEASED HUMAN OMENTUM STAINED WITH ELEMA. A. Connective-tissue framework. B. Cells distended with fat showing fat crystals. C. Cells from which the fat has been dissolved by ether. D. Cells faintly seen below the more sharply focussed plane. X 400. 56 PRACTICAL MICROSCOPY. CAETILAGE. Cartilage consists of a dense 'basis substance, in which, cells or chon droblasts are imbeded. It presents in three forms. HYALINE CABTILAGE. The matrix of hyaline cartilage is transluscent, dense, and appar- ently structureless. Minute channels in certain instances, and deli cate fibrillae in others, have been demonstrated. Fia. 40.— SECTION OF HYALINE CARTILAGE FROM A HUMAN BRONCHUS. The ground substance is apparently structureless, and it contains the membrane-lined exca- vations in which one, two, three, or more cartilage cells appear. These cells show a well- marked intra-cellular network, x 400. The basis material contains excavations, generally spherical, called lacunce. They are lined with a delicate membrane and contain one, two, three, and perhaps as many as eight cells — the cartilage corpus- cles. Hyaline cartilage is found covering joints generally, where it is termed articular cartilage. It is also found in the trachea, the bronchi, the septum narium, etc. Fig. 40 shows a section from one of the rings of a large bronchus. FIBRO-CARTILAGR. Fibrous connective tissue predominating largely in the basis sub- stance, produces a structure of great strength — fibro-cartilage. The ELASTIC OR RETICULAR CARTILAGE. 57 intervertebral discs afford an example of this variety, from a section of which Fig. 41 has been drawn. The membrane lining the lacunae FlO. 41.— FlBRO-CARTILAGft FROM AN INTERVERTEBRAL PLATE OR DlSC. The ground-substance, unlike that of the hyaline variety, consists of dense fibrous tissue with little calcareous matter, x 400. is much thicker than in the previous example, and the fibrous tissue is a very prominent feature of the ground substance. ELASTIC OR RETICULAR CARTILAGE. As the name implies, yellow elastic tissue is an important element of the ground substance of elastic cartilage. It presents in the form FIG 42.— ELASTIC CARTILAGE PROM EAR OF BULLOCK. The ground-substance consists largely of a network of coarse, yellow elastic tissue X 400. 58 PRACTICAL MICROSCOPY. of a reticulum, as shown in Fig. 42. It is not extensively distributed in the human being, although the cartilages of the external ear, Eusta- chian tube, etc., are of this variety. Cartilage should be hardened by the chromic acid and alcohol pro- cess. The sections from which the illustrations have been drawn were cut without the microtome. They should be cut extremely thin, not necessarily large. We frequently succeed in getting good fields from the thin edges of sections which may be elsewhere too thick. Stain with haema. and eosin. The differentiation will be excellent. The delicate nutritive channels in the matrix connecting the lacunae may be demonstrated in the cartilage of the sternum of the newt; the xiphoid appendix is sufficiently thin without sectioning. BONE. Bone consists of an osseous, lamellated matrix, in which occur irregularly shaped cavities — la>'unce. The latter are connected by means of exceedingly fine channels — canaliculi. The lacunae contain FIG. 43.— PORTION OF A TRANSVERSK SECTION FROM A DRIED FEMUR SHOWING PART OF THE WAIJU OF AN HAVERSIAN SYSTEM. A, A. Bony laminee. B, B, Lacunse. C, C. Canaliculi. X 400. BONE. 59 the fione corpuscles, the bodies of which are projected into the cana- liculi. In compact bone, the blood-vessels run in a line parallel with the long axis of the bone, in branching inosculating channels (averaging one-five hundredth of an inch) — the Haversian canals. The lamellae of osseous tissue are arranged concentrically around these canals. A single Haversian canal with the lamellae surrounding and belonging to it constitute an Haversian system. The lamellae beneath the periosteum are not arranged as above, but FIG. 44. — TRANSVERSE SECTION OF PORTION OF A DRIED LONG BONE, SHOWING THE HAVERSIAN SYSTEMS. A, A, A. An Haversian system. B. Haversian canal. The lacunae, canaliculi, and Haversian canals all appear black in the section, as they are filled with air and the bony fragments resulting from grinding of the section, x 60. parallel with the surface of the bone. These plates are perforated at right angles, and obliquely by bloo>l- vessels from the periosteum, as they pass on their way to the Haversian canals. These lamellae are also perforated by calcific connective tissue — the perforating fibres of Sharpcy. An Haversian canal contains (Fig. 44) an artery, a venule, lymph 60 PRACTICAL MICROSCOPY. channels, and a nerve filament. The whole is supported by connective- tissue cells with delicate processes. The walls of the lymph spaces are prolonged into the caualiculi and thus placed in connection with the elements of the surrounding lacunae. FIG. 45.— DIAGRAM OF AN HAVERSIAN CANAL. A. Artery. B. Vein. C. Nerve. D. D, D. Lymph-channels. Each lacuna contains a bone corpuscle, the protoplasmic body of which sends prolongations into the contiguous canaliculi. In the adult bone, the cell is shrunken ; and the processes just mentioned are not readily demonstrable. FIG. 46. - DIAGRAM OF A BONE LACUNA. A, A. Ground-substance of the bone. B, B. Limiting membrane of the bone corpuscle within the lacuna. C, Nucleus and nucleolus of the corpuscle. D, D. Projection of the cell-body into the canaliculi. " Fig. 44 has been drawn from a section of dry bone which has been sawn as thin as possible, and afterward rubbed down on a hone with water. It is a tedious process, and shows little but the osseous ma- trix. Bone should be decalcified for microscopical work, and it may SPECIAL CONNECTIVE TISSUES. 61 then be readily cut in thin sections with a razor. The process is as follows; To four ounces of the dilute chromic-acid solution add a drachm of 0. P. nitric acid. The bone, previously divided into slices not over one-fourth cf an inch in thickness, is then placed in the fluid, and should be completely decalcified in a week or ten days. Examine the pieces after twenty-four hours by puncturing with a needle. Should the action proceed too slowly, add a few drops more of the nitric acid from time to time. The bone eventually takes on a green color. After complete decalcification, wash the pieces for twenty-four hours in clean water, and preserve them until required, in " B " alcohol. Small pieces of young bone may be decalcified in a saturated aqueous solution of picric acid. The process is slow, but it leaves the tissue in excellent condition. Sections cut in the usual way may be stained with carmine and picric acid, and examined in a drop of glycerin. They should not after the staining be placed in the oil of cloves, as they would curl and become hard. Transfer them to equal parts of glycerin and water, from which they are to be carried to the slide. Add a drop more of the dilute glycerin if necessary and put on the cover-glass, carefully avoiding air bubbles. If you desire to make a permanent mounting, the edge of the cover must be cemented to the slide. Thoroughly wipe the slide, around the cover, with moistened paper, until every trace of glycerin is removed. Then with a sable brush, paint a ring of zinc cement (vide formulas) around the slide just touching the edge of the cover-glass. Repeat the cementing in twenty-four hours. A turn-table will be a useful aid in this work. Dr Carl Heitzmann, who uses glycerin as a universal mounting fluid, prefers ordinary black (asphalt) varnish as a cement. SPECIAL CONNECTIVE TISSUES. Connective Tissue of the Lymphatic System. — The matrix of lym- phoid or adenoid tissue consists of a network of branching cells, which support the lymph corpuscles. It is distributed extensively in organs, and where it appears in stained sections, the lymphoid cells are so numerous as to obscure the reticulum almost entirely. The structure will be minutely described in connection with the lym- phatic system. The Connective Tissue of the Central Nervous System (neuroglia) consists of branched connective-tissue cells, which are supported in 62 PRACTICAL MICRO-SCOFF. 3-n intimate network of exceedingly fine elastic fibrillae, and will re- ceive attention later in our work. Embryonic Connective Tissue presents a homogeneous, mucoid matrix containing branched cells. It is not found normally in the adult. MUSCULAR TISSUE. This tissue is found in three varieties: 1. Non-striated or involun- tary; 2. Striated, red, skeletal, or voluntary; 3. Cardiac. N02ST- STRIATED MUSCLE. The histological element of non-striated muscle is a spindle-shaped cell from one-tenth to one-five hundredth of an inch long. The cell body presents longitudinal striae, and contains an ovoid nucleus. The nucleus contains a reticulum which is probably in connection with the FIG. 47.— WALL OF THE FROG'S BLADDER, STAINED WITH A. A. Bands of involuntary muscular fibre, recognized by the spindle-cell sarcous elements. B. A small arteriole, showing the same muscular element. C. Scattering muscle cells. D. Connective tissue cells. X 400. fibrillae, which produce the longitudinal striation of the body. The cells are not unfrequently bifid at one or both extremities. A trans- parent cement substance serves to unite these cells in forming, with connective tissue, broad membranous plates, bundles, plexuses, etc. STRIATED MUSCULAR TISSUE. DO It serves to afford contractility, especially to the organs of vegetative life. Kill a good-sized frog by decapitation, and open the abdomen on the median line. Fill the bladder with air, after the introduction of a blowpipe into the vent. Remove the inflated bladder with a single cut with the curved scissors, and place it in a saucer of water. Pro- ceed to brush it, under the water, with two earners hair pencils so as to remove all of the cells from the inner surface. It will bear vigor- ous rubbing with one of the brushes, holding it at the same time with the other. Transfer to alcohol for ten minutes, and afterward stain with haema. and eosin. While in the oil, cut the tissue into small pieces, and mount flat in dammar. Examine L. and H. Observe the bands of involuntary muscle crossing in various direc- tions. You will distinguish (Fig. 47) between the muscle and the connective tissue cells by their nuclei. STRIATED MUSCULAR TISSUE. A skeletal or striated muscle consists of cylindrical fibres, one-three hundredth to one-six hundredth of an inch in diameter, and one to two inches long. These primitive fibres are supported by a delicate, FIG. 48.— DIAGRAM INDICATING THE MINUTE STRUCTURE OF STRIATED MUSCULAR FIBRE. A, A. Sarcolemma. B, Krause's line connecting with the sarcolemma and dividing the fibril into compartments. C, C. The rod-like contractile substance. D , Hensen's middle disc. 64 PRACTICAL MICROSCOPY. transparent sheath — the sarcolemma. They are aggregated, forming primitire fasciculi, which are again united to form the larger bundles of a complete muscle. The connective tissue uniting the primitive fibres is termed endomysium ; while that uniting the primitive bun- dles is the perimysium. The primitive muscular fibres exhibit marked cross striations with FIG. 49.— STRIATED MUSCULAR FIBRES PROM THE TONGUE, TEASED AND STAINED WITH A. A fibre, with the muscle substance wanting, from stretching during the teasing, the sar- colemma alone remaining. B. Partly separated disc of Bowman. C. Ultimate fibrillae. D. A blood capillary. X 400. faint longitudinal markings, the former being produced by alternate dark and light spaces. Fig. 48 illustrates diagrammatically the theory of the structure of a primitive fibre: A indicates the sarcolemma. The dark substance B, B (Krause's membrane) divides the fibre completely, and is united with the sarcolemma. The light spaces C, C, between Krause's membranes, containing the contractile substance, are termed the muscular compartments or discs of Bowman. This contractile sub- CARDIAC MUSCULAR FIBRE. 65 stance in the living muscle is semi-fluid, but in hardened tissue it can be split up, as indicated at 0, into rods, the sarcous elements. A transparent line, D, in this contractile substance can sometimes be demonstrated; it is known as Hensen's middle disc. Macerate human muscle, preferably that from the tongue, in dilute chromic acid for twenty-four hours; wash, tease in water, cover and focus H. Fig. 49 was drawn from such a preparation. The sarcolernma is best seen where the contractile substance has been broken. The muscle nuclei are seen at various points beneath the sarcolemma. Portions of a fibre have been split off transversely in places, indicating the discs of Bowman. Sarcous elements are in- dicated where the fibre has been split during the teasing. The capillaries are arranged in a direction parallel to the fibres, with fre- quent transverse connections. CARDIAC MUSCULAR FIBRE. It presents the following characteristics: 1. The fibres are smaller than those of ordinary skeletal muscle, 2. They are striated both transversely and longitudinally, 3. They branch, forming frequent inosculations. FIG. 50.— TEASED CARDIAC MUSCULAR FIBRE. Stained with heema. X 400 and reduced. 4. They are divided by distinct transverse lines into short prisms. 5. Their nuclei are situated within the fibre. 6. They present no distinct sarcolemma. 5 66 PRACTICAL MICROSCOPY. Fig. 50 has been drawn from fresh cardiac muscle, teased in nor- mal salt solution, and tinted with eosin. BLOOD-VESSELS. Blood-vessels include arteries, arterioles, capillaries, venules, and veins. They are all lined with flattened endothelial cells cemented by their edges; and their walls are constructed from non- striated muscular, yellow elastic and fibrous connective tissues, in proportions varying according to the size and function of the vessel. Arteries are the active, while the veins are comparatively passive agents in the circulation of the blood. The large arteries are eminently elastic, from preponderance of yel- low elastic tissue; while the arcerioles are eminently contractile, from excess of muscular fibre. FIG. 51.— TRANSVERSE SECTION OF A MEDIUM-SIZED ARTERY, PARTLY DIAGRAMMATIC. A. The endothelial cells in profile. B. Elastic and connective tissue supporting the endothelium. C. The internal elastic lamina or fenestrated membrane. A, B, and C constitute the INTIMA of the artery. D. The MEDIA. It consists of muscular and elastic tissues in alternating layers. E. Points to one of the elastic layers. F. The ADVENTITIA. Loose, connective tissue, with few elastic fibres. Arteries possess three coats: the intima (internal), media (middle), and adventilia (external). Fig. 51 represents a medium-sized typical artery. The intima, or internal coat (1), consists of a layer of flattened endothelial cells, which rest upon fibrous connective tissue, with a few elastic fibres. The last is surrounded by a layer of elastic tissue, the elastic lamina or fenestrated membrane, which is the external limit of the intima. It presents in a transverse section as a wavy (from contraction of BLOOD-VESSELS. O7 the media) shining line; and is an important element, from its rela- tion to certain abnormalities of the blood-vessels. The media (2) consists of alternate layers of elastic and muscular tissue. The ad- ventitia (3) is composed of fibrous connective tissue, containing some elastic elements. As we approach the larger arteries, the muscular tissue diminishes in quantity and the elastic tissue is increased. On the other hand, the elastic element diminishes with preponderance of muscle as we approach the smaller arteries, until we meet the arterioles, the walls of which are made almost exclusively of involuntary muscular fibre. The walls of capillaries consist of a single layer of flattened endo- thelial cells cemented by their edges. The union is not quite con- FIG. 52.— ISOLATED BLOOD CAPILLARIES. A. Plexus from a pulmonary alveolus, stained with ssilver. X 350. B. Capillary from omentum, stained with silver and hsema. X 700. In A, the cells are outlined by the silver; while in B the nuclei in addition are brought out by the haema. tinuous, as minute openings (stomata) are to be seen at irregular in- tervals. The walls of veins are much thinner than those of arteries. The intima presents an endothelial lining, but no fenestrated membrane; and the line of demarcation between this coat and the media is often indistinct. The media contains muscular, but little elastic tissue; and the adventitia, usually the most prominent of the three coats, is composed largely of fibrous connective tissue. I shall defer the microscopical examination of blood-vessels until we meet them in future sections of organs, as they are best studied in such connection. PART THIRD. ORGANS. THE SKIN". The skin consists of (1) the epidermis (or scarf skin), which every- where covers and protects (2) the derma (corium or true skin). The epidermis varies greatly in thickness in different locations; and in the thicker portions several layers may be differentiated. It is composed entirely of cells, while the derma is fibrous. Stratum Gorneum, \ H L , | /Stratum Luciaum, } Stratum Granulosum } Malpighian Layer \ I Stratum of Prickle Cells, \ W ,| Muoomim 5. Stratum of Elongate (Pigment) Cells, ) ° becomes straight, and passes into a pyramid of Malpighii. It reaches sometimes just into the pyramid , more frequently, however, passing deeper than this — often descending two-thirds of the distance to the apex; and is called the descending limb of Henle. Henle's descending limb suddenly turns upon itself, FIG. 86.— DIAGRAM SHOWING THE DIVISIONS OP A KIDNEY TUBULE. forming a loop; and, widening (ToVg- ")> returns upon its course as the ascending limb of Henle. It again enters the cortex, keeping in a pyramid of Ferrein, and passes outward until it approaches the outer limit of the cortex, near the capsule of the kidney. Here the ascend- ing limb of Henle widens (-5-^3- ")> forming the distal convoluted, which pursues a tortuous course in the outer cortex. The distal con- 124 PRACTICAL MICROSCOPY. voluted then re-enters a pyramid of Ferrein, narrows (-g-J-g- "), and passes a second time into a Malpighian pyramid, under the title of straight or collecting tube, or tube of Bellini. The last, after reach- ing very nearly to the apex of the pyramid, unites with others of a like character, and forms principal tubes (TUT")- Several principal tubes unite to form a papillary duct (T-J-¥ ")• From 100 to 200 of the last open upon the surface of the apical portion of a Malpighian pyramid. It must be borne in mind that, in describing the tubular system, although such terms as "convoluted tube," " looped tube," etc., are employed, these are not separate tubes, but only names applied to different portions of one long tube. A single tubule, then, com- mences at Bowman's capsule, becomes narrowed like the neck of a flask; courses as the proximal convoluted and spiral; descends into, turns, and emerges from a Malpighian pyramid, as Henle's looped por- tion; reaches the extreme cortex, and swells as the distal convoluted; and here ends as a single or isolated tubule and enters a straight tube. The straight tubes receive several distal convoluted termini, at the cortical periphery, and pass in small bundles (forming the pyramids of Ferrein) directly onward toward the apex of a Malpighian pyramid; uniting with one another at very acute angles; the resulting trunks uniting until the tube terminates as a papillary duct. The tubes are lined with epithelia; and these cell elements con- stitute the parenchyma of the kidney. The lining cells are, as a rule, of the columnar variety. Two exceptions are presented, one of which appears in the flattened cells lining Bowman's capsule, and the other in a like form, in the descending limb of Henle's loop. The parenchyma will receive attention in our practical work. BLOOD-VESSELS. The vascular arrangement is complex. The most prominent and essential feature is afforded in the existence of two distinct capillary plexuses. The renal artery, as already described, sends branches into the substance of the kidney. These pass between the Malpighian pyramids, and.m the cortical columns. These arterial trunks arch over the bases of the pyramids of Malpighii, forming the arterial arcade. From these arches small straight branches are sent outward toward the capsule of the kidney, occupying a position midway be- tween the pyramids of Ferrein, in the labyrinths. The last are the interlobular arteries. During their course, they send off side arterioles which penetrate the capsule of the Malpighian bodies. THE KIDNEY. 125 Each afferent arteriole breaks up into a capillary plexus — the tuft or glomerulus. The glomerulus does not entirely fill the capsule, so that a space remains between the spherical mass of capillaries and FIG. 87.— DIAGRAM SHOWING THE ARRANGEMENT OF BLOOD-VESSELS IN THE KIDNEY. After Ludwig. the flattened cells lining the body. The glomeruli are enveloped with a single layer of flattened epithelial cells. The blood escapes from the glomerulus by one or two efferent 126 PRACTICAL MICROSCOPY. orterioles which emerge from the capsule close to the afferent vessel. The latter is the more noticeable, as it is usually much the largest. The efferent arteriole immediately breaks up into a second capillary plexus, which courses between the uriniferous tubules of the labyrinths and of the pyramids of Ferrein. This second plexus also descends between the elements of the pyramids of Malpighii. From the arteries forming the arcade another set of branches — the arterialce rectce — is given off; which, descending into the Malpighian pyramids, provides another and direct arterial supply to the tubular elements by elongate capillary loops. The course of the venous trunks is not unlike that pursued by the arteries. Interlobular veins pass into a venous arcade ; the former lying in the cortical labyrinths parallel with and close to the arteries. In the medulla the venous blood is collected from the capillaries and carried to the bases of the Malpighian pyramids in small veins— venulae rectae. The blood from the cortical intertubular capillaries is col- lected in the interlobular veins. A peculiar vascular arrangement exists just beneath the capsule of the kidney, consisting of scattered venous plexuses, the stars of Verheyen. They contain blood collected from contiguous inter- tubular capillaries and are in connection with the summits of the interlobular veins. From what has been said, it will be seen that the cortical and medullary blood- supplies are, to a certain extent, independent of one another. The arteriolae rectae provide a vascular supply to the elements of the Malpighian pyramids even after many of the glomer- uli have become obliterated by disease. Nerve and lymphatic elements are not very prominent features in sections of the kidney. Small medullated nerve trunks may be easily demonstrated in transverse sections of the cortex, especially near the bases of the medullary pyramids, where they will be seen, in company with the blood-vessels of the arcades. Lymph channels are also to be seen in the vicinity of the vessels of the hilum, and in the connective tissue of the capsule. The nervous system of the kidney would prove a valuable field of labor, and would well repay the advanced student's patient and earnest investigation. ' The histology of the kidney will be better comprehended by a reference to its functioning. The separation from the blood of a quantity of water, together with certain excrementitious matters, is effected, partly in the Malpighian bodies, and partly in the tubules. The vascular tuft — the glomerulus— is covered with a close fitting membrane composed of flat cells. The blood in this plexus parts THE KIDNEY. 127 with a certain amount of its water, which passes through the walls of the capillaries and through the cells covering them. Whether this be due to osmosis or to some selective power of the cells we have no concern — suffice it that certain salts afterwards appearing in the urine do not leave the blood at this point. The efferent glomerular arteriole, it will be remembered,, breaks into a second capillary plexus, which brings the blood close to the walls of the convoluted tubules. We believe that the cells lining these tubules select from the blood, circulating in the contiguous capillaries, such effete materials as es- caped elimination from the glomeruli. Moreover, that some of the water, together with serum albumin, which escaped in the first in- stance and entered the proximal convoluted tubules, is here returned to the blood by the intervention of the same tubular lining cells which excrete the salts. That in the cells of these tubules there exist currents in opposite direction — one from the intertubular capillaries into the proximal part of the tubule; and one from the dilute urine in the tubule into the capillaries. Without referring to any further work on the part of the kidney, I wish to impress this part of the structual scheme : That the first part of the uriniferous tubule is the prominent excreting part. That the latter portion of the tubule — the portion in the Malpighian pyramids, the straight tubule — is for the collection and drainage of the urine already ex- creted. And that between the excreting first part and the draining second part, there exists a narrow looped tubule — the loop of Henle. The effect of this narrowing and tortuosity of the tubule will be to present a resistance to the outflow of urine from the proximal portion of the tubule. The dilute urine, excreted in the Malpighian bodies, is held back for awhile in the proximal convoluted, and time given for the completion and perfection of the excretory processes by the tubular parenchyma. PKACTICAL DEMONSTRATION. The human kidney is rarely found in a perfectly normal condition. The demonstration can be made from the kidney of the pig, except as regards certain features. The medullary pyramids do not exist in the domestic animals, and the parenchyma presents very essential differences from the cells of the human kidney. Still, very much can be learned from the organ of the pig, dog, and rabbit. The tis- sue should be divided so as to permit sections to be made parallel with the medulla, and to include both it and the cortex. The hard- ening is best by Miiller's fluid. Small pieces hardened quickly in strong alcohol, however, stain very finely with haema. and eosin. Very pleasing differentiation may also be secured by staining slowly 128 PRACTICAL MICROSCOPY. in weak borax-carmine, clearing with glycerin, and mounting in the same medium. HUMAN KIDNEY. SECTION PAEALLEL WITH MAL- PIGHIAN PYRAMID. STAINED WITH H^MA. AND EOSIN. (Fig. 88.) OBSERVE: (Naked eye.) 1. The thickness of the cortex, and its granular appearance as compared with the medullary portion. FIG. 88.— SECTION OF HUMAN KIDNEY, CUT PARALLEL TO THE PYRAMIDS OF FERREIN. SHOWING THE CORTEX AND PART OF A MALPIGHIAN PYRAMID. X 30. A, A. Capsule of kidney. B, B. Pyramids of Ferrein. C, C. Cortical labyrinths. D, D. Malpighian bodies. Many of the glomeruli drop out in the course of preparation, and such empty capsules of Bowman appear as light circular spots. E, E. Interlobular arteries. F, F. Boundary region. G, G. Transverse sections of vessels of the arcades. H. Base of a Malpighian pyramid. THE KIDNEY. 2. The "markings of the cortex." These consist of alternat- ing light and dark lines, radiating from the bases of the Malpighian pyramids. The lighter masses consist largely of collecting tubes, together with ascending limbs of Henle's looped tubes — otherwise called medullary radii. Between these lighter areas the dark labyrinths appear ; in which, by careful attention, the Malpighian bodies may be made out as minute red dots. 3. A region just outside the medullary pyramids — not as well marked as the outer cortex, in which few Malpighian bodies present — the boundary region. 4. The finely striated medullary or Malpighian pyramids. (The section will usually include portions of two of the last.) 5. That the bases of the pyramids do not appear as a sharply defined line, but fade into the boundary region ; while the union of the latter with the cortex proper is equally ill-defined. (L.) Fig. 88. 1. The cortical labyrinths, in which search for : (a) Portions of the interlobular arteries, together with the smaller twigs of the arterial arcade. (b) The Malpighian bodies. (The tuft or glomerulus which, with this power, appears as a granular mass, is wanting in numerous places — as indicated by the empty capsules.) (c) The remaining area occupied largely by the convoluted tubes, proximal and distal. 2, The pyramids of Ferrein. (Observe that, as they pass into the pyramids of Malpighii, they are well defined, but that they are lost as they approach the region of the capsule of the organ.) (H.) Fig. 89. 1. A Malpighian body. (Select, after searching several fields, a specimen which shows either the afferent or efferent vessel of the glomerulus. It will be very difficult to find a capsule con- nected with the neck of a proximal convoluted tube, as they rarely happen to be so sectioned. You may indeed be obliged to ex- amine a dozen slides before you succeed. ) Note — (a) The capsule (of Bowman or of Muller). (Observe its thick- ness ; as this becomes important in connection with the pathology of the kidney.) (b) The flattened cells, lining the capsule. (Many of them will have become detached hTthe preparation of the section.) (c) The glomerulus. (The great number of nuclei obscures the loops of capillaries. Kemember that the nuclei belong partly to the. 130 PRACTICAL MICROSCOPY. vessels, and partly to the flattened cells covering the glomerulus. Endeavor to find transversely divided loops of the vessels, showing blood within.) (d) That the glomerulus does not, entirely, fill the capsule. (e) That the tuft is frequently divided. (f) That the tuft is usually in contact with the capsule at some one point, where search may be made for PIG. 89.— PART OP THE CORTEX OF HUMAN KIDNEY. HIGH POWER. SAME SPECIMEN AS FIG. 88. X400. A. Ascending limb of Henle's loop. B. Collecting tubule— longitudinal section. C. Collecting tubule. The upper part of the tube is not sectioned , but shows the attached bases of the lining cells; and thus simulates pavement epithelium. A, B, and C are in a pyramid of Ferrein. D. Capsule of a Malpighian body. The emerging tubule is not shown, as the body is in T. S. E. Flattened lining cells of D. F. Glomerulus. G. Efferent arterioles. . H. Afferent arteriole. I. Convoluted tubules. J. Ascending limb of Henle. K. Intertubular capillaries. (g) The afferent and efferent arterioles. (The afferent is more frequently demonstrable ; and may be differentiated by its THE KIDNEY. 131 large size and the connection, which can often be traced, with the interlobular artery.) 2. Convoluted tubules. (The convoluted tubes found just be- neath the capsule of the kidney generally belong to the distal variety ; and they are not as favorable specimens as the deeper proximal portions, on account of the crowding of the tubular ele- ments in the outer cortical regions. Select a transverse section and observe :) (a) The thin membrana propria, or wall of the the tube. (It does not appear to be made up of fibrillated connective tissue ; but, rather, presents a homogeneous structure. Nuclei, however, may occasionally be seen, which apparently belong to this tissue.) (b) The peculiar lining cells. (They are unlike any other paren- chymatous elements found in the body. Note that, while they are evidently of the columnar or cylindrical type, they differ greatly in form and size. The protoplasm is hazy, granular, and frequently striated. They take a dirty brick-red hue from the eosin.) (c) The lumen. (Compared with the diameter of the tube-wall, the lumen is very small, and presents a stellate figure. The urine, in passing through the tubule, is, consequently, brought in contact with a very considerable portion of the parenchymatous lining.) 3. The large proportion of the cortical area occupied by the convoluted tubules, and the exceedingly small amount of intertubular connective tissue. 4. The intertubular capillaries. (These are exceedingly small, and difficult of demonstration unless they be filled with blood. The nuclei of the endothelial wall are frequently seen. The cells of the convoluted tubules are not infrequently detached from the membrana propria, and the space so formed may be mistaken by the careless observer for longitudinal sections of capillaries. These vessels are much better seen in an injected kidney; although if an organ be selected containing considerable blood, and the corpuscular elements have their color preserved (as in bichromate hardening), the vessels will be easily demonstrated. 5. Ascending limb of Henle's loops, in the cortical labyrinths. (The general course of these tubules is confined to the pyramids of Malpighii and Ferrein; but occasionally one of them may be seen passing in a tortuous course toward the outer cortex, running be- - tween the proximal convoluted elements. They are easily recognized by their small size and relatively large lumen. They are lined with short columnar or cuboid cells, which stain deeply blue with the haema.) 6. The pyramids of Ferrein. 132 PRACTICAL MICROSCOPY. (a) Collecting tubes. (These will be generally recognized by their large size and the blue color of the staining. They are lined with columnar cells, which are hexagonal in transverse section; and this gives an appearance of pavement epithelium,, when they are seen from above or below. Endeavor to find a tube split through the cen- tre longitudinally and note the typical columnar cells, as they pro- ject inward from the membrana propria, toward the now open lumen.) (V) The spiral tubules. (These resemble somewhat the convo- luted tubules, especially as their cells take much the same dirty red Fio. 90.— MEDULLARY PORTION OF SPECIMEN SHOWN IN FIG. 88. X 400. A. Collecting tubule in L. S. B. Collecting tubule from above, showing attached bases of lining cells. C. Collecting tubule presenting different appearance of lining cells, according to mode of section. D. Ascending limb of Henle's loop. E. Same as last. The upper end of the tubule not sectioned. F. Descending limb of Henle's loop. Below may be seen the loop and ascending limb. Gr. Oblique section of large collecting tubule. H. Basal, attached extremities of cells lining a large collecting tubule. I. Intertubular capillaries. color. The cells however, plainly columnar, are large and hexagonal in transverse section. The lumen is small.) THE KIDNEY. 133 (c) Ascending limbs of Henle's loop. (These are small tubes and have already beeu described.) (d) The intertubular capillaries. (Inasmuch as, in the speci- imen under consideration, the vessels of the pyramids are mostly in transverse section, they are not readily made out. Especially is this true if the blood-corpuscles have their color discharged.) 7. Elements of the medullary portion. Fig. 90. (a) Collecting tubes. (These tubes constitute a large proportion of the medulla of the organ. They have been already described. As PIG. 91.— TRANSVERSE SECTION OF PYRAMID OF MALPIGHII. SAME TISSUE AS SHOWN IN FIG. 88. Stainad with Haema. and Eosin . x 400. A. Group of intertubular blood-vessels. B. Collecting— straight— tubules. C. Descending limb of Henle's loop. D. Ascending limb of Henle's loop. E. Principal— collecting— tubule. F. Principal tubule. Lower portion near the papillary duct. The ring of cells will be seen detached from the membrana propria in some instances. This is due to contraction of the tissue during the hardening. the apex of the Malpighian pyramid is approached, and the straight unite to form the principal collecting tubes, these again uniting to 134: PRACTICAL MICROSCOPY. form the papillary ducts, the lining cells will be seen to get shorter, and the lumina larger.) (b) Spiral tubes. (These can be, in many instances, followed down from the pyramids of Ferrein; and examples are frequently seen very near the pelvis of the kidney in the cortical columns.) (c) Descending limbs of Henle's loop. (These tubes are the most difficult of all the tubuli uriniferi to demonstrate. The sec- tion must be very thin, and, even then, they may be mistaken for blood capillaries. Their peculiar feature consists in the wavy lumen, which is produced by the alternate disposition of the lining cells.) (d) Loop of Henle. (The loops will be recognized by the curv- ing of the tube. They are lined with short columnar cells which are sharply brought out by the haema. On account of their course, but few complete sections are seen. ) (e) Ascending limbs. (Conveniently traced from the loop.) (/) Intertubular blood-vessels. (Do not mistake tubules con- taining blood, for capillaries. The human kidney is rarely absolutely normal; and blood is frequently found outside the proper channels. The vessels will be differentiated by the histology of their walls. Quite a number of venules will be seen running in groups in the medulla — the venulce rectce.} 8. The same elements as in 7. (shown in a transverse section of the middle of a Malpighian pyramid, Fig. 91). GENITO-URIKARY ORGANS. 135 EPITHELIUM OF THE GENITOURINARY TRACT. URETER, BLADDER, UTERUS, VAGINA, ETC. The lining membrane of the genitourinary apparatus is of interest to the medical man; particularly in connection with diseases, whose diagnosis may be largely determined by a microscopical examination of urinary deposits. In the preparation of this subject, I have confined myself, rather closely, to the consideration of such portions of the lining of the genito- urinary tract as may be recognized and differentiated, as they occur in urine and other fluid discharges. The limit prescribed for these pages will permit little beyond this. It is not always possible to determine the origin of detached cells, for two reasons, viz. : First, certain widely separated portions are very similarly cell covered; and, secondly, cells which are the result of proliferation accompanying diseased processes, are quite frequently un- like the original type. Still, certain portions of the genito-urinary apparatus have a distinctly characteristic epithelium; and to such will our present notice be directed. PEACTICAL DEMONSTKATION. From the body of a (preferably young) human female, as soon as possible after death, remove half-inch cubes of the organs required, observing that the lining is included. The outer portions are of very little moment comparatively. Secure pieces from the os, cervix and fundus of the uterus, the base of the bladder, the wall of the vagina near the cul-de-sac, the ureters, and the pelvis of the kidney. We desire to prepare the tissue so as to keep the original form of cell elements — to avoid contraction; and the Miiller process will accomplish this perfectly. Allow the pieces to remain for two weeks in the bichromate solution, with an occasional change. Complete the hardening in Nos. 3, 2, and 1 alcohol, as usual. Infiltrate with eel- loidin or bayberry tallow, and let the sections be vertical to the mucous surface. The tissues should not be handled with the fingers, otherwise the epithelial lining cells will be detached. Stain with haema. and eosin; mount in dammar. 136 PRACTICAL MICROSCOPY. UTERUS AND VAGINA OF THE HUMAN FEMALE AT PUBERTY. VERTICAL DEXTRO-SINISTRAL SECTION OF THE RIGHT LIP OF THE OS, AND INCLUDING PART OF THE VAGINAL CUL-DE-SAC. OBSERVE: (L.) 1. The outline of the section. (Commencing at D, Fig. 92, which is placed in the internal os, follow downward, out upon the FIG. 92.— VERTICAL DEXTRO-SINISTRAL SECTION OF THE RIGHT HAND SIDE OF THE Os UTERI. SHOWING THE INTERNAL Os, THE EXTERNAL Os, THE VAGINAL CUL-DE-SAC, AND THE UPPER PORTION OF THE VAGINAL WALL. X 60. A. The letter is placed in the internal os. B. Vaginal cul-de-sac. C. Vaginal wall. D. Columnar epithelium of the internal os. In the upper portion the tubular glands are well E. Stratified epithelium of the vaginal lining. F. Change at the external os from stratified flattened, to columnar epithelium. external os, curve upward reaching, at B, the vaginal cul-de-sac. Descend along the right vaginal wall.) 2. The irregular surface of the internal uterine wall. (Caused UTERUS AND VAGINA. by longitudinal section of the glandulae uterinae or g. utriculares, branched tubular glands. These are increased in depth during preg- nancy, and are most prominent in the lower portion of the organ.) 3. The epithelium, (a) The deeply stained layer lining the vagina, cul-de-sac, and external os. (b) The wavy course of a as it covers the irregularly formed and often imperfect papillae of the mucosa. (c) The lighter appearance of the lining of the inter- nal os. (d) Projection of the last into the glands, (e) The sharp line of separation between the deeply stained lining com- mon to the vagina and the lighter lining of the uterus at the ex- ternal os (Fig. 92, F). 4. The mucosa of the uterus. (There are no sharply defined regions in the genito-urinary tract corresponding to the mucosa and submncosa of typical mucous membranes. The arrangement gener- ally is : 1, an epithelial lining; 2, a subepithelial structure, consisting of a more or less prominent or abundant plexus of capillaries sup- ported by delicate connective tissue, and which corresponds to the mucosa of typical structures; 3, loose connective tissue, with more or less muscular tissue, containing larger vessels, not separated from the mucosa by any well-defined line or muscularis mucosa3, which represents the submucosa; 5, the muscular walls proper, consisting of layers in different directions, frequently irregularly disposed and sel- dom in distinct fasciculas.) 5. The mucosa of the vagina (less distinct than that of the uterus). 6. The uterine and vaginal walls (consisting largely of involun- tary muscular fibrils, recognized by the elongate and deeply stained nuclei, and containing numerous thick-walled arteries and irregular lymph spaces). (H.) 7. The uterine epithelium (Fig. 93). (a) That it consists of a single layer of cells, (b) That the cells are columnar, not cylin- drical, (c) The cells in transverse section are polygonal, (d) They are ciliated. (This demonstration is not always made; but if the section has been properly prepared from uninjured tissue, the cilia will be seen without difficulty, and especially in the depressions where they are somewhat protected.) (e) The cell body and nucleus. (Note the elongate, clear, free portion and the frequent curving of the whole. Near the attached extremities, which often appear pointed, note the small deeply stained nuclei.) (/) The large mu- cous cells. (These singular cells appear scattered between the cyl- inders, with a clear bulging body, often six times the breadth of the 138 PRACTICAL MICROSCOPY. ordinary elements.) (g) The absence of any special basement membrane. 8. The abrupt transition from columnar to flattened cells in the epithelium of the external os. (a) The shortening of the columnar cells as the point of change is approached. (Sections must be examined until one is found showing this point well. The illustration (Fig. 93) is not exaggerated, and a properly cut and selected specimen must exhibit clearly the last columnar and the adjoining flattened cell. I know of no location in the human body FIG. 93.— EXTERNAL Os OF FIG. 9,2. MORE HIGHLY MAGNIFIED. X 400, A. Muscular tissue of the os uteri, with numerous blood-vesseis. B. Capillary plexuses of subepithelial tissue — mucosa. C. Ciliated columnar cells covering the os. D. Vacuolated cells. E. Shortening of the columnar cells preparatory to F. Change from typical uterine epithelia— ciliated columnar cells— to flattened, stratified cells. G. Papillary structure of the mucosa of the external os, after the change of epithelium. where the change in form of cell covering approaches this in abrupt- ness.) 9. The vaginal epithelium (Figs. 93 and 94). (a) That it is of the stratified variety, (b) The deepest line of cells following the UTERUS AND VAGINA. 139 sinuous line formed by sectioning the papillary mucosa. (c) That the cells are more or less flattened, (d) That their edges, excepting those of the surface, are serrated. (The union is by a cement be- tween the interdigitating cell bodies.) (e) The change in form as the surface is approached. (/) The surface cells. (These are very much flattened, and so fused as to resemble, in longitudinal sec- tion, fibres.) (g) Detached surface cells. (At H, Fig. 94, these are shown in plan, having been torn off; those intact are, of course, seen in profile. Fig. 97 represents the same elements as they gener- FIG. 94.— VERTICAL SECTION OF THE VAGINAL LINING AT PUBERTY. Stained with Haema. and Eosin. x 400. A. Sub-epithelial capillary plexus. B. Papillary arrangement of the mucosa. C. Large blood-vessels in the submucosa. D. Muscular wall of vagina. E. Deep cells of the lining epithelium. F. Middle strata of lining stellate cells. Gr. Surface cells in profile. H. Surface cells in plan — detached. ally appear in a film of urine.) (h) The nuclei, evenly granular, usually larger than a red blood-corpuscle, (i) Vacuolated cells. 10. The subepithelial vaginal structures, (a) The large and abundant capillaries of the mucosa. (b) The submucosa, not 140 PRACTICAL MICROSCOPY. clearly separated from the superior coat, but easily recognized by the large vessels and the abundant connective tissue, (c) The muscular vaginal wall. (Here the muscular bundles are much better defined than in the uterine walls.) PELVIS OF THE KIDNEY AND UKETEK. TRANSVERSE SECTION OF THE URETER NEAR THE PELVIS OF THE KIDNEY, AND DETACHED CELLS FROM THE EPITHELIAL LINING OF PELVIS. (Figs. 95 and 97.) (The arrangement and form of the cells lining the pelvis of the kid- ney and the ureters are precisely similar, and so far Fig. 95 will represent both.) FIG. 95.— TRANSVERSE SECTION OP THE URETER, NEAR THE PELVIS OF THE KIDNEY. Stained with Haema. and Eosin. x 400. A. Rich capillary plexus of the niucosa. B. Internal circular muscular coat. C. External longitudinal muscular bundles. D. Large vessels of the areolar adventitia. E. Deep layer of somewhat cubical cells. F. «' Tailed cells " of the middle epithelial lining. G. Surface cells in profile. H. Surface cells in plan— detached. THE URINARY BLADDER. 141 OBSERVE: (L.) 1. The relative thickness of the epithelium. 2. The narrow mucosa. 3. The internal circular muscular belting. 4. The transversely divided bundles of the external longitudi- nal muscular layer. 5. The large arteries between the muscular bundles. 6. Adipose tissue, more or less abundant in the loose cellular tis- sue surrounding these canals. (This element will afford a prominent feature of a section of the pelvis of the kidney, while the muscular tissue will be seen to a limited extent only.) (H.) 7. The epithelium, (a) That it is of the stratified type, though poorly demonstrated, (b) The broad basal attachment of the deep cells, (c) The elongate form of the cells generally, (d) That the borders are smooth and closely adherent, unlike those of the vagina, (e) The more flattened surface cells. (/) The outline of the last, as seen in the detached specimens, (g) The very large and finely granular nuclei. (These cells contain peculiarly large nuclei, as compared with the size of the body» The deeper examples present tapering prolongations, generally at one end only, and are hence called " tailed cells/' They will not be confounded with similarly shaped, though much larger cells from the bladder. The surface elements, while sometimes nearly circular, generally pre- sent one or two incurvations of the periphery, indicating their con- nection with the neighboring cells. These peculiarities are best exhibited in Fig. 97.) 8. (Keview the objects previously examined with low power.) THE URINARY BLADDER. VERTICAL SECTION OF IKNER PORTION OF WALL. (Fig. 96.) OBSERVE: (L.) 1. The epithelial lining, (a) That it is formed after the strati- fied type, (b) That, as compared with other previously studied por- tions of the genito-urinary tract, the epithelium is thin. 142 PRACTICAL MICROSCOPY. 2. The thin mucosa and its small capillary supply. 3. The dense muscular portion, not arranged in bundles. 4. The epithelium, (a) The magnitude of the cells. (Z>) The broad based cells without any special basement membrane. FIG. 96. — VERTICAL SECTION OF THE LINING PORTION OP THE BLADDER (MALE) BEHIND THE TRIGONE. Stained with Hsema. and Eosin. X 400. A. Connective tissue of subepithehal region, containing large amount of muscular fibre. B. Scant capillary supply of subepithelial region. C. Muscular wall of bladder. D. Large basement cells of the epithelial lining. E. Middle region of lining. F. Detached surface cells showing processes beneath. G. Thin surface cells in profile. H. Squamous surface cells, seen detached, in plan. I. Vacuolated cells. (c) The three regions, viz., basal, middle, and superficial. (<$) The form of the middle cells; not unlike in outline, though larger, those of the corresponding region in the ureter. (This is best shown in Fig. 97.) (e) The large, scaly, and often fused surface epithe- THE URINARY BLADDER. 143 lia. (Note that while these, when seen in plan, all appear flat, it is only those of the extreme surface that are simple scales; the less superficial examples show, when viewed in profile, prolongations FIG. 97.— CELLS FROM GENITOURINARY TRACT. ISOLATED BY TEASING, AS IN TEXT, x 400. A. Surface bladder cells. B. The same seen partly in profile. C. C. Bladder cells from deeperjayers. D. Surface vaginal cells. E. Vaginal cells from deeper layers. F. Superficial cells from pelvis of the kidney or upper ureter. G. H. From the same as F — deep layers. " Tailed Cells." G is the more usual form. I. Ciliated vaginal cells. J, K. Cells from collecting tubules of the kidney. L. Pus-corpuscles. Not stained. M. Red blood-corpuscles. L and M are introduced as measurement standards of comparison. from the under surface, by means of which union is effected with the deeper cells.) (/) Vacuolated cells. (These vacuolations do not occur in the basal layer.) 144 PRACTICAL MICROSCOPY. THE OYART. The ovary consists of a stroma or ground substance of connective and smooth muscular tissue, in which are scattered various sized spherical bodies or Graafian follicles. The stroma is divided into three layers or regions, which are not very sharply differentiated. The ovary is covered upon its free surface with a single layer of cells which in early life are cylindrical, becoming shortened with ad- vancing age until after the menopause, when only flattened scales can be demonstrated. Immediately beneath the epithelium a thin layer of fibrous tissue presents, with a free admixture of smooth muscle cells, and is termed the tunica albuginea. The cortex proper, or second layer, is distinguished by the Graafian follicles, which will be described later. The central portion of the organ, the zona vasculosa, is largely occupied by thick-walled blood-vessels, among which the extremely tortuous arteries are specially evident. Occasionally may be seen in this region somewhat ovoid nodules in varying degrees of retrograde change — the corpora lutea. They present the phenomena resulting from the maturation of the follicle during menstruation. The accom- panying illustration was drawn from a corpus luteum which had formed in the site of a Graafian follicle, the contents of which had escaped at some menstrual epoch, and been followed by impregnation. PEAOTIOAL DEMONSTRATION. The ovary of a young animal is to be preferred. If the organ can- not be obtained from the human subject, the female of almost any domestic animal will provide an excellent demonstration for the his- tological elements. Let the tissue be hardened with strong alcohol, and sections be cut vertically to the free surface and stained with hsema. and eosin. The sections should include at least one-half the the depth of the organ, so as to exhibit all of the regions. SECTION OF THE ADULT HUMAN OVARY. (Fig. 98.) OBSERVE: CM 1. The tunica albuginea. (Note that the layer is not of uniform thickness, and is composed largely of smooth muscular tissue, as THE OVARY. 145 shown by the numerous elongate nuclei. Search particularly for and note the character of the epithelial covering.) 2. The cortical layer, containing numerous Graafian follicles, and possibly a corpus luteum. (Note the aggregation of the smaller fol- licles in the extreme outer portion of the region.) 3. The zona vasculosa. (Note the unusual thickness of the vas- cular walls and the irregular outline of section, on account of their tortuous course.) FIG. 98.— SECTION OP THE OVARY FROM A WOMAN 35 YEARS OLD. Stained with Haema. and Eosin. x 250. A. Surface of the ovary. B. Muscular stroma. C. Large tortuous, thick-walled arteries of the central portion of the organ. D. D. Small Graafian follicles of the superficial zone. E. Larger follicles of the deeper portion. F. Membrana propria of a Graafian follicle. G. Membrana granulosa of the follicle. The line leads to the discus proligerus. H. An ovum. I. Germinal vesicle. J. Germinal spot. K. An old corpus luteum. (H.) 4. The Graafian follicles, (a) Their diameter, varying from one-one hundredth to one-one thousandth of an inch, (b) The mem- 10 146 PRACTICAL MICROSCOPY. brana propria. (This is difficult to separate from the stroma of the ovary itself, except in more mature follicles than shown in the sec- tion.) (c) The membrana granulosa. (This, in general, appears to be the outer layer of the follicle, on account of the difficulty of separating the membrana propria from the stroma proper of the ovary. Note that it is composed, in the smaller and less mature fol- licles, of pavement cells, and that the cells become thicker with matu- ration, until columnar cells in single layer result.) (d) The ova. '(These are contained within the follicles, excepting they may have become detached during manipulation of the section, and occupy the :greater area of the same.) (e) The zona pellucida (the thin wall of the ovum). (/) The discus proligerus. (This will be recognized ;as a mass of polyhedral cells, connecting the ova at one side with the columnar cells of the membrana granulosa. These cells will prolif- erate later in the development, and completely inclose the ovum.) (g) The germinal vesicle. (Contained within the ovum. The con- tents appear granular; it, as well as the ovum, is fibrillated; but this demonstration cannot be made excepting the animal be killed for the purpose, and the tissue elements fixed before changes, which quickly follow death, occur.) (h) The germinal spot. (Appearing as a small dot within the germinal vesicle. The ovum presents the char- acteristics of what it indeed is— a typical cell, with wall, body, nucleus, and nucleolus. ) 5. The corpus luteum. (The example shown in the drawing, as I have already said, was developed after the contents of the Graafian follicle, which it represents, had suffered impregnation; and it has arrived at the later stage of the series of phenomena connected with its development — the stage of cicatrization. The cicatricial tissue, to which the letter K points, indicates the remains of the membrana granulosa. Outside is seen the thickened membrana propria, while among the contents will be found pigment granules and fat globules, imbedded in a structureless, gelatinous stroma. This material results from changes in the clot of blood effused after the escape of the ovum. I do not tabulate these elements, as it is extremely improb- able that the student will find a corpus luteum in precisely the condi- tion of the one represented until he has examined a large number of specimens. ) DEVELOPMENT OF THE OVUM. 14Y DEVELOPMENT OF THE OVUM. As has been previously shown, the ovary is covered with epithe- lium; and singular as it may appear, the fifty thousand Graafian fol- licles, which it is estimated are developed during the life of the human female, have their origin in these cells. During foetal life, this surface epithelium undergoes a very rapid proliferation, and chains of cells are imbedded in the stroma of the ovary. A little later in the development, separate portions or links of these chains are cut off by the ingrowth of the stroma. The little groups of cells thus isolated become each a Graafian follicle. Scattered among the columnar cells, larger, more nearly spherical cells are also found, the primordial ova. These are also imbedded in the substance, and one at least will always be found among the minute collections of cells which have been isolated. In the process of development, each group of cells becomes a Graa- fian follicle with its contained ovum, the columnar cells forming the wall proper, and the primordial cell the ovum with its vesicle and germinal dot. PEACTICAL DEMONSTRATION. The ovary from a still-born babe is to be removed with the scissors, exercising the utmost care that the surface be not touched. Place immediately in strong alcohol, and in twenty-four hours it will be fit for cutting. Cut extremely thin sections at right angles to the free surface and including the same. Stain with hsema. and eosin. Mount in dammar. OYARY OF HUMAN FCETUS OF EIGHT MONTHS. (Fig. 99.) OBSERVE: (L.) 1. The free surface. (Note the occasional depressions which mark the involution of epithelia.) 2. The layers. (Note the absence of demonstrable tunica albu- ginea and the great area occupied by the cortex. The vessels of the central portions are unlike the ovary of mature life; large, not numerous, and thin-walled. ) 14:8 PRACTICAL MICROSCOPY. (H.) 3. The primordial ova of the surface epithelium. 4. The projecting lines or chains of epithelium undivided. (Here the cells seem rather elongate.) 5. Chains which are in process of subdivision. 6. Young Graafian follicles in columns at right angles to the surface of the ovary. 7. The discus proligerus, in many instances yet composed of flat- tened cells. FIG. 99.— SECTION OF OVARY OF CHILD. DEATH TEN DAYS AFTER BIRTH, x 350. A. Germinal epithelium, covering surface of the ovary. B. Primitive ova. C. C. Projection of surface epithelium within the organ. D. Constriction of the projected chain or cord of epithelium and isolation of portions to form Graafian follicles. E. Chain of Graafian follicles. The stroma is seen filled with previously formed follicles which have become now isolated. F. A large Graafian follicle. It has been cut in half; the ovum has fallen out; and the mem- brana granulosa is seen lining the cup-shaped cavity. G. Large arteries of the central portion of the ovary. 8. Follicles showing discus proligerus as columnar cells. 9. Follicles showing great proliferation of discus prolig- erus. DEVELOPMENT OF THE OVUM. 149 10. Ova in early development from primordial cells, with gran- ular vesicle. 11. Instances of development of two, possibly three, ova in a single follicle. 12. Large blood capillary supply of cortex, vessels generally parallel with the Graafian chains. 150 PRACTICAL MICROSCOPY. THE SUPRARENAL CAPSULE. These bodies are attached by areolar tissue to the summit of the kidneys, and consist of several folia or leaflets. An examination of one of these leayes will give us an idea of the organ as a whole. The plan of structure seems to be as follows: In the connective tissue which supports the folia are found arterial branches derived from the phrenic (and sometimes from the renal before it enters the kidney). These arteries penetrate the organ, break up immediately into capillaries, which finally converge toward the centre of the leaflet; the blood is here collected in thin-walled veins, by which it is drained into the suprarenal vein, thus leaving the capsule. The capillary meshes vary in form and size, according to their position. Near the circumference of the leaves the meshes are small and ovoid, while, as the centre is approached, they become elongated. These spaces between the capillaries are filled with compressed, glob- ular, nucleated cells, the smaller containing only perhaps six or eight, while the longer may be occupied by thirty or forty of these cell elements, which constitute the parenchyma of the organ. This variation in size of the cell compartments, contributing, as it does, to alter the appearance of the different zones of the tissue, has given rise to a division into cortex and medulla, with subdivisions even of these. There is no histological or physiological difference, as we believe, between the different parts of the folia of the suprarenal cap- sule, except as has been indicated. The structure is exceedingly simple, although its function is not settled beyond question. PKACTICAL DEMONSTKATION. The tissue is best hardened in strong alcohol, and should be cut as soon as the hardening is complete. It will be sufficiently firm to- ad mi t of the thinnest sections being made free-hand or with a simple microtome. The cuts, stained with haoma. and eosin, give excellent differentiation. HUMAN SUPRARENAL CAPSULE. 151 HUMAN SUPRARENAL CAPSULE. (Figs. 100 and 101.) SECTION OF A SINGLE LEAFLET, CUT TRANSVERSELY TO THE CEN- TRAL VEINS. STAINED WITH H^MA. AND EOSLN; MOUNTED IK DAMMAR. OBSERVE: (L.) 1. Section of arterial twigs on the border of the leaflet. 2. The convergence of the parenchyma toward the centre. FIG. 100. — VERTICAL SECTION OF A SINGLE LEAFLET OF THE SUPRA-RENAL CAPSULE. Stained with Haema. and Eosin. X 60. A. Fibrous tissues surrounding and connecting the leaflets. B. The outer portion, consisting of small compartments— the so-called cortex. C. The central, elongated cell-compartments — medulla. D. Large thin- walled central veins. E. Arteries ramifying in the outer fibrous tissue which supply the parenchyma. 3. The large and thin-walled central veins. 4. The small size of the parenchymatous areas on the outer borders and their elongation within. 152 PRACTICAL MICROSCOPY. (H.) Fig. 101. 1. The capillary plexus, forming ovate or elongate meshes. 2. The compressed globular cells of the parenchyma. (Note that the cells are small in the small compartments, as though crowded. This is due, in a measure, to the contraction of the tissue from the rapid hardening. ) .— SAME SECTION AS FIG. 100, MORE HIGHLY AMPLIFIED. REGION MIDWAY BETWEEN THE FIBROUS INVESTMENT AND THE CENTRE OF THE LEAFLET, x 400. A. Blood capillaries, arising from the arteries seen in the preceding illustration; and ramify ing in the connective-tissue framework. B. Compartments— lobules— formed by delicate connective-tissue prolongations from the fibrous capsule. C. Lobular parenchyma. These large somewhat rounded cells are generally mono-nucleated, contain fat globules, and are frequently pigmented. 3. The minute fat globules in the parenchyma. (This I believe to be physiological, and not unlike the fat storing observed in the parenchyma of the liver.) 4. Yellow pigment granules in the parenchyma. SALIVARY GLANDS. 153 THE SALIVARY GLANDS. PANCREAS. PLAN OF GLAND STRUCTURE. GLANDS. A gland is an organ — frequently subsidiary to and located within other organs — whose cells manufacture from the blood products to be utilized in the maintenance of physiological integrity. Glands are tubes or cavities, with connective-tissue walls lined with cells of a columnar type. Around, and in close proximity to the lin- ing, is spread a plexus of blood capillaries. The essential parts of a gland are, therefore: 1. A duct) or efferent conduit for the secretion. 2. Parenchyma, cells engaged in secretion. 3. A blood-vascular supply. TUBULAR GLANDS. The simplest gland structure is presented in the form of a tube. Ci-lands are, frequently, little more than tubular depressions in mucous FIG. 102.— DIAGRAM. SIMPLE TUBULAR GLAND. A. Lining cells— parenchyma. B. Capillary plexus, supplying the parenchyma. C. Connective tissue supporting capillaries. D. Arterial supply. 154 PRACTICAL MICROSCOPY. surfaces. Examples are found in the uterus, stomach, small in- testine, etc. COILED TUBULAR GLANDS. Tubular glands are often greatly elongated, with the blind ex- tremity coiled. This variation presents the simplest differentiation between the part of the tube which is secretory, and the duct, or drainage part. With this change in function of the different ex- tremities of the tube will occur a change of epithelium. The cells belonging to the duct-end will usually retain the columnar form; while the actively secreting elements will become enlarged, more nearly filling the tube, and assume a polyhedral form from pressure. FIG. 103.— DIAGRAM. COILED TUBULAR GLAND. Same references as Fig. 102. Examples have already been seen in the sweat tubes of the skin — sudoriferous glands — and the mucous glands of the submucosa of the larger bronchi. BRANCHED TUBULAR GLANDS. With the branching of the duct portion of gland tubules, there usu- ally occurs a dilatation of the extremities into alveoli, although pure examples of branched tubular glands are afforded in the gastric and in- testinal glands, those of the cervix uteri, etc. The most nearly typical branching of gland-like tubules is afforded by the tubuli uriniferi of the kidney — although not contained in a ACINOUS GLANDS. 155 true gland. The tubules here present other features peculiar to hem, which will be referred to under the proper head. FIG. 104.— DIAGRAM. BRANCHED TUBULAR GLAND. References same as Fig. 102. ACINOUS GLANDS. The dilatation of branching tubules, referred to under the previous heading, results in the formation of acinous glands. They are formed by the subdivision of a main tube or duct, with repeated branching of the secondary tubules. Collections of terminal branches often result in globular masses which are more or less perfectly isolated from one another by connective tissue. In this way compound acini are pro- duced, such as the pancreas, the salivary, mammary, and buccal glands. The acini may be developed into alveoli — as in the active mammary, and in the sebaceous glands. These are usually filled with polyhedral cells, or with the products of fatty degeneration of the same. 156 PRACTICAL MICROSCOPY. FIG. 106.— DIAGRAM. ILLUSTRATING THE PLAN OF ACINOUS GLANDS. References same as Fig. 102. FIG. 106. —SECTION OF A SMALL PORTION OF THE PAROTID GLAND. Stained with Haema. and Eosin. x 250. A. Narrowing of the duct from a small lobule, before entering a larger duct. B. Dilatation of a duct after leaving a small lobule. C. Primary lobules, in nearly L. S. D. Acini in T. S. showing the minute lumen. E. Connective tissue supporting the gland. F. Striated muscular fibre adjacent to the gland. G. Adipose tissue in the loose areolar tissue. THE PAROTID GLAND. 157 THE PAROTID GLAND. The Parotid, Submaxillary , SuUingual, and' B'ueeal Salivary Glands are typical glandular structures, with individual peculiarities only in respect to the cell elements; these vary according to the nature- of the secretion formed in each. The parotid is a compound acinous gland, leading from which is a principal duct — lined with tall columnar cells — which collects the fluid saliva from the different divisions of the organ. As the duct penetrates the gland it branches freely, the lumina becoming smaller and the cells shorter as the deeper parts are approached. Each terminal duct is in connection with several acini. The con- nective-tissue adventitia of the duct becomes the thin wall of the FIG. 107.— SECTION OF PART OF THE SUBMAXILLARY GLAND. X 250, A. Narrow duct from terminal lobules. B. Small duct in T. S. C. Small duct in oblique section. D. Transversely divided acini, showing large lumen. E. Mucus remaining in the lumina. F. Striated muscular fibres G. Adipose tissue. 158 PRACTICAL MICROSCOPY. acinus, and the lining cells broaden, frequently become polyhedral, and are bluntly pointed. The cells so nearly fill the acini as to leave a small and not easily recognized lumen. The gland is richly supplied with blood-vessels. THE SUBMAXILLARY GLAND. The submaxillary is presented as an example of a typical mucous gland. As I have previously said, the general arrangement is not unlike that of the other salivary glands. Similar structures are found in the submucosa of the mouth, tongue, fauces, trachea, and the larger bronchi. Its peculiarity appears in the parenchyma, and will be noticed later. FIG. 108. — SECTION FROM THE PANCREAS. A. Wall of a large duct. B. The somewhat cubical lining cells. C. Arteries. D. Lumen of the acini, T. S. E. Terminal duct entering a lobule. F. Acini in L. S. THE PANCREAS. 159 THE PANCREAS. The histology of the pancreas is, in general, that of a true serous ^gland — e. g., the parotid. It has been called by physiologists the abdominal salivary gland. The cells, constituting the parenchyma, are somewhat smaller; the lobules less regular in size and form; and the lumen of the acini much less easy of demonstration, in an ordinary hardened section, than the same in the parotid. The vascular supply is also more abundant. The branches of the pancreatic duct are provided with a very thick adventitia, are lined with short columnar cells, and seldom present the dilatation, which generally occurs in a serous gland, on entering the lobule. PEACTICAL DEMONSTRATION. PAKOTID AND SUBMAXILLARY GLANDS, AND THE PANCREAS. The tissue must be fresh, divided in small pieces — not larger than a quarter of an inch cube — and hardened by placing in ninety-five per cent alcohol for twelve hours, after which fresh spirit should be sub- stituted. If, after the lapse of another twelve hours* the tissue should not be sufficiently firm, it should be placed in a small quantity of ab- solute alcohol for three hours. Sections should be made immediately after hardening — as more prolonged action of the strong spirit will cause the tissue to contract. Sections may be cut with or without a simple microtome — the desideratum being thin, rather than large cuts. Stain lightly with haema., and deeply with eosin. After sections of hardened tissue have been examined, the glandular parenchyma may be profitably studied in teasings from tissue which has been in Miiller twenty-four hours. Wash the teasings on the slide with a liberal supply of water, removing the same from time to time with blotting paper. Add a drop of haema, solution; and, after washing this away, add a drop of glycerin, and cover. This method is very generally useful for teased or scraped fragments of glandular structures. (Figs. 106, 107, and 108.) OBSERVE: (L.) 1. The connective tissue. (Most abundant in the parotid, and least so in pancreas. ) 2. The ducts. (Note the flattening of the lining columnar cells, as the ducts approach the acini, until mere scales result. 160 PRACTICAL MICROSCOPY. Also the thick connective-tissue adventitia, especially demonstrable in1 the pancreas.) 3. The lobules. (These are formed by several acini, and are typical only in the parotid — at least they only here appear well formed. It must be remembered that only one plane is visible, and that there is little perspective.) 4. The acini. (Note the lumina — large in the submaxillary, less so in the parotid, and least, and often difficult to make out, in the pancreas. 5. The blood-vessels, muscular and adipose tissue. (The two latter are demonstrable only in the salivary glands, and do not belong properly to the gland itself. The capsule of the pancreas, in common with such structures in general, contains adipose. The abundant inter-acinous capillary plexuses of the pancreas require the high power for satisfactory demonstration.) (H.) 6. The parenchyma, (a) The small but distinct shortened columnar cells of the acini of the parotid. (Observe that they are frequently so formed that the convexity of one cell fits into the con- cavity of its neighbor. Where seen in transverse section, the outline is a polygon. Nbte especially the change in the parenchymatous elements as the terminal duct merges into an acinus.) (#) The large, swollen cells of the mucous gland— submaxil- lary. (Observe the comparatively clearness of the cells. They contain a very delicate reticulum, and their nuclei are often obscured and frequently seen to be placed at the junction of the cells.) (c) The rounded, often polyhedral cells of the pancreas. (They resemble the parotid elements, although smaller and less granular.) THE LYMPHATIC SYSTEM. 161 THE LYMPHATIC SYS The Lymphatic System is a circulatory apparatus of exceedingly complicated arrangement. It comprises: 1. — A system of irregular clefts and cavities which are of almost universal distribution in the more solid tissues, in the framework and parenchyma of organs, around tilood-vessels and viscera. 2. — Nodules of sponge-like tissue, improperly called lymphatic glands. 3. — Channels of communication, consisting of capillaries and ducts. 4. — A central reservoir — the receptaculum chyli. 5. — Large efferent ducts, by means of which the contents of the system are, eventually, poured into the blood, in both sides of the neck at the junction of the internal jugular and subclavian vein. 6. — A fluid, lymph, containing numerous nucleated bodies or lymphoid cellst and various substances in solution. The whole provides a channel for the introduction of formed and nutrient elements into the blood; as well as affording drainage for the tissues, the products of which are also emptied into the blood-vascular system, to be afterward eliminated by special organs. The circulating lymph always passes in a direction toward the ven- ous system. This current is established in some of the lower animate by means of distinct, pulsating, hollow organs, or lymph hearts; but no corresponding structure exists in man, and the system becomes here subordinated to the blood-vascular apparatus. In man, the maintenance of the lymph-flow is due largely to a negative pressure, consequent upon the connection between the termini of the lymph-vessels and the veins. Without doubt the pumping motion of the intestinal villi presents a factor in the establishment of a current in the lacteals toward the mesenteric vessels. The perivas- cular lymph receives an impetus with each cardiac systole. The muscular contractions of inspiration contribute motility to the con- tents of the diaphragmatic lymph-channels, in a direction against gravity. Indeed, the contractions of nearly every muscular fibre, whether skeletal or organic, lend their aid to lymph propulsion. The direction of the lymph-current is determined by valves which resemble, somewhat, those of the veins. Cavities lined with so-called serous membranes, may be considered as expanded lymph-channels. II 162 PRACTICAL MICROSCOPY. LYMPH CHANNELS. The larger and more regularly formed channels for lymph circu- lation, such as the mesenteric and thoracic ducts, do not differ, materially, in structure, from correspondingly sized veins. The irregular clefts in the interstices of fibrous tissues, serving as the primitive lymph-containing channels, have been already, and repeatedly, noticed. Fig. 109, although purely diagrammatic, will serve to show the relation of this system to the blood-vessels. A perivascular lymphatic channel is a sort of tubular investment of the MEDIA ADVENTITIA ERIVASCULAR FIG. 109.— DIAGRAM. ARTERY IN TRANSVERSE SECTION, SHOWING THE PERIVASCULAR LYMPH-SPACE. blood-vessel, lined with flattened endothelia sending prolongations inward; these prolongations branch, and are finally in communication with a layer of cells covering the adventitia. In this manner, in close apposition to parts of the vascular system, a system of channels is provided, within which the lymph may sloivly percolate. The largest lymphatic channels in the human body are the cavities of the peritoneum and pleurae. They are in connection one with the other, and with the lymphatic system generally; and these channels of communication between the great abdominal and thoracic lym- phatic cavities present, perhaps, as the most convenient and typical for demonstration. LYMPH CHANNELS. 163 PRACTICAL DEMONSTKATION. LYMPHATIC VESSELS OF THE CENTRAL TENDON" OF THE DIAPHRAGM. (Figs. 110 and 111.) This demonstration had best be made with tissue from the rabbit, inasmuch as the slightest decomposition of the epithelium would be fatal to success. A small (preferably white) rabbit should be quickly killed by de- capitation, and immediately suspended by the hind legs, so as to thoroughly drain the body of blood. As soon as the blood has ceased dripping, open the thoracic cavity by slitting up the skin along the median line, pushing it to the sides and removing the sternum. In this operation, work rapidly and avoid soiling the internal parts. Then with the fingers of one hand raise the lungs and heart from the diaphragm, and with a large camel's-hair brush proceed to quickly, and quite forcibly, pencil the white glistening surface of the central diaphragmatic tendon, moistening the brush from time to time in the lymph of the pleural cavity. Should the quantity of fluid be small, add a little distilled or previously boiled and filtered water. The object of the brushing is to remove the epithelial cells which cover the surface, and which would otherwise hide the lymph spaces. After the pencilling, drain away the fluid, and pour over the brushed sur- face a solution of one grain of nitrate of silver to an ounce of distilled water.* Allow the silver solution to remain for twenty minutes in contact with the tissue, the body meanwhile being kept away from the bright sunlight; then pour off the solution, wash the surface twice with distilled water, and afterwards allow water from the tap to flow over the parts for at least five minutes. If yon observe the directions carefully, the surface of the tendon will lose its original glistening appearance and become whitish and opaque. The tendon, or such portion of it as you wish to preserve, may be cut out with the scissors after the washing, thrown into glycerin, and placed in the sunlight until the surface becomes brown. With the forceps tear off small pieces of the stained side, say one-half inch square, and examine in glycerin, or mount them permanently in the same medium. The demonstration of the channels of the lymphatic system is based upon the following: 1. Lymph channels are always, however small or irregular, lined with flattened cells in a single layer, i. e., pavement endothelium. 2. The lining cells are cemented together with an albuminous sub- stance. * Water which has been well boiled in a clean vessel, and afterward care- fully filtered, may be generally employed in histological work when distilled water is not available. 164 PRACTICAL MICROSCOPY. 3. Nitrate of silver combines with the cement, forming albuminate of silver, which becomes dark brown when exposed to light. If you have been successful, the silver will have penetrated the ten- don, and mapped out the lymph channels, indicating an outline of every lining cell by means of a dark border. Failure will result only from non- attention to cleanliness in the handling of the tissue; the silver in which case becomes deposited generally over the surface. The margins or outlines of the cells, it must be remembered, are stained with the silver. The nuclei may be demonstrated by after- staining with dilute haema., or better, borax-carmine. The mounting may be done in dammar, although the elastic fibres, of which the matrix of the tendon is composed, will become stiff during immer- sion, and show a tendency to curl and contract. If glycerin be used after carmine staining, tissues should be washed thoroughly in water, subsequently to the oxalic acid bath, transferred to equal parts of glycerin and water, and allowed to remain for an hour, at least, before mounting. CENTRAL TENDON OF THE DIAPHRAGM. SILVER STAINING. (Vide Figs. 110, 111.) OBSEBVE: (L.) 1. The division of the specimen into dark and light areas. (The dark areas represent the more solid portions of the tissue or the partitions between the channels, and the light spaces are the lymph paths.) 2. The lymph paths — the light spaces. (These show, with this amplification, as irregular, winding, and anastomosing courses, marked with very delicate lace-like tracery — the silver lines.) 3. Valves of the lymph paths. (At points, the paths will be crossed by dark curved lines. These are imperfect valves, not unlike a single cusp of an aortic valve. ) (H.) 4. Outlines of the cells lining" the larger excavations (lymph paths) in the tissue. (Note that the cells are generally elongate in the direction of the lymph path. The edges are frequently serrated.) 5. Stomata, minute openings at the junction of several cells. 6. The construction of the valves. (These are curved against the lymph flow, and covered with cells like other parts of the channeL CENTRAL TENDON OF THE DIAPHRAGM. 165 Note the change in form of the cells approaching and covering the valves.) 7. Elastic fibres of the more solid parts of the tendon. 8. Lymph capillaries. These will be seen in the partitions be- tween the larger paths. In places they may be observed emptying into the paths, and again will appear as simple cavities, according to the manner sectioned.) 9. The deeper capillaries. (Careful focussing the portions of FIG. 110.— LYMPH-CHANNELS. CENTRAL TENDON OP DIAPHRAGM OF RABBIT. SILVER STAINING. K60. The dark portions represent the more solid portions of the tissue. The light areas are the lymph -channels; and the direction of the flow is shown by the Arrows. The minute lines in the lymph-spaces are the silver-stained cement boundaries of the pavement cells lining the channels. The valves appear as curved lines in the lymph-spaces. the tendon which appear most solid will reveal minute cell-lined channels or capillaries. The student must remember that we cannot penetrate tissues with the microscope to any considerable depth, but are restricted to nearly a single plane. If it were posssble to penetrate 166 PRACTICAL MICROSCOPY. with the eye the entire thickness of the tendon, we might trace the lymph paths or channels from the abdominal to the thoracic surface.) FIG. 111.— A SMALL PORTION OF SPECIMEN SHOWN IN FIG. 110, MORE HIGHLY MAGNIFIED, x 350. A, A, A. Large lymph-channel. B. Valve in the course of last. C, C, C. Lymph capillaries in the more solid parts of the tendon. D. Pavement cells upon which a large amount of silver has deposited. Failure to follow the instructions for the staining frequently results in a like deposition of silver over the whole sur- face. LYMPHATIC NODES OK GLANDS. 167 LYMPHATIC NODES OE GLANDS. At numerous points along the course of lymphatic vessels they pen- etrate small nodules of so-called adenoid tissue, which have heen termed lymphatic glands. They are frequently microscopic; others, again, not unusually attain the size of a large pea. They secrete nothing, hence are not glands. They are somewhat sponge-like in structure, and the lymph filters slowly through them. Most frequently several ducts enter one of these larger nodes, while perhaps only a single efferent will be found. The histology of a lymph node is not always easily comprehended by the student, and I have endeavored to make a diagram (Fig. 112) that would simplify the matter somewhat. They are enveloped by a capsule of connective and involuntary muscular tissue, which sends trabeculm into the body of the organ, and these branching posts sup- port the structure as a framework. The interstices are quite small in the more central portion and larger toward the periphery; this has resulted in the application of the terms medullary and cortical to the respective parts. The nutrient blood-vessels are contained in the framework. The compartments contain the structure peculiar to the lymphatic system, viz., adenoid tissue. Adenoid tissue consists of a mass of flattened cells, with numerous delicate fibrillar prolongations, which branch and anastomose so as to form an interwoven structure — the adenoid reticulum. Klein regards the cells as forming no essential part of the structure, but considers them as flattened plates attached to the fibrils. The meshes of the adenoid reticulum are in connection with the fibres of the trabeculae and, with exception of the portion next the latter, are filled— crowded, in fact — with countless small spherical lymphoid cells. Those por- tions of the tissue which contain the cells are termed follicular cords. The lymph path is the portion between the fibrous trabeculae and the follicular cords. When we learn that the trabeculae, follicular cords, and lymph paths each pursue very tortuous and branching routes, we can appre- ciate the complexity of the organ as a whole. The blood-vessel arrangement presents no anomalies. The small arterial trunks enter within the trabeculae, finally break into capilla- ries which supply the follicular cords, etc., and the blood is then col- lected by the venules for the efferent veins. 168 PRACTICAL MICROSCOPY. Small diffuse collections of adenoid tissue have already been seen in many organs. These do not differ essentially from the tissue just described, excepting that there is no definite arrangement of trabeculss and lymph paths, as in the compound lymph node; the lymph simply filters through the reticulum, the same being a part of the lymph- channel system of the tissue in which the adenoid structure may occur. FIG. 112.— DIAGRAM. PERIPHERAL PORTION OF A LYMPH-NODE. A, A. Afferent lymph-vessels. B. Capsule of the node, with lymph-spaces C. C. D, Trabecula of connective tissue. E, E, E. Lymph path in the node. F, F. Follicular cords. G, G, G. Lymphoid cells in the cell -network of the paths. H, H. Blood capillaries of the cords. The arrows show course of lymph. SECTION OF MESENTERIC LYMPHATIC NODE. 169 PEACTIOAL DEMONSTRATION. The mesenteric lymphatic nodes present the most typical structure, may be obtained from the human subject, if fresh, although those from the dog are preferable, on account of the better condition of the tissue as usually secured. The nodes should be sliced in half, placed in Miiller for a week, and then hardened by two days' immersion in strong alcohol. Sections should be mounted, of two kinds, viz. : Those including the whole area of the node — which need not be very thin — for demon- stration of the scheme or plan of structure, and excedingly thin ones, even though they may include only a small part of the organ, for study of the details of the adenoid reticulum. The latter purpose will be subserved by shaking a number of thin cuts in a test-tube with alcohol for a few minutes, and with considerable violence, even to the sacrificing of most of the sections. The agitation will dislodge the lymph cells, which otherwise would obscure the histology of the follicular cords. Stain deeply with haema. and eosin, and mount the thicker sections in dammar, and those especially thin in glycerin. SECTION OF MESENTERIC LYMPHATIC NODE. (Figs. 113 and 114.) OBSERVE : (L.) 1. The fibrous capsule. (Note the elongate dots in the deeper parts of the capsule — the nuclei of the smooth muscular tissue, the thick-walled arteries, the lymph spaces.) 2. The trabeculae. (Trace these as they penetrate the organ and observe that they frequently end abruptly, on account of having curved, so as to leave the plane occupied by the section. The tra- beculse are not partitions, like the iuterlobular pulmonary septa or the prolongations from the capsule cf Glisson in the liver; they are not unlike rods or posts, making a framework and not producing alveoli. Find one divided transversely.) 3. The follicular cords. (They are recognized as granular masses between the trabeculae. Observe the varying forms, largest and more spherical or ellipsoidal, near the periphery — cortex. The smaller ones of the central region (medulla) must not be overlooked, as the differentiation is sufficiently marked between them and the variously sectioned trabeculae.) 4. The lymph paths. (These can be appreciated by remembering 170 PRACTICAL MICROSCOPY. that the follicular cords do not entirely fill the spaces between the- trabeculae, and that the area between the two, i. e., outside the cords, is the more open in texture, and contains the filtering lymph. They are more distinct in the cortex.) FIG. 113.— VERTICAL, SECTION OP A LYMPH-NODE FROM THE MESENTERY, x A. Capsule of node. B. Lymph -spaces in the last. C. C. Trabeculse, L. S. D. D. Follicular cords, L. S. E. Obliquely sectioned trabecula. F. F. Large blood-vessels of the central portion of the node. G. Trabecula in T. S. H. Follicular cord in T. S. I. Small and irregular cords of the centre of the node. J. Obliquely sectioned trabecula of the centre of the node. K, K, K. Lymph-paths- 5. The histology of the capsule, (a) The closely united con- nective tissue with the scattering elastic fibres of the external layer, (b) The smooth muscle of the deeper portions, (c) Sec- tions of arteries. (These may present of considerable size.) (d) The lymph spaces. (The differentiation is by the flattened endo- thelia of spaces which otherwise would be supposed mere rifts in the tissue, inasmuch as no definite or special wall can be detected.) 6. The structural elements of the trabeculse. (They are simi- lar to those of the capsule, excepting the elastic element, which can- SECTION OF MESENTERIC LYMPHATIC NODE. 171 not here be demonstrated. Note the variously sectioned small arteries.) 7. The follicular cords. (In the thicker section, the field will be completely crowded with lymphoid cells. Select a thin field and observe: (a) The lymphoid cells. (These will be found varying in size from a very small red blood-disc to that of a large white corpuscle; some are filled with granules only, and others with one, two, and even three nuclei.) (b) The branching endothelioid cells, (c) The delicate fibrillae of the adenoid reticulum. (You may endeavor to FIG. 114.— FRAGMENT OP SECTION SHOWN IN FIG. 113. MORE HIGHLY MAGNIFIED, x 350 A. Trabecula. B. Follicular cord. C. Lymph-path. D. Large branching cells of the path-network. E. Capillaries of the cord. determine whether this reticulum exists as an offshoot of the endothe- lioid cells, or whether the latter are simply adherent to the broadened plates of the former.) 8. The reticulum of the lymph paths. (Observe that this is pre- cisely like the reticulum of the follicular cords, as demonstrable after 172 PRACTICAL MICROSCOPY. shaking out most of the lymph corpuscles of the last.) (a) The connection between the fibrillae of the paths and those of the trabeculae. 9. Capillaries of the paths and cords. (These will be recog- nizable only by the regular succession of the contained red blood- corpuscles. ) THE SPLEEN. 173 THE SPLEEN. The spleen presents no regular subdivision of parts which may be studied separately and combined afterward, as we are able to do with organs like the lung, liver, etc. The spleen is a ductless organ or so-called gland, and the plan or scheme may, perhaps, be best comprehended by following the blood distribution. The splenic artery enters the organ, supported by a considerable amount of connective tissue, and rapidly breaks into smaller branches, from which the arterioles leave at right angles. The arterioles quickly merge into capillaries, which form plexuses throughout the #ip^ufo i£ii* %afyl$u^ tuft*" Artery FIG. 115.— DIAGRAM. SHOWING THE COURSE OF BLOOD IN THE SPLEEN. different portions of the organ. Here we meet with an anomalous structure. The capillaries, instead of uniting to form venules as in the usual vascular plan, empty their contents into small chambers or sponge-UJce cavities — the venous spaces. The blood, after filtering through these venous interstices, is collected in larger, irregular, vein-like channels, which finally conduct the blood into the veins proper and out of the spleen. The tissue, containing the vascular arrangement described in the last paragraph, is called spleen pulp. 174 PRACTICAL MICROSCOPY. The fibrous capsule which envelopes the spleen sends trabeculae within, which form a framework; and from this fibrils are sent off which branch, broaden, and inosculate to form t/ie venous chambers of the pulp. The arteries are frequently surrounded by nodules of adenoid tissue, sometimes globular, more frequently considerably elongated, and fol- lowing the vessel for a considerable distance. These nodules are called Malpighian bodies. They bear no resemblance to similarly named structures in the kidney, excepting, perhaps, when seen in transverse section by the naked eye. The spleen will thus be seen to consist of a fibrous trabeculated framework, the pulp, blood-vessels, and more or less isolated nodules of adenoid tissue. PKACTICAL DEMONSTKATION. The organ must be absolutely free from decomposition. If human tissue cannot be obtained in good condition, recourse may be had to the ox, which will provide an excellent substitute. The small super- numerary spleens, not infrequently found during post-mortem work, are most desirable, as sections can be easily made through the entire organ. Pieces of tissue half an inch cube, including a portion of the cap- sule, should be hardened as directed for lymph nodes. Sections are easily made without the microtome, as the mass is very firm; they should be thin and stained with borax-carmine, and mounted in dam- mar or in glycerin. SECTION OF HUMAN SPLEEN, CUT AT RIGHT ANGLES TO AND INCLUDING THE CAPSULE. (Fig. 116.) OBSERVE: 1. The fibrous capsule, (a) Its division into two very distinct portions or layers, (b) The clear translucent appearance of the tissue (elastic) of the outer layer, (c) The darker deep layer with elongate nuclei. (The elastic element of the capsule not infrequently becomes, in the human subject, considerably increased; and this de- velopment occurs irregularly, sometimes in the form of minute nodules. I do not know that they present any pathological signifi- cance. ) 2. The trabeculae. (The depth to which they may be traced will depend largely upon the direction of the section.) (a) That these are not bands, but bundles, more or less circular, in transverse sec- SECTION OF HUMAN SPLEEN. 175 tion. (b) Their irregular course, quickly after leaving the sur- face, (c) That occasionally a small artery may be found within them, though they are usually destitute of large vessels, (d] The elongate nuclei of the muscular fibre largely forming the trabeculae. 3, The large blood-vessels, (a) The arteries more frequent FIG. 116. SECTION OF THE SPLEEN. X 60. A. Elastic portion of the capsule. B. Lymph spaces of last. C. Involuntary muscular portion of capsule. D. Deeply pigmented portions of capsule. K, E. Trabeculse from C. F. Trabeculae in oblique section. G, G. The spleen pulp. H, H. Large arteries in T. S. I. Arteries in L. S. J. Adenoid nodule, not connected with an artery. K. A.denoid nodule.— Malpighian body— along course of artery. L. Adenoid nodule in T. S. M. Vein. than veins. (&) Their very prominent adventitia. (c) Their tor- tuous course. 4. The adenoid tissue. (This you will be enabled to recognize by the great number of lymphoid cells of the adenoid structure, the nuclei of which become stained very deeply blue with haema., giving 176 PRACTICAL MICROSCOPY. a very distinct differentiation. At this point, examine every part of the specimen, and endeavor to detect even the most minute collection of this tissue.) (a) Around arteries, constituting the so-called Mal- pighian bodies, (b) Transverse sections of Malpighian bod- ies, noting that the vessel is seldom in the centre of the nodule. (c) Nearly longitudinal sections of Malpighian nodules, observ- ing that the adenoid tissue usually follows or surrounds the artery for a short distance only, (d) That the distribution is not confined to the arteries, but is quite common around trabeculae and beneath the capsule. 5. The Spleen-pulp. (This will be found in those portions of the- section not occupied by structures previously demonstrated; and will be determined by its light color. Keview the whole area, and en- deavor to differentiate every portion of the adenoid and pulp tissue.. The staining will have been your principal guide thus far, the pulp- elements appearing in strong contrast by their pink eosin color.) (H.) 6. The structural elements of the capsule, (a) The nu- merous minute lymph-spaces and the imperfect vascular sup- ply, (b) The nuclei of the peritoneal cell covering. (This pre- supposes that the section has been selected so as to include the peri- toneal investment.) (c) The abundant and closely packed elastic fibrillae. (d) The muscle nuclei of the deeper parts, (e) Cells containing granular yellow pigment. (The quantity varies largely with different specimens.) 7. The Malpighian nodules, (a) The arterioles — very small and apt to escape attention unless filled with blood corpuscles. (#} The adenoid reticulum. (This will be difficult of satisfactory demonstration, excepting the section be thin.) 8. The elements of the pulp, (a) Large flattened cells, the- branches forming the mesh work of venous channels. (These are only susceptible of very satisfactory demonstration in the spleen of leucocythcemia.) (b) Red blood-corpuscles. Very numerous and often broken and distorted, (c) Blood pigment, (d) Lymphoid or white blood-corpuscles. SECTION OF THE THYMUS BODY. 177 THYMUS BODY. The thymus body (frequently and improperly called a gland) is an adjunct to the lymphatic system of — in man — foetal and infantile life ; disappearing, by an atrophic process, at or before the age of puberty. It is enveloped by a fibrous capsule, partitions from which sub- divide the organ into lobes and lobules. The lobules are generally subdivided into follicles, which are irregularly sized and shaped, while tending to an ovoid form. It is in connection with the general lymphatic system by peri- pheral, afferent lymph-channels ; and by efferent vessels which emerge from the hili of the lobes — the lymph having meanwhile traversed the mesh-like structure of adenoid tissue composing the follicles. The blood-vascular system is in the form of a nutritive supply; the larger vessels occupying the fibrous framework, and sending branches into the follicles. The capillary plexuses are more abundant in the peripheral portion of the follicles. The blood is collected in the venous channels of the central or medullary area, and emerges from the organ by the veins which accompany the efferent lymphatics. PRACTICAL DEMONSTRATION". The organ should be obtained from a still-born infant, divided in small pieces, and hardened rapidly in strong alcohol. Sections may include an entire lobe, and be stained with hgema. and eosin. SECTION OE THE THYMUS BODY FROM AN INFANT AFTER DEATH ON THE SIXTEENTH DAY. (Fig. 117.) OBSERVE: (L.) 1. The fibrous capsule. 2. Division by prolongations of 1 into somewhat spherical lobes. 3. Subdivision of 2 into lobules. 4. Subdivision of 3 into follicles. (Note that these are not uni- formly outlined by the connective tissue.) 12 178 PRACTICAL MICROSCOPY. 5. The subdivision of the follicles into an outer, deeply-stained! cortex, which completely surrounds a light centre, the medulla. 6. The larger lymph-spaces and arteries of the capsular and trabecular tissue. FIG, 117.— SECTION OP A PORTION OF THE THYMUS BODY, FROM A CHILD, SIXTEEN DAYS AFTER BIRTH. X 60. A, A. Capsule which divides the organ into lobes. Portions of six lobes are visible in the section. B, B. Lymph-spaces. C, C. Trabeculae dividing the lobes into imperfect lobules. D, D. Subdivisions of the last into follicles. E, E. Central light portion of the lobules. (H.) 7. The cortex of the follicles. (a) The numerous deeply- stained lymph-corpuscles, (b) The network of the adenoid tissue. (This will be greatly obscured by the lymphoid cells.) (c) The blood capillaries. Only recognized by the contained cor- puscles, (d) Minute trabeculae of connective tissue projected from the capsule. 8. The medulla of the follicles, (a) The sparsity of lymph- corpuscles as compared with the cortical portions, (b) Large SECTION OF THE THYMTJS BODY. 179 mono nucleated cells, (c) Still larger multinucleated cells. (d) Larger — though varying in size — spherical bodies, Hassall's corpuscles. (These are composed of epithelioid cells, arranged con- centrically, and are unlike any other structure found in the normal tissues of the body. They resemble very closely the smaller " brood nests " of epithelial cancer. ) (e) Small thin- walled venules. 180 PRACTICAL MICROSCOPY. THE NERVOUS SYSTEM, STRUCTURAL ELEMENTS. The elements of the nervous system are : 1. Nerve Fibres. 2. Nerve Cells. 3. Connective Tissue. 4. Peripheral Termini. i NERVE FIBRES. A typical nerve fibre consists of three portions, viz.: a central conducting portion, the axis cylinder ; the medullary sheath, or white substance of Scliwann ; and the enveloping connective-tissue substance, the neurilemma. This constitutes a medullated nerve- fibre, and is found largely in the trunks of the cerebro-spinal FIG. 118.— SEPARATED NERVE FIBRES. X 400. A. Neurilemma of a fibre. B. White substance of Schwann, stained with osmic acid, which hides the axis cylinder. C. Nucleus of the neurilemma. D. One of Ranvier's nodes in an osmic acid stained fibre showing the axis cylinder between the separated portions of Schwann's sheath. E. A medullated fibre, teased in normal salt solution. The medullary substance has be- come coagulated on exposure and removal. The axis cylinder is faintly seen. F. Axis cylinder at torn extremity. G. Non-medullated fibre. H. Fibres without neurilemma. Small clusters of medullated substance are seen covering the axis at irregular intervals. NERVE CELLS. 181 system. The trunks of the sympathetic system are composed prin- cipally of fibres destitute of the white substance of Schwann — non- medullated nerves ; while fibres minus the neurilemma exist in the trunks belonging to some organs of special sense. After treatment with reagents, the axis cylinder (one-two thousand five hundredth to one-fifteen thousandth of an inch) may be split up longitudinally, and is found to be composed of fine (one-twenty-five thousandth of an inch) primitive or ultimate fibrillae, which present minute varicosities or swellings at irregular intervals. The white substance of Schwann presents under the microscope the most prominent feature of medullated nerves, affording a nearly com- plete investment of the nerve axis. The neurilemma is an elastic connective-tissue envelope, which completely invests the medullary substance. This tubular membrane is nucleated, and at irregular intervals is constricted so as to reach very nearly the axis cylinder. These constrictions are called by Ran- vier nodes, and it is believed that the perineurium presents a single nucleus between each of these nodal points. The constrictions do not, however, affect the even calibre or continuity of the axis cylin- der. A typical nerve fibril has been described as resembling, structu- rally, a doubly insulated telegraphic cable, but the comparison is un- fortunate and misleading, as the functioning of the nerve bears no- resemblance to the phenomena exhibited by electrical conductors. NERVE CELLS. Nerve cells are usually grouped, and are the essential feature of nerve centres, otherwise called ganglia or gray matter. Ganglion cells are among the largest cell elements of the body, and consist of a dense, reticulated, and frequently pigmented ground work, inclosing a large translucent nucleus, and usually a single nucleolus. One or more prolongations, poles or horns, are sent from these cells, and hence they have been classified as unipolar, bipolar, tripolar, quadri- polar, and multipolar, according to the number of projections. The cell prolongations generally divide soon after leaving the body, and subdivision continues until exceedingly minute fibrils result, which serve as connecting links of the elements of a ganglion. Usually one (the larger) pole is projected which remains unbranched. This be- comes the axis cylinder of a nerve fibril, and affords connection between the elements of a ganglionic centre and the conducting por- tion of the nervous apparatus. 182 PRACTICAL MICROSCOPY. Ganglion cells are surrounded by irregular channels or lymph- spaces, and are thus in intimate relation with the lymphatic system. CONNECTIVE TISSUE OF THE NERVOUS SYSTEM. The connective tissue, which serves to unite the elements of a nerve trunk, does not differ materially from the susteutacular tissue of other organs. Different terms are applied, according to its use and loca- tion, as follows: EPINEURIUM. — Forming the sheath of the entire nerve trunk. PERINEURIUM. — Surrounding the bundles composing the nerve trunk. ENDONEURIUM. — Permeating and uniting the elements of the bun- dles. Fia. 119.— TRANSVERSE SECTION OP THE A.NTERIOR CRURAL NERVE. X 250. A. The epineurium. B. Adipose tissue in the loose areolar tissue of the sheath. C. Lymph-spaces of the epineurium. D. Large blood-vessels of epineurial sheath. E. Perineurium surrounding nerve bundles. F. Lymph-spaces of last. G. Medullated nerves in T. S. supported by connective tissue— endoneurium. NELTKOGLIA. 183 NEURILEMMA. — Surrounding the individual nerve fibres of a bun- dle. The formula E. P. E. N., composed of the initial of the name of the investments from without inward, will aid the memory. The epineurium serves to protect the organ in its passage, and to support the nutrient blood-vessels and the channels of lymphatics. The fibres run both longitudinally and transversely. The perineu- rium, arranged in dense bands, forms distinct sheaths for the nerve bundles, the fibres running, for the most part, circularly. The endo- neurium not infrequently divides the nerve bundles into smaller or primitive bundles. It supports the blood capillaries, contains small lymph-spaces, and its nuclei are frequently large and prominent. The final distribution of the elements of a nerve trunk is effected by subdivision; first, of the large, and afterward of the primitive bundles or fasciculae. The perineurial sheaths are prolonged, fur- nishing the dividing bundles, even to the final distribution, where, .around terminal and single medullated fibres, the sheath remains as a layer of exceedingly delicate flattened cells. The necessity for the endoneurium ceases with the ultimate subdivision of the nerve fas- ciculus. NEUROGLIA. The sustentacular or supporting tissue of the brain and spinal cord differs materially from ordinary connective tissue. It presents an FIG. 120. — NEUROGLTA, FROM BENEATH THE PIA MATER OF THE SPINAL CORD. A. Network of neuroglia fibrils. B. Spider (Deiter's) cells. C. Nerve fibres in T. S. X400. 184 PRACTICAL MICROSCOPY. interlacement of fibres which, even with the highest powers of the- microscope, appear ,of exceeding tenuity. The neuroglia mesh sup- ports the nervous elements, scattering branched (Deiter's) cells, and small round cells. Peripheral Termini. (The demonstration of peripheral nerve apparatus should not be attempted until additional work, in the lines hereafter indicated, has secured for the student a degree of perfection in technique which he is not at present supposed to possess. ) SPINAL COED. 185- SPINAL COED. The membranes covering the cord will be discussed later. The spinal cord is composed of gray (cellular) and white (fibrous) nerve matter, and serves as a medium of communication between the brain and peripheral nerve apparatus. The arrangement of its sev- eral parts will be best understood by the study of a transverse section,, of which Fig. 121 is a diagrammatic representation. The gray substance occupies the central portions of the structure,. FIG. 121.— DIAGRAM. CERVICAL SPINAL CORD IN TRANSVERSE SECTION. A. Anterior median fissure. B. Posterior median fissure. C. Anterior cornu— gray substance. D. Posterior gray cornu. E. Point of emergence of anterior root of spinal nerve. F. Posterior root of spinal nerve. G. White commissure. H. Anterior gray commissure. I. Posterior gray commissure. J. Substantia gelatinosa. The tracts which are named on the diagram have no definite boundaries histologically. They are physiological areas. 186 PRACTICAL MICROSCOPY. and consists of two lateral masses and a connecting link or commis- sure. Near the central portion of the figure, a small circular opening presents — the transversely divided central canal. This is in commu- nication, in the medulla, with the fourth ventricle, and will serve as a starting-point for our study. The gray matter completely surrounds the central canal, and its outline resembles the capital H. The bars or columns each present anteriorly a blunted extremity, horn or cornu, while the posterior cornua are pointed. The lateral bars or columns are connected, as we have seen, a portion of the connecting substance passing in front and a portion behind the central canal — the anterior and posterior gray commissural lands. The white substance is divided anteriorly by the anterior median jissure, which sections the cord nearly, but not entirely, to the ante- rior gray commissure. A corresponding division appears posteriorly (the posterior median -fissure) which does not divide the cord posteri- orly as completely as does the previously named fissure anteriorly; but the divison is indicated by a band of neuroglia, which penetrates entirely to the posterior gray commissure. The two masses of white substance thus indicated by more or less complete central division are termed lateral white columns, and these are united just in front of the anterior gray commissure by white nerve tissue— the white com- missure. The spinal nerves take, origin from the gray cornua, the anterior roots from the anterior and the posterior roots from the pos- terior cornua. The white substance consists essentially of medullated fibres which, with the exception of the anterior spiral nerve roots and the commissural fibres, pass mainly in a longitudinal direction. PRACTICAL DEMONSTRATION. Nerve tissue should, under all circumstances, be hardened in Miiller's fluid. The cord should be obtained as nearly fresh and un- injured as possible; cut transversely with a sharp razor into pieces half an inch long, and placed immediately in the fluid — in the pro- portion of a pint of the mixture to two ounces of tissue The solution should be thrown away after twenty-four hours, and a fresh supply provided. It should be again changed after three days, and again after another week. After four weeks the bichromate should be poured off, and the tissue rinsed once with water; after which the hardening is to be completed with alcohol in the ordinary manner, i. e., commencing with the weak spirit. After hardening, pieces from the different regions should be cut, and this is best effected by the infiltration methods. Transverse .sections are the most instructive, although the student should after- ward study longitudinal cuts. The sections must be thin, but not HUMAN SPINAL CORD. 187 necessarily large, and they may be stained by the method of Weigert, or with haema. and eosin. Weigert's method requires very careful manipulation, and is of more special value in pathological research. If human tissue cannot always be procured in suitable condition, the cord of the ox, pig, sheep, cat, or rabbit will serve well. The ox, especially, provides a means of securing tissue of surpassing excellence, particularly for demonstration of the ganglion cells. The cord of the smaller domestic animals is, in nearly every respect, as valuable for study as that of man, especially as the latter cannot usually be gotten before the serious putrefactive changes, to which nerve tissue is prone, have made marked progress. HUMAN SPINAL COED. CERVICAL REGION. TRANSVERSE SECTION. (Fig. 122.) OBSERVE: (L.) 1. General arrangement of gray and white substance, with the latter surrounding the former, which resembles in outline the letter H. 2. Subdivisions of white substance, (a) Anterior median fis- sure. (Note its passage inward and its cessation before reaching the gray substance.) (b) Posterior median fissure. (Note its shallowness as a true fissure, and the extension of the connective tissue from the bottom inward, until the gray substance is met. Compare the two median fissures.) (c) The emergence of the anterior nerve-roots. (This provides the external or lateral boundary of anterior white columns or direct pyramidal tracts, the internal boundaries being provided by the anterior median fissure.) (d) The lateral columns. (These contain the fibres of the crossed pyramidal tract, and include the white substance between the anterior nerve-roots and the posterior gray cornu. Each lateral column contains nerve fibres which pass to the cerebellum — direct cerebellar tract; observe that these tracts have no internal histological boundary. Note the numerous prolongations of the pia mater inward in the lateral columns.) (e) The postero- internal or column of Goll — -funiculus graoilis. (These columns present on either side of the posterior median fissure, and are bounded laterally by a prolongation from the pia mater.) (/) The postero- external columns — -funiculus cuneatus. (Bounded internally by the postero-internal columns, and externally by the posterior gray cornua.) (g) The white commissure. (Note the absence of a white commissure posteriorly, the posterior median septum reaching the .gray substance.) 188 PRACTICAL MICROSCOPY. 3. Subdivisions of the gray substance, (a) The central canal. (Should the section have been taken from the extreme lower cervical cord, this canal as such will be difficult of demonstration, a number of deeply stained cells only remaining.) (b) The gray commissure, anterior and posterior, (c) The gray columns, (d) The anterior gray cornua, broad and not reaching the periphery of the cord sec- tion, (e) The posterior cornua, narrow and passing completely out, posteriorly, to form the posterior root of a spinal nerve. FIG. 122.— TRANSVERSE SECTION OF THE SPINAL CORD. MIDDLE CERVICAL REGION. X 60. A. Anterior. B. Posterior. The references in Fig. 131 apply also to this illustration. Also vide text. This section was made from the cord of a man who died at the age of 75 years, from senile dementia. The gray substance is perfectly normal, but of somewhat diminished area. (H.) 4. The white substance (select a field, e. g., in the anterior median column, and observe the transversely divided nerves), (a) The nerves are not collected into fasciculae, but each fibre pursues an independent course, (b) The axis cylinders, stained lightly with the eosin. (Note the great variation in size. ) (c) Most of the axis cylinders surrounded by more or less concentric rings of translu- HUMAN SPINAL COED. 189 cent, unstained white substance of Schwann. (These are medul- lated fibres.) (d) The few and scattering axis cylinders without surrounding white substance. (Non-medullated nerves.) (e) The neurilemma, appearing as a thin, violet ring around the white sub- stance of Schwann. (Most medullated nerves of the cerebro-spinal .system are provided with this sheath.) (/) The small, about one- three thousandth of an inch, deeply haema. -stained cells of the neuroglia. (g) The neuroglia substance, finely granular or fibrillated, between the nerve fibres, (h) The spider cells (Deiter's) of the neuroglia. (These are not numerous, but easily found near the periphery.) (i) The longitudinal nerve fibres passing from the an- FIG. 123.— SAME SPECIMEN AS SHOWN IN FIG. 122. MORE HIGHLY MAGNIFIED. REGION OF AN- TERIOR CORNU. x 350. A. Medullated filaments passing out from the gray substance to form the anterior root of a spinal nerve. B. Ganglion cells. C. Neuroglia nuclei. D. Blood-vessels. E. One of the transversely divided medullated fibres of the white substance, anteriorly to the anterior gray cornu. The line leads to the neurilemma. F. White substance of Schwann — of last. G. The axis cylinder of E. terior gray cornu to form the anterior root of a spinal nerve, (j) The different size of the nerve fibres in different areas of the section. (Note the small fibres of the postero-internal column.) (k) The blood-vessels. (These vessels are largely confined to the neuroglia- septa, which pass in from the pia. These septa contribute to the 190 PRACTICAL MICROSCOPY. formation of the nearest approach afforded by the cord of nerve bundles.) 5. The gray substance, (a) The central canal. (The canal is lined with columnar ciliated cells in single layer. The cilia are rarely demonstrable in the human cord, on account of changes which occur very quickly after death. The canal is usually broadest in its lateral diameter, viz., at upper portion of cervical region, from one-one hundredth to one-two hundredth of an inch, (b) The ground sub- stance. (This consists, 1st, of exceedingly minute fibres, formed by the repeated subdivision of the axis cylinders — the primitive fibrillae ; 2d, of the delicate neuroglia fibres. It is usually difficult in a section to differentiate between the two. Attempts have been made, with more or less success, to differentiate by means of staining agents.) (c) Large ganglion cells. (Select a field in the anterior horn. The straight, unbranching axis-cylinder process can frequently be distinguished. Note the large, shining nucleus and the deeply stained nucleolus. These cells are frequently deeply pigmented.) (d) Small ganglion cells. (Best seen in the posterior horn. In the dorsal cord a collection of medium-sized cells presents, just within the poste- rior commissure and encroaching upon the white substance posteriorly to this, the column of Lockhart Clarke.) (e) Lymph spaces. (Ob- served as a somewhat clear space around the ganglion cells.) (f) Blood-vessels. (These are much more numerous here than in the white portion; and arteries may frequently be found of considerable size.) (g) Peri-vascular lymphatics. (Find an artery in trans- verse section, and observe the clear space around it, which may be mistaken for the result of contraction of the tissue in hardening. Careful study will reveal minute branches of cells, passing between the adventitia of the blood-vessel and the wall of the lymph space.) THE BRAIN AND ITS MEMBRANES. 191 THE BRAIN AND ITS MEMBRANES. The brain and spinal cord are surrounded by three connective- tissue layers — the dura mater, arachnoid, and the pia mater. The dura is the most external and the thickest of the three mem- branes, and constitutes the periosteal lining of the cranial and spinal cavities. It consists largely of elastic tissue, the laminae and blood- vessels of which are supported by connective tissue. The outer sur- face is in more or less intimate connection with the bone, and both surfaces are covered with a single layer of thin pavement cells. Beneath is a space — the subdural — containing lymph. The arachnoid, exceedingly thin, presents an outer, glistening, pavement-cell covered surface (isolated from the dura by the sub- dural space), from the under (inner) side of which short fibrous trabeculae are projected to the pia. The subarachnoidal space is thus seen to consist of numerous communicating chambers, and these spaces are everywhere lined with flat cells, and contain lymph, as does the subdural space. The pia mater consists of fibrillated connective tissue, usually in intimate connection with the arachnoid externally, by means of the trabeculae of the latter. The pia is exceedingly vascular, and every- where covers the brain and cord, and, unlike the arachnoid, pene- trates the sulci of the former and the fissures of the latter, becoming continuous with the neuroglia tissue. The subdural and subarachnoidal spaces are lymph-cavities, and, while not in direct connection one with the other, belong to the general lymphatic system, and are in eventual connection. The two spaces are projected, independently, in the sheaths of the cranial and spinal nerves — the subdural communicating with the lymph channels of the epineurium, and the subarachnoidal with those of the peri- neurium. The arrangement of gray and white nerve substance in the brain is precisely the reverse of that of the cord. The gray matter forms an external covering or layer of varying thickness, while the white matter occupies the more central regions. Collections of gray matter — gang- lia— are also situate in the deeper parts of the brain substance, the study of which does not come within the limits of this work. The brain substance does not differ, essentially, from the cord, except in the arrangement of its parts. The nerve fibres are largely 192 PRACTICAL MICROSCOPY. medullated, but have no neurilemma. The neuroglia and ganglionic tissues do not here differ in structure from that previously de- scribed. The gray substance is arranged in layers, which are, in .some instances, quite sharply defined, and again demonstrable only with considerable difficulty. PRACTICAL DEMONSTRATION. The tissue is to be prepared in the manner usual with nerve sub- stance — hardened with Miiller, followed by alcohol. Thin sections, stained deeply with haema. and eosin, may be mounted in dammar or, if preferred, in glycerin. SECTION OF HUMAN CEREBRUM. CUT PERPENDICU- LARLY TO THE SURFACE. (Fig. 124.) OBSERVE: 1. The membranes. (In the drawing only the arachnoid and pia are shown.) (a) The fine fibrillar mesh of the arachnoid. (b) The nuclei of the flattened cell-covering, (c) The large blood-vessels, (d) The pia. (e) Its continuity with the neuro- glia of the cerebrum. 2. The outer layer — the first — of the gray substance. (This layer is poorly defined, but can usually be made out. It consists of primitive nerve fibrillae, neuroglia fibrils, and scattered spherical cells.) 3. The second layer. (This layer presents about the same thick- ness as the preceding, and will be recognized by the numerous small, triangular nerve-cells. Indeed, these afford the only means of dis- tinguishing the boundary between the two layers, as the stained ele- ments of the outer layer are seldom pyramidal.) 4. The third layer. (This layer — the thickest of all the gray laminae — has been shortened in the drawing, on account of lack of space. It is three or four times as thick as the first layer, and will be readily made out by the large, elongate, pyramidal cells, with their long axes at right angles to the brain surface. Medullated fibres, in more or less distinct bundles, pass between the column-like ganglion cells.) 5. The fourth layer. (The large cells of the third layer are seen to stop abruptly, as we pass inward, and give place to small, triangular nerve-cells. This brings us to the fourth plane. Between the cells of this layer, bundles of nerve-fibres are seen, as they radiate toward the cerebral surface.) THE BRAIN AND ITS MEMBRANES. 198 6. The fifth layer. The line of demarcation between this and the fourth layer is feebly shown; but, on close attention, it will be ob- served that the small cells of the fourth layer rather abruptly give place to elongate ones, not unlike those of the third stratum. The nerve-bundles are here more plainly indicated. 7. The white matter. (The ganglion cells here cease, and the field is occupied with medullated fibres and neuroglia, the spherical nuclei of the latter becoming prominent from the deep haema. staining.) FIG. 134.— VERTICAL SECTION OF CEREBRAL CORTEX. SUPERIOR FRONTAL CONVOLUTIO N. X 250. A. Arachnoid. B. Pia mater. C. D, E, F, G. First, second^third, fourth, and fifth layers of gray matter. H. White brain substance. 8. The nutrient blood-vessels. (The capillaries projected from the pia are especially to be noticed, often of the diameter of a single blood-corpuscle, and presenting as branching lines, composed of these elements— indeed difficult of demonstration when empty. Note the light, perivascular lymph-spaces, well seen around the larger arteries in transverse section.) 13 194: PRACTICAL MICROSCOPY. VERTICAL SECTION OF HUMAN CEREBELLUM. PRE PARED AS LAST SPECIMEN. (Figs. 125 and 126.) OBSERVE : 1. The arrangement of the cortex in the form of leaflets. 2. The extension of the gray laminae within even the minutest folds of the leaves, so as to completely envelop the central white nerve- FIG. 125.— LONGITUDINAL SECTION OP ONE OF THE FOLIA OF THE CEREBELLUM. X 60. A, A. Line of pia mater. B, B. Sulci. C, C. Outer layer of gray matter. D, D. Inner layer of gray matter, including Purkinje's cells. E, White nerve substance. substance. (The staining has heen so selected by the tissue as to divide the outer gray matter into two prominent layers. The ex- planation of this will follow increased amplification.) 3. The central white matter. (The fibrillar character can be made out, and the general plan seen to consist, as in the cerebrum, of VERTICAL SECTION OF HUMAN CEREBELLUM. 195 central nerve fibrillae radiating toward the cells of the cortical gray substance.) 4. The outer gray layer. (This is the thickest of the three layers. The prominent elements to be observed are : the scattering spheri- cal neuroglia and small nerve-cells, nerve-fibrils passing at right angles to the surface and lost as the outer region is approached, the prominent blood-vessels which pass in from the pial investment.) FIG. 126.— VERTICAL SECTION, CORTEX OF CEREBELLUM. PORTION OP SECTION SHOWN IN FIG. 125, MORE HIGHLY MAGNIFIED, x 250. A. Outer layer of gray matter. B. Layer of Purkinje's cells. G. Inner gray layer. D. White nerve substance. 5. The ganglionic layer. (Directly beneath the outer layer the section becomes deeply stained, from the presence of numerous small cells, among and partly concealed by which are the large ganglion cells of Purkinje. These are flask-shaped, and are arranged in a single plane, with their long axes vertically. A thread-like prolon- 196 PRACTICAL MICROSCOPY. gation may be seen penetrating the layer beneath, providing the cell has been centrally sectioned. Large horns are projected from the outer extremity of the cells, branches from which provide the nerve-fibrils seen in the last-observed layer. The cell bodies take the- eosin, and the nuclei the logwood.) 6. The granular layer. (This is the layer seen so distinctly with the low power. It consists of innumerable small, deeply hsema.- stained bodies, usually spherical, which are, as is believed, mostly neuroglia cells. These nucleated elements are embedded in an ex- ceedingly fine matrix of neuroglia [Klein] fibrils. Search carefully for the axis cylinder processes of the Purkinje cells which pierce this layer, and follow them into the white matter below.) 7. The white subtahce. (This consists of medullated nerves which arise largely from the cells of the second gray layer. Klein has also traced fibres into the nuclear layer, and demonstrated their dis- tribution to the small ganglion cells of the lamina, and to the network of the outer gray substance.) FORMULAE. 197 MISCELLANEOUS FORMULAE. DAMMAR MOUNTING VARNISH. No. 1. Gum Dammar, 4 ounces. Gum Mastic (in "tears"), ... 2 " ' Spirits of Turpentine, .... 8 " Chloroform (Squibb's), .... 4 " Mix the dammar with about an equal bulk of clean broken glass. Put in a wide-mouth bottle, and, having added the turpentine, set in a warm place. The glass is added for the purpose of separating the lumps of the resin, and thus hastening the solution. Stir the mixture occasionally. Add the mastic to the chloroform in a well-stoppered bottle. When the solution of the resins is complete add the chloroform mixture to the dammar, and keep in a bottle covered with a plate of glass. After the dirt from the gum has thoroughly settled, decant into small bottles for use. Do not attempt to filter. I have histological mounts which were made with this medium nearly ten years since, and the tissues have shown no deterioration whatever. DAMMAR MOUNTING VARNISH. No. 2. Bsst dammar varnish of the paint-shops, diluted with a sufficient quantity of turpentine. I do not know that this is in any way inferior to the last, but histol- ogists generally have a preference for media of known composition. They may both be diluted with turpentine, chloroform, ether, or pure benzole; or thickened, by removing the stopper of the bottle until a sufficient amount of the solvent has evaporated. XYLOL BALSAM. Xylol is preferred by some histologists as a solvent or diluent of Canada balsam for general mounting purposes. Xylol dissolves the balsam very readily without heat, and evaporates more rapidly than 198 PRACTICAL MICROSCOPY. most solvents. A thin solution — say xylol two parts to Canada balsam one part — should be first prepared, and, after filtering through paper, it may be placed in an unstoppered bottle until, by evaporation, it be- comes sufficiently thick for use. Xylol is miscible with strong alcohol, oil of cloves, etc., but not with water. VARNISHES AND CEMENTS FOR RINGING MOUNTS. 1. Dammar. — I believe a clean dammar mount, with circular cover, neatly labeled, cannot be improved in appearance by painting rings of colored varnish around the specimen. Nevertheless, the beginner will purchase and use a turn-table, and must be therefore directed in its employment. A ring of dammar, thinned with turpentine so as to flow readily from the brush, makes a very neat border to the cover-glass of a specimen mounted in this medium. Let the layer be quite thin. 2. Zinc Cement. — To a small quantity of thin dammar varnish, add q. s. of pure dry zinc white. Mix thoroughly on a glass plate with paint-knife or spatula. The consistency should be such as to flow readily from the brush. Before adding any cement containing pigment or color to a dammar mount, a protecting covering should be applied; otherwise the cement will eventually creep in between the cover and the slide and mix with the original mounting varnish. It may proceed very slowly; but in time the specimen will be surely ruined. This is best avoided by painting a thin ring around the edge of the cover of liquid glue. Cooper's and LePage's are both excellent. Let this dry, and any amount of varnish may be subsequently applied without disaster. Aniline Colors. Red, blue, etc., maybe employed by dissolving the dry color in a little thin shellac varnish. This dries quickly and may be used over the zinc. Oil colors. The artists' colors which are sold in tubes may be thinned with dammar. Black varnish. This is the ' ' Black Japan " of the paint shops. It may be made by dissolving gum asphaltum in turpentine. The gen- uine asphaltum is very difficult to procure, as coal tar is generally sold under this name. It is therefore best to purchase the 'varnish, thin- ning with turpentine or pure benzol if necessary. Shellac Varnish. — The best orange shellac dissolved in strong alcohol, in quantity sufficient to make a varnish of the consistency of treacle. FORMULAE. 199 This is an excellent cement for glycerin mounts. It should be considerably thinned for use as a vehicle for color. PRESERVATIVE FLUID. Acetate of Soda, 1 Ib. Distilled Water, 8 ounces. Scraping or teasings from tissues, morphological elements from urinary sediment, casts, in fact, almost any histological element may be indefinitely preserved in this solution. It is important that the tissues, etc., be freed from organic matters in solution, before being placed in the preservative; for example: urine must be carefully decanted from the sediment to be preserved before the acetate solu- tion is added. I am enabled to preserve the different forms of cells from year to year, for my classes, in small bottles of this fluid. When a demonstration is required, it is simply necessary to transfer a drop of the sediment, by means of a pipette, to a slide, and apply the cover-glass. , If it be desired to preserve such a mount, wipe the edges of the cover with a bit of blotting paper, and seal with a ring of shellac or asphalt um varnish, or zinc cement, applied with a small brush. NORMAL SALT SOLUTION. Chloride of Sodium (common salt), . * 7 grains. Distilled Water, 2 fluidounces. A medium for the temporary examination of fresh tissues — scrap- ings, teasings, etc. RAZOR-STROP PASTE. Sulphate of iron and common salt equal parts. Calcine in a sand crucible at a dull red heat for ten minutes. When cold, grind lightly in a porcelain mortar and pass through fine gauze. Preserve dry in a well-corked bottle. A few grains dusted on the surface and mixed with a minute quan- tity of tallow will add greatly to the efficiency of the strop. Another. — Equal parts of opticians' rouge and the finest washed flour of emery, mixed with a sufficient quantity of vaseline to make a stiff paste. 200 PRACTICAL MICROSCOPY. SILVER STAINING SOLUTION. Nitrate of Silver, 5 grains. Water (distilled), 4 ounces. Used for the demonstration of cement substance between cell elements. OSMIC ACID SOLUTION. Osmic Acid, 1 gramme. Water, 100 cubic cent. The acid is found in market sealed in glass tubes holding one gramme each. The utmost care should be taken to avoid inhaling the vapor from the pure acid as it produces an intense inflammation of the respiratory surfaces. The best method of handling the material is to measure 100 c.c. of water, place it in a strong bottle, and then drop in the tube containing the osmic acid. Then with a glass rod break the tube. The bits of glass need not be removed. Keep the bottle, tightly stoppered, in a cool dark place. Osmic acid is used in histology on account of its action upon fatty substances, i. e., staining them of a deep brown or black. WEIGERT'S H^EMATOXYLIN STAINING FOR MEDUL- LATED NERVE TISSUE. Formula. 1. A saturated aqueous solution of neutral acetate of copper. 2. Strong haematoxylin staining fluid, vide text. 3. Borax, ferridcyanide of potassium, aa 1 drachm. Water 4 fluidounces. Process. 1. Allow the sections to remain in 1 for twenty-four hours.* 2. Wash in clean water for a few moments only, and transfer to 2. Brain sections will require from two to three days, and spinal cord twenty-four hours for complete staining. They must appear almost black. If the hardening has been effected with the bichromate solu- tion as given in the text (vide hardening fluids), maceration of the sections in the cupric acetate may be omitted. 3. Wash in water for five minutes and transfer to 3. They may be * The haema. solution gives the best results, in this staining, if kept at tem- perature of about 100° F. KAKYOKINESIS. 201 allowed to remain for twelve hours without harm. Generally an hour will be sufficient. 4. Wash in water. 5. Dehydrate gradually, first placing in dilute alcohol, and after- ward in stronger. The sections can now be kept in alcohol indefinitely* If the tissue has been infiltrated with celloidin, the sections must not be held in ninety-five per cent alcohol longer than five minutes, as the infiltrating medium will be dissolved. 6. Clarify with oil of cloves — oil of bergamot for celloidin speci- mens— and mount in dammar. The method does not produce bright colors, but it gives a very remarkable differentation of nerve tissue, by staining the medullary substance violet, and the axis cylinders brown. It is particularly valuable in pathological histology. BAYBERRY WAX INFILTRATING METHOD. In answer to many inquiries regarding the material used in this process, I may say that Messrs. Eimer & Amend, chemists, of New York, from whom my supply was originally obtained, state that the article furnished me was the Japan wax. Dr. J. W. Blackburn, of Washington, D. C., kindly informs me that this is the product of Rhus succedanea. The material with which I have had the best re- sults w