! -l COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD HX00032735 A Text Book OF Practical Histology, WILLIAM STIRLING. M. D. > ^' -^ ') ^1 i i' , ■>' " 'i k: >-'t' '^^^{v .•*? ^^' '/■:-.,l (Z Lf- Sb Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons http://www.archive.org/details/textbookofpractiOOstir PRACTICAL HISTOLOGY Frontispiece Mirvtem BrOSf'litK ; I THE DETAILS RLLED IN . A TEXT-BOOK OF PRACTICAL HISTOLOGY WITH OUTLINE PLATES BY WILLIAM STIRLING, M.D., Sc.D., RR.S.E. ' REGIUS PROFESSOR OF THE INSTITUTES OF MEDICINE IN THE UNIVERSITY OF ABERDEEN SKilb 30 (!3«tlxitt ;platcs, 1 Colourti) |3lHtf, ai>b 27 aJloob 6ngrafatngs PHILADELPHIA J. B. LIPPINCOTT & CO. LONDON : SMITH, ELDER, & CO. i88i \All rights reservi:d\ <.. St ^ TO CARL LUDWIG PROFESSOR OF PHYSIOLOGY IN THE UNIVERSITY OF I.EIPSIC AND L. RANVIER PROFESSOR OF HISTOLOGY IN THE COLLEGE DE FRANCE, PARIS TO WHOM THE AUTHOR IS INDEBTED FOR HIS EARLIEST TRAINING IN THE TRUE METHOD OF MAKING AND RECORDING ORIGINAL OBSERVATIONS IN PHYSIOLOGY AND HISTOLOGY, HE, WITH HEARTFELT PLEASURE, DEDICATES THIS WORK. In OFFERING TO THEM THIS TRIBUTE OF HIS ADMIRATION AND REGARD, THE author desires to express his sense of the inestimable advantage which they have conferred on physiology and histology. Their labours, characterised as they have been by enthusiasm and single- minded DEVOTION TO THE ADVANCEMENT OF SCIENCE, HAVE NOT ONLY ADDED GREATLY TO OUR FUND OF KNOWLEDGE, BUT HAVE ALSO DEMONSTRATED THE SUPERIORITY OF THAT METHOD OF SCIENTIFIC OBSERVATION WHICH CONSISTS IN A PATIENT AND INTELLIGENT SCRUTINY OF NATURE. To THIS METHOD THEY HAVE ATTRACTED MANY DISCIPLES WHO ACKNOWLEDGE THE INCALCULABLE VALUE OF THEIR PERSONAL INFLUENCE, THE CORDIALITY WITH WHICH THEY HAVE ENCOURAGED AND DIRECTED THEM IN THEIR PURSUIT OF ORIGINAL INVESTIGATIONS, AND THE GENEROSITY WITH WHICH THEY HAVE LAID OPEN TO THEM THE RICH STORES OF KNOWLEDGE ACCUMULATED DURING A LIFE-LONG STUDY OF THE PROBLEMS OF ANLMAL STRUCTURE AND FUNCTION. PREFACE. The ruKPOSE of this work is twofold : first, to give plain, definite, and precise directions for the preparation and examination of the animal tissues ; and, secondly, to ensure that the student executes a drawing of the majority of the microscopic specimens which he makes for preservation. For this purpose a series of Outline Plates is issued with the text. The author has endeavoured to give a faithful account of the methods which he has found to be most useful for the preparation of each of the tissues and organs of the body for microscopic purposes. No method is introduced which he has not found from repeated trials to be successful. The methods described are those which, after nine years' experience in the teaching of practical histology, he has found to be really reliable. In the introductory chapter a short account is given of the microscope and how to use it ; but no attempt is made to explain the optical principles on which it is constructed, as the author believes that such details are better omitted in a work like the present. The reader is therefore referred for such details to any of the numerous works upon that instrument. The student is advised to read the intro- ductory chapter before beginning the practical portion of the work. The author, from long experience in the teaching of histology, has learnt that the reluctance shown by students in making drawings of their microscopic prepara- tions renders it advisable to facilitate as much as possible this method of exact representation. This he does by giving to each student a series of outline plates, in which the main features of the chief microscopic sections are indicated. For two sessions he has done this with his classes of practical histology in the University of Aberdeen, and he finds that the students rapidly acquire the art of fillin^T in the necessary details. At the end of the course of histology, therefore, each student is in possession of a complete set of microscopic preparations, which illustrates the viii PREFACE. whole range of normal histology, and which he can permanently preserve and take away with him. He has also an atlas representing the details of the more impor- tant preparations ; and, lastly, in the text-book he has a record of how each specimen was prepared, and a description of the chief points to be observed in each microscopic preparation. It is to be remembered, however, that no mere instructions or directions given in books can ever supersede that form of informa- tion which is imparted viva voce by a teacher, and which is elicited by the difficulties that meet the student of histology at the beginning of his course. He is indebted to Mr. E. A. Schafer for the use of several of the woodcuts employed in the introductory chapter ; and for fig. 3, to Messrs. Baillicre, Tindall, & Cox. The outline figures were drawn by the author from the microscopic specimens with which each student is supplied or makes for himself. He has also to express his sincere thanks to Dr. Robert Lawson, Deputy Commissioner in Lunacy, and to Dr. De Burgh Birch for revising the proof-sheets. WILLIAM STIRLING. Physiological Laboratory, University of Aberdeen : March, 1881. CONTENTS. INTRODUCTION. Histological requisites The use of the microscope Artificial illumination . Cleaning of the objectives Method of focussing an object . Rules for examining an object Preparation of a simple object . Methods of applying a cover-glass . Method of cleaning a cover-glass Measuring cover-glasses Placing a section on a slide Delineation of an object . . . . Methods of studying living protoplasm Methods of applyitig heat to miscroscopic objects . ■ . . . . . Methods of applying vapours to objects PAGE xix xxi xxiii xxiii xxiii xxiv xxiv xxiv XXV XXV xxvi xxvi xxvii xxviii xxix Preliminary preparation of the tissues Methods of hardening the tissues Methods of softening the tissues Dissociation of the tissues . Methods of cutting sections . Microtomes .... Methods of staining the tissues Aniline dyes .... Metallic solutions . Double staining Treble-staining Staining reagents Methods of mounting sections Labelling and preserving sections Methods of injecting blood-vessels Methods of injecting lymphatics PAGE XXX xxxi xxxiii xxxiv -xxxvi xxxvii xlii xliv xlv xlvi xlvii xlvii xlviii y.lix 1 Iv Blood of newt or fiog Human blood Blood-crystals Epithelium Endothelium Cartilage Connective tissue Bone Muscular tissue . Nerve-tissue . Heart and blood-vessels Circulation of the blood . Larynx, Trachea, and Lungs Lips and Tongue . Tooth Salivary glands Tonsils .... Oesophagus and Stomach intestine .... Liver .... Pancreas .... Lymphatic system . Lymphatic glands Spleen .... Thymus .... PRACTICAL WORK. PAGE I 5 6 13 14 20 30 36 42 47 52 S3 60 62 64 65 65 67 71 75 76 77 78 PAGE Ductless glands . 82 Supra-renal capsules .... 82 Kidneys • H Ureter . SB Bladder . 89 Skin . 90 Spinal cord and medulla oblongata . 96 Cerebellum and cerebrum . 100 Nerve-ganglia . 102 Eye . 104 Cornea . 104 Nose . Ill Semicircular canals and cochlea . 113 Testis ....... 116 Epididymis ..... 118 Ovary ....... 120 Fallopian tube ..... 121 Uterus ... ... T2I Mammary inland .... 122 Milk . ' 122 Placenta ...... 122 Deciduse ...... 124 .Amnion ....... 124 Umbilical cord ..... 124 INDEX 125 ABBREVIATIONS USED IN THE TEXT. (H) means /lig/i power, i.e. a magnifying power of 300 diameters linear. Such a magnifying power is obtained by using a No. 7 objective, and a No. 3 eyepiece of Hartnack with the tube drawn out ; or an English |-inch objective and a medium eyepiece. (L) means l(nv power, i.e. a magnifying power of 65 diameters linear. Such a magnifying power is obtained by using a No. 3 objective and a No. 3 eyepiece of Hartnack with the tube drawn out ; or an English one-inch objective and a medium eyepiece. T. S. and L. S. mean transverse and longitudinal section respectively. EXPLANATION OF THE OUTLINE PLATES. Plate I. BLOOD. ( To face /. 4. ) Fig. 1. Coloured blood-corpuscle of a newt or frog on the flat. 2. Coloured blood-corpuscle of a newt or frog on the edge. 3. Effect of acetic acid on a newt's corpuscle. 4. Effect of syrup on a newt's corpuscle. 5. Effect of tannic acid on a newt's corpuscle. 6. Effect of water on a newt's corpuscle. 7. Effect of osmic acid and picrocarmine on a newt's corpuscle. 8. Colourless blood-corpuscles of a newt or frog. 9. Human blood-corpuscles (coloured and colourless) and fibrin. 10. Human coloured blood-corpuscle on edge. 1 1. Human coloured blood-corpuscle on the flat; centre in focus and out of focus. 12. Human coloured blood-corpuscle crenated. 13. Human colourless blood-corpuscles. 14. Effect of acetic acid on these corpuscles. 15. Effect of a magenta solution on these corpuscles. Plate II. EPITHELIUM. (To face t>. 12.) 1. Squamous epithelium and salivary corpuscles (mouth). 2. Squamous epithelium cuticle of newt (intra-cellular plexus of fibrils) 3. Silvered epithelium (mesentery of a rabbit). 4. Columnar epithelium of the intestine. 5. Vertical section of the cornea. 6. Chalice or goblet cell from the small intestine. 7. Columnar epithelium from a newt's intestine. 8. Ciliated epithelium of the trachea. 9. The free ends of the cells of fig. 8. 10. Bars of the gills of a mussel. 11. Transitional epithelium. 12. Secretory epithelium. Xll EXPLANATION OF PLATES. Fill. J- 4- 5- 6. Plate III. CARTILAGE. {Tof.uep. .6.) Hyaline cartilage. Transverse section of a rib of a small mammal. White fibro-cartilage (intervertebral disc.) Transverse section of a rabbit's or cat's ear (L). Transverse section of a rabbit's or cat's ear (H). Vertical section of articular cartilage and subjacent bone. Plate IV. CONNECTIVE TISSUE AND TENDON. (To face p. 24.) Fibres of elastic tissue (Lig. nuchse of ox). Transverse sections of these fibres. Fresh areolar tissue. Effect of acetic acid on fig. 3. Longitudinal section of a tendon (H). Transverse section of a tendon (L). Transverse section of a tendon (H). Tendon of the tail of a rat (L). Tendon-cells from the tail of a rat (H). Plate V. OMENTUM AND FAT {To face p. 28.) 1. Omentum of a rabbit silvered. 2. Omentum of a cat silvered. 3. Vertical section of foetal skin (developing fat-cells (L)). 4. Fat-cells (H). 5. Fat-cells after the action of ether. 6. Mucous tissue. 7. Adenoid tissue. Plate VI. BONE. [To face p. 32.) I T. S. of the shaft of a decalcified bone (L). 2. Part of fig. I with (H). 3. L. S. of a bone with its blood-vessels injected. 4. T. S. of dense dry bone. 5. Lacuna of dense dry bone on the flat 6. T. S. of the shaft of a foetal bone. 7. V. S. through the line of ossification. Plate VII. MUSCLE. (To face p. 40.) 1. Non-striped muscle of the small intestine. 2. Isolated non-striped muscle-cells. 3. Transverse section of non-striped muscle. 4. Cement-substance of non-striped muscle, silvered, 5. Non-striped muscle-cells from the mesentery of a newt. EXPLANATION OF PLATES. xiii Fio. 6. Sarcolemma of striped muscle (effect of water). 7. Effect of acetic acid on striped muscle (frog). 8. Isolated striped muscular fibre. 9. Muscle-discs. 10. Muscular fibre splitting into fibrils. 1 1. Striped muscle of a crab. 1 2. T. S. of a striped muscle. 13. Blood-vessels of an injected muscle. Plate VIII. NERVE-FIBRES. (Tofacep. iA-) 1. Fresh meduUated nerve-fibres (myeline drops). 2. Fresh medullated nerve-fibres (nodes of Ranvier). 3. Effect of osmic acid on nerve-fibres (H). 4. The incisures of Schmidt. 5. Effect of osmic acid (L). 6. Non-medullated nerve-fibres. 7. Nerve-fibre (medullated) after chromic acid. 8. Small nerve silvered (Ranvier's crosses) (L). 9. Nerve-fibre silvered (Ranvier's crosses) (H). 10. T. S. of a sciatic nerve (L). 11. T. S. of a sciatic nerve (H). 12. Concentric lines in the myeline of nerve-fibres. Plate IX. HEART AND BLOOD-VESSELS. (To face p. SO.) Muscular fibres from the heart of a mammal. T. S. of the muscular fibres of the heart of a mammal. Muscle-cells from Purkinje's fibres of a sheep's heart. Capillaries silvered. Small artery silvered. Non-striped muscle in a small artery. Small artery after acetic acid, or stained. V. S. of a middle-sized artery. Plate X. TRACHEA AND LUNGS. (To face p. 56.) T. S. of the trachea (L). Part of Fig. i with (H). T. S. of a bronchus and blood-vessels at the root of a lung. Plate XI. LUNG. (To face p. 58.) Part of a bronchus (H). V. S. of the pleura and air-vesicles. (L). Air- vesicles of a lung stained. Air-vesicles of a lung silvered. Injected blood-vessels of a lung (L). V. S. of a foetal lung. XIV EXPLANATION OF PLATES. Fia I 2 3 4 Plate XII. TONGUE AND TOOTH. (To face p. 62.) T. S. of a tongue — general view (L). V. S. of a tongue with papilla; (H). Taste-bulbs in the papillaj foliatEE (L). Taste-bulbs (H). V. S. of a decalcified tooth and jaw. Pl.\te XIII. SALIVARY GLANDS AND STOMACH. (To face p. 66.) Sub-maxillary gland (mucous) of a dog. V. S. cardiac end ot the stomach (cat) (L). 3. V. S. pyloric end of the stomach (cat) (L). 4. Peptic gland (H). T. S. of the peptic glands (H). Plate XIV. SMALL AND LARGE INTESTINE. (To face f. 70.) V. S. of the small intestine (cat) (L). V. S. of an intestinal villus (H). v. S. of a Lieberkiihn's follicle. T. S. of a Lieberkiihn's follicle. V. S. of the vermiform appendix (rabbit). V. S. of the mucous membrane large intestine (L). I. 2. 3- 4- 5- 6. Plate XV. LIVER. (To face p. 72.) T. S. liver and its capsule (pig) (L). T. S. liver and its capsule (rabbit) (L). Columns of liver-cells (H). Columns of liver-cells (newt) (H). Plate XVI. LIVER AND PANCREAS. (To face p. 74.) 1. Blood-vessels of a liver (rabbit) injected (L). 2. T. S. of a portal canal and Glisson's capsule. 3. Injected bile-ducts. 4. Alveoli of pancreas. Plate XVII. LYMPHATICS. (To face p. 76.) Silvered lymphatics (small intestine). Stomata from the wall of a frog's great lymph-sac. V. S. of a lymphatic gland (L). V. S. of the cortex of a lymphatic gland (H). V. S. of the medulla of a lymphatic gland (H). Adenoid tissue (H). EXPLANATION OF PLATES. XV Plate XVIII. Fig. I. 2. 3- 4- SPLEEN AND THYROID GLAND. (To face p. 8o.) T. S. of a spleen (cat) (L). T. S. of a spleen (H). T. S. of a Malpighian corpuscle (H). T. S. of the thyroid gland. Plate XIX. KIDNEY. ( To face A 86. ) 1. L. S. of a kidney (L). 2. V. S. of the cortex of a kidney (L). 3. Bowman's capsule and glomerulus (H). Plate XX. KIDNEY AND SUPRA-RENAL CAPSULE. (To face p. 88.) 1. V. S. of the cortex (straight and convoluted tubules). 2. Straight tube of the medulla. 3. Injected blood-vessels of a kidney. 4. V. S. of a supra-renal capsule (L). 5. V. S. of a supra-renal capsule (H). Plate XXI. SKIN. (To face p. 92.) 1. V. S. of the skin, uninjected and injected (L). 2. V. S. of the skin (H). 3. Sweat-glands. 4. T. S. of a Pacini's corpuscle. Plate XXII. SKIN AND HAIR. (To face p. 94.) V. .S. of the scalp with a hair-follicle (L). A sebaceous gland (H). A hair without medulla. A hair with medulla. Plate XXIII. SPINAL CORD. (To face p. 98.) 1. T. S. of the spinal cord. 2. Anterior horn of the spinal cord (H). 3. T. S. of the white matter of the cord (H). Plate XXIV. BRAIN AND NERVE-GANGLIA. (To face p. 102.) V. S. of the cerebellum (L). V. S. of the cerebellum (H). 3. V. S. of the cerebrum (L). 4. Nerve-cell of the cerebrum (H). 5. L. S. of the Gasserian ganglion (L). 6. Nerve-cell of fig. 5 without its capsule (H). Nerve cell of fig. 5 with its capsule (H). xvi EXPLANATION OF PLATES. Fig I 2 3 4 5 6 7 Plate XXV. CORNEA AND CHOROID. {To face p. I06.) V. S. of the cornea. Corneal corpuscles stained with gold chloride. Cell-spaces in cornea after silver nitrate. Lens-fibres (man or dog). Lens-fibres (cod-fish). Branched pigment-cells of the choroid. Hexagonal pigment-cells of the retina. PLATE XXVL RETINA AND CILIARY MUSCLE. {To face p. no.) T. V. S of the retina (cat) (L). 2. V. S. of the retina (frog) (H). 3. Cones of retina of a codfish (H). 4. Ciliary muscle (L). Plate XXVII. NOSE AND COCHLEA. (To face p. 114.) 1. V. S. of the nasal mucous membrane (H). 2. Isolated epithelial cells of the olfactory region (H). 3. T. S. of a turn of the cochlea (L). 4. Part of fig. 3 with (H). Plate XXVIII. TESTIS, OVARY. {To face p. liS.) 1. T. S. testis (L). 2. Part of a seminal tubule with developing spermatozoa (H). 3. T. S. of the epididymis (L). 4. Part of a tubule of the epididymis (H). 5. A spermatozoon. 6. V. S. of the ovary (L). Plate XXIX. OVARY, FALLOPIAN TUBE, UTERUS. (To face p. 120.) 1. T. S. of a Graafian follicle with an ovum (H). 2. V. S. of an ovary (H). 3. T. S. of a uterus (L). 4. T. S. and V. S. of the uterine glands (H). 5. T. S. of part of a Fallopian tube (H). Plate XXX. MAMMA, MILK, PLACENTA. {Tofacep. 122.) 1. Alveoli of a mammary gland (H). 2. Milk fresh and after acetic acid (H). 3. Placental villus (L). 4. Placental villus (H). 5. T. S. of the umbilical cord (L). 6. Part of fig. 5 with (H). DIRECTIONS FOR FILLING IN THE DETAILS IN THE OUTLINE PLATES. As a rule only the outlines of the leading features in the sections are indicated. The outlines are not drawn to any definite scale, but where practicable the figures are of the actual size as seen under a linear magnifying power of three hundred diameters ( x 300) or (H), such a relation being noted on one side of the plates devoted to the elementary tissues. The details are to be filled in by the student in pencil (H or HB) and coloured, the latter being done with coloured pencils or water colour. Sufficient dexterity in the manipulation of water colour is, I find, readily acquired by the student, as very few pigments suffice. All the necessary colours are contained in the shilling boxes of moist colour made by Reeves and Sons. A preliminary ground colour or light wash can be laid on in most cases to great advantage. Evenness of colouring is ensured by operating with a full brush upon the paper placed at a gentle slope. Commencing at the top of the figure, carry the brush from side to side with a steady even stroke until the whole is coloured. Invariably allow one colour to dry before another is applied. It is sometimes convenient to use a dilute solution of picrocarmine as a groundwash, whilst the nuclei and other parts stained red may be tinted with solution of carmine. A logwood tint is obtained by mixing a small quantity of carmine with a good blue, such as ultra- marine. The leading parts to be filled in are indicated in the text. ILLUSTRATIONS IN TEXT o>»;o FIG. I. 4- 5- 6. 7- 9- lO. II. 12. 13- 14. 15- End of a mounted needle Scissors curved on the flat Ranvier's arrangement for holding Drop-bottles with Reagents Hartnack's Microscope, No. III. A. Swift's College Microscope . Nosepiece made by Swift Microscopic Paraffine Lamp . Pocket Sheet - metal Gauge for Measuring Cover-glasses Cover-glass Measurer of Zeiss Camera Lucida of Chevalier . Camera Lucida (Zeiss) . Simple Warm Stage Warm Stage and Apparatus for maintaining a constant tempera- ture under the Microscope . Section of a Gas-chamber Ranvier's Photophore . PAGE FIG xix 16 xix 17- XX 18. xxi 19. xxi xxii 20. xxiii 21. XXV XXV 22. xxvii 23- xxvii 24. xxviii 25- xxviii 26. xxix XXXV 27. 16. Swift's simple Dissecting Micro- scope ..... Mr. A. B. Stirling's Microtome Ranvier's Hand Microtome . Dr. Rutherford's Freezing Micro- tome ...... 20. Williams' (Quecket Club) Freezing Microtome .... The Ether Freezing Microtome of Dr. Bevan Lewis Modified Turntable Cannulse for Injecting . Ludwig's Mercury Pressure-appa- ratus ...... Simple Mercurial Injection-appa- ratus ...... Injection -apparatus with water- pressure Subcutaneous Syringe for Interstitial Injections, and the Cannula PAGE XXXV xxxvii xxxvii .xli xlix Hi liii liv Iv INTRODUCTION. ■■^^^XKo HISTOLOGICAL REQUISITES. The student of Histology requires the following apparatus : — 1. A compOTmd microscope, capable of magnifying from fifty to three hundred diameters linear. 2. Two short, strong, sharp-pointed needles, fixed in wooden handles, at least five inches long, leaving about half an inch of their point pro- jecting (fig. i). Each student ought to make these needles for himself Ordinary sewing needles may be conveniently fixed in crotchet-holders. 3. A pair of steel forceps, which must not be too broad at the points. 4. A hollow ground razor, for making sections with the hand (p. xxxvi). The razor ' made for the army ' by John Heifor is the best. 5. A pair of sharp-pointed scissors. It is advantageous for some purposes to have a pair curved on the flat (fig. 2). Fig, «, End of a Mounted Needle. Natural Size. Fig. 2. Scissors Curved on the Flat. Natural Size. 6. Three or four small camel-hair brushes — duck size — for putting on the cement used to seal up the preparations. It is convenient to keep Nos. 2, 3, 4, 5, and 6 in a small box or in a small drawer in one's vvork-table. 7. Glass slides. — It is desirable to have two sizes. The glass should be free from specks and not too thick, and the edges ought to be ground. {a) Six dozen ground-glass slides 3x1 inch. {b) Three „ „ 3 x I2 » S. Cover-glasses. — I prefer the circular form, though many observers use squares. The one point of importance about them is that they cannot be too thin. It is well to measure their thickness, and this can be easily done as described at p. xxv. Messrs Chance, of Birmingham, make cover-glasses of three thicknesses, which are numbered Nos. i, 2, and 3. b2 XX INTRODUCTION. No. I is the thinnest, and is what ought to be used. They vary in thickness from -004 to 008 inch in thickness. (a) \ oz. extra thin f inch circles, or No. i. {b) \ oz. extra thin i inch circles, or No. i. {c) \ oz. extra thin \\ inch circles, or No. i. These covers ought to be arranged, according to their size and thickness, in a small box divided into compartments by means of small pieces of pasteboard. This the student can easily make for himself. 9. A pine-wood box, or cabinet, provided with trays, so that the specimens may lie on the flat. The trays ought to be made both for large and small slides, and the cabinet ought to contain at least si.x dozen slides. Each student in the class of practical physiology mounts over one hundred slides in a session. 10. Reagents. — Each student ought to be provided with a small wooden framework for holding bottles, containing the solutions in common use. It is not of much importance what kind of bottle is employed. One-ounce bottles with moderately wide mouths, and fitted with a glass rod passing through the cork, answer ad- mirably. The solutions most commonly employed are : — 1. \ pey cent, salt solution (p. xxx). 2. Dilute Acetic Acid, i part to 10 of water. 3. Solution of Picrocarniinc (p. xliii). 4. Solution of ordinary Carmine (p. .xliii). 5. Solution of Logwood {t^. xlii). 6. Clove oil. — It is advantageous to use a camel-hair or sable brush fi.xed to the end of a piece of wood thrust through the cork. 7. Dammar Mounting Fluid (p. xlviii). 8. Glycerine. 9. Farranfs Solution (p. xlviii). The other solutions are supplied as they are required. Ranvier uses a very convenient arrangement for holding the reagent bottles (fig. 3). It consists of a circular glass vessel, about four inches in diameter and two inches deep. Into its mouth a perforated plate of cork is fitted, and in this cork small ' drop bottles ' are fixed, and the whole is covered with a glass shade to keep out dust. Anyone can easily make such an arrangement for himself. 10. Narrow strips of blotting paper, which ougKt to lie in a small tray attached to the stand containing the reagents. They are of use to soak up surplus fluid, and also serve as a white background, when such is required, as in teasing certain coloured preparations. Watch-glasses and small glass capsules are required, and the student will find the instru- ments of his dissecting case very useful. Glass pipettes are occasionally required to remove fluid. They are easily made by heating a narrow glass tube in a gas flame, and then drawing it out. Fig. 3. Ranvier's Arrangement for Holding Drop-Bottles with Re- agents. INTRODUCTION. XXI ON THE USE OF THE MICROSCOPE. GENERAL DIRECTIONS. The microscope used in the Physiological Laboratory of the University of Aberdeen is Hartnack's, model No. III. A. (fig. 4) with an eyepiece, or ocular, No. 3, and two objectives, or lenses, Nos. 3 and 7. The combination of ocular No. 3 with objective No. 3, gives a mag- nifying power of about 65 diameters linear, and ocular No. 3 and objective No. 7 about 300 diameters, with the tube drawn out. These powers are quite sufficient for beginners, and, indeed, for most of the ordinary work. This microscope costs about 7/. Higher magnifying powers aie supplied when they are required. Fig. 4. Hartnack's Microscope No. III. A. One-fiflli Natural .Size. The Condenser is attached to the Body of the Microscope. Fig. 5. .Swift's College Microscope. With a Condenser attached to the Stage for examining opaque objects. The magnifying power can be diminished or increased by pushing or pulling out the draw- tube. In fig. 4, the draw-tube is represented as drawn out. In Hartnack's microscope, the stand has a horse-shoe form, but many prefer the tripod form (fig. 5) as made by Messrs. Swift and Son, University Street, London. In this microscope the tube is cloth-lined, so that it moves easily, and the stage is covered with a glass plate which enables the slide to move easily on it, and prevents it from being acted on by acids. The object-glass, objective, or lens, is the most important part of a microscope, and it is necessary to see that a good lens be procured. The lens should be tested by a competent microscopist before it is purchased. Coloured blood-corpuscles and salivary corpuscles are xxii INTRODUCTION. better ' test objects' than the markings of diatoms. It is most important to have a good high power such as Hartnack's No 7, equal to about an English \ inch. If a higher power be required, Zeiss' E = g inch, and his F = j\7 inch, are excellent lenses. IMMERSION LENSES. Ordinary lenses are ' dry,' that is, air is the medium between the object and the objective. For higher powers, lenses are specially constructed so that a fluid medium is placed between the object and the objective. The effect of this is to collect a larger number of rays of light passing through the object, and so increase the illumination of the field of the microscope. Water was formerly employed, and lenses were specially constructed for use with water as the medium. Recently, however, oil immersion lenses have been constructed by Zeiss of Jena, and his Jy inch is the best lens. These lenses are rapidly taking the place of water immersion lenses. Similar lenses are made by Messrs Powell and Lealand. Cedar oil is the medium employed for these lenses. A drop of oil is placed on the lens, and the tube is then lowered till the oil comes in contact with the slide, and the object is focussed in the ordinary way. They increase the sharpness and brilliancy of objects immensely. As the cedar oil dissolves dammar, all preparations to be examined with oil lenses must be sealed up with marine glue, which is not acted on by cedar oil. It is not necessary for the student to get these expensive lenses ; they are supplied when they are required. A nosepiece is a simple device (fig. 6) by means of which the unscrewing of one objective to use another is obviated. Either the high or the low power can be u.sed. It is necessary that both lenses be accurately centred. The nosepiece is screwed to the tube of the microscope, and to it the lenses are screwed. A nosepiece may likewise be made to carry three or four lenses. Eyepieces. — Nos. 3 and 4 are supplied with Hartnack's microscope but it is well always to use the deep eyepiece, or No. 3, i.e. the less powerful of the two. THE ADJUSTMENTS OF A MICROSCOPE. In every microscope there are two adjustments : — {a) The coarse adjustment, which serves to bring the Fig. 6. Nosepiece made by Swift, ^^ject roughly within focal distance. In Hartnack's micro- scope, this is accomplished by rotating — not pushing — one tube inside another with the hand. This is b)' far the most convenient method. As the tube of this microscope is not cloth-lined, the two surfaces ought to be kept perfectly clean to ensure easy motion. If the tube does not move easily enough, rub it with a little almond or watchmaker's oil, and move it upwards and downwards till it glides easily. Wipe off the surplus oil with a cloth. {b) The pine adjustment, is accomplished by rotating a milled head or screw placed at the upper part of the pedestal of the microscope. It serves to bring the object accurately into focus after its outline has been brought into view by means of the coarse adjustment. The stage, on which the object is placed, ought to be fixed, and at least three inches in breadth. In Hartnack's microscope it is placed at a very convenient height above the table, so that when the outer surface of the left hand is resting on the table, the thumb and fore- INTRODUCTION. xxm graduated apertures, are finger can be used to move the slide on the stage. In this way all unnecessary fatigue of the arm is avoided. In Swift's microscope it is glass-plated. The diaphragm is placed under the stage, and consists of a circular plate, with a graduated series of round apertures in it. When using a high power, turn round the diaphragm until a small aperture — usually the second smallest — is directly under the hole in the centre of the stage. A small catch indicates when this is accomplished. When using a low power, employ a large aperture of the diaphragm. On the more expensive microscopes small stops, or tubes with supplied with the lenses. They are slipped into a hole in the stage from below, but care must be taken that they are accurately centred, so that their apertures are exactly under the objective. The illumination. This is accomplished by means of a mirror placed under the stage. Never employ direct sun-light ; rather employ light from a white cloud. In selecting a site to work with the microscope, try to secure a north light. ARTIFICI.A.L ILLUMINATION. Under certain circumstances it is necessary to use a lamp which burns either gas or paraffine. I find that an ordinary gas jet, fitted with a Sugg's burner, surrounded by a chimney of pale blue glass, answers admirably. Figure 7 shows a form of microscopic lamp which burns paraffine. It is also very convenient. The funnel. It is made by Swift, ight is better when it is transmitted through a light blue glass An inexpensive convenient light is obtained from an ordinary Fig. 7. Microscopic Paraffine paraffine lamp with a flat wick. ' ( «'i )■ CLEANING OF THE LENSES OR OBJECTIVES. The lenses are optically the most important part of a microscope. Never unscrew the separate parts of the objective or lens. If there be any dimness, it is sure to be due to dirt on the outer lenses. Carefully rub the lens with a piece of chamois leather, which should be kept ready at hand. A convenient way is to tie it to the stand of the microscope. If a drop of dammar chance to be on the lens, it must be cautiously removed by applying a drop of clove oil and removing it quickly with chamois leather. It is necessary to remove the clove oil at once, as the glasses which compose the lens are soldered together with Canada balsam which would be dissolved by the clove oil. A dilute solution of ammonia applied to the lens with chamois leather or cotton wool is an excellent method of removing the greasy film which sometimes coats glass. . The surface of a freshly-fractured piece of elder pith answers the same purpose admirably. METHOD OF FOCUSSING AN OBJECT. It is extremely desirable for the student of histology to begin at once by learning to keep botli eyes open when examining an object. After putting on the proper lens, arrange the mirror under the stage so as to direct a beam of light up the tube of the microscope and illuminate the field. See that the field is brightly illuminated, and that no specks are present. xxiv INTRODUCTION. If specks are present, it may be easily determined as to whether they are on the ocular (eye- piece) or on the objective ; if they arc on the eyepiece, they will necessarily move when it is moved. They must be removed by gentle cleaning with chamois leather. (rt) Low Power (L).— With the No. 3 objective. Place the tube of the microscope so that the lens is at least an inch and a half above the object. Keep looking through the micro- scope, and, whilst doing so, keep the slide on the stage in motion with the left hand, and rotate or twist the tube downwards — always from right to left — until the outline of the object comes distinctly into view. Then employ the fine adjustment, and accurately focus the object. (1^) High Power (H). — With the No. 7 objective. Place the lens a quarter of an inch above the object, and then slowly rotate the tube downwards, keeping the object on the stage in motion all the time with the thumb and fore-finger of the left hand, until the object just comes into view, then employ the fine adjustment. As already indicated the magnifying power may be increased by pulling out the draw-tube. Movement of the Slide on the Stage. — This is done most conveniently by means of the thumb and fore-finger of the left hand. Never employ two hands to move a slide ; it is quite unnecessary. By practice wonderful precision of movement is acquired. Two small clips are placed on the stage, which serve to fix the slide, so that it remains in any position. All the cumbrous mechanical appliances formerly in fashion for moving the slide on the stage are quite unnecessary. GENERAL RULES FOR THE EXAMINATION OF AN OBJECT. Always begin the examination of an object with a low power, in order to obtain a general survey of the arrangement of the parts. If it be desired to study any particular part, place it directly in the centre of the field of the microscope by means of the low power, and then ex- amine it with a higher power, taking care in the latter instance to use a small aperture of the diaphragm. PREPARATION OF A SIMPLE OBJECT. A drop of blood or milk serves the purpose very well. Clean a slide carefully. Dip the slide in water, and rub both surfaces at once with a towel which has been washed several times, so as to get rid of all adherent threads. It is often convenient for the beginner to use a coloured towel — say, a red one — for if he finds a coloured thread in the field of the micro- scope, he knows that it has come from the towel. Place a small drop of the fluid to be ex- amined on the centre of a slide by means of a clean glass rod. Apply a clean cover-glass, taking care to prevent the entrance of air-bubbles. MEHODS OF APPLYING A COVER-GLASS. Seize by the edge a previously cleaned cover-glass with forceps, and allow the drop of fluid to come in contact with the edge of the cover farthest removed from the forceps ; gradually and slowly allow the cover-glass to descend obliquely until the fluid forms a uniform stratum under the cover. There ought to be just a sufficient amount of fluid to fill out the space between the slide and cover-glass. When forceps are not at hand, the cover may be applied with the fingers. Take a cover by the margins, rest one edge on the slide close to the drop, allow the fluid to touch the under surface of the edge of the cover, and slowly permit the cover to descend obliquely. INTRODUCTION. XXV METHOD OF CLEANING COVER-GLASSES. Not unfrequently covers are coated with a dull film, which obscures the passage of the light. To remove this film place the cover-glasses for an hour or two in a beaker containing strong sulphuric or nitric acid. Pour off the acid, and wash the glasses thoroughly with water until all the acid is removed. They may then be placed in methylated spirit, and as each cover-glass is taken out it is cleaned with chamois leather or a well-washed silk hand- kerchief This is accomplished by rubbing the surfaces of the cover-glass with the hand- kerchief, held between the thumb and forefinger of the left hand. The cover ought always to be lifted by the edges, and never allowed to lie on the flat after it is cleaned, as particles of dirt are apt to adhere to it. Tilt it up against something until required. For inch-and-a-quarter cover-glasses a good plan is to have two perfectly smooth pieces of wood, covered with chamois leather, and to place the cover-glass between them, and rub it until cleaned. ON MEASURING COVER-GLASSES. As already indicated. No. i cover-glasses do very well for the ordinary purposes of a student. When very high powers are used, it is well to know the thickness of the cover-glass employed. Cover-glasses may be rapidly measured in two ways. {a) Micrometer caliper. — This little American instrument (fig. 8) is sold by Messrs. Chas. Churchill and Co., 28, Wilson Street, London. It is used for measuring the thickness of fine brass plates. The piece in the form of the letter U has a projecting hub, a, at one end. Through the two ends are tapped holes, in one of which is the adjusting screw, B, and in the other the gauge screw, C. Attached to the screw, C, is a thimble, D, which fits over the exterior of the hub, a. The end ofthis thimble is bevelled, and Fig. S. Pocket Sheet-metal Gauge for Measuring Cover-glasses. Natural Size. Fig. 9. Cover-glass Measurer of Zeiss. the bevelled edge graduated into twenty-five parts, and figured O, 5, 10, 15, 20. A line of graduation, 40 to the inch, is also made upon the outside of the hub, a, the line of these divisions running parallel with the centre of the screw, C, whilst the graduations on the thimble are circular. The pitch of the screw, C, being 40 to the inch, one revolution of the thimble opens the guage i-40th or 25-ioooths of an inch. The divisions on the thimble are then read off for any additional part of a revolution of the thimble, and the number of such divisions are added to the turn or turns already made by the thimble, allowing 25-1000 for each graduation on the hub, a. c xxvi INTRODUCTION. Measure an ounce of No. i cover-glasses, and put aside all those under "004 inch for use with high powers. {J}) The cover-glass measurer (Deckglass-tastcr) of Zeiss (fig. 9) is a very convenient appa- ratus, which has a movable disc, d, divided into degrees. The cover-glass, c, is placed between the ends of two screws, and the disc is rotated till the screw just holds the cover. By an ingenious arrangement of teeth the handle is thrown ' out of gear ' when this occurs. It gives the thickness of a cover-glass in parts of a millimetre. HOW TO PLACE A SECTION ON A SLIDE. The section ought to be placed in a bowl of water — preferably a white bowl — as it admits of the section being easily seen. Take a slide, and plunge it for three-quarters of its length into the water, and with a needle in the right hand gently guide the section on to the slide, which ought to be held in a slanting direction. The section is thus easily _/?i3i7/'(:rt' on to the slide, and thus all unnecessary tearing of it is avoided. The needle is used to hold the section on the slide whilst the latter is being removed from the water. Remove the surplus water with a towel. To the section on the slide the necessary staining reagents may be applied. Sections may easily be treated with alcohol and clove oil on a slide. This method is infinitely preferable to lifting sections from clove oil, etc., even with the most approved form of ' section-lifters.' LABELLING OF MICROSCOPIC OBJECTS. This is most important. Every preparation, when mounted, ought to be properly labelled. Small paper labels are affixed to the slide, and on these are marked the nature of the section, whether it is a transverse (T. S.) or a longitudinal section (L. S.) ; any special points in its struc- ture ; how it was prepared and in what medium it is mounted. The thickness of the cover- glass in special cases ought to be indicated. By using a series of symbols, as Pc for picrocarmine, Lg for logwood, G for glycerine, D for dammar, and F for Farrant's solution, much important information can be put upon one small piece of paper. ON MAKING DRAWINGS OF MICROSCOPIC PREPARATIONS. It is of the utmost importance that each student should make a sketch of many, not neces- sarily of all, the preparations he mounts. This is the only method of ensuring that he sees what is to be seen. The outline plates are designed to aid the student in this task. Once the student has made a drawing of the details of a preparation he is not likely to forget them. It is far more important that each student should thoroughly understand a selected series of pre- parations than that he should carry off with him a couple of hundred slides, few of which he understands. Various instruments are used for making drawings of microscopic preparations, but it is to be remembered that they are in many cases more useful for giving the actual size of an object as seen with a certain combination of lenses, and for giving the general outline of a section, than for the individual details. INTRODUCTION. xxvu Camera Lucida of Chevalier. — This instrument (fig. lo) is made by Hartnack. Instead of the eyepiece, the tube s' is fixed in the tube of the microscope. The observer looks through the small prism, and sees the outline of the section thrown on the table, or on a piece of paper laid on the table, close to the microscope. A shade is placed in front of this, and must be so adjusted that too much light does not fall on the paper. The contour is then mapped out with a pencil. Fig. 10. Camera Lucida of Chevalier. a the Instrument ; l> a section, showing the prisms. Fig. II. Camera Lucida (Zeiss). The ring of brass fixes it to the tube of the Microscope, and the aperture a is placed over the Eyepiece. Other Cameras. — Much simpler forms are the camera lucida of Nachet, or the one made by Zeiss (fig. 1 1). The ocular is left in its place, and the camera is fixed to the tube of the microscope by the ring of brass. The aperture a is placed immediately over the centre of the eyepiece. On looking through this aperture the surface of the table in front of the micro- scope is seen at the same time as the object. The drawing-paper is placed on an inclined plane on this part of the table, and the outline of the object is sketched, whilst the details are afterwards filled in without the camera. Neutral tint reflector. — This simply consists of a small cap, which fits on the end of the microscope, and in it is placed a piece of glass, at an angle of 45°. The microscope tube must be horizontal. On looking through the glass the image of the object is seen on the paper. The same precautions with regard to the modification of the light on the paper must be taken as for Chevalier's camera. It is not even necessary to have a brass cap to fit on to the microscope ; an ordinary cover- glass, fixed to the eyepiece, by means of modeller's wax, at an angle of 45°, answers the purpose admirably. METHODS OF STUDYING THE PROPERTIES OF LIVING PROTOPLASM. THE MOIST CHAMBER. Moist chamber of v. Recklinghausen. — This is easily made by cutting off the lower end of an ordinary glass cylinder, such as is used for a paraffine lamp. Through the lower part of the cylinder the tube of the microscope is placed. The lower end of the cylinder is ground flat, XXVIU IN TROD UC TION. and rests on a plate of glass, to which it is fitted air-tight with oil. The inner surface of the cylinder is partly coated with filter paper moistened with water, so that the air in the enclosed space is always kept saturated with watery vapour. To close the space all that is required is to tie a layer or two of sheet caoutchouc over the lower end of the tube of the microscope, and the upper end of the glass cylinder. The object to be examined is placed on the floor of this chamber, and may be examined either with or without a cover-glass. Simple moist chamber. — A very simple chamber may be made by fixing a ring of glass, a quarter of an inch in height and seven-eighths of an inch in diameter, to an ordinary slide by means of dammar solution. A drop of water is placed in the chamber, which is then covered with a cover-glass, on which the fluid to be examined is placed. The cover-glass is so applied that the drop of fluid hangs into the chamber, as is shown in fig. 14. METHODS OF APPLYING HEAT TO MICROSCOPIC OBJECTS. Various forms of warm stages are in use ; Max Schultze was the first to use them. Simple form of zvarm stage. — This consists of a thin sheet of copper, three inches long and one and a half inches broad (fig. 12). In the centre is a hole half an inch in diameter. From one side of the plate a narrow copper arm, five inches in length, projects. This is placed on the stage of the microscope, and on it is placed the preparation to be observed. If it be blood (p. 4), the cover-glass must be sealed round with oil to prevent evaporation, and the whole is fixed by the clips on the microscope. Heat is applied to the brass arm by means of a i ^iH mm Fig. 12. Simple Warm Stage. Half Natural Size. Fig. 13. Warm Stage and Apparatus for Maintaining a Constant Temperature under the Microscope (Schafer). spirit-lamp, and, in order that it may not be over-heated, a piece of a mixture of cocoa- butter and white wax is placed on the copper plate near the preparation. This mixture is so made as to melt at a temperature of 30° C. When it melts the source of the heat must be removed. This does very well for the purposes of the student ; anyone can make it for himself from sheet copper. For accurate experiments it is necessary to have a thermometer, so that the degree of heat can be accurately measured. Such an arrangement is found in Strieker's warm stage. INTRODUCTION. xxix Mr. Schdfer's constant temperature apparatus. — The following apparatus, devised by Mr. Schafer, is an excellent one (fig. 13). It consists of a brass box, a, just like a Strieker's warm stage, having a hole in its centre to admit light to the microscope. In it is a ther- mometer, b. This box lies on the stage of the microscope. It is connected by elastic tubes with a hollow jacket,/, and the whole system is filled with water previously boiled to expel the air. The water is warmed at ^ by a small gas-flame, the size of which is regulated by a modified Bunsen's regulator, (/. By means of the screw e the temperature is regulated at starting. METHOD OF APPLYING VAPOURS AND GASES. A simple apparatus is described at p. 11 for applying the vapour of ether or chloroform to an object, e.g. to cilia. It is made in the same way as the moist chamber described at p. xxviii. If gases, such as o.xygen or carbonic acid, are to be applied, two tubes require to open Fig. 14. Section of a Gas Chamber, a. Entrance ; b, Exit tube ; c, Object ; d. Cover-glass. Natural size. into the glass cell, one for the entrance of the gas, and the other for its exit. Fig. 14 shows a section of such an arrangement. The cover-glass, with the object on it is inverted over the cell. Strieker's warm stage is usually so made that it can be used for this purpose. XXX INTRODUCTION. ON THE PREPARATION OF TISSUES FOR MICROSCOPIC EXAM IN A TION. FRESH TISSUES. Fresh tissues as a rule are unsuited for examination with the microscope, though under certain circumstances it is advisable to examine a fresh tissue. When this is done, the tissue must be examined in a fluid which will alter its characters as little as possible ; such fluids are called NORMAL OR INDIFFERENT FLUIDS. They closely resemble in composition some of the fluids in which the tissues of the body are bathed. 1. Aqueous humour of the eye. — This is easily obtained by puncturing the cornea of an eye- ball removed from an ox newly killed. 2. Blood-serum.— Pour blood into a tall vessel, and allow it to coagulate. After the blood coagulates, run a knife between the upper margin of the clot and the vessel, to permit the clot to contract and sink in the serum, which will be squeezed out of the clot. After twenty-four hours draw off the yellow-coloured serum with a pipette. 3. Iodised serum. — Add iodine to blood-serum, prepared as above, until the fluid is of a distinctly yellow colour. This fluid alters the tissues slightly, however, and colours them yellow. A similar solution may be made by adding iodine to amniotic fluid. 4. Salt solution (three-quarter per cent.). — Dissolve 7-5 grammes sodic chloride in 1,000 c.c. of distilled water. This is by far the most convenient fluid to employ. Its composition is so near that of lymph — the fluid normally bathing the tissues — that it alters fresh tissues very slightly. HOW TO HARDEN TISSUES. GENERAL DIRECTIONS. Tissues, however, generally require to be hardened before they can be cut into sections for examination. This is accomplished by exposing the tissue to the action of certain fluids. The Material to be employed. — The tissues should in all cases be as fresh as possible. When possible the tissues ought to be obtained from the human body, though these in all cases may not be available. It is therefore necessary to use the corresponding organs of some of the lower animals, e.g., cat, dog, rabbit, or pig. Practically their structure is identical with INTRODUCTION. xxxi that of man. The tissue to be hardened ought to be cut into small pieces— half to one inch square — by means of a sharp razor. If foreign matters are to be removed from the tissue — e.g. the contents of the gut — do not wash them with water, but with a stream of normal salt solution (p. xxx). Place the tissues in a relatively large quantity of the hardening fluid, at least twenty times the bulk of the tissue. Change the fluid at the end of the first, third, seventh, and tenth days. It is better to place the tissues in a shallow but wide bottle. See that the tissues do not stick to the wall of the vessel, else the hardening fluid is prevented from having access to them. Cotton wool moistened with dilute alcohol answers admirably as a bed for the tissues to rest on and to separate one piece from another. Place the bottle in a cool place. The hardening will be effected in from one to four weeks. At the end of this time remove the tissues ; wash them thoroughly in water, or let them steep in a large volume of water for several hours to get rid of the hardening reagent, as in some cases — e.g. chromic acid — its presence interferes with the process of staining. Place the tissues in equal parts of methylated spirit and water for two days, and then transfer them to methylated spirit to complete the hardening. If the spirit becomes cloudy it must be changed. This keeps the preparations until they are required for sections. Instead of merely placing the tissue in the hardening fluid, in many cases it is advisable to suspend it by means of a thin cord in tJie fluid, which permits the fluid to penetrate the tissue more rapidly. The process of changing the various hardening fluids on certain days appears tedious, but it is not so in reality, if the tissues to be hardened are placed on a shelf by themselves. Chromic acid preparations require to have the fluid changed more frequently than when Miiller's fluid is employed. For convenience, therefore, they may be placed by themselves. On all occasions label each bottle, and note the tissue, from what animal it is taken, the name and strength of the hardening fluid, and the date on which it was changed. Attention to these apparently small details is the necessary condition for successful histological work. HARDENING SOLUTIONS. The following are the solutions in most common use. A. THOSE WHICH HARDEN TISSUES WITHOUT COLOURING THEM. 1. Chromic acid 1 per cent, solution. — Dissolve lo grms. chromic acid in i,ooo c.c. water. So strong a solution is not used, as it renders the tissues too brittle. It is convenient to keep a strong solution, which can be diluted as required. A ;^ or ^ per cent, solution is generally used. 2. Chromic Acid and Spirit Solution. — This is one of the most useful solutions. Make a i per cent, solution of chromic acid, either by diluting the above, or dissolving one gramme of chromic acid in 600 c.c. 'of water. Take one part of this, and add two parts of methylated spirit. It is better to prepare this mixture as it is required. As heat is developed by the mixing, the tissues must not be put into the mixture till it becomes cool. It hardens in from seven to ten days, according to the size and nature of the tissue. 3. Potassic Bichromate Solution.— Dissolve 20 grms. in 1,000 c.c. water. The solution is most easily made with warm water, but it must be allowed to cool before it is used. If the solution is changed every four days it hardens tissues in three to four weeks, but usually a longer time is required, as it is not necessary to change the fluid so often. xxxii INTRODUCTION. 4. Ammonium Bichromate Solution. — Dissolve 20 grms. in 1,000 c.c. water. This is very useful for hardening the brain and spinal cord, and nervous structures generally. 5. Ammonium Chromate Solution. — Dissolve 50 grms. of ammonium chromate in 1,000 c.c. water, i.e. a five per cent, solution, or it may be made by adding one ounce of the salt to twenty fluid ounces of water. Filter and preserve in a stoppered bottle. It hardens fresh tissue, such as the mesentery, in twenty-four hours. After hardening, the tissue must be washed until no more colour is removed by the water. It may then be stained and mounted in gly- cerine. For other cases, the kidney for instance, small pieces are hardened for forty-eight hours, and after being thoroughly washed are then treated with spirit, as directed above (p. xxxi). This substance is of the utmost value for revealing the rod-like structure in the renal epithelium, and for demonstrating the existence of the intra-cellular and intra-nuclear ple.xus of fibrils in cells. 6. Miiller's Fluid. — Dissolve 25 grms. potassic bichromate and 10 grms. sodic sulphate in 1,000 c.c. water. This is a most useful reagent, as it penetrates more readily than chromic acid, though it takes longer time to harden tissues — usually from five to seven weeks. It is frequently advantageous to combine the use of two of the above fluids, thus: place the tissue for a week in Miiller's fluid, and complete the hardening in No. 2. After hardening, the tissues are well washed, and preserved in spirit. 7. Miiller's Fluid and Spirit. — Mix 3 parts of Miiller's fluid and i part of methylated spirit. This does very well for nerve-tissues, muscle, and the retina. It ought to be prepared fresh when required, and the tissue ought not to be placed in it till the mixture cools after the addition of the spirit. To prevent the separation of the chromium salts, which is apt to occur, keep the mi.xture in a dark place. 8. Methylated Spirit is sometimes, though rarely, used alone. 9. Absolute Alcohol of S. G. 0'795. — This hardens very rapidly — in twenty-four hours — but it causes considerable shrinking, though it is invaluable for gastric mucous membrane, and for secretory glands generally, e.g. the salivary glands and pancreas. Tissues become stained ver>' readily after hardening in pure alcohol. B. THOSE WHICH HARDEN, AND AT THE .SAME TIME COLOUR THE TISSUE. 10. Picric Acid. — Make a watery solution saturated in the cold. Keep crystals in the bottle to ensure saturation. Tissues ought to be left in this only for a day or two, else they are rendered too brittle. Its stain is of a bright yellow colour, which is easily removed by prolonged washing in water (Ranvier). Tissues hardened in this fluid become easily stained with picrocarmine. 1 1. Kleinenberg's Picric Acid is a modification of the above. To 100 c.c. of a cold saturated watery solution of picric acid add 2 c.c. of strong sulphuric acid, which throws down a yellow precipitate. Filter, and to the filtrate add 300 c.c. of distilled water. This solution is most valuable for fcetal tissues, and especially for early embryos. It produces its effects in from three to ten hours. 1 3. Osmic Acid. — A one per cent, waterj' solution is most useful, which can be diluted as required. A ^ per cent, solution, prepared with distilled water, is usually employed. It decomposes very rapidly when in contact with organic matter and exposed to light ; and as it is very volatile, it must be kept in a glass-stoppered bottle, covered with paper, to protect it from the light. It hardens in from six to twenty-four hours, and acts specially on fatty matters, which it blackens, and is very useful for tracing the course of medullated nerve-fibres, INTRODUCTION. xxxiii as it blackens the myeline. Small pieces of tissue exposed to the vapour of a one per cent, solution for a few minutes, are easily stained with picrocarmine, though the prolonged action of a strong solution renders subsequent staining difficult. It is one of the most useful of all hardening reagents (M. Schultze). It is not necessary to mount preparations treated with osmic acid in a saturated solution of acetate of potash. METHODS OF SOFTENING TISSUES. Certain tissues, as bone and tooth, which contain calcareous matter, require to have the calcareous matter removed before they can be conveniently cut for the microscope. This is done by placing them in a decalcifying solution. Some solutions remove only the calcareous matter —e.g. dilute hydrochloric acid — without at the same time hardening the tissue; others, as a mixture of chromic acid and nitric acid, remove the lime salts, and at the same time harden the tissue, and generally the latter are to be preferred. DECALCIFYING SOLUTIONS. 1. Hydrochloric Acid. — Mix one part of strong acid with ten parts of water. This does very well for the removal of lime salts from an injected bone. 2. Chromic and Nilsric Acid Fluid. — Make a \ per cent, solution of chromic acid, and to every lOO c.c. add i c.c. of strong nitric acid. The chromic acid solution is easily made by adding an equal volume of water to a one per cent, solution (p. xxxi). This fluid takes two or three weeks to remove the lime-salts from a small piece of bone, but if the fluid is frequently changed the result is accomplished more rapidly. The tissue is then thoroughly washed to get rid of the acids, and then hardened, first in weak and then in strong methylated spirit. The advantage of this fluid is that the chromic acid hardens the parts, whilst the nitric acid removes the lime-salts. Bones, after being exposed to its action, assume a green colour, owing to the formation of a sesquioxide of chromium. 3. Picric Acid Solution. — A saturated watery solution takes several weeks to act on a moderately large piece of bone. It is of great value for fcetal bones. 4. A 10 per cent. Solution of Common Salt and Hydrochloric Acid. — This is most valuable for showing the matrix of bone, which consists of ordinary fibrous tissue, and swells up in the ordinary acid media. A 10 per cent, solution of salt prevents this (v. Ebner and De B. Birch). The bone is placed in a 10 per cent, solution of common salt, to which i to 3 per cent, of hydrochloric acid is added. Add from day to day as much acid as will decalcify the bone. When the bone becomes flexible It is placed for several hours in water, to remove all the acid. Leave it for several days in 10 per cent, salt solution, which must be changed repeatedly. When the reaction of the bone becomes neutral the bone is white and opaque. Sections are made, and mounted in a 10 per cent, solution of salt. They show the fibrillar structure of the matri.x (p. 32) (v. Ebner). xxxiv INTRODUCTION. METHODS FOR DISSOCIATING TISSUES. Various solutions dissolve or soften certain parts of a tissue whilst other parts are left un- affected. The result is that the component parts may be readily separated by teasing. The piece of tissue ought not to be larger than a pea. The result is usuall)' effected in from t\\'cnt}-- four to thirty-six hours, though much less time may suffice. DISSOCIATING SOLUTIONS. 1. Iodised Serum. — Add iodine to blood-serum or amniotic fluid, till the fluid is of a dis- tinctly yellow colour. This fluid dissolves the cement-substance between cells in from one to two days. 2. Dilute Chromic Acid (-oi per cent.). — Dissolve i grm. chromic acid in 10,000 c.c. water, or dilute a one per cent, solution. This does excellently for isolating the fibriUa; of muscle, and for the nerve-cells of the spinal cord. Two to three daj-s' maceration serves to bring about the result. 3. Dilute Alcohol (' Alcool au tiers '). — Mix 2 parts of water with i of rectified spirit. This is one of the most useful dissociating fluids, and requires one to tv/o days for its action (Ranvier). 4. Saturated Aqueous Solution of Baric Hydrate requires about twenty-four hours to act on the fibrillae of tendon. 5. Caustic Potash. — Dissolve 40 grms. of caustic potash in 100 c.c. water. This isolates muscle-cells in from twenty to thirty minutes. 6. Ten per cent. Solution of Common Salt is useful for dissolving the cement of white fibrous tissue. It takes several days to act. It is very useful also for showing the fibrillse of the matrix of bone. 7. Nitric Acid and Glycerine. — Mix one part of strong nitric acid containing nitrous acid with 3 parts of water and i part of glycerine. The object is placed in this mixture for two or three days and then in water. It is specially useful for isolating nerve-structures and lens- fibres (Freud). DIGESTION AS A HISTOLOGICAL METHOD. This method was introduced into histology by the author several years ago. It has re- cently been employed by Kuhne for investigating the structure of nerves, and by De Burgh Birch in his investigations on the composition of the matrix of bone (p. 32). Either an arti- ficial gastric or pancreatic juice may be employed. Artificial Gastric Digestion. — This method is fully described at p. 92 (W. Stirling). Artificial pancreatic, i.e. trypsin, Digestion. — Either an aqueous or glycerine extract of the pancreas may be used. The latter is the more convenient. It is made thus by v. Wittich's method. The pancreas of a dog is chopped up and is dehydrated with absolute alcohol for twenty-four hours. The alcohol is removed and sufficient pure glycerine is added to cover the gland, and it is allowed to stand for three weeks. Press the glycerine through muslin to remove the gland tissue. The glycerine is a solvent for the trypsin of the pancreas, just as it is INTRODUCTION. XXXV for other soluble ferments, i c.c. of the glycerine filtrate is added to 19 c.c. of 1 per cent, solution of sodic carbonate. The fluid becomes turbid, but after filtration a pale yellow fluid is obtained. The tissue to be digested is placed in this fluid, and the whole is kept at a tem- perature of 40° C. in a water bath as directed at p. 92. Sections of softened bone digested by this method are preserved in a 10 per cent, solution of common salt (Birch). METHODS OF TEASING A TISSUE. Take only a small' piece of tissue, and place it on a slide in a small drop of the fluid in which it is to be mounted, e.g. glycerine. Fix one end of the tissue with a strong needle in a handle, and tear in the direction of the fibres with the other needle. The low power must be used from time to time, to ascertain when the component parts are sufficiently isolated. It is very desirable to have a proper background, so that the object to be teased may be distinctly seen ; for a coloured object a small piece of white bibulous paper answers admirably, and a trans- parent object is generally best seen on a dark surface. A good plan is to have a narrow white line painted along the edge of the work-table. The same result is obtained by having a small slab, one-half of which is glazed white and the other black. Into Fig. 15. Ranvier's Photophokf.. One-si.\th Natural Size. r.a.nvier's photofhore. The following little arrangement devised by Ranvier is of great value for this pur- pose. It is called a photophore (fig. 15). It consists of a small mahogany box about four inches square ; one side is open and so is the top. The lid is formed of a glass plate, the bo.x a mirror is fixed at an angle of 30°-3S°. This arrange- ment reflects the light through the preparation. If it be desired to work with a black background, all that is required is to place a black card over the mirror. This simple arrangement is of the utmost value, and when in use has simply to be directed with its mirror towards a window. It is well to have a bent piece of wire, with a ring formed on it and attached to the back of the box, for receiving a small magnifying lens. Each student should be provided with such an arrangement. He can easily make one for himself by taking an ordinary small cigar-box without a lid, and placing in it a mirror at an angle of SO'-SS". On the upper side make a square hole, and into this fit a glass plate. SIMPLE OR DISSECTING MICROSCOPE. In teasing or dissociating a tissue it is often of great value to use an ordinary dissecting microscope. The form devised by Bri.ickc and made by Hartnack is useful, but it is expensive. For practical purposes I find that the form, fig. 16, made by Swift, is a most useful and handy instrument. Fig. 16. Swift's Si.mi'i.e Dissecting Microscope. One-sixth Natural Size. d2 xxxvi INTRODUCTION. METHODS OF CUTTING SECTIONS. It is of the utmost importance that the student should thoroughly master the details of cutting sections by hand, and also with the aid of certain instruments called microtomes. I. SECTIONS OF UNHARDENED TISSUES. If it be desired to examine only a small piece, snip off a thin fragment with a pair of scissors curved on the flat, or cut off a slice with a Valentine's knife. II. SECTIONS OF HARDENED TISSUES. I. Cutting sections by hand with a razor. — Tissues hardened by any of the above-mentioned methods are difficult to cut unless they are clamped or imbedded in some easily cut material, which must have nearly the same consistence as the tissue itself As to the kind of razor or knife to employ, the ' army razor ' answers admirably, though some prefer specially constructed knives, which, I believe, possess no advantages, whilst others prefer the razor to be ground flat on the side directed towards the tissue to be cut. If the piece of tissue be sufficiently large, seize it between the thumb and fore-finger of the left hand. Hold the fore-finger horizontally, so that its outer surface forms a rest on which the blade of the razor may glide. Keep the handle of the razor in a line with the blade, and hold it firmly in the right hand. Draiv it (from heel to tip) through the tissue towards yourself Keep the razor ivcll ivetted ivitli. spirit, and float the section off into spirit or water, with a camel-hair pencil. It requires very considerable practice before one becomes expert at making sections of sufficient thinness to be of use. The razor must be kept perfectly sharp, and after making a few sections should always be ' stropped.' If the tissue be too small or too delicate to hold in the hand, it must either be clamped or imbedded in some substance. {a) A piece of amyloid or waxy liver, or the liver of a pig hardened in alcohol, serves the purpose admirably. Make a slit in the piece of liver, and clamp the tissue in it, and cut both liver and tissue as directed above. (J?) Carrot or elder-pith may be used instead. Make a slit in the carrot or pith, and clamp in it the tissue to be cut. IMBEDDING MIXTURES. {a) Melt together with the aid of a gentle heat four parts of ordinary solid paraffine with one of pure lard. A quantity of this mixture should be kept ready for use. When required, the mixture is to be dissolved in a vessel heated in a water-bath at the lowest temperature possible. INTRODUCTION. XXXVll Method of Imbedding. — Press the tissue to be imbedded between folds of blotting-paper, to remove all the alcohol, and see that its surface is thoroughly Axy. It is a good plan to dip it for a moment into a weak solution of gum arable, and allow the gum to dry as a film on its surface. This prevents the paraffin from penetrating the tissue, and also causes it to separate more readily from the section. Fold a piece of paper in the form of an oblong box, and pour into it a little of the previously melted paraffine. Place a needle into the tissue, fix it conveniently in the paraffine mixture in the paper box, add more paraffine until the tissue is well covered. Use the needle to keep the tissue in its place until the mixture sets. The paraffine block is then removed from the paper, and sections cut through it and the imbedded tissue. The sections are floated off into spirit or water. {J}) Other imbedding mixtures are sometimes used, as two parts of paraffine mixed with one of vaseline, which gives a transparent mass, in which the position of the tissue can be detected. MICROTOMES. When a large number of sections is required, or when a complete section is wanted through an organ, there can be no doubt that a microtome of some kind is required. One of the earliest microtomes introduced (fig. 17) is that of Mr. A. B. Stirling, of the Anatomical Museum, Edinburgh University. It consists of a strong brass tube, with a broad, smooth, metal plate fi.xed at right angles to it at one end. In this cylinder a plug is moved by a very fine- threaded screw, which works in the lower end of the tube. The instrument is so made that it can be clamped to a table. The tissue to be cut is imbedded in the well of the microtome in paraffine and lard, in the same way as a tissue is imbedded for cutting hand sections. Melt the imbedding mi.xture, and fill the well of the microtome, and whilst still fluid put in the tissue to be cut — using the same precautions as to drj-ing — and hold it in position till the mixture sets. By turning the screw the mass and included tissue are elevated ; and a sharp knife carried along on the flat brass plate shaves off a section, which can be made as thin as desired by having a screw of sufficient fineness. The knife must be kept flooded with spirit, and the sections are floated off by means of a camel-hair brush into spirit or water. Fig. 17. Mr. a. B. Stirling's Microtome. Fig. iS. Ranvier's Hand Microtome. Half Natural Size. Ranvier's Microtome is simply a smaller instrument, working on the same principle, and designed to be held in the hand (fig. 18). The whole apparatus can be inverted in spirit, if it be desired to make sections of the same tissue from time to time. XXXVlll INTRODUCriON. Sections to be cut in Ranvier's microtome are imbedded just as in Mr. Stirling's form, with a paraffinc and lard or other mixture. Elder-pith, however, may be used. The tissue is packed in with dry elder-pith, and then the whole apparatus is placed in spirit, which causes the pith to swell, and so to fix the tissue sufficiently firmly for a section to be made of it. FREEZING MICROTOMES. Dr. Rutherford's Freezing Microtome. — This microtome (fig. 19) is essentially a modification of Mr. Stirling's microtome. The hollow brass cylinder is surrounded with a large trough (g), which is filled with a freezing mixture composed of equal parts of salt and ice. The trough is provided with a tube (h) to permit of the outflow of the water resulting from the melting of the ice. It is a great improvement to have the cutting- table covered with a thick plate of glass, as this is not worn away so readily as the brass. This microtome can be used as an ordinary microtome, as well as for freezing. METHOD OF USING A FREEZING MICROTOME. Preparation of the Microtome. — Clamp the instrument to a table. Turn the screw (d) until the plug has descended a good distance into the well, and to prevent the screw freezing pour a little alcohol into the well and let it run through the tube ; dry the well thoroughly, and smear a little lard round the top of the plug, to prevent the escape of any of the imbedding mixture between the plug and the wall of the cylinder, which would interfere with the action of the screw. Preparation of the Tissue to be Frozen. — The tissue has first been properly hardened by one of the foregoing methods. If it has been kept in alcohol, it must be placed in a large quantity of water — to be changed several times — during twenty-four hours, to remove all the alcohol. It is then placed for twenty-four hours in gum-mucilage (B. P.). The well is then filled with gum (B. P.), and equal quantities of powdered ice and salt are packed into the trough with a piece of stick until the edge of the gum is observed to freeze, when the piece of tissue is placed in the gum in the side of the well nearest the side from which the person makes the sections, by means of a needle. The freezing process is continued until the whole mass of gum becomes solid, the gum becoming of such a consistence that it cuts like a piece of cheese (U. Pritchard). The Section Knife. — The ordinary razor does not answer well for cutting sections with the microtome. Its blade is too thin and too short. It is necessary to have a stout blade, seven or eight inches long, with a tJiick straight back — at least a quarter of an inch thick — fixed in a straight handle-. The blade must be so strong that it cannot be bent whilst pushing it along the glass plate. The surface next the glass plate ought to be slightly concave. The surface of the knife ought to be moistened with gum solution, which prevents the sections from curling up. In cutting, keep the back of the knife directed towards the operator, and push the blade obliquely from tip to heel, guiding it with the thumb and forefinger of the left hand through the tissue. Each section as it is made must be floated off into a bowl of water with the aid of a large camel-hair brush. (See p. xl for how to preserve them.) Fig. 19. Dr. Ruthekkord's Freezing Microtome. B, Plate of brass with aperture A leading into the well of the Microtome; c, Lower end of cylinder in which the screw D works, moving a brass plug up or down ; G, Trough to hold freezing mixture ; n. Exit tube for water; F, Clamp to fix the instrument to a table. INTRODUCTION. XXXIX MODIFICATIONS OF THE ABOVE METHOD. In the preparation of tlie Tissues (a). — For some tissues — e.^. brain — it is necessary to leave them forty-eight hours in the gum before cutting. (/;) The following method is an excellent one, and is of the greatest value for very delicate tissues as the retina, ear, brain. After soaking the tissues in water, place them for twenty- four hours in syrup made by dissolving four ounces of crystallised sugar in two ounces of water. They are afterwards placed in gum for twenty-four hours, and are then read}' for cutting. With such a microtome as described above there is no difficulty whatsoever in making the most perfect sections. The well can be made of any size, and, indeed, D. J. Hamilton, of Edinburgh, has had a large freezing microtome constructed which enables him to make microscopic sections of an entire brain. \VILLIAM.S' FREEZING MICROTOME. Some histologists prefer this form of microtome (fig. 20). It is made by Swift, of London, and consists of a wooden tub for the freezing mixture. In this is an upright, brass bar, into whose upper end the circular brass plates, on which the tissue is frozen, are screwed. A glass top with a hole in its centre, through which the circular plate projects, forms a lid for the box. The knife is fixed in a triangular frame, provided with screws by which it can be raised or lowered. METHOD OF USING WILLIAM.?' MICROTOME. Remove the lid of the box and fill the chamber with equal parts of pulverised ice and salt, care being taken not to allow the mixture to touch the under side of the cover, which, when Fig. 20. Williams' (Quecket Club) Freezing Microtom^. replaced, must be firmly secured by the clamp screw for that purpose. The substance to be cut is placed on the surface of the central circular brass piece, and surrounded with a solution of gum, which readily congeals, and will thus hold the specimen firmly in position, xl INTRODUCTION. which will solidify shortly after the gum has frozen. It is advisable to cover the apparatus with baize, to facilitate the freezing process. The edge of the razor must be elevated to the required height for cutting the section by means of the three screws supporting the frame. After the first cut, each end of the razor must be again presented to the surface of the specimen, when either end of the blade can be adjusted by one of the back screws until its entire length is level ; then by turning the large screw in the front part of the frame it can be lowered for each successive section required. Preparation of the Material for Freezing. — Follow exactly the same methods as are in- dicated at p. xxxviii. A piece of tissue, a quarter of an inch in thickness, is quite sufficient to freeze at one time. The tissue to be frozen is, after the above preparation, placed on one of the circular plates, and is fixed to it with gum solution. The razor ought to be moistened with the same solution, which prevents the sections from curling up. TREATMENT OF THE SECTIONS. From the bowl of water, into which they are put at first, they are transferred to a conical glass filled with water, in which they gradually subside. All the gum must be got rid of, which is accomplished by changing the water several times. When all the gum is dissolved, transfer the sections to one or other of the following fluids till they are required. Keep them in glass- stoppered bottles. PRESERVATIVE FLUIDS. 1. Ordinary methylated spirit. 2. Glycerine i oz., water i oz., carbolic acid 4 minims. DR. BEVAN lewis's ETHER FREEZING MICROTOME. It is not always possible to obtain ice for freezing, and hence it is necessary to have another medium for freezing. This is supplied in the most perfect form by the above in- strument (fig. 21), devised by Dr. Bevan Lewis, of the West Riding Asylum. This microtome is specially valuable for rapid freezing. The lower half or body of the instrument {a) is a slightly modified Stirling microtome; the upper part {p) consists of a freezing-chamber and a section-plate. The Body.— 'Y\\& framework of the body {a) is a solid brass casting, which can be secured to a table firmly by the coarse screw (w). Through its central aperture works the brass plug {li), driven by a spring and fine micrometer screw ik), the pitch being fifty threads to the inch, the screw having a diameter of ^\ths of an inch with a milled head i^ inches across. Section-Plate. — The section-plate {c) is a circular plate of zinc f ths of an inch thick, raised upon a strongly vertical arm of brass or zinc — preferably of the latter metal. In the section- plate there is a circular central aperture through which the freezing-chamber works. Lately the efficiency of the instrument has been much improved by capping the zinc plate [c] with a square of plate-glass |-inch thick, extra polished and drilled with a central aperture («) corre- sponding in size to that in the zinc plate, and which in my own instrument measures 2^ inches in diameter. For cutting sections of frozen tissue, the glass-section plate is a great advantage, and when once used will never be dispensed with. It renders the movements of the knife absolutely free and easy, whilst, on the other hand, the blade never gets injured from scratches which sooner or later invariably appear upon a metal section-plate, and which turn or indent the edge. INTRODUCTION. xli Freezing-Chamber. — This consists of a zinc cylinder ip) \\ inches deep by 2\ wide, closed above and below. An aperture (/), | of an inch across, is made on either side of the cylinder to allow the admission of the nozzle of the spray-producer, as also for free evaporation of ether, a large proportion of which, however, condenses on the sloping false bottom i^g) and is conveyed off by the tube (/) into a bottle attached to it. Fig. 21. The Ether Freezing Microtome of Dr. Bevan Lewis. This instrument is made by Mr. Gardner, South Bridge, Edinburgh. The instrument is simple, efficient, and compact, and enables one with a minimum expenditure of ether to obtain the most delicate sections of any tissue not more resistant than cartilage. A little experience in the use of the instrument enables the operator to freeze so rapidly that he will scarcely feel that it repays him to collect the condensed ether. xlii INTRODUCTION. METHODS OF STAINING TISSUES. Tissues may be stained with certain reagents, e.g. eosin, which give a uniform colour to the section, whilst other substances derive their value from staining different parts of a section unequally ; nuclei, for example, are usually stained of a deeper red than the surrounding protoplasm. For the method of transferring large sections to a slide, see p. xxvi. GENERAL DIRECTIONS. It is always advisable to use rather a weak solution of the staining agent, as this stains slowly and gives the dye time to completely permeate the section. When practicable the sections should remain in the fluid for several hours. The proper depth of tint can easily be ascertained by lifting out a section and washing it in water, and examining it on a slide with a low power. A little practice soon enables one to tell when the staining is deep enough. For class purposes, however, it is often necessary to use strong solutions of the dyes, and the results are very satisfactory. For class purposes staining with a strong dye is readily accomplished. Float a section on to a slide (p. xxvi), remove all the water from the slide and around the section. Place on the section a large drop of the staining fluid, and leave it 'until the section is properly stained. Wash off the surplus pigment in a bowl of water, float the section on to the slide again, and mount it in glycerine or other mounting fluid. STAINING SOLUTIONS. LOGWOOD SOLUTIONS. {a) A. Dissolve '3 grms. of haematoxylin in lo c.c. of absolute alcohol. B. Dissolve '3 grms. of alum in lOO c.c. water. A few drops of A added to a few c.c. of B gives a solution of a beautiful violet colour, which rapidly stains tissues. {b) A strong infusion of logwood chips is made ; to this reddish-coloured fluid, after filtra- tion, alum is added, until a bluish-violet solution results. The violet colour deepens by ex- posure to the air for several days. (c) Take 60 grms. of extract of hEematoxylin and 180 grms. of alum. Rub them toge- ther in a mortar and slowly add 280 c.c. distilled water. Filter, and to the filtrate add 20 c.c. of absolute alcohol. Keep all of the above in glass-stoppered bottles. Logzvood solutions ought always to be filtered before they are used. I have found solutions prepared by any of the above methods to give excellent results. They act very rapidly on nuclei, giving them a bright violet colour, softer and pleasanter to the eye than the bright red stain of carmine. Many, however, prefer a solution which contains INTRODUCTION. xliii alcohol ; such a solution is the following, which has the advantage of not requiring to be filtered so frequently. id) Kleinenbergs Logzvood Solution. — Make a saturated alcoholic (70 per cent.) solution of crystallised calcic chloride, and a similar solution of alum. Add 8 parts of the alum solution to I of the calcic chloride. To this mixture add, drop by drop, a saturated solution of haemato.xylin in absolute alcohol, until the whole fluid assumes a deep purple colour. Use only a small quantity of alcohol, as the haamatoxylln is very soluble. The colour deepens and improves by keeping. When it is desired to stain a section, this solution must be diluted by adding a few drops of it to a watch-glass full of water. {e) Logivood solution foi- rapid staining. — This solution, devised by E. A. Cook, yields good results, but it has no advantages over any of the above. The ingredients are extract of log- wood and alum, of each, 6 parts, cupric sulphate i part, and water 40 parts. Grind in a mortar the alum, logwood, and cupric sulphate, all of which must be iron free, and when powdered add sufficient water to form a thin paste. Leave this for one or two days, then add the remainder of the water and filter. To the filtrate add a crystal of thymol, to preserve it from mould. For tissues hardened in chromic acid add 8 drops of this solution to 120 drops of water, and add one drop of a tenth per cent, solution of potassic bichromate just prior to use. CARMINE SOLUTIONS. Strong Carmine Solution for rapid staining. — Rub up two grms. of the best carmine in a mortar with a few drops of water, add 4 c.c. of liquor ammoniae to dissolve the carmine, then add 60 c.c. of water. Filter the solution and keep it in a stoppered bottle. The solution may be diluted, if it be desired to stain a tissue slowly. Carmine is not employed so frequently as it used to be. A strong solution, such as the above, is apt to give a somewhat uniform tinge to the section. Better results are obtained by using either the above fluid diluted, or the following : — Weak Carmine Solution. — Rub up i grm. of best carmine in a mortar with a few c.c. of distilled water ; add 3 c.c. of liquor ammonia, and, when the carmine is dissolved, add 1 50 c.c. of distilled water, and filter into a stoppered bottle. Add 30 c.c. of glycerine and 100 c.c. of rectified spirit. This is a modification of Beale's carmine fluid. All carmine-stained preparations, after being thoroughly washed in water, are improved by placing them for a few minutes in a one per cent, solution of acetic acid. This brightens the colour and fixes the carmine in the nuclei, and also differentiates the stained from the unstained parts. The stain thus becomes more selective. PICROCARMINE, OR PICROCARMINATE OF AMMONIA. Picrocarmine Solution. — Make a solution of carmine by rubbing up i grm. of best carmine with 10 c.c. of water, and adding 3 c.c. of liquor ammoniae. Add this solution to 200 c.c. of a cold saturated solution of picric acid. Leave the mixture exposed to the air until it evaporates to one-third of its bulk. The same result may be obtained by evaporating it slowly in a hot chamber, or over a water-bath at a low temperature. Filter to get rid of the precipitated carmine. This substance was introduced by Ranvier, and is one of the most valuable dyes one can employ. It is especially used for ' double staining,' containing as it does two pigments, one yellow and the other red — eg. the perinuclear part of a frog's coloured blood-corpuscle is xliv INTRODUCTION. stained yellow, the nucleus red. Connective tissue is stained red and elastic tissue yellow. It has this further advantage — that though sections are left for several hours in the solution, they seldom become overstained. It is not necessary to remove all the picrocarmine from the section ; in fact, it is advisable to leave a little adhering to the section, for after being mounted in glycerine or Farrant's solution, the trace of dye left is gradually absorbed by the section. If it be desired to retain the yellow and red colours it is desirable not to wasli the section after staining, because the yellow picric acid stain is readily removed thereby. In some cases it is advisable to mount the section in glycerine, containing one per cent, of formic acid. All picrocarmine preparations improve by keeping. After several days nuclei become stained that at first were unaffected ; so that sections should invariably be examined several days after they are mounted. Picrocarmine may be purchased as an imperfectly crystalline body. If the crystals arc used, make a one per cent, solution. Purpurine. — The dye obtained from madder has no advantages over any of the other methods. It can be dispensed with. ANILINE DYES. {a) Magenta Solution.— Dissolve i decigrm. of the sulphate or nitrate of rosanilin in lOO c.c. of water, and add lO c.c. of rectified spirit. This does very well for fresh tissues. The stain is not permanent, though it may last for many months. For blood-corpuscles 2 decigrms. are dissolved in 40 c.c. of a mixture of equal parts of water and glycerine. {U) Aniline Blue, soluble in Water. — Make a one per cent, solution in water. This is sometimes used for fresh tissues. (c) Aniline Blue-Black. — Make a one per cent, solution in water. If the section is over- stained the excess may be removed by steeping it in a two per cent, solution of chloral hydrate. For the nervous system an alcoholic solution is most useful. Dissolve i decigrm. in 4 c.c. of water, add to this lOO c.c. of rectified spirit and filter. Preserve in a stoppered bottle. This solution stains rapidly and gives a pleasant slate-grey colour, which fatigues the eye very slightly. It is specially useful for nerve-centres. Sections may be mounted in dammar without any fear of removing the dye. (d) Methyl-Aniline. — Make a strong watery solution and stain the tissue deeply. Wash away the superfluous pigment in water acidulated with acetic acid. The acetic acid dissolves out much of the pigment, and the washing must be continued till the proper tint is obtained. The sections must then be thoroughly washed in water, and mounted in glycerine or Farrant's solution. Dammar is unsuitable, as clove oil discharges the colour. This is a most valuable dye, for in contact with certain tissues it gives a ' double-stain.' It decomposes into two colours — one a red-violet, the other a blue-violet — each of which acts on different tissues. It is useful for hyaline cartilage ; the red-violet attaches itself to the matrix, and the blue-violet to the corpuscles. It is in the pathological change known as amyloid or waxy degeneration, however, where it is most useful. All parts of an organ — e.g. the liver — affected with amyloid degeneration are stained red-violet, whilst the neighbouring unaffected elements are coloured blue-violet. For fresh tissues it also forms a useful dye, and in the form of a one per cent, watery solution it may be used instead of magenta. It stains certain parts of a beautiful violet. It is very useful for showing the corpuscles in connective tissue, or the nuclei in fresh cells, or for mucous tissue. It is best to mount a tissue in a saturated watery solution of potassic acetate. INTRODUCTION. xlv {/) Iodine Green. — A saturated watery solution may be made and diluted as it is required, or a one per cent, solution may be prepared. It stains rapidly, and the stain cannot be removed by washing. It is one of the most useful of the aniline dyes, especially for double- staining (p. xlvi). It stains mucous glands green, and acts similarly on unossified cartilage- Preparations stained with it must be mounted in dammar. Do not leave the stained sections long in spirit, which partially removes the dye. (/) Rosein is soluble in spirit, and so is useful for double-staining when the sections are to be mounted in dammar. See retina. Ordinary methylated spirit does quite well as a solvent Many other aniline dyes are employed, but the above are certainly the most useful. {g) Eosin, though not an aniline dye, may for convenience be taken here. It yields a rose-coloured dichroic fluid, when dissolved. Dissolve i part in 1,500 of water. Tissues become stained in a few seconds. After staining, place them in water slightly acidulated with acetic acid (one per cent.), to remove the excess of pigment and to 'fix' the remainder. Sections may be examined either in glycerine or in dammar. For some purposes, as for staining the haemoglobin of coloured blood-corpuscles, it is used as an alcoholic solution. It is very useful in the study of developing coloured blood-corpuscles. METALLIC SOLUTIONS. {a) Silver Nitrate. — Dissolve i grm. of silver nitrate in 100 c.c. of distilled water. For use dilute this to g^ or ^ per cent, solution. Wash the tissue to be stained {ivhick must be quite fresh) in distilled water to remove chlorides, place it in the silver solution for five minutes, or until it becomes of a whitish appearance, then remove it, wash it in ordinary water, and expose it in water, or alcohol and water, to diffuse daylight till it becomes brown in colour. Silver nitrate solution is used where it is desired to bring into view the existence of epithelial cells — e.g. on serous membrane, lining arteries, lymphatics, capillaries, or the air-cells of the lung. It acts upon the intercellular substance, or cement, and when the tissue is exposed to the action of light, it is precipitated in the intercellular substance as the black oxide, in fine black lines, and thus the cells are mapped one from another. These lines are known as ' silver lines.' It also stains the intercellular substance of cartilage and the cornea. The prepara- tions may be preserved in glycerine or dammar, and ought to be kept in a dark place, as they are apt to spoil when long exposed to light. {b) Gold Chloride. — Solutions from \ per cent, to 2 per cent, are employed. 1. Place the perfectly fresh tissue in the gold solution (^ per cent.) for twenty to thirty minutes, wash it in water, and then expose to daylight in water slightly acidulated with acetic acid (2 or 3 drops of acetic acid to i oz. of water) until it becomes of a purplish colour. 2. Or the following method may be employed, especially where the tissue is dense and it is not desirable to retain the epithelium. Cut a fresh lemon in two, squeeze out the juice and fil- ter it through muslin. Place the perfectly fresh tissue — e.g. cornea — in the juice for five or seven minutes, wash it in wafer to remove the juice, and steep it in a one per cent, solution of gold for twenty minutes, or half an hour. Wash off the surplus gold, and place the object in a mixture of one part of formic acid to four of water. Put the bottle in a dark place. After twenty- four hours the gold is completely reduced. All the epithelium, however, is removed by the formic acid. This method is the best for demonstrating the terminations of nerves in muscle. Gold is especially useful for staining nerve-fibrils — e.g. in the cornea — and also for connec- tive tissue-corpuscles. Under each tissue where gold is useful, special directions are given for its employment. xlvi INTRODUCTION. (c) Osmic Acid. — Make a one per cent, solution which can be diluted as required — to i or i per cent. The method of using and preserving this substance has already been referred to under Hardening Solutions (p. xxxii). It is useful for blackening fat-cells, and for fixing the white substance of Schwann in nerve-fibres, though it has many other important applications. ON DOUBLE-STAINING OF TISSUES. By using two differently coloured solutions, it is found that in certain tissues one part will take up one colour, whilst another part takes up another. Some glands stain of a green colour with iodine-green — eg: the mucous glands of the tongue — whilst the other glands, the serous glands, are not affected by it. This method is of the greatest value, and I find it can be practised very successfully by students even in large classes. riCROCARMINE. As already indicated, this dye stains certain parts yellow and other parts red, hence its value. PICROCARMINE AND LOGWOOD. Stain the sections slightly with picrocarmine, and after washing them in water, place them in dilute logwood solution, in which they must not remain too long. They soon be- come lilac-coloured. Wash them and mount them. This method is useful for the mesentery of the newt (p. 38), for developing bone, epiglottis, &c. PICROCARMIME AND IODINE GREEN. This is one of the most useful combinations I know. Stain the sections in picrocarmine, and wash them well in water slightly acidulated with acetic acid, and then stain them in a watery solution of iodine-green (p. xlv), taking care that they do not become over-stained, which can easily be ascertained by washing them in water. If a section of the posterior third of the tongue be so stained (p. 61), all the muscles and connective tissue are red, whilst the mucous glands and adenoid tissue are green. The serous glands do not take up the green stain. This combination is, therefore, of the utmost value for gland-tissue. All the mucous glands and adenoid tissue of the intestinal tract become green, whilst the connective tissue is red, and the non-striped muscle of a light yellow or brown. Most exquisite effects are produced in the cerebellum, bone, and intestine by this method. The sections must be mounted in dammar. EOSIN AND LOGWOOD. Stain the sections lightly in eosin and then in logwood. This does very well for the brain. GOLD CHLORIDE AND SILVER NITRATE. This method is sometimes employed for the cornea, which is first impregnated in the usual way with gold chloride and then with silver nitrate. The method is very difficult to be suc- cessful with and often fails. INTRODUCTION. xlvii TREBLE-STAINING. This method has recently been practised by Dr. Heneage Gibbes. The process is tedious, and not so satisfactory as double-staining. A combination of picrocarmine and iodine-green for sections of the small intestine, including a Peyer's patch, gives an excellent and most instructive effect (p. 69), in fact a perfect treble stain. Other combinations, as picrocarmine, eosin, and iodine-green, have been tried. GOLD CHLORIDE AND AN ANILINE DYE. This process is alluded to in connection with the study of the parts in the tail of a rat (p. 25). The tissue is first treated with gold chloride and then decalcified, and the sections are stained with rosein, and then with iodine-green, or with iodine-green alone. The effects produced on the developing bone especially are most remarkable and striking. This method is capable of further extension. LIST OF STAINING REAGENTS. Ordinary staining reagents. 1. Logwood or HEematoxylin. 3. Picrocarmine. 2. Carmine. 4. Eosin. A niline dyes soluble in water. 1. Magenta, or Rosanilin. 3. Aniline blue-black. 2. Aniline blue. 4. Methyl-aniline. 5. Iodine Green. Aniline dye soluble in spirit. Rosein. Metallic Substances. I. Silver Nitrate. 2. Gold chloride. 3. Osmic Acid. Many other dyes, as iodine, indigo-carmine, &c., are recommended, but the above arc what the author has found to be really useful. xlviu INTRODUCTION. METHODS OF MOUNTING SECTIONS. Most sections, even when very thin, are too opaque to be examined, and must therefore be placed in some medium or mounting fluid which will render them more transparent. MOUNTING FLUIDS. 1. Glycerine. The section must be floated on to a slide from water, and, after removing the water, a small drop of glycerine is applied. The drop ought to be of just such a size that when the cover-glass is applied, it accurately fills the space between the cover and the slide. Apply a cover-glass, as directed (p. xxiv). A little practice is required to gauge the size of the drop required. If too much glycerine be added, the excess is most conveniently removed by placing several small pointed pieces of bibulous paper round the margin of the cover. All surplus glycerine must be carefully removed, else the cement to be afterwards applied will not adhere to the glass. Glycerine has this great disadvantage, that the cover- glass moves so easily that it becomes difficult to seal up the preparation. This is entirely obviated by mounting sections in the following fluid, which has the advantage, that after exposure to the air for three or four days, the cover-glass becomes firmly fixed to the slide, so that the coat- incT of cement can be applied without any fear of displacing either the cover or the section. 2. Farranfs Solution. — Take equal parts of glycerine and a saturated watery solution of arsenious acid. Add gum arable in large quantity, and allow the mixture to stand for several weeks, stirring it from time to time, until it ceases to dissolve more of the gum. Filter through paper. This takes a considerable time, but one is rewarded by the beautifully clear fluid obtained. The above fluids are suitable for mounting sections — stained or unstained — from water. Unless for very delicate tissues, such as isolated epithelial cells, Farranfs solution is in all cases to be preferred. It is always advisable, when wishing to study the minute relations of parts— except those of the nerve-centres— to mount sections in either of the above fluids. 3. Glycerine Jelly. This is sometimes employed to mount softened bone and tooth, and some urinary deposits. It must be melted in a water bath before being used. As it sets rapidly, the cover-glass must be applied at once to the preparation. It is far better to buy it ready- made than to take the trouble of making it. Rimington's is the best : it contains, in addition to gelatine and glycerine, a trace of carbolic acid. 4. Dammar Solution. — Mix in an earthenware vessel 2 oz. of mastic and 2 oz. of dammar with 2 oz. of chloroform and 2 oz. of turpentine. Shake or stir the mixture from time to time until the substances are dissolved. Filter through paper. Formerly Canada balsam was employed, but the above fluid is certainly to be preferred. If Canada balsam be thought preferable, it may be easily prepared by heating some balsam in a capsule in a warm chamber at 65° C. for twenty- four hours, and, after it cools, dissolving the dry yellow mass in as much benzole as will give a fluid of a syrupy consistence. INTRODUCTION. XllX Both dammar and Canada balsam render tissues much more transparent than glycerine. All traces of water must be removed from the sections before they can be mounted in dammar or Canada balsam. This is done by immersing them for five or ten minutes in absolute alcohol. METHOD OF MOUNTING IN DAMMAR. When a section is to be mounted in dammar all water must be got rid of. Place the section in absolute alcohol for five or ten minutes, until all the water is removed. Transfer it for the same period to oil of cloves or turpentine, — preferably the former, which expels the alcohol, and renders the section quite transparent. These operations can all be accomplished on a slide thus, — Float out the section on to the slide in water (p. xxvi) ; remove the water with blotting-paper, add absolute alcohol to remove the remainder of the water ; after a few minutes soak up the alcohol with blotting-paper, and with a stiff brush insinuate a drop of oil of cloves under the section. The clove oil gradually rises through the section, expels the alcohol, and renders the section quite transparent. This process ought to be obser\ed with a low power under the microscope. Remove the surplus clove oil with blotting-paper, add a drop of the dammar mounting fluid, apply a cover, and the preparation is finished. LABELLING AND PRESERVING SECTIONS. After mounting, each section must be properly labelled (p. xxvi), and \a.\d flat in a tray, and preserved from dust. iMETHODS OF SEALING UP THE PREPARATIONS FOR PRESERVATION. Each preparation has still to be sealed. This is done by placing some kind of cement over and round the margin of the cover-glass. After several days the specimens mounted in dammar and Farrant's solution will be ready for sealing up. This is best done by means of a turntable, which consists of a brass disc, at least three inches in diameter, and rotating on a pi\ot. The upper surface of the disc has on it a series of concentric rings, corresponding to the different sizes of cover-glasses used. The disc is rotated by means of the forefinger of the left hand acting on a small milled head placed underneath. The slide is placed on the disc, accurately centred and fi.xed in position by two clips, which ought to be made of bent steel spring in preference to brass. A camel-hair or goat-hair brush is dipped in the cement, and whilst the disc is rotated a ring of the cement is run round and -over the margin of the cover. This ought to be done twice or thrice, until a sufficiently strong ring is formed. In selecting a turntable, see that the disc is heavy and wide enough to admit the largest slide under the clips. The disc must be heavy, and should rotate steadily and easily ; and this is best secured by having the pin or pivot on which it rotates made f Fig. 22. Modified Turntable. 1 INTRODUCTION. ^pin-pointed', i.e. the upper half of the pin much finer than the lower half. When square cover-glasses are used, the cement is simply painted on with a brush. Fig. 22 shows a turntable invented by Mr. Dunning. It is so made that any non-central position may be obtained, even with slides two inches in width, thus facilitating the appli- cation of the cement to any slide the cover-glass of which has not been accurately centred. I. METHODS FOR TREPARATIONS MOUNTED IN DAMMAR. It is not necessary to apply any cement ; but the preparation looks neater if a ring of the ordinary dammar mounting fluid is run round the margin of the cover-glass. After using the brush wash it in benzole to remove the dammar. II. FOR PREPARATIONS MOUNTED IN GLYCERINE OR FARRANT'S SOLUTION. After removing all the surplus mounting fluid, paint a ring of luarinc ghte ox painter s gold- size round the cover-glass, so as to fix it down, and after it dries (twenty-four hours) put on a tliick ring of zinc-white cement. It may be necessary to put on more than one ring of zinc cement. This is by far the best way of sealing up these preparations. When the zinc-white dries it forms a ring as hard as enamel. The brush used for the zinc-white ought to be washed at once in benzole, and the one used for gold-size in turpentine, and the one for glue in water. These simple precautions ought never to be neglected. Various other substances, as asphalt and melted paraffine are recommended for sealing up preparations, but none of them are equal to the above described method. The jEsthetically inclined student can easily tint the white cement by adding to it a little solid eosin or any other dye. It is, however, quite unnecessary to spend time in adorning the outer surface of preparations. FLUIDS FOR SEALING UP PREPARATIONS. 1. The ordinary dammar solution may be used. 2. Zinc-white Cement. — (Gum dammar, 8 oz. ; zinc oxide, i oz. ; benzole, 8 oz. Dissolve the dammar in the benzole and add the zinc oxide, and then strain through muslin.) 3. Marine Glue (Hollis'). 4. Painter's Gold Size. Number 3 or 4 is applied first, and when it becomes dry it is covered with a ring of zinc- white cement. In my opinion gold size is preferable to marine glue. It is not so liable to crack. METHODS OF INJECTING THE BLOOD-VESSELS. The ordinary method of hardening tissues in chromic acid does not suffice to reveal the arrangement of the blood-vessels of a part, hence we must have recourse to filling the blood- or lymph-vessels artificially with a coloured mass. The coloured fluids at present employed are of two kinds — one kind which \s. fluid, the other which is solid at ordinary temperatures. The chief medium of the former is water or water and glycerine, and of the latter gelatine. INTRODUCTION. li INJECTION FLUIDS. (a.) injection-masses pxuid at ordinary temperatures. 1. Watery Solution of Briicke's soluble Berlin-blue. — Dissolve lo grms. of Briicke's soluble blue in 500 c.c. of distilled water. Briicke's soluble blue can be purchased in the market ; but if it be desired to make it, the following is the formula. The process is rather tedious, {ci) Dissolve 217 grms. of potassic ferrocyanide in i litre of distilled water, {b) Make one litre of a 10 per cent, solution of ferric chloride. Add to each of these solutions {a and b) two litres of a saturated solution of sodic sulphate. Then add the ferric chloride to the ferrocyanide, and stir the fluids whilst they mix. The precipitate is filtered through a conical bag of new flannel, and washed with a small quantity of water. The first washings are returned. The washing is repeated for several days, until the solution when passed through filter paper is of a deep blue colour. It is then soluble. The precipitate is collected from the flannel, pressed between folds of blotting paper, dried, and broken into small pieces. After the blood-vessels of an organ have been filled with this solution it is placed in equal parts of methylated spirit and water, to which a trace of hydrochloric acid is added, as this prevents the diffusion of the blue ; or the organ may be hardened in Miiller's fluid or picric acid. In some cases it is advisable to heat the injection-fluid to a temperature of 40° C. before injecting it into the vessels. A little glycerine added to the mass makes it flow more easily. 2. Richardson's Blue. — {a) Dissolve 10 grains of ferric sulphate in 10 oz. of water, [b) Dissolve 32 grains of potassic ferricyanide in i oz. of water. Mix a and b, and then add water to 20 oz. (b.) injection-masses not fluid at ordinary temperatures. 1. Soluble Blue and Gelatine. — Soak 50 grms. of the best gelatine (Cox or Coignet) in water for several hours. Pour ofl" the water which is not absorbed by the strongly swollen-up gelatine, and melt the gelatine over a water-bath. The gelatine may be placed in an ordinarj- jelly can or tin vessel, and this placed in a pan of water on the fire, if a water-bath is not at hand. A two per cent, watery solution of Berlin-blue is prepared (p. li.), and heated to the same temperature as the gelatine. Take 250 c.c. of this fluid, and gradually add to it the solution of gelatine, the mixture being stirred all the time. Whilst still hot the mixture is filtered through flannel, and then kept at a temperature of 40° C. until it is injected. After the injection is finished, place the organ in alcohol for twenty-four hours, to coagulate the gelatine. 2. Carmine and Gelatine Mass.— («) Take i oz. of the best gelatine, and make a solution of it as described above. Strain while hot through flannel, and make up the solution to 2 oz. {b) Place I drachm of the best carmine in a mortar, to it add i drachm of liquor ammonia; and 2 oz. of water, and leave it for twelve hours (if in a hurry, place the carmine solution near the fire for an hour). Filter, and add about 80 minims of glacial acetic acid, drop by drop, stirring all the while, until the ammonia is completely neutralised. As the odour of the ammonia becomes faint the acid must be added very cautiously. As long as there is free ammonia the fluid is of a dull red, but it becomes of a bright, florid, cherry colour the moment the ammonia is neutrali.sed. Add the florid red carmine fluid (Ji) to the 2 oz. of gelatine solution {a), and keep at a temperature of 40° C. until the mass is required. f 2 lii INTRODUCTION. The principle to be attended to in making the mass is this :— The carmine, if alkaline, would diffuse through the tissues ; if acid, it would be deposited in fine granules, which block up the capillaries— hence the necessity for having a neutral fluid. The best guides are the colour and odour of the fluid. The injected organ must be placed in equal parts of methylated spirit and water, to which a few drops of acetic or hydrochloric acid (one per cent.) are added, to prevent the carmine from diffusing into the tissues. APPARATUS EMPLOYED FOR INJECTING THE BLOOD-VESSELS. Injections are made either with a syringe, or with .some form of apparatus, which will give a constant pressure. Whatever apparatus be employed it is necessary to insert a cannula into the blood-vessel by which the injection will flow, e.g. the aorta. The cannulae may be made of brass, such as are ordinarily supplied with a syringe, or they may be made of glass, with a short piece of india- rubber attached to them. The advantage of a glass cannula is that one can see when air-bubbles are present. For the method of making these cannula see fig. 23. The cannula ought to have a shoulder, over which the thread is applied, so that when tied it cannot slip out of the vessel. Method of inserting a Cannula into a Blood-vessel. — Expose the artery of the part to be injected, clear a small piece of it from the surrounding tissue, and place a ligature around it by means of an aneurism-needle, or a narrow-pointed pair of forceps. With sharp scissors make an oblique slit in its wall, and insert the nozzle of the cannula, directing it towards the periphery. Tie the ligature firmly over the artery, and behind the shoulder on the cannula. Fig. 23. Cannul.e for Injecting. Natural Size. r' f- 1-^ Glass Cannula; of different sizes; a and b show the method of making a cannula : a glass tube is heated in a flame and drawn out lo the size required, as at a. It is then heated in the middle and slightly drawn, to make it thinner there. After it cools it is cut by means of a triangular file at /, and then each half is ground down obliquely on a hone. The sharp edges are got rid of by heating it in a flame for a few seconds. A piece of india-rubber tubing is tied to the end of each and the cannula is complete. c\ A metal cannula ; cl, A steel clamp for clamping a small artery (Schafer). HOW TO INJECT BY MEANS OF A SYRINGE. The syringe is made of brass, and is provided with several cannulae, of different sizes, and a separate piece with a stopcock to connect the cannula with the syringe. Tie the cannula in the vessel, as directed above, and fill it with salt solution, by means of a pipette. Fix the stopcock-piece into the cannula, and fill it in the same way with salt solution. Then fill the syringe with the injection-mass, taking care not to suck up any air. All air ought to be expelled from the syringe by holding it vertically, nozzle uppermost, and expelling a few drops of the injection-mass. The syringe ought to be filled and emptied several times before doing this, to get rid of air. Insert the syringe in the stopcock-piece, and proceed slowly with the injection. The piston must be rotated very slowly; so that the mass is driven on slowly and steadily. Half-an-hour is not too long for the injection of a rabbit. One judges of the com- pleteness of the injection by observing the colouration of such vascular parts as the tongue, lips, toes, or ear. After the injection is completed, turn the stopcock to prevent the escape of fluid, remove the syringe, and place the part injected in equal parts of alcohol and water, or any of the other fluids described at p. xxxii. If a gelatine mass be employed, it is better to INTRODUCTION. liii. inject the animal after it has been killed by bleeding. The animal or part to be injected must be kept warm by immersing it in water at 40° C. Even when a watery injection is emploj-ed, it is as well to warm it to 40° C, as while hot it causes less spasm of the small arteries than a cold mass. The art of injecting can only be acquired by practice. The part to be injected and the mass must be at the proper temperature, no air must be allowed to enter the blood-vessels, and the mass itself must not contain any particles which will block up the capillaries. One of the great difficulties is to keep the pressure constant ; this may in part be arrived at by fi.x- ing a tube at right angles to the side of the stopcock-piece, and bringing in connection with it, by means of a caoutchouc tube, an ordinary mercurial manometer, viz., a bent tube filled with mercury. A pressure of two to five inches of mercury is usually sufficient for injecting small animals. In selecting a syringe sec that you get one that is capable of holding from three to six ounces. The barrel ought to be /c//^— six or seven inches. For corrosive (or metallic) sub- stances, a glass or vulcanite syringe must be employed. INJECTING APP.\RATUS FOR OBTAINING A CONSTANT PRESSURE. Ludwig was the first to introduce this arrangement. Sufficient pressure may be obtained either b\' means of a column of mercury or a column of water. Fig. 24. Ludwig's Mercury Pressure-api'aratu.s. a. Reservoir bottle for mercury; b, Pressure bottle inio which the mercury of a flows; c. Bottle containing the injection-mass, connected with b by one india-rubber tube, and by another with the cannula f, and by a glass tube with a small manometer ; d. Handle of screw for raising the wooden shelf on which a rests ; e, A screw clip, and g, a spring clip. Ludwig's Mercury Pressure-apparatus (fig. 24). — The apparatus consists of two Wolff's bottles, one of which, a, contains a reserve of mercury, and ^>, into which, owing to the bottles being at different levels, the mercury flows. This compresses the air in d, and so acts on the liv INTRODUCTION. injection-fluid contained in the bottle c, to which a mercurial manometer is attached. From this an exit caoutchouc tube passes, which is attached to the cannula fixed in the artery. The pressure-bottle a rests on a wooden support, which is raised by means of a screw, and in this way sufficient pressure is obtained. In injecting with any pressure-apparatus, begin with a pressure of one inch of mercury, and slowly increase it to four inches. Of course if a gelatine mass be employed the bottle c must be kept warm, by placing it in warm water to keep the gelatine in a fluid condition. Before beginning the injection, the cannula and the exit-tube from c must, of course, be filled with the injection-mass, to avoid the entrance of air. This apparatus is expensive, and therefore an apparatus that one can make for himself is to be preferred ; such as the following simple mercurial injection-apparatus (fig. 25). It works on exactly the same principle, and Fig. 25. Simple Mercurial Injection-apparatus. fig. 25 explains itself. One of the bulbs (c), containing the mercury, is stationary, whilst the other (a) rests in a wooden framework, or shelf, which moves freely in a slot (d). The part of this movable shelf which is fi.xed in the slot is so made that it works very freely, and when it is loaded at the free end of the horizontal arm, it becomes self-fixing at any height. The pressure is very easily regulated by raising or depressing the movable shelf (a). By means of a divided, or Y-shaped tube, two injection-fluids (b, r) may be used simul- taneously. This arrangement, I find, works admirably. Simple Water Pressure-apparatus. — Mercury is not always to be obtained, hence it is sometimes convenient to employ water-pressure. Satisfactory results are not obtained by connecting a water-tap to a system of pressure-bottles. The following arrangement (fig. 26) is simple, cheap, easily made, and effective. It consists of a bottle, C, which holds the injection mass, a much larger glass bottle, B, with a manometer (m) attached ; and communicating I NT ROD UCTION. Iv with this is a cyhiider of tin (a), which is filled with water, and is raised or lowered by means of a cord which passes over a pulley (p) fixed to the ceiling of the room. INJECTION OF THE BLOOD-VESSELS OF AN ENTIRE ANIMAL. Having determined which injection-mass is to be used, prepare the cannula;. The animal —a rabbit or guinea-pig— whose blood-vessels are to be injected, is killed by chloroform. As soon as it ceases to breathe, the thorax is opened by making an incision along the middle of the sternum, so as to expose the heart. Pull the heart forward, and open the pericardium. Snip through the wall of the right auricle, and allow as much blood to flow out as possible ; the animal may even be suspended by the hind limbs to expel as much blood as possible. Expose the aorta, and tie into it a glass cannula (fig. 23, c) as directed at p. lii. The cannula is now filled with salt solution, and is connected either with a syringe or one of the constant-pressure arrange- ments described above. Take care that there is no air either in the cannula or in the india- rubber tube coming from the injection-bottle. At first a pressure of two inches of mercury is suffi- cient, but gradually the pressure may be raised to four inches. As the injection is forced into the aorta, and gradually fills the blood-vessels, blood and then blood and injection-mass flow out of the right auricle. Continue the injection until the injection alone flows from the hole in the right auricle. It is then closed by a broad ligature, and the injection continued until the vascular system is filled with the injection, which may be judged of by the colour of transparent parts, as the ears, lips, &c. After the injection is complete, ligature the vessels at the base of the heart, or a glass stopper ma)^ be put into the caoutchouc tube attached to the glass cannula. The after-treatment of the in- jected animal is described at p. lii. If an animal be allowed to cool after death, it must be thoroughly warmed before using a gelatine mass. This is done by placing it for an hour in w'arm water not above 40° C. Fig. 26. Injection Apparatus with Water Pressure. A, Vessel filled with water; B, Bottle for compressed air ; c, Bottle for injection mass ; M, Manometer. INJECTION OF THE LYMPHATICS. Puncture Method. — This is very easily done by the ' einstich ' or ' puncture ' method of Ludwig. A small subcutaneous (Pravaz) syringe (fig. 27, p. 22) is filled with a two per cent. Ivi INTRODUCTION. solution of Briicke's blue (p. li), and the nozzle is thrust into the pad of a dog's or cat's foot, and the fluid injected into the pad, which is then compressed, and the limb stroked from below upwards. The coloured mass finds its way from the connective-tissue spaces into the lymphatic vessels, and runs along them very earily. In this way one can easily inject the glands of the leg and abdomen, and even the thoracic duct. The lymphatics of the testis are injected in the same way. It is easy to demonstrate the lymphatics in the intestinal wall and in the liver by the same method. Instead of a syringe, one of the constant-pressure arrange- ments may be used. PRACTICAL HISTOLOGY. o»{o BLOOD. When examined microscopically blood is seen to contain corpuscles floating in a fluid — the plasma or liquor sanguinis. The corpuscles are of two kinds, (i) coloured and (2) colour- less, and in addition (3) minute granules are to be seen. Besides these, there fall to be examined (4) the fibrin and (5) the colouring matter of the coloured corpuscles and (6) various pigments derived therefrom. (A.) BLOOD OF A FROG OR NEWT. PREPARATION. — Give a frog or newt a smart blow on the head. If a newt be taken, carefully wipe its tail dry by means of a cloth and snip off the point of the tail. Then squeeze out a drop of blood from the stump on to a glass slide, and at once apply a cover- glass. If a frog be used a toe should be cut off, when a drop of blood can easily be squeezed out of the stump. In cither case the blood thus obtained will be mixed with a considerable quantity of lymph. If a drop of blood without any admixture of lymph is required, the heart of the animal must be exposed, and when this is done the auricle should be snipped through and a drop of blood allowed to fall on the glass slide. EXAMINATION. — i. Observe the coloured corpuscles (H). They are seen to be very numerous, of a yellowish straw colour, uniform in size and elliptical in shape when seen on the flat (PI. I., Fig. i).* A few of them may also be seen edgewise, and they then appear narrow, somewhat pointed at the ends, and slightly thicker in the middle (PI. I., Fig. 2). Sometimes one corpuscle lies above another, when the outline of the undermost one may be distinctly seen through the one lying above it ; it is thus seen that each corpuscle is transparent. At first each corpuscle may seem homogeneous, but in a very short time an elliptical slightly granular body shows itself in the centre of each as seen on the flat. This body, which is somewhat lighter in colour than the rest of the corpuscle, is called the )U(deiis (PL I., Fig. i). EFFECTS OF REAGENTS on the coloured corpuscles. {a) One per cent. Hydro cMoric Acid. — Squeeze out a fresh drop of blood on a clean slide, quickly apply a cover-glass, and then place a drop of a one per cent, dilution of hydrochloric acid at one edge of the cover-glass (taking care that none of the acid gets on the top of the cover- » Where a simple reference is made to a plate and figure (as PI. I., Fig. i), it is intended that the student shall insert by delineation and colouring such appearances and changes as are indicated in the text as the result of reagents or otherwise. 2 PRACTICAL HISTOLOGY. fjlass). Place a small piece of blotting-paper on the slide at the edge of the cover-glass opposite to the spot where the drop of acid was placed, which will have the effect of drawing the acid under the cover so as to ' irrigate ' the blood-corpuscles with the reagent. Steadily observe one or two corpuscles ; they will gradually enlarge, become globular and all of a sudden swell up and become distinctly spherical, and then as suddenly collapse and ' shoot ' or discharge their hasmoglobin. The nucleus comes clearly into view during the process. This process ought to be diligently looked for, because the phenomenon is so striking. It is as if the corpuscle burst and discharged something. After the collapse, the residue of the stroma adheres to the nucleus. In other cases the corpuscles do not collapse, but remain globular, clear, and transparent. The peri-nuclear part becomes decolourised, and appears to be limited externall}^ by a membrane from which fine shreds — the residue of the stroma — stretch to the nucleus, which is now clearly brought into view, and has assumed a distinctly granular appearance, due to the existence of a fibrillar network in its interior. If the acid used be too strong this action may be missed, and the outline of the corpuscles becomes very faint, and the shrivelled nucleus may appear excentrically, or may even escape altogether outr-ide the corpuscle. These phenomena are best observed with frog's blood. (/;) Acetic Acid. — Shows the same phenomena, though in a less degree. The corpuscles swell up and become clear, so that their nucleus becomes very obvious, and the nucleus not unfrequently passes out of the corpuscles. It is to be noted that all the corpuscles are not so affected, which is perhaps due to difference in the chemical constitution of the corpuscles. In many corpuscles distinct vacuoles are formed in the peri-nuclear part. The coloured pig- ment may be seen gradually to leave the wall of the corpuscle, and become heaped up round the nucleus. The fluid in which the corpuscles float becomes slightly coloured by a diffusion of the pigment of the blood-corpuscle (haemoglobin), and sometimes the nucleus takes up some of this pigment and becomes of a distinctly yellow colour (PI. I., Fig. 3). (c) Water. — Squeeze out another drop of blood on to a slide, cover, and irrigate with water in the manner just described. The corpuscles and their nuclei both swell up and become globular, owing to endosmosis, and at the same time the haemoglobin diffuses into the sur- rounding fluid (PI. I., Fig. 6). {d) Syrup. — Squeeze out a fresh drop of blood, add a drop of strong syrup of white cane sugar, and mix the two drops thoroughly with the point of a needle, and apply a cover-glass. Owing to the greater specific gravity of the syrup the corpuscles lose fluid, and, on examina- tion some of them present a shrivelled appearance, and resemble thin misshapen biscuits, while others are but slightly affected. Here and there a reddish tinge is seen in a corpuscle (Pi. I., Fig. 4). {e) Dilute Alcohol. — One part of rectified spirit to two of water, applied in the same way as acetic acid, decolourises the corpuscles, causes them to swell, and brings a nucleolus into view (usually placed towards one end) in each nucleus (Ranvier, Stirling). (/) Magenta. — Mix a drop of blood with a drop of a solution of sulphate or nitrate of rosaniline (p. xliv), cover and examine. The nuclei of the coloured corpuscles become stained of a deep red colour, the peri-nuclear part remaining unaffected, though of course the magenta has to traverse it to reach the nucleus. All the corpuscles are not, however, equally affected. In many the indications of the existence of an envelope with one or more thickenings in it are observable. This last appearance is even better seen if the magenta be added after the blood has been irrigated with dilute alcohol. [g) Tannic Acid. — Mix a drop of the blood with a drop of a freshly prepared two per cent, aqueous solution of tannic acid (made with hot water and allowed to cool) ; cover and BLOOD. 3 examine. The tannic acid acts on the serum-albumen of the plasma and forms a finely granu- lar precipitate which somewhat obscures the field, but it also acts (like some other acids) on the corpuscles, causing them to become globular and the colouring matter to separate from the stroma. The separated colouring matter tends to pass out of the corpuscle, but as it does so it becomes coagulated by the acid, and remains attached to the corpuscle in the form of one or more granular-looking buds (PI. L, Fig. 5). None of the preparations of the blood of the frog or newt hitherto described can be permanently preserved ; the two following preparations may, however, be kept as permanent slides. (Ji) Picric Acid and Picrocarmine. — Place a drop of blood on a slide, and add a drop of a saturated solution of picric acid, put the slide aside and allow it to remain for five minutes, and at the end of that time, when the acid has ' fixed ' the corpuscles (that is, has coagulated their contents), the excess of acid should be removed by means of a narrow slip of blotting-paper. A drop of a solution of picrocarmine (p. xliii) should now be added, and a trace of glycerine to prevent evaporation, and the preparation set aside for an hour. At the end of that time, remove the picrocarmine solution by means of a slip of blotting-paper, and add a drop of Farrant's solution or glycerine (p. xlviii) and apply a cover. The preparation may then be ex- amined, when the peri-nuclear part of some of the corpuscles will be seen to be highly granular and of a deep yellow colour ; while the nucleus is stained red. In some of the corpuscles there may also be seen delicate yellow-coloured threads, extending from the nucleus to the envelopes. In others the peri-nuclear part remains uniformly homogeneous. («') Osmic Acid and Picrocarmine. — Mix a drop of blood with a drop of a one per cent, aqueous solution of osmic acid (p. xlvi), and allow the slide to stand. This ' fixes ' the corpuscles without altering their shape. At the end of five minutes remove the excess of osmic acid with blotting-paper, add a drop of solution of picrocarmine and a trace of glycerine to prevent evapo- ration, and set aside for three or four hours (or even longer, as no over-staining takes place). At the end of this time, the preparation is treated as iji) and examined. The nucleus will now be found to be stained red, and the peri-nuclear part homogeneous and yellow. If a drop of blood (taken from a frog which has been kept through the winter) be prepared by this method, some of the corpuscles may show ' vacuoles ' in the peri-nuclear part. (PI. I., Fig. 7)- (j) Osmic Acid and Logwood. — A similar preparation may be made by using a solution of logwood (p. xlii) as the staining agent instead of picrocarmine, with the advantage that the staining of the nucleus takes place much more rapidly — in about five minutes. ik) Ammonium Chromate and Picrocarmine. — Mix a few drops of blood with a small quantity of a five per cent, solution of ammonium chromate in a small glass vessel, and leave the mix- ture for twenty-four hours, taking care to prevent evaporation. Then pour off the chro- mate solution and substitute picrocarmine solution for another twenty-four hours. Mount a drop of the deposit in glycerine and examine it. The effects of this substance are most remarkable. Some corpuscles retain their shape though the nucleus enlarges, and shows an intra-nuckar plexus of fibrils. In others the haemoglobin is arranged in groups around the enlarged nucleus ; while others have their wall partially dissolved on one side, with part of the cell-contents extruded through it. The preparations ij.) and (y) show colourless corpuscles with their nuclei stained. 2. Observe the Colourless Corpuscles (H). — Prepare a drop of blood as for the examination of the coloured corpuscles. On careful examination there may be seen, scattered amongst u 2 4 PRACTICAL HISTOLOGY. the coloured corpuscles, colourless, irregular, granular bodies. They arc few in number compared with the red, and by careful searching three distinct varieties may be made out as follows : — (a) Finely granular Corpuscles.— These have no definite shape, are colourless, and have a faint, finely granular appearance. They are usually slightly smaller than the coloured corpuscles, and irregular in outline, with fine projecting processes. Careful observation may discover nuclei, which arc irregular in shape, and sometimes subdivided. (y8) Coarsely granular Corpuscles. — These also are indefinite in shape and colourless, but contain a large number of highly refracted granules, usually lying at one side of the corpuscle, whilst the subdivided nucleus occupies the other side. They ma)' be either larger or smaller than (a) and their processes are usually blunt and rounded. (7). The third variety consists of small nucleated masses of protoplasm about the size of the nucleus of a coloured corpuscle ; and as these latter sometimes escape from the corpuscles, care must be taken not to confound the one with the other. Amoeboid movements of Colourless Corpuscles. — The finely granular corpuscles (a) may be seen to change their shape and place (in the newt even at the ordinary temperature of the air) in the same manner as an amceba does, hence the term amoeboid ; and the coarsely granular corpuscles (/3) behave in a similar manner. In these latter, the granules, being more distinct, may be seen as it were to rush from one part of the corpuscle to another. In this change the granules are passive, they arc carried by the protoplasm. The small nucleated masses ^7) undergo similar changes of site and form, but, on account of their smallness, these phenomena are not so easily observable. The best way to notice these changes is to make drawings from time to time- — say every two minutes — of a colourless corpuscle. The change of form is some- times so great that a corpuscle may actually be seen to divide into two. At other times a transparent spot --a vacuole — which contains fluid may be seen in some corpuscles. All these changes take place more rapidly when the slide is warmed on a hot stage (p. xxviii) ; and when this is done, it is necessary, in order to prevent evaporation, to surround the cover with a little oil or, better, with a ring of melted paraffin. {Sketch the changes of shape in PI. I., Fig. 8). Feeding Colourless Blood-corpuscles. — That the colourless corpuscles can take small par- ticles into their interior is easily demonstrated, thus : all that is required is to rub up a little china ink or vermilion in a three quarter per cent, salt solution, and to mix a drop of this with the blood and examine the preparation from time to time on a hot stage, as indicated above. The preparation requires to be sealed up with oil to prevent evaporation. For newt's blood 30° C. is sufficient. The EFFECTS OF REAGENTS on the colourless corpuscles should next be observed. {a) Water. — When the blood is irrigated with water, the protoplasm of the white corpuscles swells up, becomes quite clear and transparent, and the nuclei, usually two or three in number, come clearly into view. Sometimes also when so treated the granules may be seen to exhibit Brovvnian movement. {b) Acetic Acid. — When blood is irrigated with a one per cent, dilution of acetic acid, the protoplasm becomes clear and transparent, and a bi- or tri-partite nucleus is distinctly seen. {Indicate this effect in one of the corpuscles sketched in PI. L, Fig. 8.) {c) Magenta. — When the blood is irrigated with magenta solution (p. xliv), the protoplasm takes on a faint red, the nuclei a deep red colour. {Indicate the effects in one of the corpuscles sketched in PI. I., Fig. 8.) Pi ATE I. Blood ■^^nt«l:T1. Bros lilh BLOOD. 5 Note. — The osmic acid and picrocarmine, and osmic acid and logwood preparations of blood are the only permanent preparations which show colourless corpuscles. A careful search is sure to reveal several corpuscles with their nuclei stained. In fresh blood an eighth-of-an-inch lens shows the intra-nuclear plexus of fibrils in the colourless corpuscles. (B.) HUMAN BLOOD. PREPARATION. — Wrap a handkerchief tightly round a finger, beginning at the base and proceeding towards the point, which will cause the point of the finger to become congested ; then prick the skin at the root of the nail with a clean sewing needle and bring a slide in con- tact with the drop of blood which exudes. Apply a cover and examine. EXAMINATION. — i. Observe the coloured corpuscles (H). Af fir.st the coloured blood- corpuscles will be seen adhering by their flat surfaces so as to form rouleaux. These piles may form a kind of network. [Indicate these rouleaux in PI. I., Fig. 9.) When so disposed it is of course their edges only that are seen ; but if the cover be gently moved with a needle-point, the rouleaux break up, and then the individual corpuscles are seen both on the flat and on edge. On the flat they appear as bi-concave, circular discs ; while edge- wise they appear somewhat dumbbell-shaped (PL I., Fig. 10). On carefully focussing a corpuscle as seen on the flat, a dark part at first appears in the centre, the margin being light (PI. I., Fig. 11); but on altering the fine adjustment the centre becomes light, and the circumference dark (PI. I., Fig. 11). These changes in appearance are due to the fact that the corpuscle is a bi-concave disc. It is not due to the presence of a nucleus, for these corpuscles are non-nucleated and homogeneous throughout. It is well to become familiar with the size of a corpuscle seen under a power of 300 diameters, for a corpuscle so magnified serves as a useful standard of comparison for other objects. The diameter of a corpuscle is ^^rVo inch. It is generally stated that the coloured corpuscles are all of the same size, but this is not strictly correct, for a careful examination shows that there is a slight variation from this standard, and this variation is greater in some individuals than in others, and becomes specially marked in some diseases. EFFECTS OF REAGENTS, [a) Acetic Acid.— When irrigated with one per cent, dilution of acetic acid, the coloured corpuscles become globular and lose their colouring matter, which diffuses into the surrounding fluid, so that it is difficult to distinguish the outline of the indi- vidual corpuscles. No nucleus is revealed. {b) Water. — Irrigation with water decolourises the corpuscles and renders them globular. The colouring matter is dissolved by the water which passes into the corpuscles by endosmosis and renders them globular. The surrounding fluid becomes yellowish in colour. {c) Tannic Acid. — If a drop of blood is mixed with a two per cent, solution of tannic acid (p. 2), one or more small refractive buds are seen to appear on the margins of the corpuscles. These buds are produced by the same causes that produce similar buds on the corpuscles of the newt. {d) Common Salt. — When blood is irrigated with a two per cent, solution of common salt, crenation of the coloured corpuscles takes place — that is to say, they become jagged at the margins and on the surface, so that they resemble small thorn-apples. At the same time, the corpuscles gradually become smaller and deepen in tint, but all the corpuscles are not affected to the same extent or with the same rapidit}-. Perhaps this difference in the action of the 6 PRACTICAL HISTOLOGY. reagent is caused by some difference in tlie age or physical properties of the various cor- puscles. {Indicate this in I'l. I., Fig. 12.) (f) Exposure to Air. — If a drop of blood be exposed to the air before the cover is applied, in some cases crenation occurs, but this phenomenon is not constant, nor when it takes place does it always occur to the same extent. None of these preparations of human blood make good permanent preparations. 2. Observe the colourless corpuscles (H). — In a fresh drop of blood they are few in number —from five to eight being usually in the field at once. They are best seen between the rouleaux of coloured corpuscles, and resemble in general characters the colourless corpuscles of the newt or frog. They are, however, smaller and do not exhibit amoeboid movements unless when kept at the temperature of the blood (38° C.) on a hot stage (p. xxviii). Some are larger than and others are about the same size as the coloured corpuscles, and for the most part they are all finely granular, though now and again a coarsely granular one is met with. If the glass cover be touched with the point of a needle the colourless corpuscles are seen to adhere to the glass, unlike the coloured corpuscles, which freely roll over one another ; and even when the coloured corpuscles impinge on a colourless one applied to the slide, they seldom displace it, so firmly do these adhere to any object. {Indicate the' characters of the colourless corpuscles in PI. I., Fig. 13.) EFFECT OF REAGENTS. — The actions of Acetic Acid, Water and Magenta on the colourless corpuscles of human blood are identical with the effects of the same reagents on the corre- sponding corpuscles of the blood of the newt or frog. {Indicate the effect of acetic acid in PI. I., Fig. 14, and magenta in PI. I., Fig. 15). 3. FREE GRANULES (H). — Besides the two varieties of corpuscles just described (r and 2) a number of very fine granules may be observed in the human blood. 4. FIBRIN (H). — Place a cover on a drop of blood on a slide and allow it to stand for a quarter of an hour. At the end of this time, on careful examination a number of delicate threads, which form an open network between the rouleaux, may be detected. These threads consist of fibrin, and are still better seen if the blood be irrigated with a drop of magenta or iodine solution. The former colours the threads red, the latter colours them yellow. {Indicate the appearance of the fibrin threads in PI. I., Fig. 9.) (C.) BLOOD-CRYSTALS. 5. H.ffiMOGLOBIN. — The colouring matter of blood, hemoglobin, may be obtained in a crystalline form, though it does not occur in that form inside the coloured corpuscles. In the corpuscles it seems to lie in the meshes of a stroma. PREPARATION. — Kill a guinea-pig and take a drop of blood from the heart or else- where, mix it on a slide with a drop of distilled water and cover. The haemoglobin diffuses from the blood-corpuscles into the water, and, as evaporation takes place, needle-shaped crystals form at the margin of the cover-glass. They cannot, however, be preserved for any length of time, so as to make permanent preparations. Or use the blood of a white rat, from which hjemoglobin crystals form in a few minutes. 6. H.ffiMIN. — Besides haemoglobin, which exists normally in the coloured blood-cor- puscles, and from which it may be obtained in a crystalline form, as above described, certain BLOOD. 7 other crystals — derivatives of haemoglobin — may also be obtained from blood. Of these (6), Haemin (Hydrochlorate of Hzematin), or Teichinaim's Crystals, are of most importance, as their presence is considered one of the surest as well as one of the most delicate tests for blood in medico-legal cases. PKEPARATION. — Take a drop of blood from the finger and allow it to dry on a slide, or take some powdered dried blood previously prepared and add to it a trace of finely powdered common salt. Moisten the whole with a drop of glacial acetic acid, apply a cover, and heat the slide gently over the flame of a spirit lamp till bubbles of gas appear. Allow the prepara- tion to cool, and apply a cover-glass. EXAMINATION. (H.) — Observe the short reddish-brown prismatic crystals scattered all over the field ; they are best seen where they are attached to small masses of blood. These are the crystals of Haemin. These make good permanent preparations. To preserve them, take off the cover-glass, and remove the surplus acid with blotting paper. Add a drop of Tarrant's solution, re-apply the cover. For the method of sealing up the preparation per- manently, see p. xlix. For the method of ascertaining the number of coloured blood-corpuscles by Malassez's method, see Archives dc Physiologic, 1874, or the Practitioner, July 1S78, where Dr. Gowers describes the hasmacytometcr. The amount of haemoglobin may be ascertained by means of the hasmaglobinometer. See Clinical Society's Transactions, vol. xii., p. 64. PRACTICAL HISTOLOGY. EPITHELIUM. Epithelium consists of corpuscles or cells with or without envelopes, of various shapes, sizes, and consistence, united by an intercellular substance or cement. The corpuscles are always disposed on surfaces, and usually contain a nucleus and sometimes a nucleolus ; they may be colourless, though they sometimes contain pigment. They may be arranged in a single layer or in several layers. Each cell regulates its own nutrition and never contains a blood- vessel. The following is a convenient classification of the varieties of epithelium met with in the human body : — (i) Squamous, (2) Columnar, (3) Ciliated, (4) Transitional, (5) Secretory. I. SQUAMOUS EPITHELIUM. PREPARATION.— With the finger-nail or a blunt knife scrape off a little of the epithelium lining the inside of the cheek or covering the tongue. Diffuse the scraping in water on a slide, cover and examine. EXAMINATION (H). — In this preparation only the superficial squames are seen. They are irregular polygonal cells when seen on the flat, and are several times the breadth of a coloured blood-corpuscle. Each cell contains a relatively small nucleus, and not unfrequently fine granules. Occasionally several cells are found adhering to each other by their margins, and sometimes micrococci are seen attached to them. If the edge of a cell be directed towards the observer, it will appear more or less spindle-shaped according to the angle which it forms with the plane of the slide. {Indicate these characters in PI. II., Fig. i.) In addition, small round and finely granular cells, about the size of a colourless blood-corpuscle and con- taining one or two nuclei, may be found here and there in the field. These are the salivary corpuscles. The included granules exhibit Brownian movement — a phenomenon which occurs when fine particles are suspended in a watery fluid, and which is probably due to differences of temperature in the several strata of the liquid. Brownian movement is easily shown by rubbing down a little gamboge in water. EFFECT OF REAGENTS.— Irrigate with a drop of magenta solution (p. xliv). The nucleus stains of a deep red colour, while the peri-nuclear part is but slightly affected. The alcohol in this solution precipitates the mucin of the saliva in the form of fine red threads or red membranous flakes (PL II., F"ig. i). Squamous Epithelium in exposed situations, as in the superficial layers of the skin, becomes hardened, and forms what is known as the horny layer of the epidermis. PREPARATION. — With a knife shave off a few of the surface scales of the skin, diffuse the scraping in water, cover and examine. EPITHELIUM. 9 EXAMINATION (H). — The squaines are flat, horny, and transparent, and usually no" nucleus is visible. EFFECT OF REAGENTS.— Irrigate with a five per cent, dilution of liquor potassae; the cells swell up and become globular. Neither this nor the previous preparation need be preserved. INTRA-CELLULAR PLEXUS OF FIBRILS. It has recently been shown that the nucleus of cells contains a delicate plexus of fine fibrils — an intra-niiclear plexus — which is continuous with a similar plexus in the perinuclear protoplasm — the intra-cellular plexus. It is this plexus, when the ends of its fibres are directed towards the observer, that gives the cells the appearance usually described as ' granular.' PREPARATION. — Keep a newt in a small quantity of water for several days, but do not change the water. At the end of this time the superficial layer of cutaneous epithelium will slough off in the form of a fine film. Harden this film for twenty-four hours in a saturated solution of picric acid, or in absolute alcohol. Preserve both in alcohol. When required for use, snip off a small piece and stain it, if hardened in picric acid, with picrocarmine for half an hour, while the piece hardened in alcohol may also be stained with logwood. Mount in Farrant's solution. EXAMINATION (H) — Observe the cells united by their margins. Within each polygonal area notice the oval red-stained nucleus : study it carefully, and a delicate plexus of fibrils — the intra-nuclear plexus— will be seen (PI. II., Fig. 2). Both in mucous membranes and in the skin, epithelium occurs in several Xdiy&x?,, constituting stratified epithelium. In order to see the deeper layers, and to study the relation of one layer to another, sections must be made. A section of the conjunctival epithelium of the cornea is convenient for this purpose. Vertical section of the conjunctiva and cornea for stratified epithelium. PREPARATION.— Remove the cornea from the eye of a cat just killed and place it in 40 c.c. of chromic acid and spirit mixture (p. xxxi) consisting of two parts of a sixth per cent, solution of chromic acid, and one part of methylated spirit. Change the fluid at the end of the first, fourth, and seventh days ; on the tenth day it will be sufficiently hardened. For preservation transfer it to methylated or rectified spirit, after washing away all the surplus chromic acid. When sections are wanted, put the cornea in a large glass of water for twenty-four hours to remove the spirit. Transfer it to a solution of gum (p. xxxviii) for twenty- four hours and then cut vertical sections in a freezing microtome (p. xxxviii). After the sections are made, they are placed in a large quantity of water for twenty-four hours, to get rid of the gum, when they may be put into the preservative glycerine fluid (p. xl) till they are required for mounting. •EXAMINATION (H).— Float a section on to a slide (p. xxvi), add a drop of solution of picrocarmine (p. xliii), and allow it to remain on the section for fifteen minutes, or until the section is sufficiently stained. Soak up the surplus staining-fluid with blotting-paper, and add a drop of Farrant's solution. Cover and examine. The fibrous cornea is stained red, but neglecting this, observe the layers of epithelial cells, stained yellow and their nuclei red, covering its anterior surface. The deepest layer consists of cells columnar in shape ; those on the surface appear as little more than lines or very elongated spindles, whilst between these extremes there are several layers of cells which show intermediate forms. The cells are developed from below— from the lowest layer — and are gradually pushed upwards, and hence the change of shape. {Indicate these chai-acters in PI. II., Fig. 5). C lo PRACTICAL HISTOLOGY. 2. COLUMNAR EPITHELIUM. A. FRESH COLUMNAR EPITHELIUM. PREPARATION. — Slit open the small intestine of a cat just killed, and wash away the mucus covering its inner surface with a stream of | per cent, solution of common salt, scrape the mucous surface and diffuse the scraping in the same strength of salt solution, and after breaking it up with needles, cover and irrigate with magenta solution (p. xliv). EXAMINATION (H). — A large number of elongated, narrow, or columnar cells are to be seen. Each cell is finely granular and contains a nucleus which is stained red. One end of the cell — the attached end — is somewhat pointed, while the free end presents a narrow, clear, unstained hem, or border, marked with fine vertical lines or striae. This indicates that the free end of each cell is covered by a transparent plate or disc. When several cells adhere to each other, and their free ends, instead of their sides, are presented to the observer, a fine mosaic is observed. The intercellular substance is small in amount, mapping out the individual cells one from another, while in the centre of each area a red nucleus is seen (PI. II., Fig. 4). In addition, here and there so-called 'chalice' or 'goblet' cells are seen (PL II., Fig. 6). These have a cup-shaped appearance, are devoid of the clear border and contain a nucleus sur- rounded by a small quantity of protoplasm near the lower pointed extremity of the cell, whilst the greater part of the body appears to be clear and looks as if it were empty. This is not to be preserved ; but, in order to obtain permanent preparations, the small intestine may be treated in one or other of the following ways. B. PERMANENT PREPARATIONS. PREPARATION {a). — Place a square half-inch of the intestine in 40 to 60 cubic centimetres of a one per cent, solution of ammonium bichromate for two days. {b) A similar piece in dilute alcohol (i rectified spirit to 2 water) for twenty-four hours. {c) A similar piece in a few c.c. (5-10) of a one per cent, solution of osmic acid (p. xlvi) for half an hour. Any of these may be used for preservation, but in every case the specimen must first be steeped in water for half an hour or longer, to get rid of the hardening reagent. EXAMINATION (H). — Scrape off a little of the mucous surface of a or b, place it on a slide, and break it up with needles. Add a drop of picrocarmine, and allow it to stain for fifteen minutes. Remove the surplus staining-fluid, add a drop of glycerine, cover and examine. In either case columnar and chalice cells similar to those already described will be found, except that in a each cell will be yellow with a red nucleus, while in b the red nucleus will be seen to contain what has been described as a nucleolus. If c be similarly treated, it must be left in the picrocarmine for twenty-four hours, when the same appearances are seen as in a, with this addition — that if the cells contain any fatty particles, these are blackened. These three preparations when sealed as described (p. xlix) are permanent. Columnar Epithelium from the small intestine of a newt. — Where the cells are relatively large, and show very distinctly the intra-cellular and intra-nuclear plexus. PREPARATION. — Kill a newt and place its small intestine in a few c.c. of a five per cent. EPITHELIUM. II solution of ammonium chromate for forty-eight hours. After washing, transfer it to picro- carmine for twenty-four hours. Open the gut, and with a knife scrape off a little of the epithelium and diffuse it in a drop of glycerine on a slide, cover and examine. EXAMINATION (H).— Observe the tall, relatively large, columnar cells, with a fine striated disc on their free ends, and a large red-stained nucleus. A careful examination reveals the plexus of fibrils. Besides these cells, look for ' goblet ' or ' chalice ' cells, which are somewhat cup-shaped, with a nucleus situated near the narrow, often tailed, end of the cell, and surrounded by a small quantity of protoplasm, while the upper part of the cell appears empty. These cells secrete mucin, so that they may be regarded as unicellular glands. The mucin seems to be derived from the interfibrillar substance (PL II., Fig. 7). 3. CILIATED EPITHELIUM. A. LIVING CILIATED EPITHELIUM AND CILIARY MOTION. PREPARATION. — Kill a frog by giving it a sharp blow on th^ head, but do not give chloroform, for it paralyses ciliary motion. Scrape the mucous membrane of the roof of the mouth or gullet, and diffuse the scraping by means of two fine needles in J per cent, salt solution. Place a hair in the fluid to prevent the cells being crushed, cover and examine. EXAMINATION (H). — Observe the groups of short columnar or nearly spherical cells with very granular contents, and having on one end vibratile cilia in active movement. This movement causes currents in the surrounding fluid, which carry along with them any suspended particles, such as granules or blood-corpuscles. Sometimes a detached cell may be observed to spin round and round by the motion of its own cilia. The cilia themselves — which are fine homogeneous projections — are best seen when their movements become languid. EFFECT OF REAGENTS.— Irrigate with a drop of magenta solution, and note that the colouring-matter does not stain the cells until the cilia have ceased to move. Thus each cell zoliilc living can regulate its own nutrition. There is a marked difference, therefore, in respect to colouring-matters between a living and a dead cell. B. CILIARY MOVEMENT. For the study of ' ciliary nwveinent ' the cilia of the common salt-water mussel are best adapted. On opening the mussel with a knife, the yellowish-coloured gills are seen, and if a small part be snipped off and placed in the salt water found in the inside of the shell, teased with needles, covered and examined with a jow power — (L) — the gills are seen as a series of bars fringed with cilia, much larger than those of the frog's epithelium. (H). Observe the long, tapering, clear, and structureless cilia bending most at their tips. Seal up this preparation by running a ring of oil round the margin of the cover-glass, and set it aside for several hours till the movement of the cilia becomes more languid (PI. II., Fig. 10). The oil prevents the access of fresh oxygen, which is necessary for the ciliary motion ; and as the oxygen in the fluid becomes used up, the ciliary action becomes slower. Place the slide on a hot stage (p. x.xviii) and gradually raise the temperature to not higher than 35° C. As the temperature rises, the ciliary movement becomes more rapid, but if the temperature be too high, so as to coagu- late the albumen, a permanent arrest of the movement results. An opposite effect is pro- duced by the action of chloroform or ether. Place a fresh piece of the gill on a cover-glass 12 PRACTICAL HISTOLOGY. in a drop of salt-water, and invert it over a gas chamber so that the gill is directed towards the chamber (p. xxix). EXAMINATION (L). —Ascertain that the cilia are active, then raise the cover-glass, put a small drop of chloroform into the chamber by means of a glass rod, and again apply the cover-glass. The ciliary movement becomes slower and slower, and finally ceases. If, how- ever, the gill is removed before the chloroform has acted too long, and is freely exposed to air, the cilia may resume their movements (Lister). None of these specimens can be pre- served. C. PERMANENT PREPARATIONS OF CILIATED EPITHELIUM. PREPARATION. — Place a small piece of the trachea of a cat, rabbit, ox, or sheep in dilute alcohol (i to 2 water) for forty-eight hours, when the inter-cellular substance of the epithelium will be so softened that the cells can be easily isolated. Then wash the whole piece in water, scrape the mucous membrane with a knife, place the scraping in a tube with a few drops of a one per cent, solution of osmic acid. The isolated cells are thus ' fixed,' and can be examined either with or without the addition of various staining reagents. EXAMINATION (H). — Place some of the cells in picrocannine {ox at least twenty-four hours, transfer some to a drop of glycerine on a slide, cover and examine. Observe the long, tapering character of the cells, with one end covered by a fringe of fine processes— the cilia — which are planted on a clear disc which is not stained. The cell-protoplasm is described as granular, but careful examination shows that it contains an intra-cellular plexus of fibrils with the fibrils arranged chiefly in the long axis of the cell. It is maintained by some observers that the cilia are actually prolonged through the clear band, so as to become continuous with this plexus. The oval nucleus is placed far down in the cell, is bright red, and shows a similar plexus, with a bright spot in its centre — the so-called nucleolus. The other end of the cell is often tapered or bifurcated (PL II., Fig. 8). Instead of the picro- carmine logwood may be used, with the advantage that a few minutes suffice for the staining process. Either of these preparations may be sealed up and preserved permanently. The ends of the cells may be directed towards the observer (PI. II., Fig. 9). 4. TRANSITIONAL EPITHELIUM. PREPARATION. — Take the bladder of a sheep, cat, rabbit, or guinea-pig, and prepare and preserve it in the same way as is directed for the preservation of ciliated epithelium. EXAMINATION (H). — Observe the great variety in the shape of the cells, some of them beino- more or less cubical, others resemble squames in character, while others again present a number of sharp angular points (PI. II., Fig. 1 1). 5. SECRETORY OR GLANDULAR EPITHELIUM. PREPARATION. — Make a cut into the liver of a dog just killed ; scrape the cut surface with a knife, so as to detach some of the liver-cells. Place these in a few c.c. of a half per cent, solution of osmic acid for an hour, then pour off" the acid and substitute picrocarmine for several hours. X 30 Plate n Epithelium ^Arvlem Bros litK ENDOTHELIUM. 13 EXAMINATION (H). — Many blood-corpuscles will be seen. Neglecting these, study a liver-cell. Notice its polygonal shape, yellowish granular protoplasm, and its nucleus deeply stained red. No cell-envelope is visible. Sometimes there are fine granules of pigment in the protoplasm, at other times fatty granules, especially if the liver of a stall-fed ox be used. In young animals (dog) the liver-cells often contain two nuclei. The ' granular ' cha- racter of the nucleus and protoplasm are due to the existence of a plexus of fibrils, which requires a higher magnifying power to make it obvious (PI. II., Fig. 12). The liver-cells of the newt are very large, and are easily prepared by placing small pieces of the liver in a five per cent, solution of ammonium chromate, and after washing, staining them with picrocarmine for several hours. When teased out in glycerine they show most admirably the intra-nuclear plexus of fibrils. The liver of the newt, especially at the end of winter, often contains particles of pigment (PI. II., Fig. 12). ENDOTHELIUM. « It is convenient to apply this term to a single la)er of squamous epithelium wherever it occurs, as the lining of serous sacs, blood- and lymph-vessels, &c. PEEPARATION BY THE SILVER PROCESS.— Bleed a rabbit, take a portion of its omen- tum and rinse it gently in distilled water to remove soluble chlorides. Place it in a quarter per cent, solution of nitrate of silver for ten minutes or until it assumes a milky appearance. Wash it thoroughly in ordinary water, and expose it to the action of diffuse sunlight, either in water or in water and alcohol, until it assumes a brownish colour. Mount a small piece unstained in Farrant's solution, and to compare with it, stain a similar piece with logwood for ten minutes, and after washing away all the surplus logwood, mount it also in Farrant's solu- tion. Picrocarmine does not stain so well after silver, but after a tissue has been in alcohol for some time it takes up this pigment more easily. The omentum of the rabbit is chosen because in it the endothelium occurs in a nearly continuous sheet. EXAMINATION (H).— The tissue will be mapped out into a series of small polygonal areas by narrow black lines — ' silver lines.' These are produced by a deposit of the reduced silver in the intercellular cement. The flattened epithelial cells adhere by their margins and form a complete epithelium or endothelial investment for the connective tissue of which the omentum consists. Focus carefully through the thickness of the membrane, and a second set of lines, whose outlines do not correspond with those lying above them, will be brought into view ; these are the endothelial cells on the other surface of the membrane (PI. II., Fig. 3). It is important to practise this method of focussing through the whole thickness of an object. Usually no nucleus is observable within the cells, but in the logwood preparation each cell will be seen to contain an oval nucleus. Preparations of other serous membranes will be made later on. 14 PRACTICAL HISTOLOGY. CARTILAGE. In every cartilage there fall to be considered {a) the matrix, and {b) the cartilage-ceils or corpuscles. Cartilages are classified according to the nature of the matrix. If the matrix be clear and like ground-glass it is called hyaline cartilage ; if fibrous it is a fibro-cartilage, of which there are two varieties : one, where the matrix resembles yellow elastic tissue, is yellow fibro-cartilage, and the other, in which the fibres resemble white fibrous tissue, is white fibro- cartilage. Where the cells are separated by a very small quantity of matrix, the cartilage is said to be cellular, as in the ear of a mouse or in the chorda dorsalis. HYALINE CARTILAGE Occurs in the articular ends of bones, cartilages of the ribs, part of the sternum, larynx, trachea and bronchi, and some nasal cartilages. A. FRESH HYALINE CARTILAGE.— Open any joint of an animal just killed, or take the cartilage of a rib from the same animal and make a thin slice of the cartilage with a razor. Place the thin slice in a drop of blood-serum or one per cent, alum solution, cover and examine. Instead of making a slice, a piece of cartilage sufficiently thin may be obtained from the sternum of a newt or the tracheo-laryngeal cartilages of a frog. EXAMINATION (H). — If a thin slice has been made, observe the hyaline matrix dotted over with corpuscles, or cells — the cartilage-cells. Study first the cartilage-corpuscles and then the matrix. The corpuscles are oval or of various shapes, granular and containing a large nucleus. The protoplasm of the cells fills the entire space or lacuna in which it lies, but — especially near the margin of the section — the protoplasm may have fallen out of the lacunae, or shrunk up so that it no longer completely fills the space in which it lies. By focussing the cells are seen to lie in several planes. The matrix is hyaline and perfectly homogeneous in its appearance (PI. III., Fig. i). EFFECTS OF REAGENTS, {a) Water.— Irrigate the section with distilled water. The protoplasm of the cell now shrinks rapidly from the cavity containing it. It becomes coarsely granular and thus often obscures the nucleus. {b) Iodine Solution. — Make a transverse section of a costal cartilage of a young animal. Stain with iodine solution for three to five minutes, add glycerine and examine (H). This stains the matrix yellow, and the corpuscles of a deeper tint. Within the protoplasm a brownish coloration may result, which indicates the presence of glycogen ; this is best seen in young growing cartilage. B. METHODS OF PREPARING HYALINE CARTILAGE FOR PERMANENT PREPARA- TIONS. — It is necessary to use reagents to ' fix ' the structural elements of the cartilage, and CARTILAGE. '5 amongst the best of these is a saturated watery solution of picric acid. Take costal and articular cartilage as types. Place small pieces — not larger than half an inch — of the costal cartilages, with their peri- chondrium, of a cat or rabbit or human fcetus, in a saturated watery solution of picric acid for forty-eight hours. Then remove them and wash them thoroughly in water till no yellow colour is given off, and place them in rectified spirit till they are required. Sections may be made either with the hand or by means of a freezing microtome (p. xx.xviii). It is well to practise making sections with a razor, and cartilage, from its consistence, is one of the easiest tissues to begin with. A similar procedure is adopted for adult hitman costal cartilage, and also {ox articular cartilage. If the costal cartilage contain any bony or calcareous matter, macerate it for one or two weeks in the acid till all the calcareous matter is removed. For articular car- tilage, if it be desired to have a section of the subjacent bone, the bone must be softened by means of an acid which removes the calcareous matter. It may be accomplished thus : — Place the articular end of, say, the femur or humerus of a recently killed cat or rabbit in a large quantity (twenty to thirty times the bulk of the tissue) of one of the following solutions: — [a) A saturated watery solution of Picric Acid. — Change the fluid at the end of a week and add fresh solution containing a few crj-stals of picric acid. The bone will be softened in from two to three weeks. (b) A quarter per cent, solution of Chromic Acid may be employed. Take care to change the fluid after three da)-s. This fluid requires also from two to three weeks to decalcify the bone. {c) A mixture of Chromic Acid and Nitric Acid (p. xxxiii) may be employed. Similar con- ditions obtain as in {a) and ib). {<£) Or a two per cent, solution of Hydrochloric Acid may be used as the decalcifying agent. («) and {c) are the methods which yield the best results. COSTAL CARTILAGE. Makeanumberof transverse sections of the costal cartilage of a kitten or young rabbit, and also of the decalcified costal cartilages of an old person, prepared as described above. These sections may be e.xamined as they are or after being stained. A. FROM A YOUNG ANIMAL. {a) Osmic Acid. — Place some of the sections of the kitten's cartilage in a one per cent, solu- tion of osmic acid for twelve hours. Wash them thoroughly in water, to remove the osmic acid. Mount one in Farrant's solution, and cover. These sections are taken, first, because they are small and one sees better the general characters of the section EXAMINATION (L). — Observe the fibrous perichondrium and the cartilage-matrix inside it, the cartilage-corpuscles, small and flattened, lying in several layers. Nearer the centre of the section are seen, as small specks, the irregularly shaped corpuscles. The corpuscles are stained of a deeper yellow than the matri.x by the osmic acid, hence its value. (H) Examine the fibrous perichondrium with its areolar tissue and sections of clastic fibres. Observe the cartilage-cells flattened towards the periphery of the cartilage, and the more rounded charac- ter of the cells placed nearer the centre. Otherwise the cells and matrix have the same characters as are indicated in fresh hyaline cartilage. (p) Carmine. — Stain a section with carmine. Lay a section on a slide and on it place a 1 6 PRACTICAL HISTOLOGY. drop of solution of strong carmine (p. xliii). Within a few minutes it will be stained of a uniform deep red colour. Wash away the surplus carmine, and if the preparation is now ex- amined (L) it will be of a nearly uniform red colour, the matrix scarcely distinguishable from the corpuscles in the depth of the tint. Place on the section a large drop of a five per cent, solution of glacial acetic acid for several minutes, examining the section with a low power all the time. The effect of the acid is to remove the surplus carmine from the matrix and leave the corpuscles deeply stained red. As soon as this is accomplished wash the section thoroughly in water, and mount it in Farrant's solution. Strong glacial acetic acid accomplishes the same result more rapidly. EXAMINATION (H). — The general characters of the section are the same as («), only the perichondrium and corpuscles are red and the matrix is colourless or only faintly stained (PL III., Fig. 2). B. FROM AN OLD PERSON. Make a number of transverse sections of the macerated tissue (p. 15). Let the sec- tions include a piece which has been calcified. {a) Osmic Acid. — Place some of the sections in a one per cent, solution of osmic acid as for foetal cartilage. EXAMINATION (L). — Observe the perichondrium and the general arrangement of the corpuscles, the corpuscles flattened at the periphery, in irregular clumps a little further in, and, as one examines towards the centre, the rows of cartilage-cells produced by transverse or oblique cleavage. The matrix, hyaline at the periphery, but finely granular in other parts. This is best brought out by slightly shading the mirror or using a small aperture of the diaphragm. (H). The corpuscles — The protoplasm is very apt to shrink within the capsules, and in it are to be seen several black spots. These are small masses of oil which have been blackened by the osmic acid, which is an excellent reagent for detecting the existence of fatty particles in any tissue, and is therefore of great value In persons above middle age, oil-drops are fre- quently to be found both in the cartilage-cells of the tracheal rings and rib-cartilages. The rows of cells are obvious. Matrix. — Examine the granular part. Between the rows of cells the matrix may be found to be distinctly ' fibrillated ' ; the fibres lie mostly parallel one with another. It is well to mount a preparation of cartilage, showing the fibrillation of the matrix in a ten per cent, solution of common salt, which prevents the swelling up of the fibrils. {U) Eosin. — Place other sections in a dilute solution of eosin (p. xlv). This substance stains the section very rapidly — a minute usually suffices. Rinse them in a one per cent, dilution of acetic acid and mount in Farrant's solution. EXAMINATION (L). — Observe a similar arrangement of the elements. The matrix is slightly red and corpuscles slightly deeper in colour. The calcareous matter deposited be- tween the cells and the fibrillated parts are more deeply stained than the hyaline matrix, and a kind of stereoscopic effect can be given to these calcified parts by using a small aperture of the diaphragm. (H). Observe the highly refractive yellow oil-globules in the protoplasm of the cells, and observe particularly the more deeply stained cell-capsule or ' cartilage capsule ' bounding the lacuna. (f) Purpnrine. — Place some sections in a solution of purpurine (p. xliv) for forty-eight hours. Plate ni Cartilage IfiT-iterr-L Bros litK CARTILAGE. 17 This substance stains the nuclei of the cells a light pink colour. It possesses no advantages over the methods already indicated. The advanced student may study the action of chloride of gold and nitrate of silver on hyaline cartilage. Action of Chloride of Gold. — Place in a one per cent, solution of chloride of gold for half an hour the articular end of the femur of a frog newly killed. Wash it thoroughly to remove the surplus gold, and place it in water acidulated with a few drops of acetic acid, and expose it to the light until the gold is reduced — usually for twenty hours — or until the cartilage becomes of a beautiful violet tint. Make thin sections of the cartilage, and mount them in Farrant's solution. EXAMINATION (H). — Observe the cells arranged in several planes and stained of a deep violet colour. Thej^ usually fill the lacunae completely. Often two cells may be seen together, in such relationship as to suggest that they have been produced by the division or ' fission ' of a single cell. The matrix is stained of a lighter colour. Action of Nitrate of Silver. — Silver the articular cartilage of the femur of a freshly killed frog, as directed at p. xlv, and after exposure to light, and when it has become brown, make a thin slice and mount it in Farrant's solution. Cover. EXAMINATION (H). — Observe the matrix stained brown, but the cells are not stained, they are merely represented by oval or round clear spaces, which correspond exactly in shape and distribution with the cartilage-corpuscles. The silver has not affected the cells, though it has darkened the intercellular substance. The gold effect represents the positive, this the negative effect. Staining with logwood reveals the presence of a cell in each apparently empty space. ARTICULAR CARTILAGE. Make a number of vertical sections of the head and subjacent bone of the femur or other long bone of a cat or other animal, decalcified according to the methods indicated (p. 15), preferably by a or c. (a) Osmie Acid. — Place sections in a one per cent, solution of osmic acid for twenty-four hours. Wash and mount in glycerine jelly (p. xlviii), for Farrant's solution renders the section too transparent. EXAMINATION (L). — If the femur be chosen, observe the general arrangement of the parts from the articular surface downwards. The superficial cells are flattened, and they always are parallel to the surface and at right angles to the axis of pressure. A little below the surface they are in irregular clumps of twos and threes, and deeper down near the bone they are in rows, parallel to the long axis of the bone. These rows are produced by transverse cleavage of the cells. Below the cartilage is the bone. The cartilage is divided into two zones, distinctly mapped off from each other by the character of the matrix. Across the sec- tion, about two-thirds or half-way below the articular surface of the cartilage, there runs a line slightly wavy. All that lies superficial to this has a Iiyaline matrix, and what lies below it, i.e. as far as the bone, is more or less granular and less transparent. In this lower part the matrix is calcified. This calcified part is distinctly mapped off from the subjacent bone ; a well-marked wavy line with very distinct undulations — the one part dovetailing into the other, indicating D i8 PRACTICAL HISrOLOGY. the line of demarcation. If the section has been made from bone softened with chromic acid the bone will have a slightly greenish tinge. (H). Examine in detail the characters of the cells, and note especially the rows of cells in the deeper part of the cartilage. These rows are produced by transverse cleavage of the cells. One part of a row may lie in the hyaline matrix, while the other half is imbedded in the granular matrix (PI. III. Fig. 6). In some sections an indistinct fibrillation is seen in the lower part of the cartilage between the rows of cells, thus resembling what obtains in calcifying costal cartilage. {b) Picrocarmine. — Put a section on a slide, and stain it with picrocarmine for twenty minutes to half an hour. Remove all the picrocarmine, and mount it in glycerine jelly (p. xlviii). The glycerine jelly must be melted in a water bath, and when fluid a drop is applied to the preparation by means of a glass rod, and covered at once, before the jelly sets. WHITE FIBRO-CARTILAGE. PREPARATION. — Place a small piece of an intervertebral disc with its adherent bone, from a newly killed sheep or ox in twenty or thirty times its bulk of a mixture of chromic and nitric acid (p. xxxiii). After two to three weeks the bone will be decalcified. Wash the pieces in water to remove all acid, and preserve them in rectified spirit, or ;— Remove the skin from the tail of a cat ; cut it into pieces an inch long, and place these in chromic and nitric acid fluid. Make with a freezing microtome vertical sections of the intervertebral disc ; including the subjacent bone, which will have a greenish colour. Make also transverse sections of a piece of the decalcified tail of a cat or other animal. Place a vertical section of an intervertebral disc on a slide. Stain it with strong carmine for ten minutes, or until it assumes a deep red colour. Wash, and mount in Farrant's solution. EXAMINATION (L).— Observe the relation of the fibrous part of the disc to the subjacent bone. (H). The matrix in the ox is distinctly fibrous ; the fibres are very fine, unbranched, and wavy. The corpuscles are nucleated, and enclosed in a distinct capsule. They lie scattered irregularly or in chains between the bundles of fibres. Sometimes the cells are seen to be dividing. Here and there fine dots, the cut ends of the fibres, may be seen (PI. III., Fig. 3)- Stain with carmine a transverse section of an intervertebral disc from the already softened tail of a cat. Remove the surplus staining fluid with blotting-paper, and mount in Farrant's solution. EXAMINATION (L). — Observe the concentric arrangement of the parts of the disc and the different directions of the fibres composing each layer of it. (H). Observe the fibrous character of the matrix and the corpuscles as before. Here and there are transverse sections of the ends of the fibres, which appear like fine granules. CARTILAGE. 19 YELLOW FIBRO-CARTILAGE. This may be easily obtained from the epiglottis, the arytenoid cartilages, or the external ear. PREPARATION. — Place the arytenoid cartilages and the epiglottis of a sheep or ox (if the latter, cut it into two pieces) in a saturated solution of picric acid. Remove it after forty-eight hours and make transverse sections of it in the usual way. Preserve it in methylated spirit. Treat the external ear of a cat or rabbit in the same way and make sections. This method is preferable to simple hardening in alcohol. Stain a transverse section of an epiglottis hardened as above with picrocarmine. A few minutes suffice to stain the parts. Remove the surplus staining fluid with blotting paper, and mount in Farrant's solution. EXAMINATION (L). — This specimen contains a section of the mucous membrane covering both surfaces of the epiglottis. This may be neglected at present. The cartilage itself is stained bright yellow (picric acid) with a perichondrium of connective tissue which is of a deep red colour. Lying in the bright yellow fibrous matrix are a number of red spots, which are the nuclei of the cartilage-corpuscles. (H). Notice the wrt//'/.!:- -yellow, distinctly fibrous, and the fibres like those of elastic tissue, and branched. If the line of .section cuts these fibres transversely they appear as fine yellow granules. Trace these fibres into the red connective-tissue perichondrium, where they become yellow elastic fibres. This continuity can be easily made out in a thin section. Study the corpuscles. Towards the periphery of the cartilage they are flattened ; but near the centre they are rounded, have a distinct capsule, and the nucleus is distinctly stained red. This forms an extremely beautiful preparation. These preparations require a day or two for the colours to become differentiated. A section may be double-stained with picrocarmine and logwood, but this method pos- sesses no advantages over the above. Stain a section of the ear of a cat with picrocarmine as above. Mount it in Farrant's solution. EXAMINATION (L and H). — The yellow mass of cartilage stands out beautifully between the red connective tissue (Pi. III., Figs. 4 and $). Sections of muscles, skin, and sebaceous glands may also be seen. The same general characters are seen with a high power as are indi- cated above. TRANSITION FROM HYALINE TO YELLOW FIBRO-C.A.RTILAGE. Make longitudinal sections of an arytenoid cartilage of an ox, and stain it with picro- carmine. The upper half shows the .structure of yellow fibro-cartilage, while the lower half is chiefly hyaline, with a few elastic fibres extending into the matrix. This shows the mode of development of yellow fibro-cartilage from hyaline cartilage. CHANNELS IN THE MATRIX OF HYALINE CARTILAGE. These are easily seen in a section of a part of the cephalic cartilage of Loligo, which has been hardened in picric acid and stained with picrocarmine. 20 PRACTICAL HISTOLOGY. CONNECTIl^E TISSUE. Connective tissue occurs in a variety of forms, as areolar tissue, or membranes, or forming fascia; and tendons. In whatever form it occurs we have to study two classes of structural ele- ments, v\z.—fibi-cs and corpuscles. The fibres are of two kinds, [a) the zvliite or gelatigejwns, so called because they yield gelatine on boiling ; (/;) the yellow or elastic, which yield elastin. The corpuscles are of three kinds : {a) the fixed connective-tissue corpuscles, which always bear a definite relation to the white fibres; (b) auiceboid ox zcawc/tv/;/^ ctV/y, which are identical with colourless blood-corpuscles or leucocytes. They wander freely in the lymph-spaces by virtue of their contractility ; {c) the so-called plasma cells, of which the exact relation is not made out. ELASTIC TISSUE. A. IN THE FRESH STATE. PREPARATION. — Tear off, in the axis of the fibres, with a forceps, a very fine piece of the fresh ligamentum nuchae of an ox. Tease it out in a drop of salt solution or water, with two needles, and cover. It is convenient to use a low power to ascertain when the piece is sufficiently teased. EXAMINATION (H).— Notice the homogeneous transparent fibres, about the breadth of a human coloured blood-corpuscle. They have a yellowish tinge, and their margins are well- defined. They branch and anastomose and may curl up at the ends (PI. IV., Fig. i). EFFECT OF REAGENTS. — Irrigate the preparation with a one per cent, dilution of acetic acid. The fibres are not affected thereby. To preserve this preparation substitute Farrant's solution for the acid watery fluid. B. PERMANENT PREPARATIONS. Excellent permanent preparations are obtained by the following method. Place small pieces — about half-inch cubes — in many times their bulk of chromic and nitric acid fluid (p. xxxiii) for a week. After that soak the pieces in water for twenty-four hours, to remove all traces of the acid, and preserve them for use in methylated spirit. Make longitudinal and transverse sections in the usual way. Stain a longitudinal and a transverse section in picrocarmine for ten minutes. EXAMINATION (H) of the longitudinal section reveals the elastic fibres stained of a deep yellow, with their characteristic branches and anastomoses. Between them a small amount of ordinary connective tissue stained red is seen. The transverse section shows the cut ends of the fibres, as solid yellow polygonal bodies in CONNECTIVE TISSUE. 2r groups of twos and threes ; when two fibres adhere their opposed surfaces are moulded to each other (PI. IV., Fig. 2). Surrounding each group of fibres is a very small quantity of connective tissue stained red. Only a very small quantity of white fibrous tissue exists in the ligamentum nucha;. If desired similar sections may be tinged with a solution of magenta (p. xliv), which stains the elastic fibres bright red, and is thus a good reagent for detecting their presence. Elastic tissue also forms part of ordinary areolar tissue. It occurs in the form of a net- work of fine clastic fibres in the skin, where it will be examined, but its characters may be well studied in the mesocolon of a rabbit. PREPARATION. —Remove the mesocolon from a newly killed rabbit. Pin it to a piece of flat cork to keep it stretched, and place it, cork uppermost, for three days in a mixture of chromic and nitric acid (p. xxxili). After washing away all surplus acid, preserve it in methy- lated spirit. Snip off a small piece, float it in water to remove the spirit, and stain it for ten minutes with picrocarmine ; mount it in Farrant's solution. EXAMINATION (H). — Observe the network of fine elastic fibres one-sixth the breadth of a coloured blood-corpuscle. The fibres are imbedded in white fibrous tissue, which is stained red. If desired another piece may be stained with solution of magenta (p. xliv) which stains the elastic network red. Elastic tissue will be further considered with arteries, and in the skin, where the method of preparing it by artificial digestion is described (Stirling) (p. 92J. WHITE FIBROUS TISSUE. IN THE FRESH STATE. PREPARATION. — Kill a rabbit or a rat, and with scissors snip off a small piece of the delicate connective tissue that lies under the skin, and which connects it to the fascia;. The piece of tissue at once rolls into the form of a ball ; but by placing it on a dry slide it can easily be spread out with needles into a fine membrane, its original form when in situ. During the process do not allow the tissue to dry, which may be prevented by breathing on it from time to time. Add a drop of normal salt solution ; cover and examine. EXAMINATION (H). — Observe the wavy fibres of white fibrous tissue running in every direction. Their outlines are indistinct, but here and there a longitudinal striation in their course is observable, indicating that each fibre is composed of fibrils. In addition to these, a few fibres which refract the light more strongly may be detected ; these are elastic fibres ; but little else is obserAable in this preparation, especially if taken from an adult animal (PI. IV., Fig. 3)- EFFECT OF REAGENTS, (a) Magenta Solution.— Irrigate the preparation with solution of magenta (p. xliv), and the existence of the corpuscular element will be revealed. The nuclei of the connective-tissue corpuscles are stained of a red colour by the dye. There are two kinds of corpuscles, one larger than the other. The larger, when seen on the flat, are like large nucleated squames, though they are sometimes branched, and when seen on edge they are fusiform. These are the ' fixed ' connective-tissue corpuscles, and the smaller ones, about the size of the colourless blood-corpuscles, with two or three small nuclei, are the wandering cells, or leucocytes, and are identical with lymph- or colourless blood-corpuscles. The elastic fibres 2 2 PRACTICAL HISTOLOGY. — which arc recognised b}- their branching and forming a network — are stained red, while the vhite fibres are but shghtly acted on by the magenta. This preparation need not be pre- served. The observer is not to suppose that the corpuscular elements do not exist before the addition of a reagent ; they do exist, but, owing to their refractive index being so near that of the fluid in which they are examined, they are not visible until some staining reagent is added. It is further to be remembered that the relation of the fixed corpuscles to the white fibres is changed in a teased preparation. Occasionally large oval granular and nucleated cells are found, especially along the course of the vessels ; they are the so-called //^ri-ww cells. (d) Dilute Acetic Acid. — Make a similar preparation, and irrigate it with a one per cent, dilution of acetic acid. EXAMINATION (H). — Note that, as the acetic acid displaces the salt solution, it acts upon the white fibres and causes them to swell up, to become transparent, so that they appear quite homogeneous and jelly like, though they are not dissolved. Some of the fibres may, however, present annular constrictions in their course. This clearing up of the white fibres brings the other elements in the tissue more clearly into view — to wit, the elastic fibres and the corpuscles. The elastic fibres are highly refractive, and their branches unite to form a net- work, and if such a fibre be broken across it curls up at its ends, though otherwise they pursue a straight course. Numerous fusiform or oval nuclei are revealed ; they are the nuclei of the fixed connective-tissue corpuscles, whilst the small bi- or tri-partite nuclei belong to the lymph- corpuscles. The acetic acid may be displaced by magenta solution, when the elastic fibres and the nuclei are stained of a blight red colour (PI. IV., Fig. 4). Do not preserve this. AREOLAR TISSUE. PERMANENT PREPARATIONS OF AREOLAR TISSUE. PREPARATION (a). — With a small subcutaneous syringe (fig. 27) forcibly inject a quantity of a half per cent, solution of osmic acid under the skin of the groin of a young dog, rat, or rabbit just killed, until an artificial Fig. 27. oedema or bulla is formed. After half an hour, snip open the bulla, taking care that no hairs get into it. With scissors snip off a small piece of the distended connective tissue and place it on a slide. Spread it out as a mem- brane with needles, and add picrocar- mine. It will require several hours Subcutaneous syringe for interstitial injections, and the cannula. The to stain, and to avoid desiccation add hollow needle ought to be made of gold, to prevent acids from acting ,, , ^ 1 ■ , on it. Natural size. a small drop of glycerme and cover. When it is sufficiently stained, remove the picrocarmine, and substitute glycerine containing a trace of formic acid. (d) A similar preparation may be stained with logwood — which acts in a few minutes ; remove the logwood solution and mount in glycerine. (c) Instead of injecting osmic acid, the subcutaneous injection of a saturated watery solution of picric acid does very well. Stain the section with picrocarmine, as directed under (<5). CONNECTIVE TISSUE. 23 EXAMINATION (H). — The corpuscles are now clearly brought into view ; their nuclei are stained and their wing-shaped expansions are distinct, — in fact, they appear as flat, nucleated plates, sometimes witlj divided expansions. Elastic fibres are yellow, and white fibres are slightly reddish. Annular Constrictions on the White Fibres. — Take a small piece of the delicate pia mater lying within the circle of Willis from the brain of a sheep just killed, or slit up the dura mater of the spinal cord of an ox, and remove some of the fine fibres that stretch across the sub-arachnoid space. Spread out the tissue on a slide, and irrigate it with a one per cent, dilution of acetic acid. The fibres swell up, but here and there they exhibit numerous annular constrictions. These constrictions are the indications of the existence of a sheath on the fibres, which is not affected by acetic acid. In some cases a fibre may be found to wind in a spiral manner round the white fibre. These preparations may be stained with picrocarmine or magenta, but they do not keep well. The fibres of white fibrous tissue are made up of fibrils, held together by a cement. — The fibrils (juflTHT inch ''i diameter) are sometimes revealed by teasing, but it is better to use some chemical reagent to dissolve the cement. PREPARATION. — Place a narrow strip of the tendo Achillis or other tendon of a rabbit or other animal in a saturated solution of picric acid (p. xxxii) for twenty-four hours After that time wash away the surplus colouring matter, tear off a small shred, and tease it in water. Cover. EXAMINATION. — The wavy, excessively delicate, non-branched, yellow-coloured fibrils (due to picric acid) are now apparent. Each white fibre is made up of a great number of these fibrils, held together by a cement. Usually no cells are seen, but irrigation with a one per cent, dilution of acetic acid readily brings the long oval nuclei of the tendon-cells into view. OTHER METHODS. — Maceration of a piece of tendon in a saturated watery solution of baryta for four or five hours, and subsequent steeping in water for twenty-four hours — or m.aceration in a large quantity of a ten per cent, solution of common salt for ten days, effect a sim.ilar result, but the picric acid method is the most satisfactory and convenient. TENDON. It is advisable to use a tendon from a young animal, for in such there is a much larger number of corpuscles than in the tendon of an adult. PREPARATION. — Place pieces of the tendo Achillis of a calf— about an inch long and a quarter of an inch thick — in many times their bulk of Muller's fluid (p. xx.xii) for fourteen days. Then remove them, and after soaking them in water to remove the chromium salts, preserve them in rectified spirit till_ sections require to be made in the usual way b}' freezing. Make both transverse and longitudinal sections. TRANSVERSE SECTION. (l) Place a section on a slide and cover it with a large drop of logwood solution ; within a few minutes it will be sufficiently stained. Wash away all the surplus logwood and mount the section in Farrant's solution. 24 PRACTICAL HISTOLOGY. EXAMINATION (L).— Observe the sheath of the tendon, composed of connective-tissue fibres disposed circularly. It sends processes into the substance of the tendon, subdividing it into polygonal areas. These areas are made up of the cut ends of the longitudinally disposed fibres which constitute the tendon. Each area has scattered in it a number of small specks deeply stained by the logwood, united by fine lines. These are the spaces — the inter-fascicular lymph-canals— between the bundles of fibres, and in each space a corpuscle may be detected (PI. IV., Fig. 6). (H). The great mass of the tendon is but slightly stained, but notice the sections of the corpuscles, which are deeply tinted. The corpuscles give off processes which may be traced a certain distance between the fibre-bundles. Amongst the cut ends of the fibres may be seen small, light, refractive points ; these are the ends of elastic fibres— few in number— which exist in tendon (PI IV., Fig. 7). The branched intcrfasciadar spaces in the transverse section are best seen in a transverse section of a dried tendon examined in water. The spaces appear branched and black and larger than natural, from the shrinking of the bundles of fibres bounding them. They are black because they contain air. The addition of glycerine renders all the parts too trans- parent, hence such a preparation is not permanent. Sometimes the logwood stains the section of an almost uniform dark violet tint, and it becomes impossible to distinguish the corpuscles from the rest of the section. Under these circumstances, irrigate the over-stained section with a five per cent, dilution of glacial acetic acid, examining the section all the time with a low power. The colouring matter will be dissolved out of the fibres and remain in the corpuscles, and if the acid is thoroughly washed out of the preparation, this may be mounted in Farrant's solution and preserved. It is well to place such a .section for a few minutes in a five per cent, solution of bicarbonate of soda to remove all the acid. This process of overstaining a section with either logwood or carmine, and getting rid of the excess by means of acetic acid, is frequently of value. LONGITUDINAL SECTION. (2) Place a section on a slide and stain it with logwood, as directed for a transverse sec- tion. EXAMINATION (H). — Observe the fibres arranged longitudinally, many of them slightly wavy, their outlines indistinct and always unbranched. Between the fibres, which are but slightly stained or not at all, rows of elongated fusiform corpuscles deeply stained are observed. Study these corpuscles. They are nucleated and the nucleus is surrounded by a granular mass of protoplasm which forms part of the body of the corpuscle. They are fixed connective-tissue corpuscles or ' tendon cells,' and are most numerous in young tendons (PI. IV., Fig. 5). (3) Tease out with needles a small piece of a section treated as (2). The fibres and cor- puscles are easily isolated. Notice the nucleated granular corpuscles in many cases adhering to and partly encircling a fibre. These corpuscles when seen on edge are fusiform, but when detached and seen on the flat they are more or less flattened, with their edges sometimes turned in. These cells partly surround and form an imperfect sheath for the fibres. Ranvier has shown that the relations of the cells to the fibres of a tendon can be more successfully made out in the fine tendons found in the tail of a young rat or mouse. X 300 Plate 'iv: Connective % r Mrtx.iem Sros lith CONNECTIVE TISSUE. TENDONS IN THE TAIL OF A RAT OR MOUSE. PREPAEATION. — Kill a rat — preferably a young one — seize the tip of the tail, and for- cibly pull it. This ruptures the caudal vertebrae, and a leash of fine silvery-looking tendon.s is pulled out. Place several of these tendons in the filtered juice of a lemon. Leave them there for three minutes. They become clear and transparent and swell up considerably. Remove them, wash them thoroughly in water, and place them in several times their bulk of a one per cent, watery solution of chloride of gold (p. xlv), and allow them to remain there for fifteen or twenty minutes. They become of a deep yellow colour, contract somewhat, and are rendered more rigid. Remove them from the gold solution, and wash away the sur- plus gold in water. Place them in an ounce of a twenty-five per cent, watery solution of formic acid. The bottle containing them must be kept in a dark place for twenty-four hours. At the end of this time, the tendons are removed from the formic acid and are thoroughly washed in water. They have now a decided purple colour throughout. The formic acid re- duces the gold on which it is deposited in the tissues, and to a greater extent in the protoplasm of the cells than in the fibres. There are other ways of employing the ' gold method,' but we have found this modification of Ranvier's very successful. [Each student cannot do the entire process ; but he is shown how to do it.] Take a small piece of one of the above tendons, and tease it with needles, in a drop of glycerine, and apply a cover-glass, or what does very well, gently press on the cover-glass until the tendon is flattened. EXAMINATION (H). — Observe the rows of flattened quadrilateral cells lying between the longitudinally disposed fibres. The central part of each cell which contains the nucleus is of a dark purple colour and finely granular. Thin slightly coloured wings may be seen ex- tending outwards from this. The position of the nucleus is often left unstained and is usually placed at one end of the cell, and the nuclei of adjacent cells in a row are usually so disposed that the two nuclei lie close together. One or more dark lines may be seen running along the centre of the cells (Boll's stripe). These are ridges projecting in a direction vertical to the plane of the rectangular plate of the cells (PL IV., Fig. 8). It is easy, by teasing out the preparation carefully, to isolate a fibre with some of the cells still adhering to and partly embracing it, so as to indicate the relation of the cells to the fibres. They can be seen to send their thin lamellar or wing-like expansions around the fibre, so as partly to embrace it (Pi. IV., Fig. 9). These cells partly occupy the branched interfascicular spaces seen in a transverse section of tendon (p. 24). DOUBLE-STAINING OF TENDONS. Gold Chloride and an aniline dye.— Place an inch of the tail of a rat in gold chloride for an hour, and then treat it as above. After the gold is reduced place the tail in chromic and nitric acid fluid, to remove the lime-salts (p. xxxiii). Make transverse sections, and stain them with iodine green and aniline blue, and mount them in dammar (p. 1). Beautiful sections of tendons stained with gold are obtained, while part of the bone or interstitial cartilage becomes green. These form most instructive preparations. CELL-SPACES IN TENDON. The above-described cells lie in spaces, which may be demonstrated by a solution of E 26 PRACTICAL HISTOLOGY. nitrate of silver — just as in cartilage (p. 17) ; the silver blackens the intercellular parts and leaves the spaces occupied by the cells clear. PREPARATION. — Take several of the freshly exposed tendons of the tail of a rat. Wash them with a camel-hair pencil dipped in distilled water. Do this several times to remove the epithelium which covers their surface. Place the tendons in a half per cent, solution of nitrate of silver for five minutes. Then wash them in water and mount a small piece of a tendon so treated in glycerine ; and expose it to the light until it assumes a brownish colour. EXAMINATION (H). — Observe the irregular spaces seen in a dark brown matrix. These spaces communicate one with another by fine clear lines. The brown stained part indicates the existence of an intercellular substance ; in the clear spaces lie the cells with their processes. EPITHELIAL COVERING OF TENDONS. Each of the fine tendons is enveloped by a layer of squamous epithelium, which is easily demonstrated by staining the tendons — witlwut p)xvious pencilling — with nitrate of silver as directed above. Mount a tendon in glycerine, and expose it to light until it becomes brownish in colour. EXAMINATION (H). — The outlines of the polygonal epithelium are distinctly seen. These indicate the existence of an epithelial investment of the tendon superficial to the fibres and cells already described. MEMBRANOUS CONNECTIVE TISSUE. THE OMENTUM. A convenient form of this tissue is found in the omentum, which differs in its characters in various animals, and also in young and in adult animals. In an adiclt cat the omentum consists of bundles of connective tissue of various sizes, cemented to each other in the same plane, so as to form a network. In the larger trabeculas blood-vessels and fat-cells are found. Each bundle of fibres is completely invested by a layer of squamous epithelium or endothelium, and the nuclei of these cells can readily be seen, on the surface or edges of the fibres when they are stained. The trabeculae consist of fibrils which can be seen in the fresh tissue. In the substance of the trabeculae and amongst the fibrils a few fusiform corpuscles — the connective- tissue corpuscles — may be seen. They are relatively few in number, and so are the elastic fibres. In a young grozving rabbit the omentum is much less fenestrated, and forms a more continuous sheet of connective tissue, covered on each surface with a layer of endothelial squames. As the animal becomes older the membrane becomes more fenestrated. PREPARATION. Silver Nitrate. — Remove the omentum from an adult cat and rabbit, and also from a young rabbit just killed. Wash each in distilled water to remove the soluble chlorides, and then place them in a quarter per cent, solution of nitrate of silver for five minutes, and, after washing them thoroughly, expose them in alcohol to the light till they become brown. Snip out a small piece of each with scissors, and float them from water on to slides in the usual way (p. xxvi). Mount in Farrant's solution and cover, or similar preparations may be stained with logwood, which stains the nuclei. CONNECTIVE TISSUE. 27 EXAMINATION (L and H). — Observe the nearly continuous layer of endothelium in the case of the young rabbit (PI. V., Fig. i) on each surface of the omentum (p. 26), the more fenestrated character in the adult rabbit, and the almost net-like condition in the cat (PI. V., Fig. 2). In all cases the silver lines, indicating the existence of the endothelium covering the trabeculae, are seen. Focus down through one of the larger trabeculae, inside which con- nective-tissue corpuscles may be found. Do not confound the large oval nuclei of the squames which are always on the surface with the nuclei of the connective-tissue corpuscles proper. Omit at present the study of the blood-vessels and the fat cells which frequently accompany them. The omentum of a young rabbit is particularly valuable for studying the development of fat-cells and blood-vessels. ADIPOSE TISSUE. This consists of small vesicles or cells filled with fatty substances. The cells are imbedded in a small quantity of areolar tissue containing blood-vessels. Each cell has a distinct en- velope, and contains a globule of clear, sometimes slightly yellow-coloured fat, which nearly fills the cell, so that the nucleus, with a small amount of protoplasm surrounding it, is pushed to one side and compressed against the cell-wall. Study fully formed cells and fat-cells developing. PREPARATION. — Snip off a small piece of fat and tease it in a drop of salt solution on a slide. Place a piece of paper between the slide and cover-glass, to prevent the latter pressing on and rupturing the envelopes of the cells. EXAMINATION (L and H).— Observe the relatively large size (3^ - ■^) of the clear, highly refractive vesicles, sometimes compressed against each other. Usually no nucleus is visible. If the cells have been ruptured, globules of fat of various sizes will be found floating about. Irrigate with magenta solution (p. xliv), which stains the nuclei of the cells. They are seen as small oval red spots compressed against the cell-envelope. Substitute glycerine for the magenta, and mount for preservation. {Indicate this in PI. V., Fig. 4.) Effect of Osmic Acid.— A similar preparation, without tlie magenta, may be irrigated with a one per cent, solution of osmic acid, which blackens fatty matter, and hence is a most valuable reagent for its detection. Examine the omentum of the cat (p. 26) and the preparations of areolar tissue (p. 22) for fat-cells. Crystals of Margarine. — Delicate, needle-shaped crystals, springing from a point within the fat-cells, often occur. They are post-mortem appearances — for the contents of fat-cells are fluid during life — and may readily be produced by steeping a morsel of fat for twenty-four hours in glycerine, or long steeping in alcohol, or after partial digestion (p. 92). The Envelope of Fat Cells is easilj' shown by steeping a morsel of fat in ether, which dis- solves out the fat and leaves the empty shrivelled-up envelopes. {Indicate tins in PI. V.^ Fig. 5.) DEVELOPMENT OF FAT-CELLS. PREPARATION. — Place a small piece of the skin of a foetus in rectified spirit for three days. At the end of that time, make vertical sections in the ordinary way by freezing. After the gum has been removed from them by steeping in water, place some of them in a E 2 28 PRACTICAL HISTOLOGY. small quantity of a one per cent, solution of osmic acid for five hours. Wash them thoroughl)^ in water, and mount one in Farrant's solution. Cover. EXAMINATION (L). — Note the skin and the subcutaneous tissue, and, scattered through- out the latter, various sized groups of black spots. These black spots are of various sizes, and are the fat-cells, which have been blackened by the osmic acid. Select a group and examine. {Indicate this general arrangenient in PL V., Fig. 3.) (H). Notice the different sizes of the various vesicles composing each group. Some of the vesicles appear to be nearly filled with a blackened globule of fat, whilst others contain only one or two small black granules of fat, the rest of the cell being occupied with a finely granular protoplasm. These cells are connective-tissue corpuscles in process of being trans- formed into fat-cells through the degeneration of their protoplasm into fat. Developing fat- cells may be well studied in the connective tissue of the infra-orbital region of a young rabbit. PIGMENT IN CONNECTIVE-TISSUE CORPUSCLES. Connective-tissue corpuscles containing pigment are seen in the skin of the frog or in its mesentery, and, in fact, in nearly all its tissues. Preparations will be obtained from the choroid coat of the eyeball of any animal (p. 107). MUCOUS TISSUE. Mucous or embryonal tissue has a close relation to adipose tissue, as in the embryo it forms the subcutaneous tissue which ultimately becomes panniculus adiposus in the adult. It is essentially an embryonal tissue, as in addition to the site mentioned above, it occurs only in the umbilical cord (Wharton's jelly), and in the vitreous humour of the eye. Acetic acid precipitates the mucin in it in the form of fine threads. It consists of fusiform and branching cells, whose processes communicate in all directions with each other, so as to form a mesh- work, whose meshes are filled with a clear transparent mucin-yielding ground substance. In it also are flat endothelial cells similar to those found in connective tissue. It soon changes its character as the embryo grows older ; white fibrous tissue appears in the matrix, so that it comes partially to resemble connective tissue, and then to pass into adipose tissue. In the umbilical cord this last change does not take place : it remains in the foetus at full time as a fibrous tissue containing a mucin-yielding fluid in its meshes. PREPARATION {a). Subcutaneous Mucous Tissue. — Inject into the axilla or groin of as young an embryo as it is possible to get a small quantity of a quarter per cent, solution of osmic acid, so as to form a bulla. Snip out a small part of the cedematous tissue, stain it with logwood or picrocarmine, and mount in glycerine. EXAMINATION (L and H).— Observe the fusiform and also the branched cells anastomo- sing with each other with their nuclei stained. Use a very small aperture of the diaphragm. {U) Umbilical Cord. — Harden part of the umbilical cord of a four months' foetus in Midler's fluid for a week, and make transverse sections. Stain a section with logwood, and mount it in Farrant's solution. Another may be stained with methyl-aniline (p. xliv) and mounted in a saturated solution of acetate of potash. EXAMINATION (L and H).— Observe the three umbilical vessels (p. 124) surrounded by 5 no ^LATE v Ome^.'Tum Fat &^ / ^ \ 'ni^' /■ / A--^^/^ .;/■ /^- c // ( ^- ^ -^ 1 ^ V "" ■--^f ^;i ^ ^^ ^n 2 4-. CONNECTIVE TISSUE. 29 mucous tissue as above described. The cells are larger and the meshwork wider in the part farthest from the vessels. The intercellular substance, even at this age, contains connec- tive-tissue fibres, and their number increases in older specimens ; hence the necessity for having as young a foetus as possible to see the uncomplicated characters of this tissue. (^Indicate these corpuscles in PI. V., Fig. 6.) If a similar preparation be teased with needles and stained with methyl-aniline before it is mounted, it is easy to isolate flattened endothelial plates with wing-like expansions. ADENOID TISSUE. This tissue forms the basis of the lymphatic glands and the spleen, and occurs widely distributed as simple lymphatic glands or in diffuse patches in many organs of the body. It was formerly believed to consist of branched corpuscles, whose processes anastomosed with the processes of adjoining cells, so as to form a network, which was filled with lymph-cor- puscles. We now know, however, that it is built on the same type as connective tissue. It consists of a ground substance, dense reticulum or network — adenoid reticulmn — made up of excessively fine homogeneous fibrils and membranes, which form a honeycomb-like open network. Transparent, flattened connective tissue or endothelial cells with oval flattened nuclei are applied here and there to these fibres, and partially embrace or enclose them just as in connective tissue The large nuclei which usually occur at the nodes of the network, and which were formerly regarded as the nuclei of the branched cells, are really the nuclei of the flattened cells applied to the fibres. This meshwork is filled with lynipJi-corpuscles. These are small cells exactly like colourless blood-corpuscles, with a small round nucleus, which is thus easily distinguished from the large oval nucleus of the endothelial plate. They are not all, however, of the same size At first sight only these lymph-corpuscles are seen, and so densely packed are they that one requires to shake or pencil them out of the mesh- work before the adenoid reticulum can be brought into view. PREPARATION {a). — Place a small piece of a fresh lymphatic gland of an ox, or an entire gland from the abdominal cavity of a cat, dog, or rabbit, for a week in Muller's fluid. Make transverse sections. Stain a section deeply with logwood, and then place it in a test-tube with water and shake it briskly for five minutes or so. This detaches the lymph-corpuscles, and the previous staining enables one to see the section. Float it on to a slide, and mount in Farrant's solution. {U) Nitrate of Silver Injection and Logwood. — Into a fresh mesenteric or lymphatic gland, thrust the nozzle of a hypodermic syringe filled with a quarter per cent, silver nitrate solution, and push in the silver solution into the gland so as to form an artificial cedema. The gland must be tinged with the silver solution. Place it for twenty-four hours in alcohol and then make sections by freezing. At first the sections will be colourless ; but on exposure to light, wherever the silver has penetrated, they will be stained brown. Stain a section in logwood, and mount it in Farrant's solution or dammar. See also lymphatic gland (p. "JJ^ EXAMINATION (L). — Indistinct indications of the adenoid reticulum here and there crowded with lymph-corpuscles stained blue will be seen. Select a spot where {^^n corpuscles are to be seen and examine it with (H). Study the excessively delicate adenoid reticulum, with here and there a large oval nucleus lying on it, especially near a node. These are the 30 PRACTICAL HISTOLOGY. nuclei of the endothelial plates. Observe the small lymph-corpuscles in the meshwork. Not unfrequcntly in the mesenteric glands of the ox certain of the lymph-cells will be found to contain a brownish-coloured pigment, and I have found multi-nucleated cells closely resembling those found in the spleen. The source of the pigment in the one case, and the origin and function of these multi-nucleated cells, have still to be investigated. In the silver preparation certain parts acted on by the silver are brown, while those unacted on are bluish from the logwood stain. The silver opens up the network somewhat, and thickens the fibrils of which it is composed, so that it is more easily seen. It forms an instructive preparation. {Indicate the characters of adenoid tissue in PI. V., Fig. 7.) BONE. PREPARATION. Methods of softening bone, {a) Chromic and nitric acid. — Dissect out a long bone — the femur or humerus of a cat or rabbit — remove the muscles attached to it, but retain the periosteum. With a saw cut the bone into pieces three-quarters of an inch in length, and place them in a bottle with 300 cubic centimetres of one-sixth per cent, solution of chromic acid (p. xxxi). After three days substitute a quarter per cent, solution of chromic acid, and after three days more a half per cent, for another three days. Then transfer the pieces of bone to a large quantity of a mixture of chromic and nitric acid (p. xxxiii). The object is to remove the calcareous matter and leave the structure of the bone intact. The chromic acid alone ' fixes ' the elements and partially decalcifies the bone, but to employ chromic acid solution alone requires a long time to complete the decalcification, hence the use of chromic and nitric acid mixture, which completes the process more rapidl}', with no risk of the more delicate structures in the bone being injured by the action of the nitric acid. It expedites the process to change the fluid frequently. At the end of three weeks the bones still retain their form, though flexible and soft, and can now be cut with a knife. By thrusting a needle into the bone one can determine when all the calcareous matter has been removed, there being an absence of grittiness. The bone will now have a greenish colour, from the deposition of a chromic sesquioxide. Place the bone in a large quantity of water, frequently changed to get rid of the acids. Either make sections (transverse and longitudinal) at once in the usual way, or place the bones in alcohol till they are required. (b) Picric acid method. — Place similar pieces of bone in a saturated solution of picric acid and add a few crystals of the acid to keep the solution saturated, as part of the acid is used up to combine with the lime in the bone. This process takes much longer time than the above, but it answers admirably for foetal bones or for very small bones. After decalcification wash the bones for a long time in water, to get rid of the intensely yellow colouring matter. Place them in spirit till they are required. Make transverse sections of the shaft of a bone, including the periosteum, decalcified in either of the above ways. Place some of the sections in a one per cent, solution of osmic acid for twelve hours. Wash them thoroughly and preserve them in weak spirit. {c) A 10 per cent, solution of sodic chloride and hydrochloric acid. — This is for showing the fibrillar structure of the bone-matrix (v. Ebner and De B. Birch). This method is fully de- scribed at p. xxxiii. The sections must be preserved in a ten per cent, solution of sodic chloride. {d) Artificial digestion with trypsin. — See p. xxxiv. OSSEOUS TISSUE. 31 T. S. SHAFT OF A DENSE BONE DECALCIFIED. EXAMINATION (L). {a) Osmic acid section. — Float the section on to a slide from weak spirit, remove the surplus spirit, add a drop of melted glycerine jelly (p. xlviii), and cover. (Glycerine and Farrant's solution render the tissues too transparent.) Observe the periosteum surrounding the ring of bone. If the section be made through the centre of the shaft there will be little cancellated tissue — only a ring of dense bone surrounding the medullary cavity. In the bone observe the Haversian canals, some divided transversely, others obliquely, and some are opened into longitudinally ; arranged with reference to these are the bone-corpuscles lying in their lacunae, which appear like little dark specks. Some lacuna; are arranged with reference to the periphery of the bone. The lamella; of bone may be faintly seen. Those arranged round the Haversian canals are the Haversian lamellae, and these with the lacunie and their cana- liculi and Haversian canal make up a Haversian system, the peripheric lamellae arranged with reference to the periphery of the bone, and lastly some segments of larger circles which lie jammed up between the Haversian systems, though not belonging to them — the interme- diary lamellae, which are segments of greater circles than the Haversian lamellae. This in- dicates that at one time they were situated under the periosteum, but in process of develop- ment they have come to lie amongst the Haversian systems. They never contain a Haversian canal. {Indicate these general characters in PI. VI., Fig. I.) (H). Observe the outer and inner layers of the periosteum and perhaps a layer of somewhat flattened cells lying directly on the bone between it and the periosteum — the osteoblasts. Observe the bone-matrix with the faintly indicated lamellae, and the bone-corpuscles — irregular, somewhat shrunken, nucleated masses of protoplasm, each lying in a space or /aciina, with faint indications of the canaliculi. The Haversian canals, opened into longitudinally, may be seen to contain osteoblasts and a blood-vessel. {Indicate these structures in PL VI., Fig. 2.) {b) Stain, a T. S. of the shaft with picrocarmine for ten minutes, and mount it in Farrant's solution or glycerine jelly. Cover. (L). The periosteum is now well defined, its outer layer being stained red, from its consisting chiefly of white fibrous tissue, whilst the deeper layer is somewhat yellower in colour. The matrix of the bone is yellow and the bone-corpuscles red. (H). The outer layer of the periosteum is now seen to consist of bundles of fibrous tissue crossing each other, mixed with a few elastic fibres ; the deeper yellow-coloured layer consists of a large proportion of elastic fibres, mostly arranged longitudinally, and so their cut ends appear as dots, and lying on the bone is a layer of flattened nucleated cells stained yellow — the osteoblasts. L. S. OF A DENSE BONE DECALCIFIED. EXAMINATION (L). — Observe the Haversian canals cut into and arranged for the most part longitudinally, with here and there transverse or oblique canals connecting them ; the rows of lacunae— five or more between two Haversian canals ; the lacuna; arc flattened, with their long axis in the axis of the bone, and from each surface they give off canaliculi. If the periosteum has been preserved the osteoblasts may be traced into a Haversian canal opening on the surface of the bone. {Iidicate the Haversian canals and lacjinw in PI. VI., Fig. 3.) (H). Observe the lacunae with their included bone-corpuscles ; in the Haversian canals the remains of blood-vessels and osteoblasts. PRACTICAL HISTOLOGY. BONE LAMELL/E. PREPARATION. — {a) With forceps pull off a thin layer of the peripheric lamellae from a bone softened in dilute hydrochloric acid (p. xxxiii) and denuded of its periosteum. Examine in water. If it is to be preserved, it must be placed for twenty-four hours in one-half per cent, osmic acid, and mounted in Farrant's solution. {b) Digestion of a piece of bone with artificial pancreatic juice (p. xxxiv), previously softened with dilute chromic acid, enables one to isolate the fibres of which the lamella; arc composed. (Birch.) The sections must be preserved in a ten per cent, solution of sodic chloride. EXAMINATION (H). — The thin sheet of tissue consists of fibres resembling white fibrous tissue, which appear to cross each other in two directions, and here and there a hole may be seen, through which a Sharpey's fibre had passed. Projecting from the surface may be seen finger-like processes ; these are Sharpey's fibres, which have been pulled out of their sockets. SHARPEY'S FIBRES. PREPARATION. — Decalcify a piece of a human parietal bone in dilute hydrochloric acid. Make vertical sections, and place one on a slide. With needles tear off the peripheric lamellae, using a dissecting microscope (p. xxxv). Some of the fibres will thus be torn from their sockets, and will project as fine processes from the lamellae. EXAMINATION (L and H). — Observe the fine elongated nail-like process, and perhaps the socket from which they were removed. In the foregoing preparation of a lamella they are seen directed towards the observer. No Sharpey's fibres, such as are described above, are found within the Haversian systems. They are found in the peripheric and intermediary lamellae. ELASTIC FIBRES IN BONE. In addition to these fibres of white fibrous tissue, elastic fibres also pass into the bone from the periosteum. These are best seen in transverse sections of a decalcified shaft of a long bone, which has been slightly stained with glycerine to which a trace of magenta solution has been added (magenta-glycerine). The magenta stains these fibres of a deep red, while the white fibres are unaffected by the dye. Further, the elastic fibres branch, which the white never do. The magenta-glycerine must be allowed to act slowly, and when the section is sufficiently stained, remove it, and substitute for it Farrant's solution, and cover. FCETAL BONE. This ought to be softened, preferably with picric acid solution (p. xxxii). Select a long bone of a human foetus or the bones of a newly born kitten. They require a shorter time for decalcification than adult bones. Make transverse sections and preserve them in preservative fluid (p. xl) until required. Stain a section with picrocarmine, and mount it in Farrant's solu- tion and cover. Plate VI Eone k r .J / (X.L.. ..flxr.'.. <:^ t^ o -^^ -^ m-'. 9 a ,^ ^NfiriemiBros lith. OSSEOUS TISSUE. ^t, T. S. OF DENSE FCETAL BONE (DECALCIFIED). EXAMINATION (L).— The bone-matrix is yellowish and the corpuscles in the lacunae red. Observe the ring of bone, not so dense as in the adult ; the Haversian canals are relatively larger, and contain a layer of osteoblasts, which line them, and also a blood-vessel. The outer la)-er of the periosteum is red and the deeper layer is yellowish, and between it and the bone a layer of nucleated, somewhat cubical cells — the osteoblasts — which may be traced into Haversian canals opening on the surface of the bone. (H). Observe the osteoblasts lying on the bone, and here and there they may be traced into Haversian canals which run horizontally and communicate with the surface of the bone. The bone-corpuscles are relatively larger_ than in adult bone. After a few days, they become quite red and are better defined. The canaliculi may also be traced. Under the periosteum giant-cells may be found here and there. {Indicate the periosteum, the osteoblasts, and bone-corpuscles in PI. VI., Fig. 6.) DENSE DRY BONE. PREPARATION. — Take a human radius which has been completely freed from grease ; with a fret-saw cut a very thin transverse section. Grind the section on a hone moistened with water. Examine with a low power to determine when the section has been ground thin enough. Rinse it in water to get rid of the debris, and allow it to dry. Take Canada balsam, which is quite hard in the cold, and place a small piece of it on a slide, gently warmed over the flame of a spirit-lamp till the balsam melts. Allow the balsam to cool, and before it has become quite hard, place on it the section of bone, and cover. This method prevents the balsam from getting into the lacunce and canaliculi. and so rendering the bone too transparent. Make a corresponding longitudinal section of the shaft of a long bone. T. S. OF DENSE DRY BONE. EXAMINATION (L).— Observe the Haversian systems, each consisting of a Haversian canal surrounded by lamelL-e disposed concentrically with relation to it— Haversian lamellae ; amongst these are seen the lacuna; which resemble black dots with fine branches— the canaliculi— proceeding from them. Observe also the circumferential or peripheric, and the intersystemic or intermediary lamellse. (H). Observe the shape— flattened ovals— and arrangement of the lacuna; ; they appear black, being filled with air. From them proceed fine, branching canals, which appear as fine black lines, running across the lamellae and uniting the lacunae by a network of very fine tubes. The canaliculi of the outermost row of lacunae of any Haversian system do not usually open into the lacuna of an adjoining system, but bend on themselves, and open into the lacuna from which they sprang, or into an adjoining lacuna of the same system. These are the so-called ' recurrent ' canaliculi (Ranvier). {Indicate the lacuna: and canaliatli in PI. VI., Figs. 4 and 5.) L. S. OF DENSE DRY BONE. EXAMINATION fL).— The Haversian canals are cut longitudinally or obliquely (with transverse or oblique branches connecting them), and they open on the surface of the bone F 34 PRACTICAL HISTOLOGY. and into the medullary cavity. The lacunae are lony, narrow ovals, parallel to the canals arranged in several rows between adjoining canals. BLOOD-VESSELS OF BONE. PREPARATION. — Place the leg-bones of a rabbit whose blood-vessels have been injected with a gelatine and carmine mass (p. li), in a three per cent, solution of hydrochloric acid for several days, until they are completely decalcified. Place them in water for several hours, and afterwards in a dilute solution of sodic carbonate, to get rid of the last trace of acid. Make longitudinal and transverse sections in the ordinary way, and mount them in Farrant's solution. EXAMINATION (L). — Observe each Haversian canal contains a blood-vessel, and note the general arrangement, which follows exactly the Haversian canals. {Indicate the longitudinal arrangement of the blood-i'csscls in PI. VI., Fig. 3.) CANCELLOUS DECALCIFIED RONE. PREPARATION. — Soften the head of a foetal bone of a kitten or new-born child in the usual way, and make transverse sections through the cancellous head of the bone. Stain a section with logwood for five minutes, and another with picrocarmine for fifteen minutes, and mount both in Farrant's solution. Cover. EXAMINATION (L). — Observe the open meshwork ; the meshes are the cancelli, bounded by trabeculee of bone, which do not contain Haversian canals. In the cancelli are osteoblasts and marrow-cells. (H). Study the osteoblasts, and search for multi-nucleated cells (myoplaques, giant-cells, or osteoclasts) — cells several times larger than the osteoblasts, containing many nuclei imbedded in granular protoplasm. They usually lie close on the bone, in a depression which they accurately fill. DEVELOPMENT OF BONE (INTRA-CARTILAGINOUS). PREPARATION. — Place the leg-bones of a rabbit, one week old, or those of a kitten, in picric acid solution, or in the chromic acid and nitric acid fluid, till they are completely softened. Make a longitudinal vertical section through the cartilaginous head and shaft of the bone. EXAMINATION (L).— Stain a section with picrocarmine. Observe the head of the bone separated from the shaft by the epiphysial cartilage, and below the cartilage the line of ossifi- cation. In the epiphysial cartilage the cells are arranged to a certain depth above the line of ossification in rows parallel to the long axis of the bone. These rows of cells are produced by transverse cleavage of cartilage-cells. Below the line of ossification, where true bone com- mences, are found irregular spaces — the primary medullary spaces — which are bounded by narrow spicules of calcified cartilage, partially covered by a laj-er of bone. In these spaces are seen osteoblasts, young marrow-cells, and blood-vessels. (H). Observe the rows of cartilage-cells, smaller above and larger below. At the line of ossification a cartilage-capsule may be found, opening and discharging its contents into a primary medullary space. The long spicules have in their centre the remains of the calcified cartilage, and on them is deposited bone, which is now stained of a deep red colour, and osteoblasts may be seen in process of becoming imbedded in this newly formed bone, to form bone-corpuscles. In the spaces observe the osteoblasts, and here and there multi-nucleated OSSEOUS TISSUE. 35 cells (osteoclasts), lying on and eroding the newly formed bone of the osseous trabeculae. {Indicate the rows of cartilage-cells, line of ossification, and other details, in PI. VI., Fig. 7.) L. S. PHALANX 01" A FINGER FROM A HUMAN FCETUS. After softening, stain with picrocarmine for fifteen minutes, and mount in Farrant's solution. EXAMINATION (L). — Observe the shape of the bone under the periosteum : on each side of the shaft is a triangular piece of bone, stained bright red ; the base of each triangular por- tion corresponds to the surface of the shaft, the apices pointing inwards. In this piece of bone are seen channels, into which vessels from the periosteum proceed. These vessels are accompanied by osteoblasts into the interior of the bone, where already the central part has been excavated to form primary meduUaiy spaces. Towards the head of the bone, the cartilage-cells, arranged in rows, are seen just as in an epiphysial cartilage. Under the perrosteum there are one or more layers of osteoblasts. DEVELOPMENT OF BONE (INTRA-MEMBRANOUS). One of the three flat bones of the skull of a fcetal kitten or other animal is taken, and after being hardened in picric acid it is stained with picrocarmine and mounted in Farrant's solution. MEDULLARY TISSUE AND MARROW. Ordinary yellow marrow consists chiefly of fat (ninety per cent.), but the spongy ends of bones generally and the medullary cavity of the long bones of some animals — e.g. guinea-pig — contain red marrow. PREPARATION. — Take the leg-bone of a rabbit or guinea-pig, and with an axe or chisel cleave it longitudinally. With the point of a knife take out a little of the red marrow, and diffuse it in a drop of salt solution on a slide, and cover. EXAMINATION (H). — Observe the marrow-cells of various shapes — many of them re- sembling colourless blood-corpuscles, though tiiey contain a large clear nucleus ; others are identical with colourless blood-corpuscles. Large multi-nucleated cells, many times the size of a coloured blood-corpuscle, may be seen. They are the myeloplaques, osteoclasts, or giant- cells, and consist of a granular mass of protoplasm, containing a large number of nuclei in their interior. Dilute alcoliol reveals these nuclei, and frequently causes a small, transparent, bulla-like mass to appear on the side of the cell (Stirling). » A permanent preparation of red marrow may be made by placing a piece of a long bone of a guinea-pig, opened, as described above, in dilute alcohol for two days. Then put a little of the marrow on a slide, and stain it wjth picrocarmine or logwood, and mount the preparation in glycerine. TAIL OF A RAT DOUBLY-STAINED. PREPARATION. Gold chloride and an aniline dye. — See p. xlvii., where this process is full}- described. EXAMINATION (L and H). — The tendons are stained with gold chloride, while the bone is stained of one colour and the cartilage of another. This is one of the most beautiful pre- parations in the whole range of histology. 36 PRACTICAL HISTOLOGY MUSCULAR TISSUE. There are two varieties of muscular tissue, the non-striped and the striped. The former may easily be obtained from certain of the hollow viscera, and the latter from the voluntary muscles of the limbs. NOX-STRIPED MUSCLE (INTESTINE). PREPARATION.— Kill a rabbit ; wash out a piece of the small intestine with salt solution, and afterwards distend the gut with absolute alcohol. Place the distended gut in absolute alcohol for twelve hours. After it is hardened, whilst still distended, with a pair of forceps strip off a thin layer of the longitudinally disposed muscular fibres, forming the outer muscular layer of the gut, and place it in water to remove the alcohol ; float it on to a slide and stain with logwood for five minutes and mount it in dammar. Cover. EXAMINATION (L). — Observe the large number of fusiform nuclei arranged more or less parallel to each other. (H). Observe the fusiform shape of the nuclei stained deep blue. These are the nuclei of the fusiform muscular fibre-cells of which the tissue is composed. {^Indicate the uitdei in PI. VII., Fig. I.) It is necessary to isolate these fusiform cells to show that the tissue is composed of a number of them cemented together. PREPARATION. — Place a small piece of the fresh muscular wall of the intestine or stomach in a twenty per cent, solution of nitric acid for twenty-four hours ; this will dissolve the cement. Place a strip of this softened tissue in water to remove the acid, and then tease a small piece on a slide. It is very difficult to stain the nuclei of these isolated cells after the action of nitric acid, but a watery solution of magenta may be tried ; or steeping in picrocarmine for forty-eight hours or longer. Mount in glycerine and cover. EXAMINATION (H).— Notice a great number of elongated small fusiform cells floating in the field. Each cell is broad at the middle and tapers towards the extremities. Near the centre is the fusiform nucleus stained red, and at each of its poles not unfrequently a few granules are to be seen. In some of the fibres corrugated bars may be seen passing across the cell ; these are where partial contractions of the body of the cell have been fi.xed by the acid. {Indicate these fibres in PI. VII., Fig. 2.) BLADDER OF A FROG. PREPARATION.— Distend the bladder of a frog with dilute alcohol (p. xxxiv), and place it distended in a large quantity of the same fluid for twenty-four hours. At the end of that time slit open the bladder, place it on a slide with the inner surface uppermost, and with a camel- hair pencil brush away all the epithelium lining the bladder, which is very easily removed. Wash the tissue in water, and stain one piece with logwood and mount it in dammar, and another for half-an-hour with picrocarmine, and mount it in Farrant's solution. MUSCULAR TISSUE. o/ EXAMINATION (L). — Observe the large number of nuclei stained either blue or red, lying usually in one plane. (H). Observe the shape and disposition of the long fusiform nuclei. Many of the cells are fusiform, but others are tri-radiate. T. S. OF NON-STRIPED MUSCLE. PREPABATION. — Place a piece of the muscular wall of the intestine of a cat in chromic acid and spirit solution, and after two weeks make transverse sections of its circular muscular fibres. Stain a section with picrocarmine for twenty-four hours and mount it in Farrant's solution. Stain another with logwood for five minutes, and mount it in dammar. EXAMINATION (H). — Observe the field mapped out into small polygonal areas of different sizes. These are the fibres cut across. These areas occur in groups of small fasciculi, sepa- rated from each other by connective tissue, which forms a sheath for them — the periniysiuin. In some a nucleus may be found, in others none, because the line of section has not been through the situation of the nucleus in the fibre. The variable size of the areas, and the absence of a nucleus in some of them, enable the observer to distinguish easily between the cut ends of non-striped muscular fibres and the mosaic formed by the ends of a number of columnar epithelium cells turned towards the observer, all of which are of the same size, and each one is nucleated. Observe the cement-substance between the fibres. This is best seen when the illumination is slightly diminished by turning off a little of the light with the mirror. Observe also that the fibres are arranged in groups or bundles, surrounded and separated from each other by connective-tissue septa, which are continuous throughout — the periinysiuin. This is continuous with the cement or endoniysiiint, which lies within the bundles between the muscle-cells. {Indicate the divided fibres in PI. VII., Fig. 3.) CEMENT-SUBSTANCE OF NON-STRIPED MUSCLE. PREPARATION. — Kill a rabbit, and wash out a piece of the small intestine with distilled water. Fill the intestine with a half per cent, solution of nitrate of silver, and place the distended intestine for ten minutes in a quarter per cent, solution of nitrate of silver. Owing to the difference of concentration of the fluids on the two sides of the gut, diffusion of the silver takes place and so complete saturation of the tissues is obtained. Wash away the silver in ordinary water and e.vpose the gut in alcohol to a strong light. After it has become brown, with a pair of forceps tear off a thin lamina of the outer longitudinal muscular wall of the gut ; steep it in water and float it on to a slide and mount it in Farrant's solution. Another strip may be stained with logwood solution and mounted in dammar, if it be desired to reveal the nuclei of the muscle-cells. Another method of obtaining the outer layer of the muscular coat is to place a small piece of the gut with its external surface next the glass slide ; and then with a knife to scrape away layer after layer from within outwards until only the longitudinal and the serous coatsTemain. EXAMINATION (H). — Obsene a large number of fine black lines disposed for the most part longitudinally. When carefully traced they are seen to bound narrow fusiform spaces or areas. These silver lines indicate the existence of a cement-substance which holds the fibres together. Lying between the fibres are elongated channels with bulgings upon them, and lined by sinuous epithelium. These are the silvered lymphatics, i.e. lacteals. We shall use this preparation for the study of the general characters of lymphatics. {Indicate the silver lines in PI. VII., Fig. 4.) 38 PRACTICAL HISTOLOGY. INTRA-NUCLEAR PLEXUS OF FIBRILS IN NON-STRIFEI) MUSCLE. The student who desires to show this arrangement must proceed as follows : — PREPARATION. — Kill a newt and place the small intestine and mesentery in a five per cent, solution of ammonium chromate for twenty-four hours. Wash in water till no colour is given off, then transfer it to picrocarmine for several hours. With scissors snip off a small piece of the mesentery and mount it in glycerine. Cover. EXAMINATION (H). — Observe the very large spindle-cells, either single or disposed in groups. Study a nucleus and observe in its interior a delicate plexus of fibrils. In its in- terstices lies a clear homogeneous substance. On carefully focussing with one-eighth of an inch objective, fibrils may be traced through the poles of the nucleus, and be found to be con- tinuous with fibrils disposed in the long axis of the cell. These muscular fibres are examined on account of their large size (Klein). {Indicate tlicsc plexuses of fibrils in PI. VII., Fig. 5.) MESENTERY OF NEWT DOUBLY-STAINED WITH PICROCARMINE AND LOGWOOD. PREPARATION. — Prepare the mesentery of a newt with ammonic chromate as directed above. After washing away all the chromate from a piece of the mesentery, stain it for fifteen minutes in dilute picrocarmine (ten drops to a watch-glass of water). Wash it in water and place it in water acidulated, with a few drops of acetic or picric acid for an hour. After washing place it in dilute logwood solution, until it assumes a faint lilac colour, taking care that it is not over-stained. Mount it in glycerine. STRIPED MUSCLE. This may be studied in any of the voluntary muscles. A muscle consists of striped muscu- lar fibres held together by connective tissue and supplied with blood-vessels and nerves. The fibres must be examined in the fresh condition, and after the action of reagents. FRESH STRIPED MUSCLE. PREPARATION. — Kill a frog, and from one of its muscles — preferably the sartorius, because it is composed of parallel fibres — snip off a small piece and tease it slightly in salt solution. Cover. EXAMINATION (H), — Observe the cylindrical fibres consisting of the sarcous substance marked with alternate light and dim stripes (PL VII., Fig. 8). In some places the striation may be obscure or the stripes very close, while here and there slight longitudinal fibrillation is obser\'able. Irrigate with distilled water and after a short time a fine bleb may be seen on the side of one or more of the fibres. This is the sarcolemnia. {Indicate the sarcolevnna in PI. VII., Fig. 6.) Water has passed through the sarcolemma and raised it from the sarcous substance. Irrigate the preparation with dilute acetic acid, when the nuclei, lying under the sarcolemma and in the sarcous substance, are revealed (PI. VII., Fig. 7). They are elongated spindles, sometimes slightly coloured, lying in the long axis of the fibres. The reason why the nuclei are not seen at first is because they have the same refractive index as MUSCULAR TISSUE. 39 the surrounding sarcous substance. Another way of showing the sarcolemma is to take a piece of fresh muscle, and after teasing it, to press a needle across a fibre so as to try and break the fibre. If this be accomplished, the sarcous substance will be ruptured, and con- tracting leaves the structureless transparent sarcolemma stretched between the ends of the ruptured fibre. CLEAVAGE OF THE SARCOUS SUBSTANCE. PREPARATION. — Place small, narrow strips of any muscle of a cat or rabbit, which has been dead for a few hours, in a small quantity of a sixth per cent, solution of chromic acid for a week. The muscular fibres will tend to split up longitudinally mtojibrils. Chromic acid seems to dissolve a cement. (2) Place similar pieces of muscle in hydrochloric acid — one part to fifty of water for a week. After this time each fibre tends to split transversely into discs. Artificial digestion (p. 92) in an acid medium produces the same result in a few hours. EXAMINATION (H).— Take a small piece of the muscle which has been steeped in chromic acid, and after washing it thoroughly tease it out with needles in glycerine. Each muscular fibre tends to split longitudinally into excessively fine threads— the fibrilhc or fibrils — each one being marked v.'ith light and dim bands like the original fibre. {Indicate the fibnlhein PL VII., Fig. 10.) Take a similar piece of the hydrochloric acid muscle and tease it. The fibres split across into thin narrow discs or ' muscle-discs.' Occasionally a muscular fibre splits up both longitu- dinally and transversely at the same time, when Bowman's sarcous elements are formed. These are sometimes seen at the end of a fibre broken across by chance. {Indicate the discs in PL VII., Fig. 9.) MUSCLES OF ARTHROPODA. To trace the finer details in the structure of muscle it is customary to use the striped muscles of insects, such as the common water-beetle— Dytiscus marginalis or Hydrophyllus piceus — or the common cockroach. I find, however, that the muscles of the common edible crab answer the purpose admirably, and are far better suited for use in a large class. PREPARATION.— (rt) Place a beetle or the amputated limb of a crab in absolute alcohol, and if possible keep some of the muscles on the stretch when so doing. Leave them there for a week, {b) A mixture of one part of methylated spirit and three parts of Mailer's fluid, for three to four weeks, hardens muscle most admirably. The tissue must be kept in the dark, else reduction of the chromic salts takes place very rapidly. Scoop out a small piece of the muscle of a crab and steep it in water. Steep one piece in picrocarmine for twenty-four hours, and stain another with logwood for a few minutes. Tease them thoroughly with needles. Mount the former in glycerine and the latter in dammar. Both preparations show much the same structure, only what is red in the one is logwood-tinted in the other. EXAMINATION of the picrocarmine preparation (H). — Notice the large size of the muscular fibre, and how easily it splits into fibrill^. Each fibre shows well-marked transverse striation. Study this. The broad, dim discs (contractile discs) are stained red, and the narrower, less refractive discs (interstitial discs) are slightly yellow. In each dim disc a series of fine vertical rods may be seen. Study carefully the light disc — across its centre runs a fine line, Dobie's line or Krause's membrane — which divides the disc into two lateral discs. This line is continuous with the sarcolemma, and is supposed to represent a membrane by whicli 40 PRACTICAL HISTOLOGY. the musclc-fibrc is divided into a series of compartments, each containing a dim disc with a lateral disc attached to each end. In this preparation no nuclei are seen. {^Indicate these discs in PI. VII., Fig. II.) Logwood and Lammar Preparation (H). — The dim discs are logwood-coloured — the light discs unaffected. In the course of the fibre are seen oval nuclei under the sarcolemma. Otherwise the details arc the same as in the above preparation. If the muscle of an insect— say hydrophyllus or dytiscus — be taken and similarly treated, a row of fine granules is observable in the lateral discs. These form the granular layer of Flogel. LIVING MUSCULAR FIBRE. PREPARATION. — This is not particularly well adapted for a large class of students. Cut off the head of a water-beetle and then open its chitinous body with a sharp-pointed pair of scissors, so as to expose the large wing-muscles. Snip off a small piece of these and tease it rapidly in aqueous humour (p. xxx) or salt solution (p. xxx). Cover and examine. EXAMINATION (H). — Observe the fibres, their striation, and that they are soft and plastic. Here and there a thickening, progressing along a fibre — a contraction-wave— with approxi- mation of the discs, may be observed. NUCLEI OF MUSCLE. PREPARATION. — Take a small piece of a muscle of a rabbit or cat which has been pre- served in alcohol, tease it out in water, and stain it in carmine till it is of a bright red colour. Wash it in water to remove the surplus carmine. Place on it a large drop of glacial acetic acid, and after doing so examine it with a low power, to notice when the nuclei alone remain stained. The effect of the acid is to remove the diffuse staining, and leave the carmine only in the nuclei. Mount it in either Farrant's solution or dammar — preferably the latter. EXAMINATION (H). — Observe the striation, and in the course of the fibres oval nuclei with their long axis in the axis of the fibres, stained deeply red. {Indicate the nitdei in PL VII., Fig. 8.) Relation of the Nuclei to the Sarcolemma. — Take a limb-muscle — or the tongue — of a cat or rabbit or other animal, previously hardened in alcohol, or chromic acid and alcohol (p. x.xxi), and make a transverse section by freezing or other means. Stain it for a few minutes with logwood and mount it in dammar. This preparation will also show the relation of the connective tissue, ox perimysium, to the muscular fibres. EXAMINATION (L). — Notice the connective-tissue — perimysium — of a light blue colour surrounding groups of muscular fibres cut transversely, which appear as small polygonal areas stained light blue. {Indicate the connective tissue, and the cut muscular fibres with their nuclei in PL VII., Fig. 12.) (H). — Observe the cut ends of the fibres, each more or less polygonal, through mutual pressure of the fibres against each other. Under the sarcolemma are seen two or more small logwood-stained nuclei in each fibre, and the cut end of the sarcous substance appears slightly dotted. The nuclei are often situated at the angles of the polygon. Trace the connective tissue in very fine strands between the fibres — forming the endomysium — and, it may be, con- taining a capillary here and there. In a transverse section of a frog's muscle nuclei are found to exist, not only under the sarcolemma, but also throughout the sarcous substance. PLAi'E V'^ MUSCLE ItntBrn Bros itk MUSCULAR TISSUE. 41 BLOOD-VESSELS OF MUSCLE. PEEPAE.ATION. — Make longitudinal and transverse sections of any muscle of a cat or other animal whose blood-vessels have been injected with a carmine or Prussian blue and gelatine mass (p. li). The sections may be very slightly stained in logwood, or mounted unstained in dammar, or a smal! piece of the muscle may be simply teased and mounted in Farrant's solution. EXAMINATION of a longitudinal section (L). Observe the capillaries arranged parallel to the long axis of the fibres —one capillary between each two fibres, with here and there trans- verse branches connecting them. They lie outside the muscular fibres and amongst the delicate connective tissue which supports them. {^Indicate the arrangevient of the capillaries in PI. VII., Fig. 13.) Transverse section (L). — Observe the cut ends of the fibres and between and outside the sarcolcmma the cut ends of the capillaries. The larger blood-vessels lie in the connective tissue of the perimysium. It is advisable to inject the blood-vessels of the lower limbs of a rabbit with a Prussian blue and gelatine mass. Place the limbs in a one per cent, solution of bichromate of potash. After the mass has set, select one of the red muscles, e.g. the semi-tendinosus. Most of the muscles of the rabbit are pale or almost colourless. The above-mentioned muscles, and some others (soleus, adductor minus, muscles of the jaw), are, however, distinctly red ; the semi-tendi- nosus, at least, has a peculiar distribution of its blood-vessels. Tease out a small piece of one of the injected red muscles and mount it in glycerine or dammar. EXAMINATION (L). — The blood-vessels have the same distribution and relations as described above, but they present this peculiarity — that on the transverse, short capillaries are small dilatations like little aneurysmal swellings. These swellings do not exist in the blood- vessels of the pale muscles of the same animal. Further, the capillaries usually pursue a more tortuous course, and the veins, also, sometimes present similar fusiform swellings in their course. The muscular fibres of the heart will be examined in connection with that organ (p. 47); and the nerves of muscles with the terminations of nerve-fibres (p. 45). Foetal developing muscle may be studied in any embryo after hardening in Miiller's fluid. It is characterised by the great number of its nuclei, the smallness of the fibres, and the im- perfect striation. In fresh fcetal muscle iodine solution reveals the presence of glycogen. REL.4TI0N OF MUSCLE TO TENDON. PREPARATION {a). Centrum tendineum of the diaphragm. — Cut out the diaphragm from a newly killed rabbit ; place it in lemon juice for five minutes, and, after steeping in one per cent, gold chloride for an hour, transfer to a twenty-five per cent, solution of formic acid for twenty-four hours. During this time the preparation must be kept in the dark till the gold is reduced. Snip out a small piece of the muscle, with the central tendon attached, and tease it in glycerine on a slide and cover ; or a section may be made parallel with the long axis of the muscular fibres. EXAMINATION (Land H). — Observe the tran.sversely .striped muscle, and trace it towards the tendon, where it ends abruptly in a conical form which is lodged in a corresponding depression in the tendon. G PRACTICAL HISTOLOGY NERVE-FIBRES. There are two varieties of nerve-fibres : — 1. The medullated or white. 2. The non-medullated or gray. MEDULLATED NERVE-FIBRES. Each nerve-fibre consists of a thin transparent structureless sheath, the primitive sheath, with here and there a nucleus lying under it. Within this is the medullary sheath or white substance of Schwann with a double contour forming the greater part of the nerve. It is interrupted at intervals by regular constrictions, or Ranvier's nodes, where the white substance is entirely absent. In the centre of the fibre is the axis-cylinder, a fine uniform cylinder continuous throughout the whole length of the fibre. The proper view to take of the axis- cylinder is that it is directly continuous at its central end with a process of a nerve-cell, and is in fact one of the processes of a nerve-cell enormously elongated and pushed outwards towards the periphery of the body, and covered in whole or part of its course with one or more sheaths. This is the only view that explains the remarkable results of degeneration in a nerve-fibre when it is separated from its so-called trophic nerve-centre (Waller) and also explains the so-called union of nerve-fibres, which is in fact no union at all, but is due to a pushing out or growth of the axial cylinder into the old sheaths of the degenerated nerve-fibres from which the axis-cylinder and most of the white substance of Schwann, but not the nuclei, have disappeared. FRESH NERVE-FIBRES. PEEPAEATION. — Kill a frog and remove its sciatic nerve. With a pair of sharp scissors cut off half an inch, and place it on a slide in a drop of salt solution. With a pair of needles gently tease out one end of the nerve, and examine it from time to time with a low power, to ascertain when it has been sufficiently teased. Cover. EXAMINATION (H). — Observe the nerve-fibres of various sizes, some of them about the breadth of a coloured blood-corpuscle, others larger or smaller. Note the double contour of the white substance of Schwann which is highly refractive. This double contour exists in the living nerve, and is not due to the action of reagents. This can easily be proved by examin- ing a medullated nerve in the lung or tongue of a living frog or the lung of a newt. Select the end of a fibre which has been broken across, and study the mycline exuding from its cut end. The myeline, or white substance, collects into drops, with concentric markings. These must not be mistaken for cells, from which they are easily distinguished by the absence of a nucleus. {Indicate the myeline exuding from a nerve-fibre in PI. VIII., Fig. i.) In the centre of each fibre is a clear uniform band, the axis-cylinder. Trace the course of a fibre carefully, and a Ranvier's node may be easily distinguished. The white substance is interrupted here ; further, if the nerve-fibre be slightly on the stretch, the oblique incisures of Schmidt and Lantermann, passing through the myeline, can also be made out. Irrigate the preparation with picrocarmine, and continue the examination after twenty minutes. Observe that a nucleus under the primitive sheath is stained red, the carmine passes in at the nodes of Ranvier and also stains the axis-cylinder red. If an axis-cylinder happen to extend beyond NERVE-TISSUE. 43 the myeline, it is quickly stained red by the carmine. {Indicate a node and the incisures in PI. VIII., Fig 2.) It is remarkable that nearly all the phenomena which have been laboriously made out by the use of special reagents can be seen in a perfectly fresh or even in a living nerve-fibre. EFFECT OF OSMIC ACID. PREPARATION. — Kill a frog ; dissect out a sciatic nerve. Tie a fine thread round each end of the nerve, carefully preserving it from getting dry, and by means of the thread stretch the nerve on a small piece of wood— an ordinary wooden match with a slit at either end to hold the thread answers admirably. Place the nerve and wood in a one per cent, osmic acid solution for ten minutes. The effect of the osmic acid is to blacken the phosphorised fats of the myeline and to ' fix ' its elements. The nerve is stretched in order that the nodes and incisures may be made quite distinct. Remove it and wash it carefully in water, and then place it, still on the stretch, in a test-tube with picrocarmine. In twenty-four hours the stain- ing is complete. I find it to be very advantageous to leave it in picrocarmine for a fortnight or longer. The effect of this is to soften the connective-tissue of the nerve, so that on teasing it at the end of this time the single nerve-fibres are easily obtained. Tease a small piece — always tearing off a fibre in its long axis — in a drop of glycerine. Cover. EXAMINATION (H). — Observe the myeline, blackened and still retaining its double con- tour. Trace a fibre and observe, at pretty regular intervals, the constrictions or nodes of Ranvier, and between each two nodes a brightly stained nucleus imbedded in a trace of pro- toplasm, will be found lying under the primitive sheath and partly indenting the myeline. {Indicate tii'o nodes in PI. VIII., Fig. 5.) Study a node. Observe the absence of the myeline, and that only the slightly red-coloured axis-cylinder passes from one segment of the nerve to another. The axis-cylinder, therefore, is the only essential part of a nerve. Some excessively delicate nerve-fibres will be seen. The delicate connective tissue supporting the nerve-fibres is easily made out. {Indicate a node, the nucleus, and the incisures in PI. VIII., Figs. 3 and 4.) Amongst the medullated fibres may be detected the grey or sympathetic nerve-fibres. They are distinguished by being delicate fibres, with a large oval nucleus here and there in their course, and are devoid of myeline, and therefore do not possess a double contour. ACTION OF NITRATE OF SILVER. RANVIER'S CROSSES. PREPARATION. — {a) Tease out roughly a small piece of a perfectly fresh sciatic nerve of a frog in a quarter per cent, solution of silver nitrate on a slide, and leave it in this solution for five minutes or so. Wash it with water and then tease it carefully in glycerine, cover and expose it to the action of light until it becomes brown. (3) The small intercostal nerves of a rat or rabbit may be excised and plunged entire into silver nitrate solution and mounted entire in glycerine, and exposed to the action of light as above. EXAMINATION (L and H). — Observe a fibre and notice the dark brown crosses seen here and there (PL VIII., Fig. 8), The long limb of the cross is produced by the silver pene- trating to the axis-cylinder, and staining it, or an albuminous material covering it, for a short distance ; whilst the transverse, more deeply stained, bar is due to the cement which unites the so-called segments of a nerve one to another (PI. VIII., Fig. 9). Sometimes the longitudinal limb of the cross may be marked transversely bylines (Frommann's lines). If a whole nerve be used, a complete investment of endothelial cells will be found outside all. 44 PRACTICAL HISTOLOGY. ARRANGEMENT OF NERVE-FIBRES TO FORM A NERVE. PREPARATION. — Place pieces of the human or other sciatic nerve, cut into pieces one inch long, into chromic acid and spirit (p. xxxi), or Miiller's fluid and spirit (p. xxxii), for ten days, and after hardening in alcohol make transverse sections in the ordinary way. Stain a section in logwood, and, if it be desired, another in carmine, and mount them in dammar. They both show the same details. EXAMINATION (L). — The various bundles composed of nerve-fibres are held together by a common framework of connective tissue — the epineurium — arranged so as to form a sheath around and between the various bundles, and in it may be found transverse sections of the large blood-vessels which supply the nerve with blood. Round each bundle observe a special sheath — \\\& perincuruiiii — composed of lamellated connective tissue with flattened connective- tissue cells between them. The spaces between the lamellae represent lymph-spaces. {Indicate these bundles in PL VIII., P"ig. lO.) Study a nerve-bundle. — Observe it is made up of many nerves cut transversely ; only the stained axis-cylinders are indicated. (H). Study the epi- and perineurium ; observe the cut ends of the nerve-fibres varying in diameter, and the section of the a.xis-cylinder stained red or violet and surrounded by a trans- parent circular area, which represents the myeline or white sheath of Schwann ; outside this a faint circle, indicating the primitive sheath. Between the nerve-fibres may be seen very delicate connective tissue — the endoneiiriuni — with here and there a small blood-vessel in it. {Indicate the stained axial cylinders and the cut ends of the nerve-fibres in PL VIII., Fig. 1 1.) If the hardening process has been continued for a month or longer, concentric rings may be observed in the white substance of Schwann. {Indicate these in PL VIII., Fig. 12.) Longi- tudinal sections of a nerve prepared as above may also be studied. THE CONNECTIVE-TISSUE ELEMENTS OF A NERVE. PREPARATION. — Take a small fragment of a nerve which has been hardened in one-si.xth per cent, solution of chromic acid for ten days, and after staining in logwood, tease it thoroughly in a drop of glycerine. EXAMINATION (H). — Observe the nerve-fibres. The axis-cylinder, stained violet, will easily be recognised amidst the coagulated myeline. It is specially interesting to trace the axis-cylinder across a node of Ranvier. Observe the connective tissue, some of it in the form of delicate lamellae, with here and there oval nuclei stained violet lying on them, and also de- tached flattened plates— the connective-tissue corpuscles — each containing a large oval violet nucleus. FRESH NERVE-FIBRES OF THE SPINAL CORD. PREPARATION. — With scissors snip off a small fragment of the white matter of a fresh spinal cord of an o.x or sheep, place it on a dry slide, do not add any fluid, put on a cover- glass, and compress it into a thin layer. EXAMINATION (H).— Observe the drops of myeline, easily recognised by their concentric markings and the absence of a nucleus, and very delicate nerve-fibres with bulgings or am- pulla: upon them, due to the pressure of the cover-glass on the nerve-fibres, which here are y. 300 L ; Plate vm ^ Nerves 12 11 V. -> n 8, ilmtem Bros lith NERVE-TISSUE. 45 devoid of a primitive sheath. There are no ampullae in the living condition. Do not pre- .serve this preparation. NON-MEDULLATEU NERVE-FIBRES. These may be detected in an ordinary nerve and studied there, but it is better to select a nerve containing these or sympathetic fibres in large quantities. The sympathetic nerve from the neck of a rabbit may be selected, but it is far better to have a larger nerve. This is easily obtained from the spleen of an ox. Accompanying the branches of the splenic vein are large nerve-trunks, the nerves of the spleen, which contain only a few meduUated fibres, the rest being non-medullated nerve-fibres. With scissors cut open the splenic vein, and then the nerve-trunk is easily dissected out. PREPARATION. — Place the nerve, extended, as for the medullated nerve, in a one per cent, solution of osmic acid, and after washing place it in picrocarmine for twenty-four hours. Tease a small fragment in glycerine, and cover. EXAMINATION (H). — Neglect the medullated nerve-fibres and observe the non-medullated nerve-fibres. They are pale, flat, slightly granular bands which have no myeline, and in their course the oval red-stained nuclei of the primitive sheath are easily seen. Sometimes the nuclei are seen on edge, and at other times on the flat. In this nerve many ' plasma cells ' (p. 20) are found. If desired, a fresh nerve may be teased in salt solution, when similar appearances are observable. Nerve-cells will be considered with the organs in which they occur. SOME PERIPHERAL TERMINATIONS OF NERVES. We shall only allude here in detail to the terminations of nerves in striped muscle by means of end-plates, and to the Pacinian corpuscles attached to the ends of certain sensory nerves (p. 46). Other modes of termination will be described in the organs where they occur. TERMIN-AFIONS IN STRIPED MUSCLE. PREPARATION. — This is by no means an easy task. In mammals the best muscles are the straight muscles of the eyeball of a rabbit — the muscular part of the diaphragm and any thin muscles such as the intercostals. In the lizard {Lacerta agilis) the end plates may be more easily found. But the best muscles are those which attach the skin to the ribs of the smaller snakes. In any case it is best to employ the following gold method. Snip out very thin pieces of the muscle to be examined, and place them in lemon-juice for five minutes, then wash in water and transfer to a one per cent, solution of gold chloride for twenty minutes or half an hour — wash again and place the prepai-ation in the dark for twenty-four hours in a twenty- five per cent, solution of formic acid. After washing thoroughly, to remove the acid, tease out a small piece of the muscle in glycerine, and search with a low power for a nerve-fibre, or trace the outline of a muscular fibre till an end plate is obtained. The a.xis-cylinder is con- tinued beyond the end plate, and breaks up into fibrils inside the sarcolemma ; their ulti- mate relation to the sarcous substance, however, is unknown. These preparations are difficult to make, and do not keep well afterwards. The terminations of nerves in unstriped muscle will be considered under the head of small intestine (p. 70 ; the same methods are applicable as for those of striped muscle nerve-endings. 46 PRACTICAL HISTOLOGY. PACINI'S CORPUSCLES. PREPARATIOIf. — These are numerous in the mescnter)' and in the mcsorectum of the cat, where they arc easily obtained free from fat. The student may also find them in the deeper layers of a vertical section of the skin of the hand or foot, or in the pad of a cat's foot, but I have found them most easily for students in transverse sections of an entire fcetal leg or arm. Being placed relatively closer to each other in the fcetus, they are, like sweat-glands, more easily observed there than in adult textures. In the cat's mesentery they can be seen as small, oval, transparent, hard bodies. Snip out one of these and examine it at once in salt solution with a low power ; the nerve entering it may be seen, as also the central core surrounded by concentric laminae. If a permanent pre- paration be desired, excise a piece of the mesorectum or mesentery containing these bodies, pin it out in a stretched condition on a flat piece of cork, and place the cork, tissue down- wards, for a week, in a two per cent, solution of potassic bichromate ; after that wash away all trace of the reagent with water, snip out a corpuscle and stain it slowly (twenty-four to forty-eight hours) in dilute logwood, which must be changed several times. Wash in water and mount in glycerine. As the corpuscles are of considerable thickness, it may be necessary to place a thin layer of paper under the edges of the cover-glass to avert pressure, and allow the cover-glass to lie flat upon the preparation. EXAMINATION (L). — Observe the nerve-fibre like a stalk approaching and entering the central core of the oval corpuscle, which latter is made up of laminse one outside the other. (H). Trace the nerve-fibre, and observe that the medullary sheath stops where the nerve enters the corpuscle. Only the axial cylinder passes into the central core. Study the con- centric laminae, which exhibit a series of nuclei on each surface. These are the nuclei of the endothelial cells which cover each lamina. To study the laminae and their endothelial covering, a corpuscle, hardened as above, must be broken up with needles, by which process the various lamina; are readily separated from each other. Transverse sections will be obtained in one or other of the preparations already mentioned. These show the concentric distribution of the laminae round the central core, and also the nuclei of the cells between each two laminae (p. 92). 47 HEART AND BLOOD- VESSELS. The heart is invested by a fibrous pericardium covered by a layer of squames, whose existence is easily demonstrated by the nitrate of silver process. It is lined by a thinner membrane also covered with squames, the endocardium, capable of demonstration in a similar manner. The muscular tissue of the heart is transversely striped, though involuntary. The strise are always more or less indistinct. The fibres branch and anastomose. They consist of short nucleated segments joined by a cement, and are devoid of a sarcolemma. ISOL.'VTED MUSCULAR FIBRES OF THE FROG'S HEART. PREPARATION. — Place the heart of a newly killed frog in a small quantity of a forty per cent, solution of caustic potash (p. xxxiv) for a quarter of an hour. Take a fragment of the tissue and tease it with needles in a drop of potash solution. Be careful to add no water. Cover. EXAMINATION (H). — Observe the isolated muscle-cells. They are fusiform, and in the broad middle part there is a well-defined nucleus, and the substance of the fibre is trans- versely striped. Do not preserve this. If it be desired to possess a permanent preparation, small pieces of a frog's heart must be placed in dilute alcohol for twenty-four or forty hours, and then stained in picrocarmine ; and, after teasing to isolate individual cells, add glycerine and mount the preparation. FRESH HEART OF A MAMMAL. Treat a small piece of the heart of a rabbit in the same way as the frog's heart. Cover. EXAMINATION (H).— Observe the oblong muscle-cells isolated. Each cell is nucleated and transversely striated. Do not preserve this. HEART FOR PRESERVATION. PREPARATION.— Place small pieces of the heart of a rabbit, or a piece of a human heart, including a piece of the pericardium, in chromic acid and spirit fluid for ten days, transfer to spirit, and then make a transverse section through a ventricle, including the pericardium. Stain a section with logwood and mount it in dammar. EXAMINATION (L).— Observe the fibrous pericardium stained of a light blue, and note its thickness. Here and there it sends fine processes into the heart, to form a perimysium for the bundles of muscle. It is important to observe the normal thickness of the fibrous covering, and also the normal amount of connective tissue present, as m every organ, to be enabled to judge when there is any alteration in the thickness of the capsule or the amount of the inter- stitial connective tissue. This point is specially important in relation to the pathological histology of organs. Not unfrequently in these trabecule are to be seen sections of the 48 PRACTICAL HISTOLOGY. coronary artery and vein and cardiac nerves, easily recogjnised by their lying together and by their structure. Trace the muscular fibres mapped out into bundles. Some are cut trans- versely, others obliquely, and some longitudinally. Many nuclei are seen, which are the nuclei of the muscular fibres. Select a transverse section of a group of fibres. (H). Observe the polygonal shape of the fibres from mutual pressure, the existence of a violet-stained nucleus in the centre of some of them — not in all, and the striae radiating from the nucleus ; a delicate endomysium will also be seen. {Indicate these in PI. IX., Fig. 2.) Select a longitudinal section, observe the faint striation, the branching and anastomosing of the fibres, and the oval, violet- stained nuclei in the course of the fibres. The places where the oblong muscular cells which make up a fibre join, indicated by a transverse line, due to the presence of a cement-sub- stance, are to be looked for. {Indicate the characters of these fibres in PI. IX., Fig. i.) Cement-substance. — The cement-substance which unites one cell to another may be de- monstrated thus. Thrust the nozzle of a hypodermic syringe filled with half per cent, solution of silver nitrate amongst the fibres < T ,-the heart, and forcibly inject the silver solution so as to separate the heart-fibres. Place the her-t in alcohol, and after twenty-four hours snip out a small piece which has been acted on by the silver, and after teasing in glycerine expose it to the light and examine for the lines of junction of the cells stained brown or black by the silver. Blood-vessels of the heart present practically the same arrangement as in striped muscle, and are prepared and examined in the same way (p. 41). Nerves and ganglia of the heart. — Sometimes preparations of these are obtained in trans- verse .sections of the ventricles ; but to see their arrangement and distribution the heart of a frog, or small pieces of any mammalian heart, must be treated with lemon-juice, chloride of gold, and formic acid, as described at p. xlv. The nerves and ganglia must then be dissected out with the aid of a dissecting microscope (p. xxxv). PURKINJE'S FIBRES. A delicate network of fine transparent lines is seen, with the naked eye, on the interior of the ventricles of the heart of a sheep or ox. These are Purkinje's fibres. They consist of imperfectly developed muscular fibres — they are, in fact, vestiges of what each heart-fibre was originally. They are made up of polygonal-shaped cells, each often with two nuclei, and only the outer half of each cell is striated ; but in the deeper layers, cells are found where the striation has involved three- fourths of a cell, and deeper down still they become continuous with the ordinary heart-muscle. Each muscular fibre of the heart, and in fact, every striped muscle, was originally non-striped, and the striation always begins at the periphery of the cell, so that the fibres made up of these cells represent an embryonal phase of muscular tissue. PREPARATION. — With scissors or a knife cut out these fine threads and macerate them for two days in dilute alcohol, stain with picrocarmine, and tease a small piece in glycerine until isolated cells or groups of cells are found. EXAMINATION (H). — Observe the isolated polygonal cells, each with two nuclei, and the outer half of each cell marked with every gradation between these and cells where the peri- nuclear part is completely striated. {Indicate these cells in PI. IX., Fig. 3.) 49 BLOOD-VESSELS. There are three well-marked varieties of blood-vessels — arteries, veins, and capillaries. A typical artery has three coats — outer, middle, and inner. In a middle-sized artery the inner coat consists of a layer of elongated squames with their long axis corresjionding to the long axis of the vessel ; outside this, delicate connective tissue and an elastic membrane or lamina, either with holes in it — ' fenestrated membrane ' — or it consists of a sheet of elastic tissue which in sections is thrown into folds, owing to the contraction of the muscular coat outside it. It is to be remembered, however, that these folds do not exist in a living vessel distended with blood. The middle layer consists of circularly-disposed non-striped muscular fibres, with a small amount of elastic and white fibres. The outer coat (adventitia) consists of fibrous tissue with longitudinally disposed elastic fibres, which are specially numerous next the muscular layer. In the largest arteries (aorta) there is relatively a large amount of elastic tissue in the inner and middle coats, and in the middle coat the non-striped muscle and elastic plates occur in alternate layers. In the veins the division into coats is not so sharply marked. The middle coat contains less muscular fibre-cells and more connective tissue, and the outer coat not unfrequently contains non-striped muscle, especially in large veins. The capillaries consist of a layer of nucleated, flattened, epithelial cells, joined at their edges with a cement-substance. Small arteries and capillaries are best studied in the pia mater. SMALL ARTERIES AND CAPILLARIES. PREPARATION. — Get the head of a recently killed sheep, and remove the brain. With a scalpel remove as much of the brain as possible, leaving the pia mater. With a stream of salt solution wash away the remainder of the brain-substance. It is most important to remove all the brain-substance, else it forms a granular deposit which obscures the prepara- tion. Place the pia mater in chromic acid and spirit solution for a week. After washing it thoroughly in water, snip out a small piece and stain it with logwood, and mount it in dammar. A similar preparation may be stained with picrocarmine, and mounted in Farrant's solution. EXAMINATION (L). — Select a small artery, an.d study its mode of branching and how these branches terminate in capillaries. Note in the artery the transversely disposed nuclei of the muscular fibres of its middle coat (PI. IX., Figs. 6 and 7). Observe the outer coat. Search for a vein with its much thinner coat and fewer transversely disposed nuclei — i.e. muscular fibres — and compare it with an artery. (H). Select a small artery. Observe its outer and middle coats, and, composing the latter, non-striped muscular fibres, disposed in one or more layers. The fusiform nuclei of the muscular coat are very apparent. The folds of the elastic lamina of the inner coat may be seen. Study a capillary. It appears as a perfectly structureless membrane, with oval nuclei imbedded in it, which bulge into its lumen. It requires the use of nitrate of silver to demon- strate its endothelial characters. H 50 PRACTICAL HISTOLOGY. DEVELOPMENT OF CAPILLARIES. The development of capillaries is easily studied in the tail of a half-grown tadpole prepared by the ordinary gold chloride method. To see the capillaries distinctly it is well to remove the superficial epithelium after staining. If the lemon-juice and gold method (p. xlv) be employed, the formic acid employed afterwards removes the superficial epithelium. The omentum of a new-born rabbit presents easily recognised whitish patches, or ' milk spots ' (Ranvier) in it, which are aggregations of corpuscles like lymph-corpuscles ; and others, which become continuous with buds given off from existing blood-vessels. These buds ultimately become channelled to form blood-vessels. FRESH BLOOD-VESSELS. Take the pia mater, washed free from cerebral matter, and tease a small piece on a slide in salt solution. Cover. An examination reveals the same general arrangement as described above, but the nuclei of the muscular coat of the arteries requires the addition of acetic acid to bring them into view. Add acetic acid, to observe this. It also swells up the adventitia, makes it clear and transparent, and the elastic lamina when present, with its folds, is brought clearly into view — and so are the nuclei in the capillary wall. Do not preserve this. AORTA. PREPARATION. — Take small pieces of the human aorta or of the aorta of an ox, and place it in picric acid for twenty-four hours. Make longitudinal and transverse sections in the usual way, and stain them with picrocarmine, taking care not to wash the sections too much in water, and mount them in Farrant's solution. EXAMINATION (L). — Observe the fusion of the different coats and the large amount of elastic fibres and plates in the different layers. All elastic tissue is stained yellow, connective tissue pink, and non-striped muscle light reddish-brown, so that these different tissues are easily distinguished (H). Note the numerous layers of elastic fibres in the inner coat ; in a transverse section they appear as bright yellow granules ; in the longitudinal as yellow lines. Note the large amount of elastic tissue in the middle coat, where it occurs in the form of elastic laminae. If it be desired to see the lining epithelium, silver (p. xlv) the inner surface of the aorta of an animal just killed, and mount a thin shaving in danuaar. ISOLATED ELASTIC LAMINA. PREPARATION. — By far the best method is the one I described several years ago for the isolation of elastic tissue, viz., to digest thin strips of the aorta in artificial gastric juice (p. 92) till everything except the elastic element is dissolved (De B. Birch). Wash the tissue, to get rid of acid, and mount in Farrant's solution. Examination reveals all gradations, from a network of elastic fibres to fenestrated membranes and sheets of elastic tissue. The elastic fibres not unfrequently present transverse markings. X300 Plate TXHe.aht 8^Blood^"essels Mirvfcarrx Bi^os .IxtK BLOOD-VESSELS. 51 MIDDLE-SIZED ARTERY. PREPARATION. — Place a moderate-sized arterj' — say the femoral or posterior tibial of a child, or the basilar artery of a sheep or man — in chromic acid and spirit for a week, and make transverse sections. Stain one section with logwood, and mount it in dammar ; cover ; another with picrocarmine, and mount in F~arrant's solution. EXAMINATION (L). — Obser\-e the inner coat, perhaps with the layer of squames lining it. Observe the elastic lamina, as a clear, yellow, bright line thrown into folds by the contraction of the middle coat which surrounds it. If a large artery be under examination, a sub-epithelial layer of delicate connective tissue may be found between the epithelium and the elastic lamina. The elastic lamina is an important guide in disease of the blood-vessels, for if changes take place internal to it, it is a disease of the inner coat ; if immediately external to it, the disease is in the middle coat. The student should, therefore, familiarise himself with its appearance, on account of its important pathological relations. The middle coat consists chiefly of several layers of circularly disposed non-striped muscular fibres. The nuclei are long ovals and of a violet colour. Between the fibres may be found elastic tissue. Outside this note the adventitia, consisting mainly of white fibrous tissue stained blue, and containing elastic fibres, especially in its inner part. {^Indicate the epitlwliiun, elastic lamina, and otiicr coats in PI. IX., Fig. 8.) (H). Study the elastic lamina ; it is a good landmark in a section. On its inner surface a profile view may be obtained of the lining epithelium. Observe the middle and outer coat's. The Epithelium and its Cement-substance in blood-vessels is demonstrated by the use of nitrate of silver. For a class this is easily managed, thus : — PREPARATION. — Kill an etherised rabbit by bleeding. Open the thorax and place a cannula in the aorta, and (after making an opening in the inferior cava), wash out the blood- vessels with distilled water. Then inject the vessels with a half per cent, solution of silver nitrate. Cut out the intestines and wash out their contents, and then expose them and their mesentery in a mixture of equal parts of spirit and water to sunlight till they become brown. Select either a piece of the mesentery which contains blood-vessels, or what answers equally well, take a piece of the small or large intestine and lay it on a slide with its mucous surface upwards. With a scalpel scrape away the mucous coat and mount the muscular and serous coats in dammar. EXAMINATION (L. and H). — Select a small artery, notice the silver lines running trans- versely, indicating the disposition of the circular muscular fibres, and inside these black silver lines mapping out narrow lanceolate areas with their long axis in the axis of the vessel which are the squames lining the artery. Try and find a vein and compare the epithelium of the two vessels. Trace an artery until it splits up into capillaries, and study the silver lines in a capillary. These indicate that it is made up of elongated flattened endothelial cells. If it be desired to reveal the nuclei in these plates, a section should be stained with logwood. {^Indicate the silver lines in a capillary in PI. IX., Fig. 4, and in the artery in Fig. 5.) VEINS. Exactly the same methods are employed for bringing into view their structure. Many sections of them will be found in other organs. They are recognised by their thin walls, the absence of so perfectly defined an elastic lamina, their thinner muscular coat, and the less distinct demarcation of their individual coats. H 2 52 PRACTICAL HISTOLOGY. CIRCULATION OF THE BLOOD. The circulation of the blood may be studied in any vascular transparent organ, e.g. web of frog's foot, lung of frog, and also in the mesentery. For the student it is best to study the circulation in the web of a frog's foot. METHOD. — Select a frog with a slightly pigmented web, and with a hypodermic .syringe inject into the lymph-sac under the skin of the back four minims of a quarter per cent, watery solution of curara, and place the frog under a bell jar till its motor nerves are paralysed, «>. till it ceases to move when it is pinched. Place the frog on a piece of card-board one and a-half inches wide and six long, with a triangular slit of one-inch base cut at one end of the card. Tie a thread round the tips of two adjoining toes, e.g. the third and fourth. Fix the threads in a slit made in each horn of the cardboard, and stretch the web gently across the slit. Moisten the web, place the card-board on the stage of the microscope, and fix it with clips. EXAMINATION (L). — Observe the arteries, with the blood moving in them from the large to the smaller vessels. In the centre of the stream a more rapid current, and between it and the wall of the vessel the slower stream or lymph-space. Select a vein, known by its thinner walls and the direction of the blood-stream, and notice the movement from the smaller to the larger vessels, and the stream slower than in the arteries. Next observe the network of capillaries, with the blood-corpuscles moving in single file. Notice also the pigment-cells, some of them branched ; but if they are contracted they look like small black specks. Cover tlie web with a fragment of a cover-glass. (H). Study a capillary ; observe its thin wall and the passage of the corpuscles in single file through it. The coloured corpuscles are elastic, as shown by the way in which they twist and easily become distorted, and as easily regain their normal shape. Select a small vein and observe the colourless corpuscles dragging lazily along in the lymph- space and adhering to the wall of the vessel. If desired, the phenomena of inflammation of the web can easily be studied by applying a small quantity of mustard moistened with water, or some otlier irritant, for a few minutes. Wash off the irritant from the web and study the effects. 53 RESPIRATORY ORGANS— LARYNX, TRACHEA, AND LUNGS. The epiglottis has already been examined (p. i8). The preparation may be referred to again. Its anterior surface is covered by stratified epithehum, and so is the posterior, but here the submucous coat contains adenoid tissue, and a large number of mucous glands (p. 54), which not unfrequently are lodged in depressions of the yellow elastic or reticular cartilage, which constitutes the basis of the organ. Not unfrequently taste-bulbs are met with amongst the epithelium on the posterior surface. The arytenoid cartilages consist of yellow fibro-cartilage, and sections of them show tran- sition stages from ordinary hyaline to yellow fibro-cartilage (p. 19). The submucous tissue contains many mucous glands. If desired the vocal cords may be hardened in chromic acid and spirit mixture, and vertical sections made in the ordinary way. TRACHEA AND LUNGS, PEEPARATION. — Kill an etherised cat by bleeding, open the thorax, and allow the lungs to collapse. Tie a cannula in the trachea, and with a syringe distend the trachea and lungs with a mixture of chromic acid and spirit (p. xxxi). Suspend the lungs in a large quantity of the same fluid. Change the fluid at the end of twenty-four hours ; after three days cut the trachea and lungs into small pieces, and place them in fresh hardening fluid for a week or ten days, and then, after washing them thoroughly, transfer them to alcohol until sections are required. Preserve a piece of human trachea in the same way. Distend a piece of human lung and preserve it as above. Make transverse and longitudinal sections of the trachea, transverse sections through a bronchus as it enters the lung, and sections through the pleura and subjacent lung, and also across a bronchus within the lung-substance. Preserve the sections in preservative fluid (p. xl) until they are required. Sections of such an organ as the lung, with its numerous open spaces, ought always to be made after steeping in gum, which fills the spaces ; and after freezing the organ is, as it were, solid, and the gum can easily be dissolved out by water. Nothing is so good as a freezing microtome for making sections of lung. The lungs are covered externally by a serous membrane, ihc pleura. Its epithelial cover- ing can easily be demonstrated by the silver process (p. xlv) on the lungs of a cat just killed. After silvering, a superficial slice is taken and mounted in dammar or glycerine. If the lung be silvered while it is distended ad maxijimm, i.e. in a state of inspiration, the cells appear as polygonal squames ; if it be collapsed, i.e. in a state of expiration, the cells appear slightly cubical ; showing that the cells are soft and accommodate themselves during life to the changes of size in the air-vesicles during the respiratory acts. 54 PRACTICAL HISTOLOGY. TRANSVERSE SECTION OF TRACHEA OF A CAT. Stain it for half an hour in picrocarmine and mount in Farrant's solution. EXAMINATION (L). — Observe the arc of hyaline cartilage forming nearly two-thirds of a circle and deficient posteriorly. The cartilage is invested by a fibrous perichondrium stained red. It is continuous with the layer of circularly disposed non-striped muscular fibres, that connect the ends of the cartilages and help to make up the posterior wall of the trachea. This muscle, the trachealis muscle, is not inserted into the tips of the cartilage-rings, but into the outer surface of the fibrous perichondrium, some distance from their free ends. The muscle is easily made out, for it has a dull red appearance. Outside the cartilage is some fat and con- nective tissue. Observe the mucous membrane with its columnar ciliated epithelium, and under it the submucous coat, containing many mucous glands, most of the acini of which lie internal to the cartilages, and it may be some adenoid tissue. The sacs of each mucous gland are en- closed in a connective-tissue capsule, and have a flattened appearance where they lie inside the cartilage, but are round or oval between the rings. Between the different groups of glands single fat-cells, or small groups of fat-cells, may be seen. {Indicate the general chaj-acters in FIX., Fig. I.) (H). Observe the epithelium in several layers, the upper layer cylindrical and ciliated. It rests on a basement-membrane not easily seen in the trachea of the cat, though easily seen in the human trachea, where it plays an important role in pathological processes. Amongst the epithelium are goblet-cells which are stained of a deep red. Under the epithelium observe beautiful layers of elastic fibres arranged longitudinally, which appear as rows of bright yellow dots lying amongst the connective tissue. Observe the mucous glands, and their acini inside the cartilages, and trace a duct to its opening on the surface in a funnel-shaped manner. The acini of the glands lie in the submucous coat, which in addition to connective tissue con- tains a very considerable quantity of adenoid tissue. As the gland-ducts pass obliquely to reach the surface, it is difficult to get a complete section of a gland with its excretory duct. The ciliated epithelium is continued a short distance into the excretory duct, but it soon gives place to moderately tall cylindrical epithelium, which lines the duct. The alveoli or sacs of the gland vary in appearance according to whether they were or were not secreting at death. If secretion had been taking place, the cells lining them will be filled with a clear transparent mucus-like mass, and few granular cells are to be seen. If there has been no secretion, the gland-sacs will be lined by a regular cubical epithelium of a more or less granular appearance. Observe the numerous leucocytes in the submucous coat. Observe the non-striped muscle — trachealis— only present posteriorly and its attachment to the perichondrium some distance in front of the free ends of the tracheal rings. Outside it may be seen transverse sections of nerves and nerve-ganglia. As the mucous membrane is very vascular, many sections of blood-vessels will be seen in it. The cartilage is ordinary hyaline cartilage, but its corpuscles present a characteristic arrangement. Under the perichondrium the cells are flattened, and lie parallel to the surface of the cartilage, whilst those in the interior are elongated or oval and are placed vertically from one surface to the other, i.e. across the long axis of the cartilage. {Indi- cate the epithelium, glands, and part of the cartilage in PI. X., Fig. 2.) Double-stain a section of a trachea with picrocarmine and iodine green (p. xlv). The mucous glands, adenoid tissue, and cartilage are stained green. Vertical section of Trachea, stained with picrocarmine and mounted in Farrant's solution. EXAMINATION (H).— Observe the elliptical pieces of cartilage— the sections of the car- tilaginous rings cut vertically. Observe the same general arrangement as described in the TRACHEA. 55 transverse section, with this difference, that the mucous glands are seen to be most numerous between the cartilages, and the longitudinally disposed elastic fibres under the epithelium can now be detected. Study these various parts under a high power. HUMAN TRACHEA. Transverse section of a Human Trachea, stained with picrocarmine and mounted in Farrant's solution. EXAMINATION (L). — Observe the same general arrangement of cartilage, glands, tracheal muscle, and mucous coat as described in the cat's trachea. Notice, however, that the ciliated epithelium is usually absent at some parts, from the difficulty of getting perfectly fresh tis- sue to preserve. An important point is the existence of a well-marked layer of tissue stained of a deep red — a ' basement membrane ' so called. It lies immediately under the epithelium inside the longitudinally disposed layer of elastic tissue. It is a marked feature in the human trachea, and plays an important part in pathological changes, especially in those resulting from chronic bronchitis. Observe the mucous glands and their ducts ; the acini of many of them lie outside the trachealis muscles, so that their ducts have to perforate it to reach the surface of the trachea. The largest glands lie posteriorly, and they present the same characters as previously described, their appearance varying with their state of physio- logical activity. (H). Where the superficial epithelium has been detached, good examples of developing epithelial cells may be studied. Study each of the other structures in detail. The so-called 'basement membrane,' it seems to me, resembles very closely a similar struc- ture which exists in the mucous coat of the cat's stomach, though there its relation to the gland- structures is different. I am unable to discover any corresponding structure in the trachea of the rabbit, cat, or dog. Its structure, relations, and homologies require further investigation. It is certainly quite different from the basement-membrane made up of epithelial cells, and described by Dcbovc as occurring under the epithelium of an intestinal villus, the bladder, trachea, &c. Double-stain a section of human trachea with picrocarmine and iodine green. TRANSVERSE SECTION OF A BRONCHUS AT THE ROOT OF THE LUNG. Place a section in a quarter per cent, solution of osmic acid for twenty-four hours, and mount it in Farrant's solution, and another ought to be stained with picrocarmine and simi- larly mounted. EXAMINATION (L). — Observe the bronchus, and note on each side of it a large blood- vessel, the one a branch of the pulmonary artery, and the other, on the opposite side, a branch of the pulmonary vein. These three structures lie in channels, and are imbedded in connec- tive tissue, and are thus accurately mapped off from the adjacent vesicular lung-tissue. Observe the bronchus. Note that several pieces of hyaline cartilage are inserted in the bronchial wall. Outside them is connective tissue often containing a few fat-cells — which are blackened in the osmic acid preparation. Besides this, note transverse sections of small blood-vessels — the bronchial arteries, and, lying near them, transverse sections of nerves, with perhaps a ganglion in their course. Observe the mucous coat thrown into folds, and under it the cut ends of longitudinally disposed elastic fibres. Outside this a continuous ring of non- striped muscular fibre — the bronchial muscle. It is perforated here and there by the ducts of 56 PRACTICAL HISTOLOGY. the mucous glands, whose acini lie between it and the cartilages, though they are most nume- rous where no cartilage exists. Perhaps a lymphatic cord may be found in the submucous coat. Observe the pulmonary blood-vessels and compare the relative thickness of their coats, {Indicate the general arrangement in PI. X., Fig. 3.) (H). The epithelium and glands have exactly the same structure as in the trachea. The cut ends of the elastic fibres, stained bright yellow, are most numerous where the mucous mem- brane is raised as a fold. Study the fibrous connective tissue mixed with elastic fibres which exists outside and around the cartilages and blood-vessels. Select a transverse section of a nerve, if possible with a ganglion in it. The ganglia and large branches of nerves will be found outside the cartilages, and usually accompanied by one or more small blood-vessels (bronchial). Observe the ganglionic cells, large oval corpuscles with a distinct nucleus and nucleolus, and between or around them the cut ends of nerve-fibres medullated and non- medullated, the whole surrounded by a fibrous sheath. {Indicate a small part of the loall of a bronchus in PI. XI., Fig. i.) Vertical Section through the Pleura and the Subjacent Vesicular Tissue.— Stain with logwood and mount in dammar. EXAMINATION (L). — Observe the pleura, made up of fibrous tissue stained blue, and note its normal thickness. From its under surface it sends into the lung, at regular intervals, fine processes of connective tissue— the interlobular septa — i.e. processes between adjoining lobules. It may be possible to trace the connection of these septa with the connective tissue around a bronchus. They contain many lymphatics, and are often pigmented from the presence of charcoal or soot, especially in the human lung. They are of great importance pathologically, and this connection of the pleural connective tissue with that which enters the lung at its root is also important, e.g. in the production of dilatation of the bronchi and other pathological processes. Here, as in other organs it is important to bear in mind the connection of the con- nective tissue of the capsule with that in the interior of the organ. Observe now the air- vesicles, cut in every direction, but notice that those placed most superficially, i.e. next the pleura, are somewhat pyramidal or conical in shape, and have their bases directed towards the pleura ; the air-vesicles elsewhere are polygonal in shape, and may or may not have a base to them, which depends on the line of section. They are separated from each other by a very small quantity of connective tissue, and by elastic fibres, which form a network round each air- vesicle. Here and there a transverse section of a small bronchus may be found. It is recognised by the cubical epithelium lining it, and by the presence of cartilage or glands in its walls. In the vesicular structure search for a transverse section of an infundibulum. The nuclei seen in the air-vesicles belong to the squames lining them, and those forming the capillaries in their walls. Search for a small bronchus opened into longitudinally, and observe its dichotomous division and its expansion into air- vesicles. {Indicate the pleura, and the shape of the air-vesicles, in PI. XI., Fig. 2.) (H). Observe thepleu7-a, which consists of two layers ; the superficial one contains a con- siderable number of elastic fibres, and the lower, or sub-pleura, consists of looser connective tissue, which is continued into the lung between the lobules as interlobular septa. In it may be seen sections of the pleural blood-vessels and lymphatics, the latter appearing as fine slits. The endothelium on the surface of the pleura is not visible. Observe an air-vesicle. Note its outline— rounded or polyhedral. If its thin edge be seen in profile, observe the squamous epithelium lining it, though it is better seen where a portion of the wall of an alveolus is flat in the field. In the alveolar wall trace the branching elastic Plate Z.Trachfa ^Ijjtig lantern, Bros .]itK LUNGS. 57 fibres, and carefully distinguish the plexus of capillaries, sometimes containing a few blood-cor- puscles. {Indicate the epithelium and elastic fibres in PI. XL, Fig 3.) In the capillary wall observe the nuclei, which are not to be confounded with those of the squames lining the air-vesicle. In the cat's lung, fusiform nuclei, indicating the existence of non-striped muscle between the vesicles, are to be detected. The amount of non-striped muscle between the air- vesicles is sometimes greatly increased in amount in certain diseases. It is easy to see this in the lungs of a cat suffering from the presence of the ova of a worm {Olhdaniis triciispis) in the lungs (Stirling). Select a very small broncluis cut longitudinally. Both cartilage and glands have disappeared from its walls, and the epithelium lining it is not so tall as in larger bronchi. Trace it into an alveolar duct, which passes into the infundibulum, into which the terminal alveoli open on all sides. In the alveolar duct and infundibulum the epithelium con- sists of low polyhedral cells without cilia. The walls of the alveolar ducts and infundibula contain many non-striped muscular fibres, disposed circularly, which are in direct continuity with the bronchial muscle. Pierocarmine Preparation. — Stain a similar section with picrocarmine, and mount it in Farrant's solution. After several days it shows the above details, even better than the dammar preparation, Osmic Acid Preparation. — Place a section of lung, hardened, as above, in chromic acid and spirit mixture, in a quarter per cent, solution of osmic acid for twenty-four hours, and, after washing, mount it in Farrant's solution. It shows the above details most beautifully ; the elastic fibres stand out very prominently, and their arrangement is easily made out. Vertical Section of Human Pleura and Lung. — Prepared as above, and similarly mounted. Note the more conspicuous interlobular septa, often pigmented from the deposition of charcoal particles in the lymphatics. Compare other parts of the section, and note especially the branching character of the elastic tissue, for the physician is sometimes called on to recognise these fibres in the sputum of a person suffering from gangrene of the lung. HUMAN FOITAL LUNG. Prepare the lung of a foetus — preferably one that has not respired — in the same way, with chromic acid and spirit mixture, as directed for adult lung. In ten days it will be ready for cutting. Or, use picric acid solution as the injecting and hardening medium, which will harden it in two days. Make sections across a lung, and, after staining with picrocarmine, mount in Farrant's solution. EXAMINATION (L). — Observe the distinct lung-lobules, each covered on its free broad end by a relatively thick pleura, and separated from its neighbouring lobule by thick interlobular septa, whose continuity with the adventitia or connective tissue of the intra-pulmonary bronchus can easily be made out. In each interlobular septum note the large open spaces, which are sections of the interlobular lymphatics. Study the small non-distended air-vesicles, and note (H) that they are lined by low cubical cells, not yet converted into squames by the distension of the lung as the result of the act of inspiration. {Indicate the pleura and interlobular septa in PI. XL, Fig. 6.) SQUAMOUS EPITHELIUM OF THE AIR-VESICLES. PREPARATION.— Remove the lungs of a cat or kitten from the thorax, and distend them with a quarter per cent, solution of nitrate of silver, and keep them distended by tying a string round the trachea. Place them, with a weight attached, in alcohol until required. When I 58 PRACTICAL HISTOLOGY. requircc!, cut the lunt^ into pieces, and make sections by freezing. Expose the sections to the action of light, until they become brownish in colour. Stain a section with picrocarmine for twenty-four hours, and mount it in glycerine ; and another with logwood for a few minutes, and mount it in dammar. EXAMINATION (L). — Observe the same general arrangement of the air-vesicles as already described. Select an air-vesicle where a portion of its wall is seen on the flat, and examine. (H). Observe the silver lines, indicating the existence of epithelium (squamous), lining the vesicle. These lines bound polygonal areas. Between these larger and clear areas notice small brown granular areas or polyhedral cells, in groups of two or three, some of them with a nucleus stained red or blue. These are young epithelial cells, and are thicker than the squames or placoids. Transition forms exist between them and the clear squames. During a maximum distension of the lung they yield and become flattened out. In a well- distended alveolus, narrow angular apertures, especially in the cement lines, are to be seen. These are the so-called pseudostomata, or small apertures which lead into the lymph-canalicular system of the alveolar wall. {^Indicate the squames and small granular cells in PI. XL, Fig. 4.) BLOOD-VES.SELS OF THE LUNGS. The branches of the pulmonary artery break up into capillaries, which are distributed on the walls of the air-vesicles. At the root of the lung the branches of the pulmonary artery and vein and bronchus (p. 55) are found together, — the bronchus in the middle, with a blood- vessel on each side of it, — within the lung the pulmonary vein pursues a separate course. The bronchial arteries enter the lung at its root, and run in the adventitia of the large bronchi, which they supply with blood. Sections of them will be found near the nerve-trunks at the root of the lung (p. 55). PREPARATION. — Make sections of a lung whose blood-vessels — pulmonary artery, or vein, or both — have been injected with a carmine gelatine (p. li) or Prussian blue (p. li) mass, and mount them in dammar. EXAMINATION (L). — Notice the extremely dense plexus of capillaries on the walls of each air-vesicle. Trace a small arteriole from a large branch of the pulmonary artery, and note that it may supply two or three adjacent alveoli ; while the efferent vein usually passes off at the other side. (^Indicate the arrangement of the capillaries in PI. XL, Fig. 5.) (H). Study the capillaries ; note the dense network of more or less wavy capillaries, and especially on a septum between two adjacent alveoli. A twisted capillary may be seen lying at one time in one alveolus, at another in the adjoining alveoli. The space between the capillaries varies according as the lung has been kept distended or not. In preparations which have been placed in alcohol, the air-vesicles shrink considerably, and hence the alveolar capillaries appear relatively close to each other. The large arterial and venous branches lie in the interlobular septa, which are continuous with the adventitia. LYMPHATICS OF THE LUNG. These are very numerous, and form three systems : — (i) the subpleural lymphatics occur in the deep layer of the pleura, and communicate with the pleural cavity on the one hand, and have a direct connection with the lymphatic canalicular system, which lies in the alveolar wall, and which communicates with the pseudo-stomata of the air-vesicles. Branches of this system pass through the interlobular septa to reach (2) the perivascular lymphatics Plate XI. LunCt iviiitem Bros.iith LUNGS. 59 which accompany the branches of the pulmonary artery and vein ; (3) the peribronchial lymphatics which occur in the adventitia of the bronchi, communicate freely with the peri- vascular lymphatics, and run towards the bronchial glands. In the walls of the bronchi of the cat large masses of adenoid tissue or lymph-cords are easily found (Klein, Sanderson, and Stirling). They are not nearly so abundant in the human lung. PEEPARATION. — The lymphatics of the bronchi may be injected by the puncture method ; but a good idea of their arrangement may be obtained by the student from the lungs of a coal-miner who has suffered from anthracosis. The particles of soot and charcoal are carried into the lymphatics, which therefore appear black. Make a vertical section through the pleura and lung-tissue of such a lung, which has been hardened in alcohol, and mount it in Farrant's solution. EXAMINATION (L).— Observe the black pigment distributed in the lung. Note that the superficial layer of the pleura has no pigment in it, though the deep layer is markedly pig- mented. Valves prevent the passage of the pigment from the deep into the superficial lymphatics of the pleura. Trace from the pleura inwards strands of pigment in the inter- lobular septa, in the outer coats of the arteries and bronchi, and it may be seen here and there between the air-vesicles, and also in the adventitia of the bronchi and arteries. (Compare Klein's 'Anatomy of the Lymphatic System,' pt. ii., 1875.) The Nerves of the Lung are very numerous, and enter it with the bronchi. As already indicated, they may be seen in a section of the root of the lung. They lie outside the cartilages in the bronchial adventitia, and are accompanied by branches of the bronchial arteries. Five or six strands of nerve-fibres may be found in one section. Many ganglia (Remak, Klein, and Stirling) are found in their course, and are often seen in section. They can be easily isolated from a bronchus with the aid of a dissecting microscope (p. xxxv). The Nerves of the Trachea. — I have recently discovered a large number of ganglia inter- calated in the course of the nerves, lying just outside the posterior muscular wall of the trachea. Some of them may be seen in sections of the trachea. 13 6o PRACTICAL HISTOLOGY. ALIMENTARY CANAL, AND THE GLANDS CONNECTED WITH IT. THE LIPS. PREPARATION. — Place small pieces of the lips of a dog or cat in chromic acid and spirit mixture for two weeks, and, when they are hardened, make vertical sections of them in the ordinary way. Stain a section with picrocarmine, and mount it in Farrant's solution. EXAMINATION (L).— Observe the hair-follicles on the outer surface of the lip, and the inner surface covered by stratified squamous epithelium, and if the animal's skin was pigmented the deeper layers of the epithelium contain black pigment or melanin. Between these two surfaces observe the transverse sections of the orbicularis oris muscle, its muscular bundles surrounded by connective tissue, continuous with the connective tissue of the rest of the lip. All the connective tissue is red, the glands and muscle yellowish, and their nuclei deep red. Numerous sections of blood-vessels and nerves are seen. (H). Place the stratified epithelium in the field of the microscope and examine it. Observe the epithelium, and study particularly the prickle-cells and the pigment in the cells of the deeper layers. The various layers of a hair-follicle are easily made out, and the sebaceous glands opening into them are easily studied (compare hair-follicles, p. 93). TONGUE. The muscular substance of the tongue is enclosed in a mucous membrane, covered with stratified epithelium. The mucous membrane on the back and sides of the tongue is provided with papillae — filiform, fungiform, and circumvalate — and on these are small secondary papillae. There is a thin sub-mucous layer, whose connective tissue is continuous with that surrounding the muscular substance. The muscular substance consists of longitudinal, transverse, and vertical bundles of striated muscle. In the posterior part of the dorsum of the tongue are to be found mucous and serous glands and adenoid tissue, and on the sides of the tongue the papillae foliatse, in which lie the taste-bulbs. PREPARATION. — Harden the tongue of a cat,and also a part of the human tongue, in chromic acid and spirit mixture for two weeks, and make transverse sections. Stain one with logwood and mount it in dammar, and stain another with picrocarmine and mount it in Farrant's solution. EXAMINATION (L). — Observe the papillae, confined to the dorsum of the tongue, beset with secondary papillae, and covered with epithelium ; beneath these the scanty sub-mucous coat, containing sections of blood-vessels. In the middle of the muscular substance a verti- cally placed septum, and muscular fibres running transversely out from it. Observe muscular TONGUE. 6 1 fibres disposed vertically, and terminating above in tendons continuous with the connective tissue of the mucous membrane. Immediately under the mucous membrane of the dorsum notice the transverse sections of longitudinal muscular fibres. Between the muscular fibres many fat-cells are seen, and excellent sections of nerves, and the branches of the ranine artery are found in the lower part. {Indicate the general arrangement of the part^ in one half of V\. XII., Fig. I.) (H). Study the papilla;, and the epithelium covering them. The upper layers of the epithelium are often horny, especially in animals. Notice the transverse sections of muscular fibres — polygonal in shape — with the nuclei placed just under the sarcolemma (PI. XII., Fig. 2). POSTERIOR PART OF THE TONGXIE.—Prepare this just as the anterior part, and make vertical sections. Here the mucous membrane is much thicker and looser, and contains many secreting glands, lymphatic follicles, and diffuse adenoid tissue. Stain a section with logwood and mount it in dammar. EXAMINATION (L). — Perhaps the section may have passed through a circumvalate papilla.. Study specially the secretifig glands, which are of two kinds, mucous and scro7is (v. Ebner). The mucous (L and H) glands are like similar glands in the mouth and oesopha- gus (p. 65). Each gland consists of a duct and a secreting part. The duct passes vertically and opens on the free surface, often with a funnel-shaped mouth. It has a wide lumen, and a membrana propria with oval nuclei, lined by a layer of columnar epithelium. The duct branches and terminates in the secretory part, which consists of more or less convoluted tubes, so that each gland is a compound tubular gland. The cells lining the secretory alveoli consist of a single layer of low columnar epithelium, and their appearance is very characteristic, especially when they are distended. They are clear and transparent, with a thin oval nucleus pushed to one side. They contain a delicate network of fibrils distended with ' mucigen,' which yields mucin. During a state of exhaustion these cells are more ' granular.' The serous glands (L and H) always bear a relation to the taste-bulbs, and these ducts open into grooves which contain these bulbs. The secreting epithelium is columnar with a spherical nucleus, and the protoplasm is very granular, due to the existence of a dense fibrillar network. This markedly granular character at once distinguishes them from the clear transparent cells of the mucous glands. They seem to secrete a watery fluid. In the same section patches of adenoid tissue may be found. Double-Staining of the Tongue with Logwood and Iodine Green. — Stain a vertical section of the posterior part of the tongue lightly in logwood, wash it thoroughly, and then stain it slightly in a weak watery solution of iodine green (p. xlv) ; mount in dammar. The striking feature is that the acini of the mucous glands are stained of a bright green, while the epithelium in the efferent duct is of a logwood tint. A high power reveals the nuclei stained with logwood. The serous glands do not take on any of the green pigment, so that the two kinds of glands stand out in striking contrast. Make a similar preparation with picrocarmine and iodine green as indicated for the small intestine (p. 69) ; mount in dammar. All the muscle will be yellowish-red, the connective tissue deep red, and the adenoid tissue and mucous glands bright green. Blood-vessels of the Tongue. — Mount a vertical section of an injected tongue in dammar. The blood-vessels present nothing peculiar. Trace branches into the papill<-E. If the papilla be simple it contains a single capillary loop ; if compound, each secondary papilla contains a similar loop. {Fill in the blood-vessels in one half of Y\. XII., Fig. i.) Nerves of the Tongue. — Many sections of the hypoglossal nerve will be found near the 62 PRACTICAL HISTOLOGY. ranine artery in any of the above sections. In the course of the Hngual and glosso-pharyngeal nerves, not unfrequently fine ganglia are seen. I have often seen them in sections, and isolated them from these nerves (Remak, Kolliker, Stirling). TASTE-BULBS.— The papilla; foliatse on the sides of the back part of the rabbit's tongue are hardened in the same way as the tongue. Make vertical sections through the leaflet-like papilhe. After staining them with logwood, mount in dammar. They are also found on the circumvalate papilla;, but the papillee foliatae are preferable. They are also found on the pos- terior surface of the epiglottis. EXAMINATION (L). — Observe the flattened papillae lying side by side ; and on each side of a papilla the four oval or flasklike taste-bulbs. At the base of the papillaj the ducts of serous glands (p. 6i) may be met with (PL XII., Fig. 3). (H). Study a taste-bulb made up of narrow epithelial cells arranged like the staves of a barrel, investing modified epithelial cells, each provided with a fine process, which projects through an opening at the upper part of the investing cells (PI. XII., Fig. 4). The 'inner' and 'outer' cells may easily be isolated by teasing out a small piece of a papilla, hardened in a quarter per cent, osmic acid solution. TOOTH. UNSOFTENED TOOTH. PREPARATION. — The same as that employed for dense, dry bone (p. n). It is better to purchase a section ready-made than to spend time in grinding down a section. EXAMINATION of a vertical section (L). — Observe the dentine or ivory forming the great mass of the tooth and surrounding the pulp-cavity in the centre, the enamel covering the crown and sides of the dentine and the crusta petrosa or cement, a layer of bone without Haversian canals covering the fang. In the enamel are a number of concentric dark lines— ' contour lines ' — running across it. In the dentine, note the wavy lines and their varying direction — the dentinal tubules. They stand out clearly because they are filled with air. Arched incremental lines may be seen running across the dentine. (H). Dentine. — In the homogeneous matrix the dentinal tubules, which open by one end into the pulp-cavity and run out with a wavy course through the dentine, dividing dichoto- mously, and giving off many anastomosing lateral branches, end in the outer part of the dentine, either in loops, or they open into irregular spaces — the interglobular spaces. Cement or Crnsta Petrosa. — Like bone but without Haversian canals. Some of the lacunae may open into the interglobular spaces. Enamel. — Observe the transversely striated polyhedral prisms set in groups on the dentine. If cut transversely, they are seen to be hexagonal. Not unfrequently cracks are seen in the enamel where it rests on the dentine, and these cracks appear black from being filled wjth air. SOFTENED TOOTH. PREPARATION. — Take the lower jaw of a cat or dog, and carefully free it from its muscles, but retain the periosteum. Cut it in pieces of an inch long with a saw, and place the pieces in Plate M Tongue & Tooth >Entern. Bi-os EtH. TOOTH. 63 a large amount of chromic acid and nitric acid fluid until they are sufficiently decalcified, which may be ascertained by piercing them with a needle. Decalcification takes place in two to three weeks if the fluid be frequently renewed. Wash the pieces frequently in water to remove the surplus acid, and then transfer them to spirit until sections are required. Make a number of vertical sections through the jaw, and the tooth implanted therein. Make a number of sections across the long axis of a tooth and keep them till they are required. In the process of softening, the enamel disappears, owing to the small amount of organic matter present. Stain a vertical section and a transverse section in picrocarmine, and mount each in Farrant's solution. Vertical section of a softened tooth. EXAMINATION (L). — Observe the absence of enamel, though the general distribution of the parts of the tooth is the same as in the unsoftened tooth. Notice the tooth planted in the alveolus, which is lined by a membrane stained red — t\\Q periodontal vianbrajie — which serves as a periosteum for the bony wall of the alveolus. Trace its continuity with the fibrous tissue of the mucous membrane of the gum. In the lower jaw-bone, observe the medullary cavity with branches of a nerve and blood-vessels. They are very apt to fall out, however. The pulp-cavity is seen to contain the pulp. {Indicate the general arrangement of the parts in PI. XII., Fig. 5.) (H). Observe the dentinal tubules, which are, however, less distinct than in the dry tooth ; the matrix is stained yellow. Here and there transverse sections of the tubules may be seen ; they appear as fine dots in a homogeneous matrix. Notice the crusta petrosa with its bone-corpuscles. In the pulp-cavity traces of blood-vessels, delicate connective tissue, and, it may be, a layer of columnar cells — the odontoblasts — may be found lying on the dentine. Place a similar section in one per cent, osmic acid for four hours, mount it in Farrant's solution, and examine. It shows the same details more clearly. Transverse section of a softened tooth. EXAMINATION (L and H). — Observe the pulp- cavity and the dentinal tubules radiating from it, and many of them cut transversely. DEVELOPING TOOTH. PEEPARATION. — Place the head of a new-born rat, kitten, or puppy in a large quantity of Mtiller's fluid for two days, and then into a sixth per cent, solution of chromic acid for two days. At the end of another two days, substitute for it the chromic and nitric acid mixture (p. xxxiii) until complete decalcification takes place. Make vertical sections through the jaw, and pass them through a five per cent, solution of sodic bicarbonate, to remove all trace of acid. Stain a section with picrocarmine, another must be left in one per cent, osmic acid for several hours, and mount both in Farrant's solution. EXAMINATION (L). — Observe the general shape of a tooth-germ still imbedded in the gum. Note the dentine and, it may be, the cap of enamel ; inside the dentine, the large mass of tissue which becomes the pulp. (H). Lining the dentine, observe a layer of columnar cells — the odontoblasts— which give off fibres — the fibres of Tomes — which pass into the dentinal tubules. 64 PRACTICAL HISTOLOGY. SALIVARY GLANDS. The salivary glands do not all possess the same structure, nor do corresponding glands in different animals exactly resemble each other ; hence the necessity for examining the various glands of different animals. There are three kinds of salivary glands, which differ in structure and in the nature of their secretion : — (a) True Salivary Glands — as the sub-maxillary gland of rabbits and the parotid of man and some mammals. {b) True Mucous Glands — as the sub-maxillary glands of cat and dog, and the human sublingual. (c) Muco-salivary Glands — as the sub-maxillary gland of man and guinea-pig. They are all compound tubular glands, made up of branched tubes of varying lengths. The ducts in all are practically the same. They are lobulated and supported by a connective- tissue framework. The nature of the lumen, and the characters of the epithelium lining the alveoli, constitute the chief differences between them. In the true salivary glands («) the lumen of the alveoli is small, and they are lined by a single layer of lovv- columnar epithelial cells, each with a nucleus near its attached end. These cells are markedly 'granular,' and, as Heidenhain has shown, they vary in appearance during rest and vi'hen they are secreting. In true mucous glands {b) there are two kinds of cells — one, mucous cells, moderate!}- tall, clear columnar epithelial cells, each with a small nucleus pushed cjuite out to the membrana propria of the alveolus. They are in fact not unlike goblet-cells, and contain a fine network, whose meshes are filled with mucigen (Heidenhain) ; this yields mucin, which stains deeply with logwood. Outside these, at various parts between them and the al\-eolar walls, are the ' crescents ' (Gianuzzi), or parietal granular cells. They are small nucleated polyhedral cells. In muco-salivary glands (c) are found alveoli, which present the characters of true salivary glands, and others exactly like those of mucous glands. PREPARATION ((?). —Place the sub-maxillary glands of a dog and rabbit in absolute alcohol for forty-eight hours. Make transverse sections. Stain with logwood, and mount one in dammar and another in Farrant's solution. This is the best method for a general survey. (/;) Harden another gland in chromic acid and spirit mixture for a week, and stain sections with logwood and mount in Farrant's solution. {c) Small pieces, the size of half a pea, may be hardened in a quarter per cent, osmic acid, and small pieces teased in glycerine, or sections may be made and mounted in glycerine. It brings out the ' crescents ' very clearly in the mucous glands. Sub-maxillary Gland of a Dog. EXAMINATION (L). — Observe the lobulated character of the section ; the fibrous capsule sending septa into the gland, and containing blood-vessels, lymphatics, and nerves. Notice the alveoli or gland-substance cut in every direction, and in the interlobular septa here and there a large duct — lobar — cut into. (H). Study a transverse section of a large lobar duct. Notice the columnar epithelium lining it, with vertical lines due to the longitudinal arrangement of the fibrils, and the nucleus placed about the middle of the cell. Within a lobule may be found sections of an intra-lobitlar djict, which have a small lumen lined by columnar epithelium. Study an Alveolus. — Observe the secretory epithelium lining it. The epithelium may be SALIVARY GLANDS. 65 quite clear, with its small nucleus near the alveolar wall. Study the lunate cells or crescents of Gianuzzi. {Indicate the alveoli, the cells lining tkein, and a lobar duct in PL XIII., Fig. i.) Sub-maxillary Gland of a Eabbit : EXAMINATION (L). — Observe the same general characters. (H). Study an Alveolus.— Observe the small lumen and the single layer of low columnar cells lining it. Compare it with the above description. TONSILS. The tonsils are folds of mucous membrane containing a large number of lymph-follicles like those composing a Payer's patch (p. 69). The mucous membrane is folded so as to eave pits or depressions visible on the surface. The epithelium covering them is stratified, and under it is a connective-tissue mucosa, which contains the closely aggregated masses of adenoid tissue. PREPARATION. — Harden the tonsils for two weeks in chromic acid and spirit mixture, or in Muller's fluid for three weeks. Make transverse sections, stain with logwood, and mount in dammar. EXAMINATION (L). — Note the stratified epithelium, and under it the oval or rounded masses of adenoid tissue, which sometimes penetrate into the epithelium. (H). The ordinary structure of adenoid tissue. CESOPHAGUS. PREPARATION. — Slightly distend the oesophagus of a dog with the chromic acid and spirit mixture, and keep it distended by placing a ligature on its upper and lower ends. Suspend it in a large quantity of the same fluid until it is ' fixed ' — i.e. for twenty-four hours — then cut it into pieces one inch long and continue the hardening for a week or ten days. Make transverse sections through the upper and lower parts of the cesophagus. Stain them with picrocar- mine, and mount in Farrant's solution. Transverse section of the (Esophagus. EXAMINATION (L). — Observe the mucous coat lined b\' stratified epithelium with well-marked mucous glands opening on its surface. The mucous membrane is beset with small papillae. In the deeper part of the mucous coat observe the cut ends of the muscularis mucosae, composed of non-striped muscular tissue arranged in bundles. In the sub-mucous coat observe the alveoli of the mucous glands and trace a duct to the surface. Observe the muscular coat — the outer or longitudinal layer — cut transversely, and the circular fibi'es inside it. These contain much striped muscle in the upper part of the oesophagus. (H). Study the stratified epithelium and the mucous glands, which are exactly like those already described in the mouth (p. 61). A preparation mounted in dammar shows their ' mucous ' characters admirably. Nerves of the (Esophagus. — The nerve-terminations are best studied in the cesophagus of a rabbit by means of the lemon-juice and gold method. The process is described at p. 71, in connection with the small intestine. K 66 PRACTICAL HISTOLOGY. STOMACH. PREPARATION. — Open the stomach of a cat, dog, or rabbit, and wash away the layer of mucus lining it with a stream of salt solution. (fl) Harden small pieces of the pyloric and cardiac ends and middle of the stomach for two weeks in the chromic acid and spirit fluid (p. xxxi). Complete the hardening in spirit. Make vertical sections of each piece. Stain sections with logwood, and mount them in dammar; and others with picrocarmine, and mount them in Farrant's solution. (/') After washing out a stomach of any of the above animals with salt solution, distend it with and place it in absolute alcohol — or merely small pieces may be hardened in absolute alcohol. These show gland-structure well, and stain readily. ic) Very small pieces, not larger than a pea, are to be hardened in a quarter per cent, osmic acid for twenty-four hours. Make sections and mount them in Farrant's solution. CARDIAC END OF STOMACH. Vertical Section. (Logwood and dammar.) EXAMINATION (L). — Observe the mucous coat with its gastric glands placed vertically. Trace a gland and note whether it branches below or not. The bases of the gland rest on a small amount of connective tissue, which sends up processes between the glands. Immediately outside this lies the muscularis mucosae, which consists of two layers — longitudinal and circular — and sends processes upwards be- tween the glands. {Indicate this general arrangcincni in PI. XIII., Fig. 2.) The sub-mucous coat composed of connective tissue containing blood and lymph-vessels, fat-cells, and nerves. The muscular coat, consisting of two coats, with perhaps a third. The appearance of these coats varies according to the plane of their section ; and outside this, the serous layer, or peritoneum. If the stomach of the cat be selected, a clear band of condensed connective tissue will be observed lying immediately above the muscularis mucosae. This preparation shows well the passage of the muscularis mucosa; fibres upwards between the glands. To see the finer details of structure study the preparation mounted in Farrant's solution. (L). Observe the same general arrangement as before. (H). Study z. peptic gland. Note the clear, tall, narrow, columnar epithelium lining the stomach, and continued some distance into it. The nuclei of the cells are placed near their attached ends. The cells appear in some cases to be open at their free ends, especially if the animal be killed during digestion, and are, in fact, mucus-secreting goblet-cells. Note that the lower part of the cell is always granular, even though the upper two-thirds be quite transparent. In the middle and lower part of the gland note the coarsely granular nucleated outer, ovoid, or peptic cells, which frequently cause a bulging on the side of the gland ; and, internal to these, small ill-defined inner or central cells, best seen at the lower part of the gland. A small narrow lumen may be detected in the gland. The cells most deeply stained are the ovoid cells. Study the mus- cularis mucosae, and note that it consists of two — sometimes three — layers of non-striped muscle, and trace processes from it upwards between the gland. Transverse sections of the glands are to be met with in the same section. They are usually in groups of four or five, and separated by a little connective tissue and a few non- Pla:te xm Sub-maxillary Gland &, Stomach Minterx-- Bros litK STOMACH. ■ 67 striped muscular fibres. Observe the relation of the outer and inner cells, and notice the small lumen of the gland. {Indicate the appearance of transverse sections of the tubes in PI. XIII.. Fig. 4.) Aniline Blue. Stain a section with a watery solution of aniline blue. Mount it in glycerine or dammar. The peptic cells are most deeply stained. PYLORIC END OF STOMACH. EXAMINATION.— Observe the mucous coat, containing glands (mucous) which branch frequently, and do not contain any large ovoid cells. The gland-ducts are relatively long, and two or three tubes open into a short neck. The gland-tube is lined by the same kind of epithelium throughout. Notice transverse and oblique sections of the gland at the lower part of the mucous membrane. Here and there, near the base of the gland, detached patches of adenoid tissue may be seen. {Indicate the general arrangement in PL XIII., Fig. 5.) Take another section, stain it with carmine, and mount in Farrant's solution. Notice the same general arrangement as above. Observe that the glands are lined throughout with columnar epithelium. Study a transverse section, and note the basement-membrane of a gland. Notice a few leucocytes lying near the bases of the glands. Isolation of the Gastric Glands. PREPARATION. — Place small pieces of the mucous membrane of the stomach of a rabbit in five per cent, ammonium chromate for three days. Wash away the colouring matter from a small piece of the membrane, and place it in picro- carmine solution for twenty-four hours ; tease a small piece in glycerine. This method shows the shape and general characters of the glands, and their cellular contents, the ovoid cells being specially distinct. The nucleus is stained of a bright red, and a beautiful intra-cellular plexus of fibrils is revealed. Note also the basement-membranes of the gland-tubes. {Indicate the cells in the gland-tube in PI. XIII., Fig. 3.) The osinic acid preparations show the peptic cells very well. They are more deeply stained than the other cells, and hence they stand out more clearly. Blood-vessels of the Stomacli. PREPARATION. — Inject the blood-vessels of the stomach of a cat or dog through the gastro-duodenal artery with a carmine gelatine mass (p. li), and place the organ in alcohol. Make vertical sections and stain them with logwood and mount in dammar. If a blue mass be used stain the sections with carmine. EXAMINATION (L). — Observe the vascularity of the mucous membrane ; sections of the large vessels will be found in the submucous coat ; and from these capillaries proceed upwards between the glands, and form a capillary plexus round them. The veins rise near the surface, and run downwards towards the submucous coat. DUODENUM. SMALL INTESTINE. PREPARATION. — Wash out the contents of the duodenum of a dog or cat with a stream of salt solution, and place pieces one inch long in chromic and spirit fluid for two weeks. If the duodenum of a pig can be obtained, it is to be preferred. Place similar pieces of the small intestine some with, others without a Peyer's patch, in the same hardening fluid for the same time. Complete the hardening in alcohol. Make transverse sections. Stain a section of the 68 PRACTICAL HISTOLOGY. duodenum with logwood, and mount it in dammar. Stain sections of the intestine — with and without a Peyer's patch — with logwood ; mount one in dammar, and another in Farrant's solution. Place sections of the small intestine in a quarter per cent, .solution of osmic acid for twenty-four hours, and mount a very thin section in Farrant's .solution. Vertical Section of the Stomach and Duodenum. — Make a vertical section through the pyloric valve, so as to include a part of the stomach and the upper part of the duodenum. Stain a .section in logwood, and mount it in dammar. EXAMINATION (L). — Ob.serve the gastric mucous membrane with its mucous glands, and trace a gradual transition from these into the compound tubular and more branched glands of the duodenum — Brunner's glands, which present exactly the same structure as the mucous glands of the stomach, with this difference, that their duct is larger, and each duct has a greater number of long branched tubes attached to it. The alveoli lie in the submucous layer outside the muscularis mucosie, and from these the duct lined by columnar cells pierces the muscularis mucosa;, and ascends between the crypts of Lieberkiihn to open on the surface. A few villi are seen on the duodenal mucous membrane. At the line of junction of the mucous membrane of the stomach and duodenum the muscularis mucosae is absent. SMALL INTESTINE. Transverse Section. (Logwood and dammar.) EXAMINATION (L). — Mucous coat.— Observe the conical pointed villi ; .some of them may be contracted and show the epithelial covering thrown into folds. Each villus consists of a central core covered with columnar epithelial cells, with their nuclei placed near the attached end of each cell. Beneath the villi the mass of the mucous membrane is made up of Liebcrkuhns follicles, consisting of simple tubular glands lined by epithelium. Note the adenoid tissue between the bases of the glands, and outside this the muscularis viucosce. The subunicous coat consists of connective ti.ssue containing blood-vessels and nerves. Muscular coat. — Note the thick inner circular layer, which will appear different according as the section has been made across or parallel to the long axis of the gut ; if the latter, note that the muscular fibres are arranged in blocks separated and surrounded by connective tissue ; outside this the narrower longitudinal layer, and outside all the serous coat. This specimen does best for a general study of the relation of parts. Select a villus and examine it with (H). Notice the long fusiform nuclei in that part of the core of the villus, next the central lacteal vessel. They are the nuclei of non-striped muscular fibres, which pass from the muscularis mucosae into the villi. (^Indicate the general arrangeincHt in one-half of Y\. XIV., Fig. i.) Examine now a similar preparation stained with logwood, but mounted in Farrant's solu- tion. Observe the same relation of parts as described above. Fix a villus in the field, and observe (H) the epithelial investment, consisting of columnar nucleated cells with a clear hem containing vertical striae (compare p. lO, on epithelium). Observe goblet-cells scattered between the epithelial cells and study them. [Indicate the covering and structure of a villus in PI. XIV., Fig. 2). Search for these cells seen en face. In the substance of the villus observe the adenoid tissue of which it consists, with nuclei in its meshes, and perhaps a lacteal vessel in its centre. Lieberkiiiin's Glands. Study one. Observe its test-tube shape, and the low nucleated columnar epithelium lining it. At their bases and outside them observe adenoid tissue. Study the two layers of the muscularis mucosa;. SMALL INTESTINE. bg Submucous coat. — Observe its blood-vessels, and perhaps nerve-ganglia {Meissners plexus), and perhaps a solitary gland. Muscular Coat. — Observe the characters of non-striped muscle cut transversely and longi- tudinally. PEYER'S PATCH. Transverse Section. (Logwood and dammar.) EXAMINATION (L). — Observe the masses of adenoid tissue in the submucous coat. Their conical points project upwards into the gut, and are covered by epithelium, but no villi e.\;ist over them. Treble-staining of the Small Intestine. PREPARATION. — Take one of the transverse sections of the small intestine, containing a Peyer's patch. Stain it with picrocarmine, wash it lightly in water slightly acidulated with acetic acid. Stain it now with solution of iodine green (p. xlv) until it becomes slightly green, wash it quickly in water, and mount it in dammar, taking care that it does not remain too long in the alcohol. EXAMINATION (L). — This is an exquisitely beautiful preparation. All the glands, and especially Peyer's patches, are green, all connective tissue is red, and so the submucous layer stands out bright red. The muscularis mucosa; and muscular coats are yellow. Observe the interruption of the muscularis mucosa, where the adenoid follicles reach the surface. A thin layer of connective tissue lies superficial to the muscularis mucosae. Such preparations keep a long time. In the preparations steeped in osmic acid, after hardening with the chromic acid mixture, the various details are seen with exquisite clearness. Blood-vessels of the Small Intestine. — Make transverse sections of the small intestine of a cat or dog whose blood-vessels have been filled with a carmine and gelatine mass. Mount in dammar. It is easy to inject the intestine from the superior mesenteric artery. EXAMINATION (L). — Observe the villi, and note their vascularity. Usually one artery ascends on one side, and splits into capillaries, which are distributed closely under the epithe- lium. The vein descends on the opposite side of the villus. Note the rich plexus of capillaries surrounding Lieberkiihn's glands, and the large arterial trunks in the submucous coat. Study the distribution of the blood-vessels in the muscular coat, and observe that it is not so vascular as the mucous coat. {Indicate the arrangement of the blood-vessels in one half of Vl. 'K.YV ., Fig. I.) Injected Villi seen from above. — Mount in dammar a small piece of the small intestine of a rabbit whose blood-vessels have been injected with a carmine and gelatine mass. Place the section in the slide so that the villous surface is uppermost. EXAMINATION (L).— Observe the leaflet-like injected villi directed towards the observer, and by focussing through the thickness of the preparation the course and distribution of the larger arterial trunks in the submucous coat can be clearly made out. Lymphatics or Lacteals of the Small Intestine. — The origin of the lacteal within the central part of a villus as a blind tube has already been noted (p. 6'&), but the branches of the lymphatics in the walls of the gut can easily be studied, after injection with Berlin-blue, by the puncture method ; an excellent view of their arrangement will be obtained in the prepara- tion of the intestine treated with silver nitrate (p. 37), to show the cement-substance of muscle. This will be referred to again under lymphatics (p. 76). 70 PRACTICAL HISTOLOGY. VERMIFORM APPENDIX. PREPARATION.— Inject the walls of the vermiform appendix of a rabbit with a two per cent, watery solution of Berlin-blue, which readily passes into the lymphatics. Harden in alcohol, and make transverse sections. Stain them with picrocarmine, and mount in Farrant's solution. EXAMINATION (L). — Observe the masses of adenoid tissue, stained red, divided into an inner and an outer set, and round each the lymph-paths filled with Berlin-blue. The masses of adenoid tissue are exactly like Payer's patches, and each arc is partially surrounded by a lymph-stream, as indicated by the blue injection, none of which passes into the interior. This space represents a lympli-simis, so that the masses of adenoid tissue are partially suspended in a lymph-stream. Exactly the same relation obtains in a Payer's patch. {^Indicate these appearances in PI. XIV., Fig. 5.) LARGE INTESTINE. PREPARATION. — After washing out the large intestine of a dog or cat, cut it into pieces one inch square and harden them in chromic acid and spirit fluid for two weeks and complete the hardening in spirit. Make transverse sections. Stain with logwood, and mount one in dammar, and another in Farrant's solution. Vertical Section of the Large Intestine. (Logwood and dammar.) EXAMINATION (L). — Observe the mucous coat thrown into folds, owing to the contraction of the muscular coat. It is devoid of villi. Observe the vertically set Lieberkiihn's glands, exactly like those in the small intestine. {Indicate one in PI. XIV., Fig. 3.) Some of them are sure to be cut trans- versely, when they present a honeycomb-like appearance. (Indicate this in PI. XIV., Fig. 4.) Observe the sub-mucous and muscular coats, very like those of the small intestine. Per- haps a solitary gland may be found in the sub-mucous coat with its apex projecting free into the mucous coat. There is a well-marked muscularis mucosae in the mucous coat, which is pierced by the solitary glands. In the preparation mounted in Farrant's solution, examine (H) the glands, both when divided vertically and transversely, and observe the adenoid tissue laden with lymph-corpuscles, between and supporting them. Solitary glands will be found in sections of the large intestine. They are simply masses of adenoid tissue placed in the sub-mucous coat. They are easily stained by the same methods as directed for Payer's patches (p. 69). Blood-vessels of the Large Intestine are prepared exactly in the same way as those of the small intestine. Several years ago it was customary to use an opaque injection, which gives a beautiful effect when the piece of intestine is mounted upon a dark ground with the mucous surface uppermost, and illuminated by means of a condenser. The mouth of each gland is surrounded by a plexus of capillaries, so that the injection presents a regular honeycomb- like appearance. Similar preparations made of the gastric mucous membrane at first sight resemble that of the large intestine. The gastric mucosa is recognised by the existence of small openings at the bottom of the depressions, and the latter are not so regular as those in the large intestine. ^=^LATE3av: Small & Lj\rge iNTEST^ffi / \ r\ 1 /. / "MitM.ei-n. Bros 1il1"i LARGE INTESTINE. 71 NERVE-PLEXUSES OF THE INTESTINE. There are two plexuses of nerves, consisting chiefly of non-medullated nerve-fibres and ganglionic cells. In the intestinal walls, one — plexiis myentericus of Meissner — lies in the submucous coat; the other — AuerbaMs plexus — lies between the muscular coats. PREPARATION. — Auerbach's plexus is best prepared in the following way. ia) Wash out a loop of perfectly fresh small intestine of a rabbit, and distend it with the juice of a fresh lemon ; ligature both ends of the gut and place it, still distended, in lemon-juice for from five to seven minutes. Open the ligatures and wash it thoroughly in water, and fill it with a two per cent, gold chloride solution ; ligature the gut as before, and suspend it for half an hour in a one percent. gold solution. Wash it thoroughly, and transfer it to a twenty-four per cent, dilution of formic acid, to reduce the gold. The preparation must be kept in the dark. After reduction, it has a beautiful, rich, reddish-brown colour. Wash it thoroughly and keep it in a preservative fluid (p. xl). With forceps it is easy to peel off strips of the outer muscular layer, to which Auerbach's ple.xus adheres. Mount in glycerine. The separation takes place more easily after maceration for a week in the preservative fluid. This is the best method. (3) Another method is to use dilute alcohol, as the distending and softening medium, for forty-eight hours ; then to peel off the outer muscular coat as directed above. Stain it with logwood, and mount in glycerine. This gives fairly good results. [c] One-twentieth per cent, acetic acid may be used in the same way to fill the gut and to macerate it, for thirty hours. After maceration and washing it is well to steep the gut for a short time in five per cent, solution of sodic bicarbonate, to get rid of the acid. Peel off the muscular coat as before, stain it v\'ith logwood and mount it in glycerine. EXAMINATION (L). — Observe the v/ide-meshed plexus of fairly regular form. At the nodes, groups of ganglionic cells are to be detected. Ganglionic cells are either in groups, or sometimes disposed lengthwise in the flat, band-like nerve-branches. Each band has an endothelial sheath. (H). The cells are very variable in size, and some of them are multipolar. Nerve-branches are given off from this plexus to supply the longitudinal and circular muscular coats, but their mode of termination is unknown. The Plexus of Meissner is prepared in a similar way, and it supplies the muscularis mucosae, the blood-vessels and glands of the mucous coat. Its meshes are much wider and less regular than Auerbach's. The nerve-cells are easily seen in it. In vertical sections of a cat's small intestine, hardened in chromic acid and spirit, groups of ganglionic cells may easily be detected in the sub-mucous coat. These are the ganglia of Meissner's plexus. THE LIVER. It is well to examine the liver of several animals as they differ in the complexity of structure. It is best to begin with a pig's liver, and to pass to that of a rabbit, a dog or cat, and then to the human organ. The liver may be hardened in several ways. PREPARATION. A. Chromic Acid and Spirit Mixture. — Place pieces of a perfectly fresh liver (pig, rabbit, cat or dog, and man), about half an inch square, in a large quantity of the above fluid. Do not wash them. As the liver is very vascular, much blood exudes and causes a precipitate in the fluid, which must be changed at the end of twelve hours. As soon 72 PRACTICAL HISTOLOGY. as the fluid appears turbid it ought to be changed. The hardening will be completed in two to three weeks. Wash the tissues well, and transfer them to spirit until they are required. B. Miiller's Fluid.— Larger pieces of the liver may be hardened in this way, as this fluid penetrates organs more readily than A. It takes, however, longer to harden (four to six weeks). C. Two per cent. Potassic Bichromate, which acts in the same way, and in the same time, as Miiller's fluid. Make sections of the liver of pig, rabbit, cat or dog, and man, by means of a freezing microtome, and see that the sections are thin and made through the fibrous capsule. Some of them are to be stained, and, for non-stained preparations, place the sections in one per cent, osmic acid for four or five hours. This is an excellent method, and sharpens the outlines of all the tissues. I. LIVER OF A PIG. EXAMINATION (L). — Stain a section with logwood, and mount it in dammar. Observe the fibrous capsule, and note the processes it sends into the organ, where they become continuous with the fibrous sheath which surrounds each lobule. Note that the section is mapped out into a number of distinct, more or less polygonal areas of nearly equal size by bands of connective tissue stained blue ; these areas are the lobules, and each one is com- pletely invested by a fibrous sheath — a continuation of Glisson's capsule. Within each of these areas is the true gland-substance, which will be considered in other preparations. This pre- paration gives the student an excellent idea of the lobular character of the liver. {Indicate the lobules ill PI. XV., Fig. i.) 2. LIVER OF A RABBIT. Logwood and dammar. Note that the liver-lobules are not nearly so accurately mapped off from each other, but still polygonal areas— sections of the lobules — can be seen mapped off here and there from each other by a very small quantity of connective tissue — interlobular comiective tissue, or Glisson's capsulc^s\.d.med blue. {Indicate these appearances in PI. XV., Fig. 2.) 3. THE LIVER OF A DOG AND OF MAN. These ought to be prepared and studied in the same way. In them the outlines of the lobules are even less distinct, owing to the lobules being more or less confluent, due to the small amount of interlobular connective tissue. It is, therefore, the amount of interlobular con- nective tissue which determines the mapping out of one lobule from another. Note particularly the thickness of the fibrous capsule, and the small amount of interlobular connective tissue in a normal human liver. This is essential, as in some diseases — e.g. cirrhosis — it is increased in amount, and it is necessary to accustom the eye to the normal amount. Continue the examination of the human liver with (L). Observe the capsule, and note its thickness, and that it consists of two layers ; the lower one sends very fine processes into the organ between the lobules. These processes are better seen in a section where the liver-cells have been pencilled or shaken out, as directed for adenoid tissue (p. 29). Study a lobule. In its centre, if it be cut transversely, observe the hepatic or intra-lobularvein, and note its size ; for it is sometimes dilated in diseased conditions, especially where there is congestion of the lungs and right side of the heart. Radiating outwards from this, observe the columns of nucleated liver-cells, united at the outer part of the lobule by transverse branches to form a Plate .Cv' li'/er Muite; n oros lith THE LIVER. 73 network, [Indicate the columns of liver-cells in PL XV., Fig. 3.) The radiate arrangement is best seen in the columns of cells nearest the hepatic vein. Between these cell-columns narrow spaces exist, which indicate the position of the intra-lobular blood-capillaries. In an unin- jected liver the capillaries appear relatively small. Search between lobules for a transverse section of a branch of the portal or interlobular vein, hepatic artery, and bile-duct, all of which run to- gether and are surrounded by connective tissue, which constitutes part of Glisson's capsule. [Indicate these structures in PI. XVI., Fig. 3.) The channels in which they lie are known as portal canals. Fix the bile-duct in a field and examine with (H). The bile-duct may be easily distinguished on account of its being lined by low, columnar, almost cubical, epithelium. In its wall may be found circularly disposed non-striped muscular fibres, and if a particularly large bile-duct be found, the walls may be seen to contain sections of mucous glands, which secrete mucin and add it to the bile as it passes along the bile-ducts. The portal vein is large, and neither it nor the hepatic artery present anything peculiar in their characters. Surrounding these is the connective tissue of the capsule of Glisson. The tissue is more or less lamellar ; the bundles run in various directions, and between them are flattened connective-tissue cor- puscles stained blue. Note its amount. A very small amount of intra-lobular connective tissue is to be found ; it is best seen around the hepatic veinlet. Study the liver-cells (H). — They are polygonal or cubical cells of nearly the same size, uniformly granular in appearance, are devoid of an envelope, and contain a spherical and deeply-stained nucleus, though some may contain two nuclei, and rare examples are met with without a nucleus. The cells at the outer part are usually slightly smaller than the others. Note particularly the network formed by these cells, and how they intertwine with the blood- capillaries between them. Each cell contains a dense intra-cellular and intra-niiclear plexus of fibrils, which give it a granular appearance (PI. XV. Fig. 4). See p. 13, where these points are alluded to. Stain similar sections with logwood and picrocarmine, and mount them in Farrant's solu- tion. In this medium the finer details can be more carefully studied. Mount an unstained section, which has been steeped in osmic acid, in Farrant's solution. This is an excellent method for sharpening the outline of the various parts. Any oil-globules present within the liver-cells are blackened, osmic acid being an excellent test for the presence of fatty infiltration or degeneration. BLOOD-VESSELS OF THE LIVER. PREPARATION. — The blood-vessels of the liver of a rabbit or other animal may be in- jected either from the portal or hepatic vein, or both. It is well to examine a liver whose vessels are injected either with a red or a blue carmine mass, or if desired the portal vein may be injected with a blue mass and the hepatic vein with a red, ox vice versd. In injecting from the portal vein the mass should flow freely from the hepatic vein, which should then be ligatured, and then the blood-vessels will fill properly. One must judge of the amount to be pressed into the vessels by the appearance and colour of the liver-lobules. Compare p. Hi for general directions. Harden the organ in two per cent, potassic bichromate and then in alcohol, or place the liver at once in alcohol. Make sections, and mount them, stained or unstained, in dammar. EXAMINATION (L). — Select a lobule which has been cut transversely, and observe the origin of the hepatic vein in its centre, and at the periphery of the lobule sections of the portal vein. Within the lobule, and connecting these two vessels, observe the blood-capillaries L 74 PRACTICAL HISTOLOGY. with a distinct radial arrangement near the hepatic vein, thou^li the ineshvvork becomes more polygonal at its outer part. Relatively the hepatic cells appear smaller than in the un- injected specimen. Sections of the hepatic vein issuing from within a lobule ought to be looked for. A double injection is, of course, very instructive, though the areas of the respec- tive veins may not be accurately mapped out. (^Indicate the general arrangcvient of the blood- vessels in PI. XVI., Fig. i.) Opaque Injections. — It is well to examine by reflected light a section of a liver whose blood-vessels have been injected by an opaque mass — say red in the hepatic vein, and j-ellow in the portal. BILE-DUCTS. PREPARATION. — Inject the fresh and still warm liver of a rabbit or guinea-pig (killed by bleeding) from the bile-duct with a cold, watery, fresh-filtered solution of Prussian blue (p. li). This is' by no means an easy task. The great thing is to use a constant and not too great pressure (p. liii). Whenever the outer part of the lobules begins to get blue, stop. It is advantageous to inject a red gelatine mass into the portal vein after the injection of the bile-ducts has been completed. Place the liver in alcohol for a few hours, then cut it into pieces, and put some in alcohol and others in Miiller's fluid for two weeks. Make sections in the usual way by freezing. EXAMINATION (L). — In a section of a piece hardened in alcohol and mounted in dammar observe the larger interlobular bile-ducts, filled with a blue mass between the lobules ; and from these fine branches can be seen to pass into the lobules, within which they form a polygonal meshwork over and between the hepatic cells. {Indicate the hilc-ducts in PI. XVI., Fig. 30 Stain a section hardened in Miiller's fluid with picrocarniine, and mount it in Farrant's solution. EXAMINATION (L). — Note the same arrangement as above, only the hepatic cells are better preserved, and their nuclei stained. (H). Select a large interlobular bile-duct and observe its lining of low columnar epithelium, and, it may be, sections of a mucous gland in its walls. The fusiform nuclei of the non-striped muscle in the wall of the bile-duct are stained red. Trace now the inti-a-lobular bile-capillm-ies or channels, and observe their polygonal shape, i.e., exactly the shape of the liver-cells, the plexus they form over and between the hepatic cells, whose nuclei are stained red. The bile- capillaries are much smaller than the uninjected spaces of the blood-capillaries. In section the blue point indicating a section of a bile-capillar)- is found in the angle where three or more cells meet. They are never found between the liver-cells and blood- capillaries, and are always separated from the capillary blood-stream by a part of one or more liver-cells. Isolated Liver-cells. — Scrape the surface of a section of a liver, and diffuse the scraping in salt solution on a slide. Compare p. 1 3, where these cells are described. Re-examine the osmic acid and picrocarmine preparations of the liver of the rabbit and newt, and in the latter study the intra-cellular plexus. Oil-particles within Liver-cells. — These are very common in stall-fed animals — e.g. ox — and are readily recognised by their highly refractive appearance, and by the action of osmic acid, which blackens them in a very short time. Plate XvT . Liver & Pancreas ^^'Iir\tert\ Brosiitk /:> PANCREAS. The pancreas in many respects resembles the salivary glands, though there are important differences. It is lobulated, and has a connective-tissue stroma, blood and lymphatic arrangements like the salivary glands. It is a compound tubular gland, and the gland-tissue has lobar and inter-lobular ducts, which terminate by means of an intermediate piece in the alveoli, which consists of wavy, branched, and convoluted tubes, each having a basement-mem- brane lined by a single layer of columnar or cylindrical cells, whose free ends are sometimes conical. The substance of each cell shows a marked division into two zones — the outer (next the membrana propria) is homogeneous, and stains easily and deeply with carmine and logwood- — the inner is coarsely granular, and does not stain readily with dyes, and in it lies the spherical nucleus. The lumen of the alveolus is small, and so are the alveoli themselves. Heidenhain has shown that the appearances and relative sizes of these two zones alter during digestion — i.e. during physiological activity, just like the salivary glands. PREPARATION {a). — Place small pieces of the freshly excised pancreas of a dog in absolute alcohol for forty-eight hours, and then make sections. Stain them with carmine, and mount in Farrant's solution, or, better still, with logwood, and mount in dammar. {U) Osmic acid preparations are also valuable. EXAMINATION (Lj.— Observe the lobules, the connective sheath and its septa, perhaps containing the section of a lobar duct. Study a lobule, and note an inter-lobular duct ; and the alveoli — relatively small — cut in every direction. (H). Study an alveolus : note the secretory epithelium, with its outer zone stained of a logwood tint, and its inner half granular and unstained. This appearance is quite characteristic. Scarcely any lumen is observable, and ver)- little interstitial matter separates one secretory cell from another. {Indicate the appearance of the alveoli, ami the epithelium lining them, in PI. XVI., Fig. 4.) 76 PRACTICAL HISTOLOGY. THE LYMPHATIC SYSTEM. The lymphatic vessels consist of capillaries and trunks of variable size. The capillaries con- sist of single layers of squames with characteristic sinuous edges, and the larger vessels have a structure resembling veins with three coats, but the walls are very much thinner. They have valves in their course. LYMPHATICS OF THE DIAPHRAGM. PREPARATION (L). — Kill a rabbit or guinea-pig by bleeding. Tie a ligature round the inferior vena cava and the gullet, and remove the abdominal viscera, so as to expose the under surface of the central tendon of the diaphragm. With a fine camel-hair pencil, brush away the epithelium covering it, and then silver it in the usual way (p. xlv). After exposure to light mount a small piece in dammar. EXAMINATION (L). — Observe the network of lymphatic vessels, some with a small, others with a large lumen. Trace the course of a large vessel, and observe the bulgings in its course with narrower parts between (PI. XVII., Fig. i). Especially at the division of a vessel a valve may be seen. (H). Study the character of the epithelium lining the vessels, and notice its sinuous outline. Lymphatics of the Intestine. — For a large class, silvered lacteals are readily seen in the preparation of silvered muscular fibre obtained from the small intestine of a rabbit (p. 37). Refer to that preparation, and observe details in the structure of these vessels similar to those described in the lymphatics of the diaphragm. The lymphatics communicate with tlu semis cavities by means of small apertures — stomata — guarded by small granular cells. The stomata are most easily obtained from the septum of the great lymph-sacs of the frog, though they may also be obtained by silvering the under surface of the diaphragm of a rabbit. Behind the stomach and on each side of the vertebral column of the frog there lies a large lymph-sac, separated only from the peritoneal cavity by a very delicate membrane or septum, which is perforated by small apertures — stomata — which bring the two cavities into direct communication. SEPTUM CYSTERN^ LYMPHATICS MAGNS. PREPARATION. — Kill a frog and carefully remove the abdominal viscera, taking care not to injure the fine septum above described. Pour distilled water over the posterior ab- dominal wall immediately behind the stomach, when a delicate membrane will be floated up and brought into view. Pour over it a half per cent, solution of nitrate of silver till it FtAiEXVII- Lymphatics ■^finterm. Bros litk LYMPHATIC GLANDS. y-j becomes slightly white ; snip out the membrane, and after washing expose it to light in the usual way. Mount a small piece in glycerine. EXAMINATION (L and H). — Observe the membrane perforated here and there by small apertures — stoinata — easily recognised by their having a brownish margin. Their margins are bounded by small, plastic, granular cells, stained brownish with the silver (PI. XVII., Fig. 2). The stomata may either be open or closed. In addition, silver outlines are seen, and by focussing through the thickness of the membrane it is seen that epithelium exists on both surfaces, and that the size and shape of the cells differ on the two sides. On the peritoneal surface the cells are elongated, while on the side directed towards the lymph-sac they are more or less polygonal and much broader. Similar stomata exist on the mesogastrium and mesentery of the frog, and in the intercostal pleura, &c., of mammals. They are prepared in the same way. LYMPHATIC GLANDS. (A.) COMPOUND LYMPHATIC GLANDS. Lymphatic glands are accumulations of adenoid tissue (p. 29) in the course of the lym- phatic vessels, and so they have afferent and efferent vessels. Each gland is surrounded by a connective-tissue capsule, which not unfrequently (ox) contains non-striped muscle, especially in the deeper layers. From its under surface a number of trabeculze carrying blood- vessels pass into the substance of the gland, thus dividing it into a number of compartments or alveoli, which are filled with a network of delicate adenoid tissue, whose meshes are in great part filled with lymph-corpuscles. The gland-substance consists of a cortical and a medullary part. In the cortex the adenoid tissue is arranged in the form of small, round, or oval nodules ox follicles, and in the medulla as interlacing cords. The lymph passes through well-marked channels in the gland — the lymph-spaces — which are spaces lying between the septa or trabeculse and the gland-substance. These spaces are lined by squamous epithelium, and across them is stretched adenoid tissue, which contains few lymph-corpuscles. The structure of adenoid tissue has already been considered (p. 29). PREPARATION {a). — Place a lymphatic gland from an o.x or other animal in Muller's fluid for a week, and then harden it in spirit and make sections. (F) Harden a gland in a saturated watery solution of picric acid for twenty-four hours, and preserve in alcohol. Stain a Mijller's fluid section with log\vood and mount it in dammar, and stain a picric acid section with picrocarmine and mount it in Farrant's solution. Lymphatic Gland. (Logwood and dammar.) EXAMINATION (L). — Observe the capsule with its septa subdividing the corte.x into a number of compartments o{ alveoli. As they pass into the centre of the gland the septa break up into trabeculcB, which run in all directions, and anastomose with each' other, forming a plexus with relatively small meshes, so that it is easy to distinguish the small-meshed medulla from the cortex. Observe the larger alveoli in the cortex. Notice the masses of leucocytes stained deep blue, and between these and the trabeculae narrow spaces, the lymph-sinuses. {Indicate the capsule, cortex, and medulla iri PI. XVII., Fig. 3). (H). Obser\-e the two layers of the capsule; the superficial layer contains connective tissue and a few elastic fibres, the deep one also non-striped muscular fibres. Study one of the trabecular, which consist of connective tissue with a few elastic fibres ; and observe the nuclei of the non-striped muscle. The trabecute arc cut in every direction, and branch 78 PRACTICAL HISTOLOGY. frequently. Continuous on each side with the trabeculse is the adenoid tissue, stretching across the lymph-space (PI. XVII., Fig. 4). Blue-stained nuclei are seen at the nodal points of the fibres (PI. XVII., Fig. 6). As already shown (p. 29), they are the nuclei of squames which cover these fibres. The me.shes contain few leucocytes. Study a patch loaded with lymph-corpuscles, and also a transverse section of a trabecula with its lymph-space around it (PI. XVII., Fig. 5). This is most easily found in the medulla. The picric acid and picrocarmine section shows similar details. Refer also to the preparations of adenoid tissue prepared by the interstitial injection of a solution of silver nitrate, which demonstrates the endothelium lining the lymph-sinuses (p. 29). Injection of the Lymph-Sinuses. PREPARATION. — With a hypodermic syringe inject into the lymphatic gland of an ox a two per cent, watery solution of Prussian blue. Place the gland in alcohol for twenty-four hours, and then make transverse sections. Stain a section with carmine, and mount it in Farrant's solution. EXAMINATION (L). — Observe the distribution of the blue deposit around the follicles in the cortex, and around the trabeculae in the medulla. This method indicates beautifully the position of the lymph-sinuses. Blood-vessels. — Study a section where the blood-vessels have been injected, and note that the large vessels lie in the trabeculae, whilst the capillaries are almost entirely confined to the glandular substance. (B.) SIMPLE LYMPHATIC GLANDS. These consist of masses of adenoid tissue occurring in the form of spherical or nodular masses, either singly or in groups, or sometimes in the form of cords. They have already been referred to as occurring in the tonsils (p. 65), at the root of the tongue (p. 61), epiglottis (P- S3), trachea (p. 54), and lungs (p. 59), (especially in the lungs of the cat), in the pyloric end of the stomach (p. 6j), throughout the intestine as solitary glands (p. 70), and in groups, as Peyer's patches, in the small intestine (p. 69). They also occur in the spleen (p. yg) as Malpighian corpuscles. They form the basis of the thymus gland (p. 81). THE SPLEEN. It is desirable to examine the spleen of one of the lower animals, say a cat, as well as that of man. (A.) CAT. PREPARATION. — Cut the spleen of a cat into five or six pieces, and harden them first in Miiller's fluid ; at the end of a week place them in chromic acid and spirit mixture, and after another week transfer them to spirit. Make transverse sections. Stain a section in logwood and mount it in dammar. (B.) HUMAN. Cut a fresh normal human spleen into small pieces, and harden them in the same way as the above, but leave them two weeks in the chromic acid and spirit fluid. Make sections of a part, and include the fibrous capsule. Stain a section in logwood, and mount it in Farrant's solution. THE SPLEEN. 79 Transverse Section of a Cat's Spleen. EXAMINATION (L). — Observe the serous covering and also the fibrous capsule, thick and firmly adherent to the subjacent organ. Trace the large coarse fibrous traheculce passing from its under surface into the organ, where they anas- tomose and form a trabecular framework, which divides the organ into compartments of unequal size, that communicate with each other, and are filled with the splenic pulp. The splenic pulp consists of a large number of cells like lymph-corpuscles, mixed with yellow blood- corpuscles. In it observe the rounded aggregations of lymph-corpuscles constituting Mal- pighian or splenic bodies. They are far more numerous relatively than in the human spleen, and lie scattered irregularly in the pulp. They are not, as occasionally described, always spherical bodies, but in reality are cord-like masses of adenoid tissue, like those of the lung, developed on the walls of the arteries, usually more on one side of it than on the other, so that one must look for a section of an artery in each mass ; and the position of the artery is eccentric. Sometimes they are oval or spherical in shape. In the trabeculae search for sections of the large branches of the splenic artery and vein, which run for a certain distance together in these trabeculae. It is to be observed that the connective tissue which passes in on the blood-vessels at the hilum becomes continuous with the trabecule of the spleen itself. There is no lymph- space between the trabecular and the pulp. (^Indicate the general arrangement iji one half of PI. XVIII., Fig. I.) (H). Observe the capsule, consisting of several layers of fibrous tissue intermixed with a few elastic fibres and some non-striped muscle. Select a Malpighian body. Observe that it is made up of leucocytes, lying in a meshwork of adenoid tissue. It has no definite wall, its outer boundary is indicated by the leucocytes being more crowded together, and so it is stained darker. It is a mass of adenoid tissue developed in the adventitia of an artery ; therefore look for a section of an artery in it, cut either transversely or longitudinally. — The Ptdp. Observe the colourless corpuscles, and note the admixture of a large number of blood-corpuscles. The fine network of delicate fibrils in which they lie imbedded may be observed. Human Spleen. (Logwood and Farrant's solution.) EXAMINATION (L). — Observe the capsule, trabeculae, and splenic pulp. In the latter notice a mottled yellow and bluish ap- pearance. The yellow streaks indicate the position of the blood-stream in the splenic pulp. All the leucocytes are stained blue. Observe the relatively small number of splenic corpuscles, which are usually ill-defined. The splenic corpuscles can usually be best seen in the spleen of a young person, especially if the spleen be congested ; small masses of yellowish-brown pigment may be found in the pulp. (H). A Malpighian corpuscle consists of a vascular mass of adenoid tissue, viz., a mesh- work of adenoid reticulum loaded with leucocytes, many of which have two spherical nuclei (PI. XVIII., Fig. 3). At their periphery they shade into the splenic pulp. The splenic pulp is the most difficult part of the spleen to understand. Select a thin fragment at the margin of the section, and observe the fine network of delicate fibrils crowded with leucocytes, and a very large number of coloured blood-corpuscles. The latter give the pulp its yellow mottled appearance. The framework or matrix of the pulp, when seen in sections, appears to consist of fine fibrils, which form a network with very small meshes, varying in size from a coloured blood-corpuscle to three or four times that size. It is probable, however, that it consists of nu- cleated, branched, cell-plates, which anastomose so as to produce a honeycomb arrangement (Klein). They not unfrequently contain (especially in the congested human spleen) small masses of fine or coarse yellow pigment. It is important to observe that this fine meshwork is directly continuous with the radicles of the veinlets. The cell-plates arrange themselves into 8o PRACTICAL HISTOLOGY. the form of a continuous tube, and at the same time they become more spindle-shaped. This is the mode of origin of a venous radicle from the pulp, so that the spaces in the splenic pulp are directly continuous with the vascular system. {Indicate these details in PI. XVIII., Fig. 2.) BLOOD-VESSELS OF THE SPLEEN. The, splenic artery enters the organ (at the hilum) along with the vein, lymphatics, and nerves, all of which are imbedded in the connective tissue, which becomes continuous with the splenic trabeculae. It runs for a short distance in these trabeculas, but it soon leaves the vein, and its branches divide suddenly into a large number of smaller branches (pcnicilli). The small branches soon become more or less completely ensheathed by a solid mass of adenoid tissue, and this tissue constitutes the so-called Malpighian or splenic corpuscles. These are supplied by distinct capillaries, which form a uniform network with wide meshes. Other arterial branches open directly into the meshwork, or ' honeycombed matrix,' of the splenic pulp, which, as above described, opens into the rootlets of the veins (venous sinuses), and these again into the larger venous trunks lying in the trabeculae. No capillaries are found in the pulp ; they are confined to the Malpighian corpuscles. At the periphery of these corpuscles the capillaries open directly into the spaces of the pulp, which are therefore the channels of communication between the terminations of the arteries and the origin of the venous radicles. The blood-stream in the splenic pulp, therefore, corresponds to the lymph-stream in a lymphatic gland. PREPARATION. Injection of the Blood-vessels of the Spleen, (a) Carmine Gelatine Mass. — It is well to use a constant-pressure apparatus, and to cease injecting whenever the mass ceases to flow into the spleen. Inject from the splenic artery a thin carmine gelatine mass. If the human spleen be taken, select the spleen of a child. After the injection is completed harden the organ in Mijller's fluid. The spleen of a rat does very well, but in all cases the animal ought to be killed by bleeding. In the case of animals it is sometimes advantageous to wash out the blood-corpuscles of the spleen with a stream of salt solution before using the gelatine mass. ip) Silver Nitrate Injection. — Wash out the blood-vessels with distilled water, and then throw in a quarter per cent, solution of silver nitrate. Harden in alcohol. Sections of this show the endothelium of the venous sinuses. Make sections of the above, stain them with logwood, and mount them in dammar. EXAMINATION (L). — Observe the Malpighian corpuscles ; oval or irregularly shaped bodies stained blue, and in each note a branch of the splenic artery and a few capillaries. The pulp contains a network of spaces (venous sinuses), filled with the red carmine mass, and in this lie the masses of logwood-coloured ' pulp ' of the spleen, which are about the same breadth as the venous or cavernous sinuses. Larger spaces — the rootlets of a vein — may be seen opening directly into these spaces. {Indicate the blood-vessels in one half of PI. XVIII., Fig. I.) (H). Note the network of spaces filled with the red mass, and between them the lymph- cells stained blue. Select a venous radicle, and note the layer of endothelium lining it. Trace the continuation of an intra-Malpighian capillary into the red network at its margin. NERVES OF THE SPLEEN. These have already been alluded to (p. 45). Amongst the non-medullated nerve-fibres, I have found a large number of ' plasma-cells ' (p. 20). PLATE X^/ffl SPHEEN &^ii-ij.r^-aE GlAED^ Mintem. Bros lith. THE SPLEEN. 8i FRESH SPLEEN. Cut across a fresh ox spleen. Observe the capsule, trabeculae, and dark grumous-like pulp. The pulp can easily be washed away with a stream of water or salt-solution from a wash-bottle ; the trabecular framework is then easily observed. Observe the Malpighian corpuscles, small white bodies about the size of the head of a pin. Do not mistake transverse sections of the trabeculae for them. Pick out one, and examine it in salt solution (H). It is seen to be made up of lymph-corpuscles, as already described. SPLENIC PULP. Tease a small part of fresh splenic pulp in salt solution, and apply a cover-glass. EXAMINATION (H). — Observe the lymph-corpuscles, perhaps the endothelial cells from a vein, also cells containing particles of fat, others with coloured blood-corpuscles, and some containing pigment, besides numerous coloured blood-corpuscles. If the coloured blood- corpuscles are so numerous as to obscure the view of the other elements, tease the pulp in water, which dissolves the blood-corpuscles ; thus the other elements can then be seen more distinctly. THE THYMUS. The thymus consists of an aggregation of lymph-follicles, or masses of adenoid tissue held together by connective tissue, which contains blood-vessels, lymphatics, and a few nerves The framework consists of a capsule of fibrous tissue, which gives off septa dividing the gland into lobes, these being further subdivided by finer septa into lobules, the lobules being subdivided by fine intra-lobular lamellse of connective tissue into follicles. The gland- substance is made up oi \hcsQ follicles, which are more or \es,s polygonal in outline from mutual pressure. Each follicle consists of a cortical and a medullary part, and the matrix or frame- work of each consists o{ 2. fine adenoid reticitliiin (p. 29), made up of nucleated branched cells, which are easily seen. In this meshwork the lymph-corpuscles lie. In the medulla are found the so-called concentric corpjiscles (Hassall). They consist of a central granular part, around which are disposed concentrically layers of flattened nucleated endothelial cells, and on section they present appearances like the ' cell-nests ' that occur in epithelioma. PREPARATION {a). — Harden part of the thymus of an infant in chromic acid and spirit mixture for two weeks, and then transfer it to alcohol. Stain a section with logwood and mount it in dammar. [li) Harden a similar piece in picr'c acid for twenty-four hours, and then transfer it to spirit. Sections of this may be beautifully stained with picrocarmine. Mount it in Farrant's solution. The thymus of a kitten or puppy may be used, but these do not show the concentric corpuscles so well. EXAMINATION (L).— Observe the capsule, the interlobular and intralobular septa of connective tissue, made up of lamellae with flattened corpuscles between them. Note the polygonal or irregularly shaped follicles. Study a follicle. Observe the cortical part stained more deeply with the dye than the medulla. (H). Search for the adenoid reticulum of M 82 PRACTICAL HISTOLOGY. branched cells, and study the concentric corpuscles. The reticulum is best seen in prepara- tions mounted in Farrant's solution or glycerine. The concentric corpuscles can easily be isolated by teasing a small part of a fresh gland in salt solution. THE DUCTLESS GLANDS. These are the thyroid gland, hypophysis cerebri, \}n' narrow lumen. The collecting tubes are lined with a single layer of homogeneous transparent nucleated cells, which vary considerably in shape. It is to be remembered that the above arrangement has only been made out after laborious investigations, and no such complete tubule was ever seen, or can ever be seen, in a section of a kidne)'. The course of the tubules can only be made out by isolating the tubules as described at p. 86. PREPARATION, a. Chromic Acid and Spirit Mixture. — Remove the kidneys from an animal just killed by bleeding — rabbit, dog, or cat ; divide one transversely into several pieces with a sharp razor, and place them in the above fluid. Take great care, in order not to separate the capsule. Divide the other longitudinally and treat it in the same way. Make both transverse and longitudinal sections of the kidney. Stain some with logwood and mount either in dammar or in Farrant's solution, whilst others may be stained with picrocarmine and mounted in Farrant's solution. b. Miiller's fluid may be employed in the same way as the above, but takes from three to four weeks to complete the hardening. c. Ammonium Chromate (Heidenhain). Harden small pieces in a five per cent, solution of ammonium chromate for forty-eight hours, then wash away all the colouring matter, and complete the hardening in dilute, and then in strong spirit. Prepare portions of a normal hiniian kidney in the same way {a and c). Compare sections of these with the above. KIDNEY OF A RABBIT OR DOG. Transverse Section. EXAMINATION.— With the naked eye note the general shape of the section, distinguishing the capsule, which is very easily detached from the cortex. Note the division of the pare?ichyiiia into three distinct regions : the cortex, the boundary layer (Ludwig) and the papillary portion, the two last forming the medulla, which terminates in a papilla, projecting into the pelvis of the kidney. (L). In the medulla observe the radiating rows of straight tubules (tubuli recti) proceeding from the pelvis outwards. As they pass outwards they divide dichotomously. When taken collectively they constitute a Malpighian pyramid.. In the r<7;Yf.r observe that on the boundary line between it and the medulla, the straight tubules of the Malpighian pyramid are continued as bundles of straight tubules into the cortex, constituting the pyramids of Ferrein, or me- didlary rays, which are the direct continuation of the straight tubes of the medulla into the cortex. As they radiate towards the capsule these bundles of tubules become smaller, and are always separated from the capsule by a layer of convoluted tubules. Observe the convo- luted tubules [tubuli contorti) confined to the cortex. They lie between the pyramids of Ferrein, and between their upper ends and the capsule. Between two pyramids of Ferrein observe the double row of Malpighian corpuscles, which consist of the capsule of Boiuman in- closing a glomerulus, or tuft of blood-vessels. Sometimes the glomeruli fall out, and then there is only a circular aperture in the section. Notice under the capsule more or less tri- angular spaces, which are transverse sections of veins. Observe how easily the capsule sepa- rates, and note the very limited amount of connective tissue which passes from its under surface into the organ. {Indicate the shape of the kidney and the general arrangement of the tubules in PI. XIX., Fig. i.) 86 PRACTICAL HISTOLOGY. HUMAN KIDNEY. Examine (L)a similar section of a human kidney, and note especially the normal thickness of the capsule and its delicate and scanty fibres of attachment to the subjacent organ. {Indicate the capsule, pyramids of Ferrein, convoluted tubules, and Malpighian corpuscles in PI. XIX., Fig. 2.) (H). Select a Malpighian corpuscle. Observe the membrane or capsule of Bowman, con- sisting of an elastic membrana propria, and note the single layer of squamous epithelium lining it. The nuclei are stained, and bulge slightly into the cavity. Outside it there is a small amount of fibrous tissue. Within it note the tuft of blood-vessels, with their nuclei stained, and the capillaries, perhaps, containing yellow blood-corpuscles (PI. XIX., Fig. 3). The capillaries are arranged in two or three conical lobules, but do not completely fill the capsule ; the space between the surface of the glomerulus and the capsule depends on the amount and nature of the secretion present in it. Between the capillaries there is a ver\' small amount of connective tissue (chiefly branched connective-tissue cells), which holds them to- gether ; and over them there is a layer ofsquames. The section may pass through the capsule in the plane of the afferent or efferent blood-vessels ; or the narrow neck which connects the cavity of the capsule with a convoluted tubule may be found. Select a convoluted tubule (H). — Observe them, one divided longitudinally and another transversely, and observe the limiting membrane, and how closely they are packed in the cortex, with very little supporting tissue between them. Study the secretory epithelium lining them, and note that the lumen of the tube is small, that the epithelial cells are ill-defined and have no cell-wall. They contain a spherical nucleus placed near the attached end of the cell, and their protoplasm is always cloudy and granular. The outer part of the protoplasm is often striated ; this is especially the case if the section has been hardened in ammonium chromate. Some tubules may be found with an angular zigzag bend in them ; these are the connecting tubules. {Indicate a convoluted tubule in PI. XX., Fig. i.) Select a Pyramid of Ferrein (H) — Observe the straight tubules, and amongst these the looped tubules of Hcnle, with their descending limb, which is very narrow and lined by flattened epfthelium, with alternately projecting nuclei on either side. They are not unlike capillary blood-vessels, though they have a well-marked basement-membrane. It is difficult to distin- guish the ascending limb, as it is so like a collecting tubule. The descending branch may be traced downwards into the medulla. {Indicate these tubules in PI. XX., Fig. i.) The Medulla (H). — Observe the straight tubules — collecting or discharging tubes — which open on the ape.x of the Malpighian pyramid. Observe their columnar or cubical epithelial covering. The cells are well-defined, and contain a well-marked spherical nucleus, imbedded in clear transparent protoplasm. There is a well-marked lumen in the centre of the tubule. Observe the basement-membrane in which the cells are placed, and note the relatively large amount of connective tissue in the medulla compared with the cortex (PI. XX., Fig. 2). Place unstained sections of a kidney in a quarter per cent, osmic acid for twenty-four hours, and mount them in Farrant's solution. ISOLATED RENAL TUBULES. PREPARATION. — The arrangement and relation of these parts one to another cannot be made out in a section ; to do so the kidney must be boiled for a long time in a mixture of alcohol and hydrochloric acid, which dissolves the connective tissue and liberates the tubules. A very good method is to place pieces of a kidney, about the size of a pea, in Plate xk.iqdney MintKrri Bros lul-i KIDNEYS. $7 pure hydrochloric acid for twelve hours, and then leave them in water for twenty-four hours. Tease out a small piece in osmic acid solution (one per cent.), and mount it in Farrant's solu- tion. By teasing part of a kidney prepared in this way it has been shown that the Malpighian corpuscle is continuous with a convoluted secreting tubule, which winds about in the cortex, and then descends in a pyramid of Ferrein into the medulla, forming the descending part of the looped tubule of Henle, and reascends as the ascending part of the loop, and after a short course as a connecting tube, with a zigzag direction, becomes continuous with a collecting tube, and descends through a pyramid of Ferrein, and in its course joins at an acute angle with similar tubules, which is thus a discharging tube, opening on the surface of a papilla. CONNECTIVE TISSUE OF THE KIDNEY. Compare this section with the above. Make transverse sections through the corte.x, and also through the medulla near the apex of a Malpighian pyramid, and stain them with logwood or picrocarmine, and mount in Farrant's solution. EXAMINATION (L).— In the cortex. Observe the convoluted tubules and glomeruli ; notice the small amount of interlobular connective tissue, and search for a transverse section of a group of straight tubules, i.e. of a pyramid of Ferrein. Study the characters of these parts (H). (L). Apex of a Malpighian Pyramid. — Observe the transverse sections of the discharging tubules lined with clear, low, columnar epithelium, and a well-defined lumen. Notice the large amount of connective tissue between and supporting the tubules, forming a marked contrast to the small amount in the cortex. It is desirable to have a transverse section through the boundary line between the corte.x and medulla. Observe the transverse sections of the collecting tubules, and here and there the cut ends of the ascending and descending looped tubules of Henle ; the narrow loop is not unlike a capillary with its bulging oval nucleus. Sections of capillaries may also be found, but they usually contain blood-corpuscles. Note the moderate amount of intertubular connective tissue — intermediate in amount between that of the cortex and medulla. Ammonium Chromate Kidney. — Heidenhain showed that the epithelial cells lining a uriniferous tubule — excepting those of the descending limb of Henle's loop — the loop itself, and all parts of the collecting tube, are made up of a number of ' rods ' or fibrils placed vertically to the long axis of the tube. These rods are most distinct in the outer half of the cells, Le. next the membrana propria. Each cell contains a spherical nucleus placed about the middle of its substarfce. EXAMINATION (L).— Select a convoluted tubule in the cortex, and use (H) to see the above-described characters. They are easily seen in a properly prepared kidney. BLOOD-VESSELS OF THE KIDNEY. PREPARATION. — Make transverse and longitudinal sections of a kidney which has been injected from the renal artery with a gelatine and carmine or Berlin-blue mass. Mount in dammar. EXAMINATION (L). — Observe large branches of the renal artery running outwards be- tween the Malpighian pyramids ; from these the interlobular arteries proceed vertically 88 PRACTICAL HISTOLOGY. outwards between the pyramids of Fcrrein. As the interlobular arteries proceed outwards, they give off on all sides small, short trunks — the afferent arteries— io the Malpia. Skin I^-Cntei-rv Bros li'-.h SKIN. 93 to it. It is then placed in some (200 ex.) of the digesting fluid, which is kept at a temperature of 38° C. in an ordinary water bath for a period varying from three to eight hours, — the time depending upon the age and size of the piece of slvin. After partial digestion the skin is placed in water for twelve hours, when it swells up and becomes extremely transparent. It may be kept most advantageously in a ten per cent, solution of common salt, or it may be hardened in one of the ordinary hardening fluids, and afterwards stained with logwood or carmine. This method is also applicable to other tissues (W. Stirling). HUMAN SCALP. Vertical Section. EXAMINATION (L). — Make the sections parallel with the hair-folliclcs. Stain a section with picrocarmine or logwood, and mount it in dammar. Observe the epidermis and corium, and in the latter the Iiair-follicles, each of which gives origin to a hair. Note its mouth, and its bulbous lower e>itremity, into which projects a vascular papilla. The follicles are usually placed obliquely, and in the scalp they lie in groups of three or four, as is easily seen when a horizontal section is made. Into the upper part or neck of the follicle one or two sebaceous glands open. Rising a short distance above the bulbous portion is the arrector pili, composed of several bundles of non-striped muscle-cells. It runs obliquely upwards through the corium, forming an acute angle with the follicle, and passes over the base of the sebaceous gland, and terminates in a plexus on the surface of the corium. {Indicate a hair-follicle and its muscle in PL XXII., Fig. i.) Study a Hair-Follicle (L and H). — Proceeding from without inwards, observe the (i) outer and (2) inner siteatk of the hair-sac, which are continuous with the papillary layer of the corium. In the outer layer the fibres are arranged longitudinally, whilst in the inner one they are transverse or oblique. Both layers are best marked at the lower bulbous portion of the follicle, where they are pushed into the lower end of the hair, to form a club-shaped /tf////rt. Internal to these tw^o layers is (3) 2. glassy hyaline mevibrane, which separates them from the epidermic coverings of the hair. Inside this is (4) the outer root-sheath, which is thickest about the middle of the follicle, and consists of several layers of cells continuous with the stratum Malpighii. Inside this is (5) the inner root-sheath, which in longitudinal sections appears as a thick glassy membrane, though it can be proved to consist of three layers, being from with- out inwards — [a) Henle's layer, then {b) Huxley's layer, and {c) the cuticle of the root-sheath, which is a very delicate membrane composed of imbricated non-nucleated scales. The inner root-sheath terminates abruptly at the neck of the follicle. Then follows (6) the cuticle of the hair and (7) the hair itself. Sebaceous Gland (L and H). — Observe the duct of one or more of these glands openino- into the upper part of a hair-follicle. The membrana propria (of the duct) is continuous with the glassy membrane (3) of the hair-sac, and the epithelium of the outer root-sheath is directly continuous with the epithelium of the duct. The duct after branching opens into flask-shaped or saccular alveoli. In these alveoli are found one or more layers of cells loaded with fat- globules (PI. XXII., Fig. 2). Arrector pili Muscle (L and H). — Observe its attachment to the lower part of the hair- follicle and trace it obliquely upwards towards the surface of the corium (p. 91). It is com- posed of non-striped muscle. Horizontal Sections of the Scalp. — Stain it with logwood or picrocarmine, and mount it in dammar. Observe the transverse sections of the hairs arranged in groups of three or four. 94 PRACTICAL HISTOLOGY. Make out the different layers of the hair-follicle. Compare also the section of a lip of a dog (p. 60) where these various coverings are exquisitely shown in sections of the large ' feelers.' HAIR. Human Hair.— Pluck a hair from the head, and place its shaft in water. Observe (H) its cuticle, consisting of imbricated scales, then the cortex, which forms the mass of the hair and is composed of horny matter made up of scales, which in pigmented hairs have fine granules of pigment deposited between them. In the centre note the medulla, or pith, which is absent in some hairs (PI. XXII., Figs. 3 and 4). A hair may be preserved dry, or simply by adding a drop of dammar solution and then applying a cover-glass, but after the addition of dammar the cuticle is not well seen. Hair of Rabbit.— Treat it similarly, but note the single or double row of oblong compart- ments in the medulla filled with air, and therefore appearing black. Transverse sections of hairs are obtained by shaving, but the hairs of the beard are usually flattened ovals, whilst those of the head are round. NAILS. Make vertical sections of a nail and its nail-bed after hardening in alcohol or chromic acid and spirit mixture. They are only peculiar modifications of the stratum Malpighii. They rest on very vascular papillae (bed or matrix). By steeping them in forty per cent, caustic potash solution, the nails can be resolved into their constituent cells (p. xxxiv). BLOOD-VESSELS OF THE SKIN. PREPARATION. — It is best to employ the skin of the extremities, for a limb is easily in- jected from its main artery. Inject a limb with a carmine-gelatine or Berlin-blue mass. Harden parts of the skin in Mijller's fluid for two weeks, and then in alcohol. Make mode- rately thick vertical sections. Stain the sections injected with a red mass with logwood, and the others with picrocarmine. Mount them in dammar. It is well to prepare similar sections of an injected scalp, to see the arrangement of the blood-vessels round a hair-follicle. EXAMINATION (L and H). — Passing from the subcutaneous tissue upwards we find the following systems (Tomsa) : — (i) Observe the fat-lobules, each supplied by a small artery which forms a dense plexus over and between the individual fat-cells, and from it emerge one or two veins. (2) A branch proceeds upwards to the coil of a sweat-gland, where it forms a plexus, whilst the duct is supplied by a branch from an artery in the corium where the capillaries anastomose. (3) Each papilla of a hair-follicle receives an arteriole, which splits up into capillaries within it. A plexus of capillaries exists between the outer longitudinal and the inner circular coats of the hair-sac. (4) A capillary plexus exists round the sebaceous gland and arrector pili. Plj^je xm . Skin & Hair ""X.,/--- / — \^y' \ ^--'''~\ /""'^ ./''""X..--'" MmLei-u Bros.nU:\. SKIN. 95 (5) Trace an artery arising from a subcutaneous trunk, and note that as it ascends it gives off branches to the sweat-duct, hair-sac, sebaceous glands, and hair-muscle, and note its termination in capillary loops in the papillae, from which a vein proceeds which opens into relatively large veins lying in the superficial layers of the corium (Klein). {Indicate the general arrangement of the blood-vessels in PI. XXL, Fig. i.) LYMPHATICS OF THE SKIN. They require to be injected by the puncture method (p. Iv), using a watery solution of Berlin-blue. But the task is by no means an easy one. Injection of the skin of the palm of the hand or sole of the foot yields the best results. NERVES OF THE SKIN. The nerves of the skin are very numerous, and the mode of termination of some of them in Pacinian corpuscles in the subcutaneous tissue (p. 46), and in Wagner's tactile corpuscles in some of the papillae of the corium (p. 92), has already been alluded to. These touch-cor- puscles are most numerous in the lips, skin of the hand, foot, and glans penis. It requires some modification of the gold method — preferably that with lime-juice and formic acid (p. xlv) — to obtain preparations of the various nerve-plexuses (sub-epithelial and inter-epithelial) in the skin. The snout of the pig and the skin of the nose of the mole may be employed. The peculiar nerve-endings recently described by Ranvier and Merkel, may be found in the snout of the pig, and especially in the soft part of a duck's bill. 96 PRACTICAL HISTOLOGY. THE NERVOUS SYSTEM. THE SPINAL CORD. PREPARATION (a). Miiller's Fluid and Spirit Mixture. — Remove the spinal cord and medulla oblongata, with the pia mater attached, from a newly-killed cat, rabbit, or dog. Indeed, all the membranes may be left attached to the cord. Take care to avoid squeezing or stretching the cord. It is not necessary to make any incisions into the cord at first. Suspend it in a wide, cylindrical vessel — such vessels as are used for water analysis do very well — filled with a mixture composed of three parts of Miiller's fluid, and one of methylated spirit. The mixture ought to be made at the time ; but as a considerable amount of heat is evolved when the fluids are mixed, it ought to be allowed to cool. Keep the tissues in a cool — in fact, a cold place. This is of great importance. Renew the fluid at the end of twenty-four hours, and then again at the end of a week. The cord may now be cut, with a sharp razor, into segments an inch or so long. The cord ought to be hardened in this mixture for three weeks. It is then transferred to a two per cent, solution of ammonium bichromate for two weeks, to complete the hardening. Preserve them either in spirit, or in a solution of chloral hydrate, of twelve grains to an ounce of water (Hamilton). A portion of the spinal cord of a man or an ox ought to be hardened in the same way. {b) Potassic-bichromate. — Cut the fresh spinal cord of an ox into pieces an inch long by means of a sharp razor. Place them in a large quantity of two per cent, solution of potassic bichromate. Do not let one piece lie on another. See that they are separated by cotton wool, previously dipped in alcohol, to avoid pressure. It requires from three to five weeks to complete the hardening. Preserve the cord in spirit. A cord hardened in this way shows the nerve-structures very well. (c) Chromic Acid and Spirit Mixture. — The cord must be cut into pieces half an inch long and placed in this fluid, which will harden them in from two to three weeks. It is not so well suited for showing the nerve-elements as for demonstrating the neurogleia. id) Spirit and Iodine, and then Ammonium-bichromate. — Suspend the cord in a long, cylindrical vessel, in methylated spirit tinged yellow with tincture of iodine. Leave it in this mixture — usually three or four days — until the yellow colour has nearly disappeared. Cut it in pieces an inch long, and complete the hardening by keeping them in a two per cent, solution of am- monium-bichromate for four or five weeks. Preserve them in spirit until required for section. METHOD OF CUTTING SECTIONS OF THE SPINAL CORD. Employ some form of freezing microtome. Steep a piece of the cord, hardened by any of the above methods, but preferably by («), in water for twenty-four hours, to remove all traces of the hardening agent, or the spirit. Soak it in syrup (two ounces of sugar to SPINAL CORD. 97 one ounce of water) for t\vcnt)--four or thirty-six hours, and then transfer it to a solution of gum for twelve hours, and then cut it in the ordinary way with a freezing microtome. Stain the sections with logwood, carmine, or aniline blue-black, and mount them in dammar. When investigating the structure of the nervous system, it is sometimes of importance to have a section only partial/)' cleared up. This may be done by examining a section in Farrant's solution, though, for general purposes, mounting in dammar is to be preferred. Make transverse sections through the hardened cervical portion of the cord of a cat, rabbit, or dog. Stain a section with carmine, or logwood, or preferably with aniline blue-black one per cent, solution, which stains the nerve-cells beautifully (p. xliv). Mount the sections in dammar. Transverse Section of the Spinal Cord. EXAMINATION (L). — Observe the pia mater investing the cord. It consists of an outer part and an inner part, which latter sends numerous processes or septa into the cord. Trace the passage of the outer layer into the anterior median fissure, and a process of the inner layer into the posterior median fissure. Observe that the cord is essentially a bilateral, symmetrical organ, its two halves being connected by a narrow bridge of tissue containing a small opening — the central canal. Note the broad, shallow, and well-defined anterior median fissure, and the narrow, deeper, and less perfectly de?meA posterior median fissure, containing a process of the pia mater, often carrying a blood-vessel. If the section has been made at the level of the origin of the nerve-roots, trace the fibres of the anterior roots of a spinal nerve, leaving the cord by several bundles, whilst those of the posterior root enter it as one bundle. White matter. — Observe the white matter, composed of the cut ends of nerve-fibres of the cord placed externally, and how each half is divided into an anterior, lateral, and posterior column by the above-described arrangement of the nerve-roots. The posterior column is ao-ain divided into two (in the cervical region), by a narrow band of connective tissue ; the narrow inner part, which lies next the posterior median fissure, is known as the fasciculus cuneatus. Grey Matter. — Observe the grey matter more deeply stained, and lying in the form of a crescent in each half of the cord. Its extremities constitute the anterior and posterior horns or cornua, though there is no well-marked limit between them. Note in the anterior horn the numerous multipolar nerve-cells deeply stained, and observe the oval cap of deeply stained matter — the substantia gclatinosa — which covers the posterior cornua. It is probably non-nervous in its nature, and consists of a peculiar accumulation of ' neurogleia.' Observe the bridge of tissue connecting the two halves of the cord, and in it the central canal lined with columnar ciliated epithelium, — in front and behind the canal, the anterior and posterior grey commissures, also the nerve-fibres crossing in front of the anterior grey commissure, forming the anterior white commissure. {Indicate the general arrangement in PI. XXIII. Fig. I.) ■ White Matter. (H). — Observe the pia mater sending in septa ; trace these septa, becomino- continuous with a fine fibrous network — the neurogleia — lying between the nerve-fibres of the white substance. Lying in the meshes of this network observe the cut ends of the nerve-fibres. They are of different sizes : some are large, others of medium size, and some are very small. Each axis-cylinder is deeply stained, and is surrounded by a clear, non-stained area, which represents the white substance of Schwann, or myeline. In sections which have been hardened for a long time in chromic acid, a well-marked concentric striation of the medulla O 98 PRACTICAL HISTOLOGY. may be observed (see Nerve, p. 42). {Indicate the nervc-fibrcs in one half and the ncurogleia in the other half of Vl XXIII., Fig. 2.) Ncurogleia (H). — This is the deHcate framework which supports the nerve-elements — fibres and cells — within the cord and brain. It fills all the interstices between the nervous elements of the cord. It consists of a semi-fluid substance — the neurogleia matrix — in which are imbedded numerous very fine neurogleia fibrils, which unite to form a network. In it are also found many nucleated, branched, connective-tissue corpuscles. The fibres seem to be allied in their nature to clastic fibres. Grey Matter (H). — Observe the large, nucleated, multipolar nerve-cells in the anterior horn ; four or five branching processes may be traced from them for a considerable distance. Each cell contains a distinct spherical nucleus, enclosed in a distinct membrane and a nucleolus. Round each cell is a space— the lymph-space. These cells are aggregated into three groups — an anterior, lateral, and median. Study the posterior horn, and notice that the cells there are much smaller in size and fewer in number. Notice the fibrillar arrangement of the rest of the grey matter, and study the substantia gelatinosa of Rolando (p. 97), which is composed of a mass of neurogleia. {Indicate the anterior horn, with its nerve-cells, in PI. XXIII., Fig. 3.) Study similar sections made from the dorsal and lumbar regions of the cord, and compare them with the above. Observe that the sections of the dorsal region are smaller than the others. Compare the absolute amount of grey matter, and observe that it is greatest in the lumbar and cervical regions. The amount of white matter is least in the lumbar and dorsal regions, and greatest in the cervical. In the upper dorsal region notice a group of cells in the grey matter, midway between the anterior and posterior cornua— the tractus inter-niedio- lateralis. Make similar sections of the spinal cord of an ox or man, or, best of all, that of a horse. It is necessary to trace the course of the fibres as seen in a section. Observe (L) the atiterior root of a spinal nerve. Trace some of its fibres. They enter the anterior cornu of its own side, and some of the fibres pass through the anterior white commissure to the anterior column of the opposite side. Others turn into the lateral column on the same side. Trace the fibres of the posterior root entering as a single bundle through the substantia gelatinosa. To study further the course of the nerve-fibres it is necessary to make antero-posterior sections through the grey and white matter. This is best done in the cervical region of the cord of an ox. Stain, and mount in the same way. Longitudinal Section of the Spinal Cord. EXAMINATION (L).— Observe the grey matter in the centre with a band of white matter— the anterior and posterior columns cut longitudinally- - on each side of it. Trace the vertically-arranged nerve-fibres in these, and note the some- what oblique entrance of the anterior roots, some of the fibres ascending, and others descending in the grey matter. In the anterior cornu note the groups of multipolar nerve-cells and also the gelatinous substance in the posterior column and the entrance of a posterior root. TO ISOLATE A MULTIPOLAR NERVE-CELL OF THE SPINAL CORD. PREPARATION, {a) Dilute Potassic Bichromate. — Macerate a small piece — an eighth of an inch long— of the perfectly fresh spinal cord of an ox or sheep in a large quantity of a one-eighth per cent, solution of potassic bichromate for two or three days. After this time, cut out with scissors a small piece of the anterior horn, and tease it on a slide in water. Stain the piece with carmine, and then continue to tease it with needles until isolated cells are Plvje X>[E. Spih^ Cord SPINAL CORD. 99 obtained. The process of teasing is best accomplished with the aid of a dissecting microscope, but the preparation ought to be examined from time to time with a low power. A hair or thin piece of paper ought to be placed under the cover-glass, to prevent the cells from being displaced. (b) Injection of Osmic Acid. — By means of a subcutaneous syringe, provided with a very fine gold nozzle, inject forcibly a small quantity of a half per cent, solution of osmic acid into the anterior horn of a perfectly fresh spinal cord. This serves to separate the parts, and at the same time to fix them. Macerate the part for two days in dilute alcohol, and isolate the cells by carefully teasing out a piece, as described under {a). (c) Dilute Osmic Acid. — Macerate pieces, the size of half a pea, of the grey matter of the anterior horn of a perfectly fresh cord in a considerable quantity of a tenth per cent, solution of osmic acid for ten days. Wash away the greyish deposit, and place the pieces in a mixture consisting of equal parts of glycerine and water, for ten days or a fortnight. Tease a small piece in glycerine. The cells are easily stained with a very dilute solution of magenta. EXAMINATION (L and H). — Observe the large multipolar cells, with many branched processes. One process — the axis-cylinder process, which is directly continuous with the axis-cylinder of a nerve-fibre — is always unbranched. Observe the large nucleus, and note the longitudinal striation of the cell-substance and that of the axis-cylinder process. BLOOD-VESSELS OF THE SPINAL CORD. An entire animal must be injected, and sections made of the injected cord. The grey matter is much more vascular than the white. The sections are mounted in dammar, and need not be stained. The connective tissue of the subarachnoid space has already been referred to (p. 23). THE MEDULLA OBLONGATA. PREPARATION. — The same methods as are employed for the spinal cord are to be used. Make transverse sections of the human medulla, some through the decussation of the anterior pyramids, and others through the floor of the medulla and the olivary bodies. Stain them with aniline blue-black, and mount in dammar. EXAMINATION (L). Transverse Section through the decussation of the Pyramids.— Observe the crossing of the fibres from one side to the other ; notice the enlarged posterior cornua (tubercles of Rolando), the central canal nearer the surface posteriorly. Through the Olivary Body. — Observe the median raphe ; the central canal has now ex- panded into the floor of the fourth ventricle. Study the columns of the medulla from the centre in front Outwards as anterior pyramids, olivary body, restiform body, and posterior pyramid on each side. Notice the folded sheet of grey matter — corpus dentatum — in the olivary body, and in it notice the multipolar nerve-cells. Observe the masses of grey matter in the floor of the fourth ventricle. loo PRACTICAL HISTOLOGY. THE CEREBELLUM. PREPARATION. — The same methods are applicable as those detailed for the spinal cord. Harden pieces — three-quarters of an inch square — of a human cerebellum in the Miiller's fluid and spirit mixture (p. xxxii), and proceed just as for the spinal cord. If a human cerebellum cannot be obtained, use that of a cat, dog, or rabbit. Make sections across the folia or leaflets. Stain some with logwood, but preferably with aniline blue-black, and mount them in dammar. Vertical Section of the Cerebellum. EXAMINATION (L).— Observe the folia or leaflets, with secondary leaflets on them. Notice the dark-stained grey matter outside, and the lighter white matter inside. The grey matter is covered with pia mater, which sends processes carrying blood-vessels into the substance of the organ. It consists of (i) a broad, outer grey or molecular layer, containing at its inner part a single row of pear or flask-shaped cells'; (2) the cells of Purkinje, which give off a number of branching processes, running outward ; inside this is (3) the inner granular or nuclear layer, about one-third the breadth of (1). Observe the large number of small deeply stained granules, arranged in many rows ; and then follows the white matter, consisting of nerve-fibres arranged more or less parallel to each other. {Indicate the layers in PL XXIV., Fig. i.) (H). (i) The outer or cortical layer contains, as a ground-work, a network of delicate nerve-fibrils, and mixed with these are the dichotomously divided branches of Purkinje's cells. It contains a few multipolar ganglion-cells (Sankey), which seem to be connected with these processes. (2) Purkinje s cells occur in a single layer. They are of a pear or flask-like shape. Each cell has a single, unbranched, or axial cylinder process, which passes centrally ; whilst from its outer surface it gives off a branched process, which splits up rapidly into branches or pro- cesses which divide again and again. They can be traced nearly to the surface of the organ. (3). The nuclear layer is a broad layer, and contains a great number of deeply-stained spherical nuclei, whose exact nature is unknown. Between them is a network of minute fibrils, which are probably nervous in their nature. {Indicate these layers in PL XXIV., Fig. 2.) In the white matter note the small nerve-tubes, and between them the neurogleia, con- taining rows of small nucleated cells. Trace a blood-vessel in the grey matter, and observe that each one is surrounded by a narrow space — the perivascular lymph-space (His). DOUBLE-STAINING OF THE CEREBELLUM. (i) Eosin and Logwood. — Stain a section in a very dilute watery solution of eosin (p. xlv) till it has a faint red colour. This it does in a few seconds. Great care must be taken not to overstain the preparation. This forms an excellent ground-colour. Wash the section in water, and then stain it with logwood, and mount it in dammar. The cortical layer is reddish, and so are the cells of Purkinje, while the nuclear layer, and all other nuclei, have a logwood tint. (2) Picrocarmine and Iodine-green.— Stain a section with picrocarmine. Wash it in water CEREBRUM. lOI acidulated with dilute acetic acid, and, after washing in pure water, stain it with iodine-^reen, and mount it in dammar. The outer layer and Purkinje's cells of the grey matter are stained red, and the inner layer is stained green. THE CEREBRUM. PREPARATION. — Prepare it in the same way as the cerebellum (p. loo), using the same precautions both for hardening the tissue and for cutting it as indicated for the spinal cord (p. 96). Select a frontal convolution of a human brain for examination. Stain the sections with aniline blue-black, and mount them in dammar. Vertical Section of a Human Cerebral Convolution. EXAMINATION (L). — Observe the shape of a convolution, and contrast it with that of the cerebellum. Notice the pia mater surround- ing the convolution, and sending fine processes carrying blood-vessels into the cortex. Notice the deeply stained grey matter or cortex, and the white matter within. Examine the layers of the grey matter from without inwards. It is possible to distinguish five layers, varying in thickness and structural characters (Meynert). These layers are not accurately mapped off from each other ; still they can be seen. It is to be remembered, however, that there are variations in different parts of the cortex. In some places only four layers can be distinguished. 1. The outer layer contains few cells of any kind imbedded in a clear matrix. It occupies about one-tenth of the entire thickness of the grey matter. 2. This is about equal in thickness to the preceding, though it is easily distinguished by its containing a large number of densely packed small pyramidal cells. Owing to the large number of cells in this lajer it always appears deeply stained. 3. This layer is much wider, and, owing to the cells occurring in it being placed well apart from each other, it appears somewhat lighter than the second layer. It consists of large and small pyramidal cells, so placed that their apices are directed towards the surface of the con- volution, and their bases towards the white matter. 4. This layer contains small irregular corpuscles with few processes. 5. This layer is broader than the one above it, and contains similar corpuscles mixed with a few fusiform cells. At its lower part it gradually shades into the wliite matter, which con- sists of very fine medullated fibres, supported by neurogleia. {Indicate t lie general arrangement of the cells in PI. XXIV., Fig. 3.) (H). Study in detail the various layers, and especially note the cells of the third layer. The cells are distinctly pyramidal. Trace the long, branching process directed towards the surface, and the branching processes from each angle of the base. Each cell has a central axial cylinder-process. Each ganglion-cell lies in a lymph-space. It is to be observed, however, that these cells only appear pyramidal when the plane of the section passes parallel to their long axis. If cut transversely they appear triangular. {Indicate a nerve-cell in PI. XXIV., Fig. 4-) Select a blood-vessel of the grey matter, and observe its perivascular lymph-space. The processes of the cells are best seen in preparations which are ox\\y partially cleared up under the influence of clove oil. This is a most important method of investigation. I have often seen in this way delicate fibrils, not unlike elastic fibres, and which are not distinct when the section is completely cleared up. I02 PRACTICAL HISTOLOGY. BLOOD-VESSELS OF THE BRAIN. The entire animal must be injected. Make vertical sections and mount them in dammar. Observe the greater vascularity of the grey matter. NERVE-GANGLIA. GASSERIAN OR SPINAL GANGLIA. PREPARATION. — With a saw divide longitudinally the head of a sheep just killed. With a scalpel remove the Gasserian ganglia from the apex of the petrous part of the temporal bone, and place them in the chromic acid and spirit fluid for eight days. Make longitudinal and transverse sections. Stain them with logwood and mount them in dammar, or in Farrant's solution. The ganglion on the posterior root of a spinal nerve of a cat or dog may be hardened by the same method. Longitudinal Section of a Spinal Ganglion. EXAMINATION (L).— Observe the sheath of the ganglion^within it are numerous large ganglionic cells — and note their arrangement. Ob- serve strands of nerve-fibres sweeping through the ganglion, and processes of connective tissue passing in from the capsule into the ganglion, and supporting the cells and nerve-fibres. {In- dicate the nerve-cells and fibres in PI. XXIV., Fig. 5.) (H). Observe the nerve-cells, with their well-marked sheath or capsule lined with squames, the nuclei of which are well-marked ; the granular mass of the cell, containing a round ex- centric nucleus with an envelope, and a distinct nucleolus. It is difficult to recognise the processes of the cell itself Note the medullated nerve-fibres. In the examination of the transverse sections note the cells as before, but between them bundles of medullated nerve-fibres cut transversely. It is also desirable to examine the cells from a fresh Gasserian ganglion. For this purpose tease a small piece of the fresh Gasserian ganglion of a sheep in a very small quantity of salt solution, and stain it with magenta solution. Details similar to those described above are seen on examination, though the processes of the cells are generally broken off, and the cells them- selves are frequently dislocated from their capsules. {Indicate an isolated cell within its capsule in PI. XXIV., Fig. 7, and without its capside in Fig. 6.) GANGLIA OF THE SYMPATHETIC NERVOUS SYSTEM. The non-medullated nerve-fibres have already been examined (p. 45). The nerve-cells of the sympathetic system have still to be considered. PREPARATION.— Remove a sympathetic ganglion from the thorax of any animal, or pre- ferably use the human superior or inferior cervical sympathetic ganglion. Harden it in Miiller's fluid, or the chromic acid and spirit fluid, for four or five days, and make transverse sections. Stain sections with logwood, and others with picrocarmine, and mount them in Farrant's solution. EXAMINATION (L). — Observe the capsule of the ganglion composed of fibrous tissue, and notice the cells and their arrangement. Place a cell in the field of the microscope. (H). Observe the cell-capsule, with its included nucleated cell, which gives off processes Plate XXiy. Brain &,Nerve Ganglia Miiiten-i. Eros hth NERVE-GANGLIA. 103 that pierce the capsule. The cells very frequently contain a yellowish-coloured pigment. Between the cells will be found transverse sections of non-medullated nerve-fibres. SYMPATHETIC NERVE-CELLS OF THE FROG. These have already been alluded to in connection with the heart. They occur in large numbers along the course of the abdominal aorta in the frog, and are easily demonstrated by the lime-juice and gold method (p. xlv). They may also be obtained from the sympathetic ganglia. It is easy to demonstrate the straight process, but by no means so easy to see the spiral one. Isolation of Sympathetic Nerve-cells. — Adopt the same methods as are recommended for the nerve-cells of the spinal cord. I04 PRACTICAL HISTOLOGY. THE EYE. THE CORNEA. PREPARATION. — Cut out the fresh cornea from the eye of a cat or rabbit. In excising the eyeball, be careful not to squeeze or injure it. For the removal of the cornea, push a narrow knife into it at its junction with the sclerotic, and then cut it out with scissors. Harden it in a two per cent, solution of potassic bichromate for ten days, or in the chromic acid and spirit mixture for a week. Make vertical sections, stain some with logwood and others with picrocarmine, and mount in Farrant's solution, and also irusdammar. EXAMINATION (L). — Observe the epithelium occurring in many layers — conjunctiva cornecu or anterior epithehmn — covering the anterior surface of the cornea proper. In the human cornea, and in the cornea of some animals, the epithelium rests on a narrow, elastic, trans- parent membrane — the anterior elastic lamina or Bowniati's membrane — but it is either absent or but feebly developed in the cornea of the cat and dog. It seems to differ from the next layer chiefly in the absence of corneal corpuscles. The substance forming the cornea proper — stcbstantia propria — is stained red, and on its posterior surface there is a section of a clear, sharply-defined membrane stained yellow — the membrane of Descemet, or posterior elastic lamina and covering the posterior surface of this membrane there is a single layer of flat- tened nucleated epithelial cells seen in profile. Examine each of these parts. {Indicate the general arrangement in one half of Y\. XXV., Fig. i.) (H). The anterior epithelium. Begin at the surface, and note the flattened cells seen in section. The deeper layers consist of two or three layers of polyhedral cells, each with a spherical nucleus. They are so arranged as to fit into each other, and the deepest layer resting on the cornea is compo.sed of a row of columnar cells placed perpendicularly. Observe how processes of the cells in the layer above this fit into depressions between the apices of the columnar cells (Cleland and Stirling). The columnar cells are not all of the same height, and their lower end possesses a flat expansion or foot-plate (Lott and Stirling), which rests on the cornea. Prickle-cells are seen in the middle layers, especially where the epithelium is in very many layers, as in the cornea of the ox. (See p. 9.) The anterior elastic lamina is a homogeneous, transparent membrane devoid of cornea- corpuscles. It is perforated here and there by a few oblique channels, which transmit fine nerve-fibrils— the rami perforantes. The substantia propria is composed of a series of layers of fibrillar connective tissue, arranged in the form of lamellae, placed one outside the other, and parallel to the surface of the cornea. These lamella:, and also the fibres which compose them, are held together by an albuminous cement-substance. Bundles of fibrous tissue pass from one lamella to another. Near the anterior surface a few fibres perforate the lamella obliquely, and constitute the CORNEA. J 05 fibm arciiata. Note the edges of these lamellas seen in section. In the ground -substance which separates two adjoining lamcUfe, there exists a series of lacuna; or cell-spaces of irregular branched form, which communicate freely by means of fine channels with lacunae in the same plane, and also with the lacunae lying above and below them. This is the lymph- canalicular system of V. Recklinghausen. Each lacuna contains a nucleated cell-plate or corneal corpuscle^ which does not completely fill the space, so that lymph and colourless blood- corpuscles can move between it and the wall of the space. These corpuscles are seen as flat, narrow, nucleated, stained spindles lying between the lamellae in oval spaces, which they do not fill completely. Each corpuscle sends processes into the canaliculi, so that they anasto- mose to form a network. They cannot be seen in this preparation ; it requires the use of gold chloride to show their processes satisfactorily (p. 105). The posterior elastic lamina is a structureless elastic membrane with sharpiiy defined margins, which stains of a deep yellow tint with picrocarmine. It may become detached, when it curls up. It consists of fibrils. Study the single layer of polyhedral, nucleated, granular, endotlielial cells on its posterior surface {jneinbrana Dcscemeti). CORNEA-CORPUSCLES, AND NERVES OF THE CORNEA. PREPARATION. — These are both demonstrated by the same method, viz. the gold method. This may be accomplished in several ways. {a) Reduction by Dilute Acetic Acid. — Remove the cornea from an animal just killed {e.g. cat, rabbit, or guinea-pig), using the same precautions to avoid stretching it as indicated at p. 104. Place it in a few c.c. of a half per cent, solution of gold chloride, and keep it in the dark. Let it remain in the gold for half an hour to an hour in the case of the cornea of a guinea-pig, and an hour and a half to two hours in that of a rabbit. Remove it, and wash it in distilled water, and place it in water slightly acidulated with acetic acid, and expose it to the light until it becomes of a decidedly purplish or slate-grey colour throughout. Make vertical and horizontal sections, and mount them in glycerine. {b) Reduction by strong Tartaric Acid. — Proceed as before up to the stage where the cornea is washed in distilled water. Expose it for a day, or two days, to the light in distilled water without the addition of any acid, until it assumes a violet colour. Place it in a mi.xture of glycerine, one part, and water, two parts, for two or three days, and keep it in the dark. Wash, and place it in a small beaker half filled with a nearly saturated watery solution of tartaric acid. Heat this in a water-bath kept about 45° C. until it assumes a deep purple colour, which it does in a few minutes. Make sections — vertical and horizontal — and mount them in glycerine. {c) Reduction with Formic Acid. — Place the cornea in the juice of a fresh lemon for five minutes. Wash it well, and transfer it 10 a one per cent, gold chloride solution for half an hour. Wash it again, and place it in a twenty-five per cent, solution of formic acid for twenty-four hours. It must be kept in the dark until the reduction is complete. This process removes all the epithelium, and is, therefore, not suited for showing the terminations of the nerve-fibrils between the cells of the anterior epithelium, but it shows the nerve-fibrils and corpuscles in the cornea admirably. Vertical Section of Cornea. EXAMINATION (L and H). — Observe the same general arrange- ment of parts as before, but note the rows of connective-tissue corpuscles, stained of a deep P io6 PRACTICAL HISTOLOGY. reddish purple, and lying between the laminae of the cornea. Trace some processes communi- cating with the cells in the same plane, and others with processes of those cells that lie above and below them. {Indicate this in one half of Y\. XXV., Fig. i.) Terminations of the Corneal Nerves. — The nerve-plexus in the cornea can be well studied in this section. Observe the primary nerve-plexus in the corneal tissue, and from it trace bundles of nerves running vertically to form the sub-epithelial plexus. It is difficult to find the very fine fibrils which proceed from this plexus to end — as an epithelial plexus — between the cells of the anterior epithelium. Horizontal Section of the Cornea. EXAMINATION (L). — Observe the branched corneal corpuscles seen on the flat. They are deeply stained, so they stand out distinctly. The primary nerve-plexus in the cornea will be only just distinguishable. (H). Cornea Corpuscles. — Note their flattened characters, their branched processes, and their frequent anastomoses with processes from adjoining cells. Each one consists of a ' granular ' plate with a nucleus (PI. XXV., Fig. 2). Nerve-Plexus. — Observe a meshwork of fine nerve-fibrils, with perhaps varicosities on them. It may be that a bundle of nerve-fibrils, constituting an axis-cylinder, and surrounded with a primitive sheath, may be found. THE CELL-SPACES OF THE CORNEA. PREPARATION. — Pith a frog, and scrape away the epithelium from the anterior surface of the cornea. Drop into the eye a few drops of a two per cent, solution of silver nitrate. Leave the silver to act until the cornea becomes of a greyish-white colour throughout, which usually takes from fifteen to twenty minutes. Excise the eyeball, and cut out the cornea, and make one or two cuts in its margin, and lay it flat on a slide in a drop of glycerine, and expose it to the light until it becomes brown in colour. Similar preparations may be made from a mammal, but of course the animal must be deeply anaesthetised to avoid all pain. EXAMINATION (L and H). — The cement-substance is stained brown, and in it is left a series of clear branched and anastomosing spaces — the cell-spaces or lymph-canalicular spaces. They correspond exactly in shape to the corneal corpuscles, which partially fill them (PI. XXV., Fig. 3). The spaces appear to be empty, because the cells filling them are not affected by the silver ; but if a section be stained with logwood the nuclei of the corpuscles are brought into view. Double-staining with silver and then gold brings the corpuscles into view. The silver gives the negative picture and the gold preparations already described represent the positive picture. The Fibrous Tissue of the Cornea. PREPARATION. — Place a small piece of the cornea of any newly-killed animal in a solution of picric acid for twenty-four hours. Tease a small piece in water or glycerine. EXAMINATION (H). — Observe the bundles of wavy fibrous tissue stained yellow. This preparation need not be kept. Plate:xx\^. Cornea.Lens &. Choroid ly lylmtem BroslitK loy THE SCLEROTIC. The sclerotic consists of bundles of fibrous tissue crossing each other in all directions in the plane of the membrane. These bundles become continuous with the substantia propria of the cornea at its margin. Mixed with them is a small quantity of elastic tissue. Sections may be made from any eye which has been hardened in Mi.iller's fluid, or chromic acid and spirit fluid. Stain the sections with logwood and mount them in dammar. THE CRYSTALLINE LENS. PREPARATION, {a) Boiling. — Boil the lens of a fish — e.g. cod — for a few minutes, until it becomes white. Peel off the outer softer part of the lens, and tease a little of the deeper, less brittle part, in Farrant's solution. (d) Glycerine and Nitric Acid Fluid. — Place the eyeball of a frog, or the lens of any other animal, in the above fluid (p. xx-xiv) for twenty-four hours, and then in water for the same time. This dissolves all -the cement, and renders the lens-fibres so tough that they do not break readily when they are teased. Tease a small piece in glycerine as above. Make a similar preparation of the lens of a dog, cat, or rabbit, and note that the lens-fibres in the case of these animals are not nearly so markedly serrated (PL XXV., Fig. 4). EXAMINATION (H). — Observe the lens-fibres of a codfish. They are long, narrow bands, with well-marked teeth or serrations along their narrow margins, the teeth of contiguous fibres dovetailing with one another (PI. XXV., Fig. 5)- (c) Potassic Bichiromate. — Transverse sections ought to be made across a lens hardened in one per cent, potassic bichromate solution for a week. Mount them in Farrant's solution. They are very apt, however, to fall to pieces. The fibres are hexagons when seen in transverse section. The capsule of the lens, and the layer of epithelium lining it, are easily obtained from the eye of a frog. The arrangement of the lens-fibres is easily made out in sections of a fcetal eye. THE CHOROID. Sections may be made through the coats of the eyeball of an ox, hardened in chromic acid and spirit mixture, to include the sclerotic and the choroid. It consists of bundles of connec- tive tissue, with some elastic fibres, many blood-vessels and branched pigment-cells. The layer of hexagonal pigment-cells occurring on its inner surface is now recognised as belonging to the retina (PI. XXV., Fig. 7). Pigment-Cells of the Choroid. PREPARATION. — From the eye of an ox, hardened as de- scribed for the ciliary muscle, remove the retina. Scrape off a little of the dark pigment-layer which adheres to the sclerotic, and diffuse or spread it out in glycerine. .Cover. EXAMINATION (H). — Observe the branched flattened cells, like branched connective-tissue io8 PRACTICAL HISTOLOGY. cells. The nucleus usually does not contain pigment, though the rest of the cell and its processes are loaded with fine granules (melanin) of a brown or black colour (PI. XXV., Fig. 6). . THE CILIARY MUSCLE. PREPARATION. — Divide the eyeball of an ox or sheep, with a sharp razor, transversely, half an inch behind the circumference of the cornea, and remove the lens, but be careful to retain the choroid and iris. Harden the anterior half of the eyeball in the chromic acid and spirit fluid for eight days. Make horizontal sections, to include the sclerotic, the cornea, and the iris. Stain a section with picrocarmine or logwood and mount it in Farrant's solution or dammar. EXAMINATION (L). — Observe the cornea in the front part continuous with the sclerotic behind. At the line of junction notice the section of the pigmented iris projecting inwards. Trace the choroid posteriorly, lining the sclerotic. Observe the fibres of the ciliary muscle arising at the junction of the sclerotic and cornea, and passing backwards over and outside the choroid, into which it is inserted. Sections of the ciliary processes may be seen. Trace the membrane of Descemet of the cornea backwards, and notice that at the junction of the cornea and sclerotic it splits into fine transparent bundles, some of which curve round towards the iris, whilst others spread out like a fan over the ciliary processes, thus forming the liga- mentum pectin at It 111 h-idis. At the junction of the cornea and sclerotic notice a small aperture. This is a section of the venous sinus, or canal of Sclilennn (PI. XXVI., Fig. 4). RETINA. PREPARATION. Miiller's Fluid and Spirit. — It is desirable to use the retinae of several animals, because each shows some special feature. (rt) With a sharp razor divide the eyeball of a pig, cat, and ox transversely, and place the posterior halves in a mixture of three parts of Miiller's fluid and one of spirit, and keep the preparations in the dark and in a cool place for a week or ten days. Make vertical sections of each of the above in the ordinary way, by means of a freezing microtome, after the tissues have been soaked in syrup and then in gum, as described at p. xxxix. This is a most important precaution to ensure good preparations. Make the sections through the sclerotic, choroid, and retina, though the retina is very apt to separate from the other coats. Stain a section of each, with either logwood or picrocarmine. Mount them in Farrant's solution. This method shows the nerve-elements best. [b) Chromic Acid and Spirit Mixture. — This may also be used, and shows the connective- tissue elements best ; besides, it does not make the retina quite so brittle as the Muller's fluid. {c) Osmic Acid. — Place the retina of a frog and that of a fish — e.g. cod— in a few cubic centi- metres of a quarter per cent, solution of osmic acid for three or four hours. The retinae are rapidly blackened and ' fixed.' Soak the retinas for several hours in water to get rid of the surplus osmic acid, and tease a small part of each in a drop of glycerine, and cover. Vertical Section of the Retina of a Cat or Pig. EXAMINATION (L). — Observe from without inwards :-- RETINA. 109 1. The uvea, ox pigmcnt-cdls of the retina, sending fine processes into the next layer. This layer is often detached, and was formerly regarded as belonging to the choroid. 2. The layer of rods and rw/^rj (Jacob's membrane). The rods are easily detected in groups, and seen to be more numerous than the cones, which are much shorter, and not so easily seen. 3. At the lower end of the rods and cones a well-defined line, indicating the position of the external limiting membrane. 4. The outer miclear layer, deeply stained — red or blue — consisting of spherical nuclei arranged in several rows. It is thicker than the inner nuclear layer. 5. The external granular or internuclear layer, a narrow layer. 6. The inner nuclear layer, also deeply stained — red or blue — and consisting of three or four rows of spherical nuclei, larger than those of the outer layer. 7. The inner granular layer, much broader than the outer layer of the same name. 8. The cellular ox ganglionic layer, consisting of a single row of large, well-marked multipolar nerve-cells, each with a well-defined nucleus. 9. The layer of optic nerve-fibres, merely indicated as fine threads, as they have no white substance of Schwann. 10. Most internally is the internal limiting membra7ie. From this at regular intervals arise, by a broad-winged base, ih& fibres of Micller {cox\x\Qctive tissue), which proceed outwards in the retina between the nerve-cells. They, may be traced as far as layer number 6. In the inner- most part of the retina, near the cellular layer, sections of large blood-vessels are to be found. Each of these parts ought to be examined separately. {Indicate the general arrangemetit in PI. XXVI., Fig. I). (H). The pigment-ceUs or uvea. — These cells are cut vertically, and are apt to be detached. They send fine processes into the next layer. Rods and Cones. — Observe the rods, more numerous than the short club-shaped cones. Each consists of an outer and an inner segment. Notice that the inner segment perforates the outer limiting membrane. Study each of the layers in succession. Notice especially the nerve-cells — their large size, their well-defined nucleus and nucleolus, the former with a distinct envelope around it ; their processes — one central, which has a connection with one of the nerve- fibres of the optic nerve, and several processes which proceed outwards and branch in the inner granular layer. Trace the outward course of the fibres of Miiller. The retina of the pig is selected because it shows so well the rods and cones ; but it is ad- visable to study also a section of the retina of an ox, because in it the ganglionic cells are specially large and easily investigated. Retina of the Frog in Osmic Acid. EXAMINATION (H). — Tease out a small piece in glycerine. The rods are very large, and so are easily seen. Both rods and cones consist of an outer and an inner segment or member. In the fresh state the outer member is transparent, bright and glistening, but it is rapidly blackened by the osmic acid, and so is easily recog- nised. It not unfrequently shows a tendency to cleave transversely. Each inner segment is pale, and more or less granular. They may be seen perforating the external limiting mem- brane, and containing one of the nuclei of the external nuclear layer in their course. {Indicate the rods and these segments in PI. XXVI., Fig. 2.) The Retina of a Codfish. EXAMINATION (H). — This is specially selected, because it shows the cones so admirably. Select a cone, and observe its large size ; notice its short tapering outer segment and its large conical inner one, and notice how readily it shows the transverse no PRACTICAL HISTOLOGY. markings on the outer segment of the cone ; sometimes it may be found split transversely. A view of the cones imbedded amongst a great number of the narrow rods may be obtained from above (PI. XXVI., Fig. 3). Many non-medullated fibres of the optic nerve will be found. DOUBLE-STAINING OF THE RETINA. Rosein and Iodine-green. — Stain a section of the retina of a cat or ox in a solution of rosein (p. xlv), and then in iodine-green. Mount it in dammar. The two nuclear or granular layers become of a greenish colour, and all the other parts are red. HEXAGONAL PIGMENT-CELLS OF THE RETINA. From an eye hardened in chromic acid and spirit scrape off a little of the black pigment lining the choroid. A sheet of flattened hexagonal pigment-cells is easily obtained. The nucleus is not pigmented (PI. XXV., Fig. 7). THE EYELIDS. The eyelids are prepared in exactly the same way as recommended for the lips (p. 60). THE LACHRYMAL GLANDS. Prepare them in the same way as the salivary glands, which they closely resemble in structure. Plate XX^/i . Retina k Qliar^ Muse lantern. Bi— s.lilli Ill THE NOSE. (A.) OLFACTORY EPITHELIUM OF THE FROG OR NEWT. PREPARATION, {a) Dilute Alcohol.— Cut off the head of a newt or frog, and slit up the nostrils with scissors ; place it in a small quantity of dilute alcohol for twenty-four hours. Stain the whole head with picrocarmine, and tease a little of the stained epithelium from the olfactory region in a drop of glycerine. This is the best method. \b) Maceration for two days, in a quarter per cent, solution of potassic bichromate, yields fair results. {c) Maceration of the entire head in a half per cent, solution of osmic acid for five hours, followed by steeping in water, also ' fixes ' the cells. EXAMINATION (H). — Observe the ordinary columnar epithelial cells, devoid of cilia, but amongst these observe the narrow, delicate, spindle-shaped cells, each with a spherical nucleus. These are the true olfactot-y cells. To their free end a bunch of fine bristles is attached. Their lower or fixed end consists of a very fine filament with varicosities on it. It is directly continuous with the terminations of the fibres of the olfactory nerve. The olfactory cells are richly scattered amongst the above-described cylindrical epithelium. {Indicate these cells in PI. XXVI I., Fig. 2.) (B.) Similar preparations may be made of the olfactory cells from the olfactory region of the nose of a mammal. In this case the olfactory cells are devoid of bristles on their free ends. (C.) OLFACTORY EPITHELIUM OF A MAMMAL. PREPARATION. — Divide, by means of a saw, the head of a freshly -killed rabbit, dog, guinea- pig, or sheep in a longitudinal direction, and parallel to the nasal septum. Cut out the nasal septum so as to expose the olfactory region, easily recognised by its brown colour. Harden this in Muller's fluid for two days, and then transfer it to the chromic acid and spirit mixture for a week. Complete the hardening in weak, and then in strong, spirit. Make vertical sections, and stain them with logwood, and mount them in dammar or Farrant's solution ; or a double-staining of logwood and picrocarmine may be employed. EXAMINATION (L). — Observe th'^ mucous membrane, covered by columnar epithelium. The upper row of nuclei seen belongs to the supporting or columnar cells ; the deeper row belongs to the true olfactory cells. In the deeper layers of the epithelium, and also in the glands, are pigment-granules. Sections of gland-ducts and alveoli, filled with granular-looking cells — Bowman's glands — are readily seen. (H). Examine the individual parts. {Indicate the olfactory epithelium and Bowman's glands in PI, XXVII., Fig. i.) 112 PRACTICAL HISTOLOGY. TRANSVERSE SECTION OF THE NOSE. The student ought to have a section through the whole nasal chamber in order to study the respiratory mucous membrane, nasal septum, the turbinated bones, and the general arrangement and comparative vascularity of the parts. PREPARATION. — Inject the blood-vessels of the head of a rabbit or guinea-pig with a carmine-gelatine mass from the aorta. Open the skull, and remove the brain ; disarticulate the lower jaw, and cut off the head. Place it in Miiller's fluid for three or four days, and then in the chromic acid and spirit mi.xture for the same time. This fixes the various elements. Place it in the chromic acid and nitric acid mi.xture, until the bones are completely decalcified. Make sections across the entire nasal chamber in a freezing microtome. Doubly stain them with logwood and iodine-green, and mount them in dammar. These form most instructive preparations. 113 THE EAR. This is the only organ of which one cannot give the student a section to carry away with him, on account of their difficulty of preparation, and also because a properly prepared cochlea yields but very i&'fi good sections. THE SEMICIRCULAR CANALS. These are best obtained from the skate, whose cartilaginous cranium should be cut away until the canals with their saccule and utricle are exposed. The canals are hardened in chromic acid and spirit mixture for a week, and then transferred to spirit. The ampullae re- ceive nerves and may be hardened in osmic acid. Sections of these canals must be made with a freezing microtome after steeping in syrup and gum. THE COCHLEA. PREPARATION (A). — The guinea-pig is the best animal to employ. Kill the animal and disarticulate the lower jaw, when the osseous tympanic bulla is exposed. It lies just behind the fossa for the condyle of the lower jaw. Cut away the external auditory meatus, and re- move the petrous portion of the temporal bone with the bulla from the other bones of the skull. Open the bulla with bone-forceps, when a conical elevation — the cochlea — is seen. Remove all the surrounding bone, so as to isolate the cochlea. Place it at once in Miiller's fluid, to harden its delicate tissues, for a fortnight. It is necessary, however, to remove the lime- salts from the bone,- which is done by transferring the organ to a mixture of chromic acid and nitric acid (p. xxxiii), or to a solution of picric acid. The fluid ought to be shaken from time to time, which greatly facilitates the process of softening. After softening preserve it, first in weak spirit, and then transfer it to strong spirit. When sections are to be made it must be steeped in water, to get rid of the spirit, and then it is subjected to the syrup-and-gum process. These substances support its delicate tissues, and sections must be made parallel with the modiolus, i.e., across the turns of the cochlea, in a freezing microtome. They must be manipu- lated with the greatest care, and ought to be stained with logwood or picrocarmine and mounted in glycerine. (B). If the human cochlea be employed, it should be obtained as fresh as possible. Cut out the part of the temporal bone containing the cochlea, from the human subject. Split this up in the axis of the meatus auditorius internus ; this gives two pieces of bone, one containing the cochlea and a part of the vestibule, and the other the semicircular canals Q 114 PRACTICAL HISTOLOGY. and part of the vestibule. Place the pieces at once in a mixture of three parts of Miillcr's fluid and one of spirit for two weeks. After that transfer them to a quarter per cent, solution of chromic acid for a fortnight. At the end of that time add a few drops of hydrochloric acid to the fluid until the bone is softened. The bone around the labyrinth is very dense, and requires a longer time than the other parts to become soft. Wash it thoroughly for twelve hours in water, and place the whole cochlea for thirty-six hours in syrup and forty-eight in gum, and freeze it in gum in a freezing microtome. The student must examine a series of preparations prepared by the above methods. Vertical Section of a Cochlea. EXAMINATION (L). — Observe the modiolns or central column in the centre, with the cochlear branches of the auditory nerve ascending within it. Note a section of one of the turns of the cochlea. Each turn is divided into two compartments by the lamina spiralis. The upper compartment is called the scala vcstihnli, and the lower one the scala tympani. Note that the lamina spiralis consists of an inner osseous portion con- sisting of two plates of bone between which runs a branch of the cochlear nerve on its way to structures which rest on the outer or membranous part of the lamina. In the course of the nerve observe the spiral ganglion. The membranous part consists of a basilar membrane, on which rests the organ of Corti and certain epithelial cells which are connected with the ter- minations of the cochlear branch of the auditory nerve. It is connected to the outer wall of the cochlea by the spiral ligament, which consists of a mass of connective tissue, continuous with the periosteum of the bone. Notice a thin membrane running obliquely upwards and outwards in the scala vestibuli, from the outer part of the lamina spiral ossea to the outer wall of the cochlea ; this is the membrane of Reissner, which cuts off a narrow triangular channel, the ductus cochlecF, or scala media, from the scala vestibuli proper. The floor of this narrow canal is made up of the outer part of the osseous lamina and the basilar membrane. At the outer extremity of the osseous lamina is a spiral groove excavated like a C, with the concavity looking outwards ; this is the sulcus spiralis. The projecting upper edge of the groove is called the crista spiralis. Observe the organ of Corti, consisting of an inner and an outer set o^ rods resting on the basilar membrane. These two sets of rods incline towards each other, their heads touching, so as to leave a triangular passage or tunnel under them, which is filled with endolymph. The outer rods are longer than the inner ones. Internal to the inner rods and external to the outer ones are a number of epithelial cells, some of them with hairs attached to them — the outer and inner hair- cells. A faintly-striated membrane springs from the upper surface of the lamina spiralis ossea, close to the crista spiralis, and may be seen partially to cover the organ of Corti. It was called by Claudius the vieiiibrana tectoria. It terminates over and is attached to the outer hair-cells. The outer and inner hair-cells send prolongations through another membrane which covers them — the membrana reticularis. {Indicate the general arrangement in PI. XXVII., Fig. 3-) (H). The membrane of Reissner consists of a very delicate hyaline-membrane covered on each surface with a single layer of epithelium. Its surface, directed towards the cochlear canal, is composed of polyhedral flattened cells, whilst the vestibular surface is covered by very thin endothelial cells. The membrana basilaris has, on its upper surface, many epithelial cells, some of them specially to form the organ of Corti, and others in which the cochlear branch of the auditory nerve probably terminates, and which are, in fact, its ' end-organs.' The basilar membrane consists of one or two layers of hyaline membrane, which are covered on the tym- panic side by a single layer of endothelial cells. On the upper or vestibular surface of the Plate XXVn . Nose & Cochlea Twfirvtem. Bros.litK COCHLEA. 1 1 5 membrane rests Corti's organ. External and internal to it lie several rows of cells. Beginning at the outer end of the basilar membrane observe the supporting cells of Hcnsen, and then a row of cells with fine bristles projecting from their upper surface — the outer hair-cells. These cells occur in three or four rows. Passing onwards we come to the arch of Corti, composed of an outer and an inner rod or pillar, each in a single row. Each rod has the shape of an /, and they are so arranged as to touch with their upper part or head, whilst the base rests on the membrana basilaris, thus leaving a triangular space between them. Each rod consists of a head, a narrow middle part or body, and a broad, wing-shaped base or foot. The head of the inner rod has a concavity on its outer surface which receives the convex surface of the outer rod. A flat process or head-plate proceeds outwards which helps to cover in the head of the outer rod. It terminates in the membrana or lamina reticularis. The outer rod also sends outwards a short process which forms the first phalanx of the membrana reticularis. The outer rods are longer, thicker, and more numerous than the inner rods. At the foot of each rod, on the surface directed towards the triangular tunnel under the rods, is a small nucleated mass of protoplasm. The inner hair-cells of Deiters lie immediately inside the inner rods. They occur in a single row, and possess fine hairs, projecting from their free surface just like the outer hair-cells. Internal to these there is a row of nucleated columnar epithelial cells, which support these cells. They are continued inwards over the membranous portion of the membrana basilaris as far as the sulcus spiralis. The lamina or meuibrana reticularis will be seen in section. It is a cuticular, hyaline, very resistant membrane which stretches outwards from the heads of Corti's rods to the outer hair-cells. In it exist a number of round or oval apertures, through which the outer hair-cells project. The parts of this membrane which exist between these holes, being shaped like a finger, were called phalanges by Deiters. {Indicate the menibrane of Reissner, Corti s rods and cells, and the membrana tectoria in PI. XXVII. , Fig. 4.) The organs of taste (p. 62) and touch (p. 95) have already been alluded to. Q2 ii6 PRACTICAL HISTOLOGY. THE MALE GENERATIVE ORGANS. THE TESTIS. PREPARATION, {a) Chromic acid and Spirit Mixture. — Use the testis of a dog, rabbit, or guinea-pig. Make several cuts across the testis with a sharp razor, taking care to squeeze the organ as little as possible, and place it in the above fluid. Change the fluid frequently, and at the end of ten days transfer it to weak, and then to strong, spirit. Make transverse sections — by freezing — through the whole organ ; stain them with logwood, and mount them in dammar. They are apt to fall to pieces, but by floating the section in water all difficulty is overcome. {J}) Miiller's fluid. — Harden the testis for three weeks in Miiller's fluid, and then in spirit. The testis is rendered very tough by this method, and it is less apt to fall to pieces. {c) Interstitial Injection of Osmic Acid. — Inject into the testis, with a subcutaneous syringe, a one per cent, solution of osmic acid, and harden the organ in alcohol. This method shows the intertubular connective tissue very well after being stained with logwood. Transverse Section of the Testis. EXAMINATION (L). — Observe the capsule, consisting of an outer layer — the tunica adnata, or the visceral layer of the tunica vaginalis — and an inner, dense, fibrous coat — the tunica albuginea. Between these two layers oval openings are seen : they are sections of lymphatic vessels. From the capsule septa radiate inwards towards the corpus Highmori, so as to subdivide the organ into a series of compartments, each one of which is filled with small convoluted tubes — the tubuli seuiiniferi — which, of course, are cut in every possible direction. These compartments, when filled with the tubules, constitute the lobules of the testis. These septa have a decidedly lamellar structure, and they carry the large blood- and lymph-vessels. Note these tubes of nearly uniform diameter, and observe the small amount of intertubular connective tissue, which takes the form of lamellae of connective tissue covered with endothelial cells. The interlamellar spaces communicate freely with each other, and form the origin of the lymphatics of the testis (p. Ii8). In some animals (dog, cat, and boar), columns of peculiar granular, nucleated cells are found between the tubules. In some animals (guinea-pig) they are pigmented, but their exact nature is unknown. The tubuli seminiferi are relatively large, wavy, and convoluted tubes of uniform diameter, and are cut in every direction. Each tube is seen to consist of a thick membrana propria, of a clear hyaline character, with flattened nuclei in it, so that it is probably endothelial in its nature. It is more or less completely filled with seminal cells. It may be possible to trace the tubules in their course towards the corpus Highmori, where they empty themselves into the vasa TESTIS. 117 recta, which in the corpus Highmori form the rete testis. These tubes are much thinner than the seminal tubes, and there is a constriction where the one tube passes into the other. They are Uned by a single layer of low, columnar cells. They lead into the wider coni vasculosi and the epididymis (p. 117). (^Indicate tlie capsule and sections of the tubules in PI. XXVIII., Fig. i.) (H). Study the tubuli seminiferi, especially in a transverse section. Observe the thick membrana propria ; within it are the seininal cells, arranged in several layers. The outer seminal cells, next the membrana propria, consist of a single layer of transparent, polyhedral, nucleated, and faintly-defined cells. Internal to these are several layers of cells — the inner seminal cells. They are polyhedral, nucleated, somewhat granular cells, though they are usually more spherical in shape next the lumen of the tube. Study the innermost cells : — some of them may be found with their nucleus partially divided, or even divided into two daughter nuclei, and then the whole cell divides, so as to give origin to two dajighter cells. These cells undergo further changes, and give origin to the spermatozoa, and are hence called spermatoblasts. These cells, after certain intermediate changes in their nuclei and cell-contents, split up into a group of young spermatozoa, which are arranged in fan-shaped groups, the head or nucleus of each spermatozoon being directed towards the membrana propria of the tube. This development of the spermatozoa in groups is quite characteristic, and several groups may be seen in a single tube ; afterwards they become detached to constitute spermatozoa. They are best seen in dammar preparations. It is to be remembered that all these stages are not found in a section of any one tube, but several tubes must be examined to see all the stages. {Indicate the cells lining a tubule and the developing spermatozoa in PI. XXVIII. , Fig. 2.) THE EPIDIDYMIS AND VAS DEFERENS. PREPARATION. — Exactly the same as directed for the testis. It is convenient to use the epididymis of a sheep. Make transverse sections, stain them in logwood, and mount them in dammar, and do the same with the vas deferens. Transverse Section of the Epididymis. EXAMINATION (L and H).— We traced the seminal tubules until they formed the rete testis. This system of tubes opens into ten or twenty vasa efferentia, which form the coni z'asculosi, which again open into the canal of the epididymis, and this again into the vas deferens. Sections of the vasa efferentia and the convoluted tube of the epididymis are well seen, the latter being much larger than the former. Each transverse section of the epididymis shows tubes with a relatively large lumen, lined with columnar epithelium, which rests on a membrana propria, supported by non-striped muscle. The epithelium is columnar and ciliated, and the cilia are relatively long. {Indicate the general arrangement in PI. XXVIII., Fig. 3, and the ciliated epithelium in Fig. 4.) At the bases 'of these cells branched pigment-cells are found, especially in the testis of the sheep. Notice the normal amount of interstitial connective tissue, in which nerves and blood- vessels may be recognised. The vas deferens has a mucous, a muscular, and an outer coat, or adventitia. The epithe- lium of this canal is columnar, and carries cilia only in that part next the epididymis. The blood-vessels of the testis are studied in the ordinary way. ]i8 PRACTICAL HISTOLOGY. THE LYMPHATICS OF THE TESTIS. PREPARATION. — Thrust the nozzle of a syringe into a perfectly fresh testicle, and inject into its substance a two per cent, watery solution of Berlin-blue. The lymphatics in the tunica albuginea and spermatic cord are rapidly filled. Harden the testis in alcohol, and make sec- tions, which are to be stained with picrocarmine and mounted in dammar, or in Farrant's solu- tion. The blue colouring matter passes into the origin of the lymphatics between the tubes, but none of it passes into them. EXAMINATION (L and H). — Observe the seminal tubules, stained of a yellowish-red colour, and between them note the distribution of the blue colouring matter, which has passed into the lymphatics. THE SPERMATOZOA. (A.) IN THE LIVING CONDITION. 1. PREPARATION in the Newt. — Kill a large male newt (Triton cristatus), which may be readily recognised by the serrated crest along its back. Remove the viscera, wlien the testes — two or three on each side— will be found as small round bodies, lying close to the spinal column. Remove one of these, make a cut into it, and press out a little of its milky contents on to a slide, add a drop of salt solution ; cover and examine. EXAMINATION (H). — Observe the seething mass of spermatozoa. Each one consists of a so-called licad, to which is attached a long, curved prolongation, the tail ox filament. Study one, and note that the tail moves with a rapid lashing motion, not unlike the action of a cilium, with the result of propelling onwards the spermatozoon. It is well to examine these spermatozoa with a higher power, with a lens of at least ^-inch focal distance. Each spermatozoon is then seen to consist of a head, and next to it is the middle piece of Schweigger-Seidel, and attached to this is the long, pointed filament or tail. A long spiral filament is attached to this by a very transparent membrane (Leydig, H. Gibbes). 2. In a Mammal, e.g. a Sheep. — Make a cut into the globus major of a perfectly fresh testicle, and press out a little of the milky seminal fluid, and examine it as above. (B.) PERMANENT PREPARATIONS. 1. Spermatozoa of the Newt, — Harden the testis of a newt in five per cent, ammonium chromate for twenty-four hours ; wash away all the colouring matter, and divide a testis, and then press out a little of the fluid, which is next mixed with a drop of glycerine, covered and preserved in the ordinary way. The spermatozoa of a newt may be stained with logwood and an aniline dye, but the process is rather difficult, and requires much time and practice. 2. Mammalian Spermatozoa, ia) — Place a little glycerine in a watch-glass, and add to it a few drops of absolute alcohol. Make a cut into the globus major of a perfectly fresh testis. Press out some of the fluid into the watch-glass, and mix it thoroughly with the glycerine fluid. Place a drop of this on a slide, and cover (PL XXVIH., Fig. 5). {b) Another way is to place a drop of seminal fluid on a slide and allow it to dry, and then cover and seal it up diy, without the addition of any fluid. PL^EJCXVm Testis & Ovary MinLem Bi'os lith 119 THE PROSTATE GLAND AND PENIS. These are best hardened in the chromic acid and spirit mixture. It is well to examine a section of a penis with its blood-vessels injected, to study the venous sinuses in the corpus cavernositm penis. The glans penis, when stained with gold chloride reduced by the acetic acid method, shows the mode of termination of medullated nerve-fibres between the epithelial cells covering it, and also their mode of termination in end-bulbs. In the prostate numerous sections of branched tubular glands lined with columnar epithelium, and supported by much non-striped muscle, are to be found. I20 PRACTICAL HISTOLOGY. THE FEMALE GENERATIVE ORGANS. THE OVARY. PREPARATION, [a) Chromic Acid and Spirit Mixture. — Harden the entire ovary of a cat, rabbit, or bitch in the above fluid for seven to ten days, and then transfer it to weak, and afterwards to strong, spirit. Make transverse sections, stain some with logwood, and mount one in dammar, and another in Farrant's solution. {b) Mttller's Fluid. — Harden the entire ovaries of any animal in Miiller's fluid. Change the fluid at the end of the first day, and continue the hardening for three weeks. The specimens must on all occasions be handled very gently, to avoid the removal of the germinal epithelium covering them. Transfer them to weak, and then to strong spirit. Both methods yield ex- cellent results. Transverse Section of the Ovary. EXAMINATION (L). — Observe the framework, which con- sists of (i) the tissue of the hilum, and (2) the stroma of the parenchyma. The tissue of the hilum consists of loose areolar tissue, with many large blood-vessels. The walls of the arteries are easily made out, because they are very thick. The stroma consists of bundles of clear fusiform nucleated cells, with a small amount of fibrous tissue. It is doubtful whether these fusiform cells are muscular or not. Near the surface of the ovary are one or two layers of these cells, which usually contain no ova. In the stroma small groups of polyhedral cells — interstitial cells — the rudiments of the Wolffian bodies, like those occurring in the stroma of the testis, may be found. Note the single layer of short columnar epithelial cells — or germinal epitJielium of Waldeyer — covering the surface of the ovary. Note the Gi-aafian follicles lying in the stroma, and observe their distribution. They are of various sizes and shapes, and the larger ones, i.e. the more developed, lie in the deeper part of the stroma, whilst the smaller ones form a layer two or three deep in the superficial part of the ovary. {Indicate the distribution of the follicles in PI. XXVIII., Fig 6.) Study a large Graafian Follicle (L). — Observe the spindle-cells arranged more or less con- centrically around it to form the tunica fibrosa. Within this a membrana propria, lined with several layers of cells, which constitute the membrana granulosa of the follicle, and placed more or less excentrically is the ovum. The ovum is imbedded in, and rests on, some of the cells of the membrana granulosa, and those cells on which it rests form the cumubis proligerus, and these cells are continued around the ovum, as the tunica granulosa, so as to embrace it. A cavity, which is filled with an albuminous fluid, the liquor folliciili, exists between these two layers of the membrana granulosa. The ovum is a complete cell, and consists externally of a hyaline cell-membrane, the zona pellucida ; within this is the transparent yolk or vitellits. Im- bedded usually in one side of the yolk is the germinal vesicle (nucleus), with its included germinal spot. {Indicate the ovum and membrana granulosa in PI. XXIX., Fig. i.) PI^TEXXK . OvARXEaLLOPIAI^ TUBEjiTERUS^ JXCCCl & (o e^ (i\ 7\ i H Mmteni. Bros.litK OVARY. 121 (H). Study each of these parts from within outwards. In the vitellus, yolk-granules and a fine reticulum of fibres may be detected. Study a Small, Unripe, Graafian Follicle. (H). — These are found' near the surface of the section. Notice the ovum, with a relatively thin zona pellucida, and the membrana granulosa, consisting of, perhaps, only two layers of cells, and not yet separated into two distinct layers by the appearance of the liquor folliculi between them. All gradations are found between these and a follicle ready to burst and discharge its ovum. {Indicate tlicsc, and the epithelium covering the ovary, in PI. XXIX., Fig. 2.) THE DEVELOPMENT OE GRAAEIAN FOLLICLES AND OVA. The -ovaries of fcetal kittens or rabbits are prepared in the same way as directed for the adult ovar>' (p. 1 20). Sections show the development of the ova from the germinal epithelium covering the ovary. CORPORA LUTEA. These are easily studied in the ovary of a cow, where they are very large. Small pieces of one of them ought to be macerated in very dilute potassic bichromate, to isolate the large branched and pigmented cells which compose them. Coloured crystals of hsematoidin may be found. Other pieces ought to be hardened in Miiller's fluid for two weeks, and sections made to study the arrangement of the cells which compose them. THE FALLOPIAN TUBE. PREPARATION.— Cut the Fallopian tube of a cat or bitch into pieces an inch long, and harden them for four days in the chromic acid and spirit mixture. Make transverse sections, and stain them with logwood, and mount them in dammar. EXAMINATION (L and H). Observe the serous, or outer coat, composed of fibrous tissue, and within this the muscuhir coat, composed externally of a few muscular fibres, arranged longi- tudinally, and therefore cut transversely, whilst within these is a much thicker circular coat. Then follows the mucous coat, consisting of fibrous tissue, containing many blood-vessels. It is thrown into longitudinal folds by the contraction of the circular muscular fibres, and is lined by columnar ciliated epithelium. It contains no glands. Do not mistake the depressions between the folds of the mucous membrane for glands. {Indicate part of tlie wall of the tube in PI. XXIX., Fig. SO THE UTERUS. PREPARATION.— Remove the uterus from a cat or bitch — preferably from an animal which has borne young — cut it in pieces an inch long, and harden them for five days in chromic acid and spirit mixture, and then transfer them to spirit. Make transverse sections, and stain them with logwood, and mou;it them in dammar. R 122 PRACTICAL HISTOLOGY. EXAMINATION (L). — Observe («) the serous coat, {b) the muscular coat, very thick, and composed of bundles of non-striped muscle, arranged in many layers. The mucous coat con- sists of fibrous tissue, and is lined by columnar ciliated epithelium (the cilia are difficult to preserve), and in it are seen sections of the uteruie glands, v/hxch are cut in every direction, but they are simple tubular glands, often branched at their lower extremities, and they are lined throughout by ciliated epithelium (PL XXIX., Fig. 3). (H). Observe specially the uterine glands, with their membrana propria lined by ciliated columnar cells. {Indicate a gland in PI. XXIX., Fig. 4.) Similar preparations ought to be made of the cervix uteri, e.g., of a cow. THE MAMMARY GLAND. PREPAKATION. — The best specimens are obtained from a gland taken from an animal near the full period of gestation. Harden small pieces for a week in the chromic acid and spirit mixture ; make sections, stain them with logwood, and mount them in dammar. EXAMINATION (L). — Observe the. framework, which consists of septa, chiefly of lamellar connective tissue, which subdivide the gland into a series of small polygonal lobules. In these septa sections of the large lactiferous or milk ducts, and large blood-vessels are found. Study a lobule (L and H). — Observe its shape, and note that it consists of a large number oi gland alveoli, supported by a very small amount of connective tissue. Study an alveolus (H). — Observe its membrana propria, lined by a single layer of short columnar epithelial cells. Sections of the large lobular ducts will be readily met with. Fat- globules, i.e. milk-granules, may be seen within the protoplasm of these cells. Osmic acid readily reveals the presence of fatty particles in the cells by blackening them. {Indicate the alveoli, with the cells lining them, in PL XXX., Fig. i.) THE MILK. Place a drop of fresh milk on a slide, apply a cover-glass and examine (H). It consists of a large number of oil-granules, of various sizes, floating in a fluid. Each globule consists of an albuminous envelope (Ascherson's membrane), enclosing a globule of oil. Add acetic acid, which partially dissolves the envelope, and then the oil-globules run into little heaps or clusters. Osmic acid blackens them. Do not preserve them. {Delineate in the upper half of PL XXX., Fig. 2, ordinary milk, and in the lower half the effect of acetic acid on it.) In the milk shortly after delivery many large nucleated cells, filled with milk-globules — the colostrum corpuscles — are to be found. Examine these (H). THE PLACENTA. PLACENTA OF A CAT OR GUINEA-PIG. PREPARATION. — The animal ought to be killed about the middle of the period of gestation. Open the abdomen and expose the uterus. The uterus is cautiously opened, PlateXXX, Mamma.MilkPlacenta. Mintem Bro B itli PLACENTA. 123 and care is taken not to sever the attachment of the placenta to the uterus. Harden the uterus, placenta, and attached foetus, which is still within the amniotic cavity, in the chromic acid and spirit mixture for two weeks, and then transfer them to dilute spirit, and afterwards to strong spirit. The foetus can be used for making sections of various organs. Make vertical sections through the uterus and the placenta, and stain them with logwood or picrocarmine, and mount them in dammar or Farrant's solution. EXAMINATION (L). — Observe the uterus, with the placenta lying internal, and firmly attached to it. The placenta consists of a narrow maternal part next the uterus, and a much thicker part — the fcetal portion. In the uterus numerous sections of the enlarged uterine sinuses are seen. The foetal portion consists of richly branched villi, which are separated from each other by a relatively small amount of delicate connective tissue, and a layer of cells, which covers each villus. Within these villi note the loops of blood-vessels. Their distribu- tion is easily seen, becau.se they still contain the coloured blood-corpuscles. FRESH HUMAN PLACENTA. PREPARATION. — Tease a small piece of a perfectly fresh placenta in salt solution. EXAMINATION (L). — Observe the large number of isolated and richly branched placental villi. Each one contains a blood-vessel, whose course is easily seen, because it is still filled with blood-corpuscles. A capillary loop is sent into each of the secondary branches on the villus. The villus is covered by a layer of epithelium, whose nuclei are easily revealed by staining the section with magenta solution. This epithelial covering varies in thickness on the same villus. A delicate mucous tissue exists around the blood-vessels. {^Indicate the shape and general characters of the villi in PL XXX., Fig. 3.) (H). Observe the capillary blood-vessels filled with blood-corpuscles, and between the vessels note the corpuscles of the mucous tissue which supports them. Study the epithelial covering. It is low, columnar epithelium, with here and there masses of protoplasm, which contain many nuclei. (^Indicate the epithelial covering and blood-vessels in PL XXX., Fig. 4.) HOW TO PRESERVE THE HUMAN PLACENTA. (1) Osmic Acid. — A small piece of the fresh placenta ma}- be teased in a one per cent, solution of osmic acid, and mounted in glycerine. (2) Dilute AIcohoL — Maceration for forty-eight hours in this fluid is an excellent means of isolating the villi. They may be stained with picrocarmine. (3) Miiller's Fluid. — Harden very small pieces of a perfectly fresh placenta for three weeks in this fluid. Make vertical sections, and treat them as directed for the placenta of a guinea-pig. R 2 124 PRACTICAL HISTOLOGY. THE FCETAL MEMBRANES. THE DECIDU^. The decidua vera and reflexa can be separated from each other near the placenta. Remove a little of the tissue from the outer surface of the decidua reflexa, and examine it in salt solution. It will be found to be nearly covered with fusiform cells, which are, for the most part, filled with fatty particles. These cells may be stained with methyl-aniline, and preserved in a saturated solution of acetate of potash. The substance of the decidua consists of a tissue closely resembling mucous tissue. If a small piece be treated with gold chloride (p. xlv) branched connective-tissue corpuscles are obtained. THE AMNION. K small piece of this membrane, after being hardened for five days in Miiller's fluid, is thoroughly washed, and placed on a slide, and stained with picrocarmine. Observe on its inner surface a layer of somewhat flattened cells. THE UMBH^ICAL CORD. PREPARATION. — Cut a fresh cord into pieces an inch in length, and harden them in Miiller's fluid for two days, and afterwards in the chromic acid and spirit mixture for a week. Make transverse sections. Stain one with logwood, and mount it in dammar, and another with picrocarmine, and mount it in Farrant's solution. EXAMINATION (L). — Observe the circular shape of the cord, containing sections of three blood-vessels, £e. two umbilical arteries and a larger umbilical vein. These are surrounded by a modified mucous tissue (p. 28), called Wharton^s jelly, and outside all is a layer of thin, nucleated, polygonal cells. The tissue is denser around the blood-vessels. In Wharton's jelly are found spaces — the sections of canals that run in the cord. They are most obvious near the circumference. Note the mucous corpuscles amongst the fibres. {^Indicate the uinbilical arteries and vein, and Wharton's jelly, in PI. XXX., Fig. 5.) (H). Examine the walls of the arteries and vein, and note their great thickness. Study the branched corpuscles of Wharton's jelly. {Indicate the spaces and the mucous tissue in PI. XXX., Fig. 6.) INDEX. o^Jo ACE ACETIC acid, xx Adenoid tissue, 29 Adipose tissue, 27 development of, 27 Adjustment, coarse and fine, xxii — of objective, xxii Air-cells of the lung, 56 Alcohol, absolute, xxxi — dilute, xxxiv — methylated, xxxii — rectified, xxxiv Ammonium bichromate, xxxii — chromate, xxxii — picrocarminate, xliii Amnion, 124 Amosboid movement, 4 Aniline blue for peptic cells, 67 Aniline blue-black for nerve-tissues, xliv Aniline dyes, xliv Aniline violet, xliv Anterior roots, 97 Aorta, 50 Apparatus necessary for histological work, xix Appendix vermiformis, 70 Aqueous humour, xxx Areolar tissue, action of acetic acid on, 22 corpuscles, 22 stained with logwood, 22 stained with picrocarmine, 22 constrictions on fibre-bundles, 23 elastic fibres, 21 — stained with magenta, 20 stained with picrocarmine, 20 interstitial injection of osmic acid, 22 interstitial injection of picric acid, 22 Arrector pili muscle, 93 Arteries, 51 Articular cartilage, 14 Artificial digestion, 92 Arthropoda, muscles of, 39 Arytenoid cartilage of ox, 18 Auerbach's plexus, 71 Axis-cylinder in nerve-fibre, 42 BRO BARYTA water, xxxiv Basilar membrane, 1 14 Berlin blue, li Bicarbonate of soda, 34 Bichromate of ammonia, xxxii — potash, xxxi Bile-ducts, 74 Birch on bone lamella, 32 Bladder of frog, 36 — of mammal, 89 Blood, circulation of, 52 — granules of, 6 Blood-corpuscles of frog, i of newt, I man, 5 effect of acetic acid, 2 of dilute alcohol, 2 of ammonium chromate, 3 of hydrochloric acid, i — ■ of magenta, 2 of osmic acid, 3 of picric acid, 3 of syrup, 2 of water, 2 of tannic acid, 2 feeding of colourless, 4 structure of, 3 Blood-crystals, 6 Blood-serum, xxx Blood-vessels, 49 — development of, 50 — injection of, Iv Bone, cancellous, 34 — dense, 33 — foetal, 32 Bone-corpuscles, 31 Bone-lamelte and Sharpey's fibres, 32 — methods of softening, 30 Bowman's glands, 1 1 1 — sarcous elements, 39 Brain, loi Bronchus, structure of, 55 Brownian movement. 126 INDEX. CRU DUC Briicke's dissecting microscope, xxxv Brunner's glands, 68 CABINET for microscopic specimens, xx Caliper micrometer, xxv Camera lucida of Chevalier, xxvii Zeiss, xxvii Canada balsam, xlix Canaliculi, 31 Cannulas for injecting, Hi Capillaries, 49 Carmine staining-fluid, xliii — gelatine injection, li Carotid gland, 82 Carrot for imbedding, xxxvi Cartilage, articular, fresh, 14 — cell-spaces of, 14 — matrix, 14 Cartilage-cells, action of water, 14 — action of iodine, 14 — preservation of, 14 — fat in, 16 — stained by gold chloride, 17 — stained by eosin, 16 — stained by silver nitrate, 17 Cartilage, costal, adult, 16 young, 1 5 — transition between hyaline and yellow, 19 — hyaline, 14 — elastic, 18 — fibrous, 18 — of Loligo, ig Cell-spaces of tendon, 25 Cells of tendon, 25 Cements, 1 Cement-substance of muscle, 37 Central tendon of diaphragm, 76 Cerebellum, 100 — double-staining of, lOO Cerebrum, loi Chalice cells, 10 Chloral hydrate for brain, 96 Chloride of gold, xlv Chloroform on cilia, 12 Choroid of eye, 107 Chromate of ainmonia, xxxii Chromic acid, xxxi and spirit, xxxi and nitric acid, xxxiii Cilia, 1 1 Ciliary motion, 1 1 on gills of a mussel, 1 1 — effect of heat, 1 1 — effect of chloroform, 1 2 Ciliary muscle, 108 Ciliated epithelium, 1 1 Circulation of the blood, 52 Circumvallate papillte, 60 Cleaning cover-glasses, xxv Cleaning slides, xxiv Cleavage of muscular fibres, 39 Clove oil, XX Coccygeal gland, 82 Cochlea, 1 13 Colostrum, 122 Columnar epithelium, 10 Commissures of the cord, 97 Condenser, bull's eye, xxi Cones of retina, 1 10 Connective-tissue corpuscles, 21 Convoluted tubules of kidney, 85 Corium, 91 Cornea, cell-spaces of, 106 — corpuscles and nerves of, 105 — method of preparing, 105 Corpora lutea, 121 Corti, organ of, 114 — rods of, 114 — cells of, 115 Costal cartilage, 1 5 Cover-glasses, xix — measuring of, xxv Crusta petrosa, 62 Crystalline lens, 107 Cutis vera, structure of, 90 Cutting sections, methods of, xxxvi DAMMAR, xlviii Decalcifying solutions, xxxiii DeciduK, 124 Deiters, cells of, 115 Delineation of objects, xxvi Dentine, 62 Diapedesis, 52 Diaphragm, of microscope, xxiii — lymphatics of, 76 — tendon of, 41 Digestion, method of (W. Stirling), 92 Dilute spirit, xxxiv Dissecting microscope, xxxv Dissociation by needles, xxxv — by teasing, xxxv — by shaking, 29 Dissociating fluids, xxxiv Distilled water, xlv Double-staining, method of, xl — cerebellum, 100 — bone, 35 — glands, 61 — Peyei^s patch, 69 — retina, 1 10 — tendon, 25 — trachea, 54 Duodenum, 67 Ductless glands, 82 Ductus cochleae, 114 INDEX. 127 EAR pAR, 113 J — ' Ebner, v,, method for bone, xxxiii Elastic cartilage, iS lamina of cornea, 104 Elastic tissue, 20 transverse section of, 21 network in mesocolon, 2 r laminK of arteries, 50 Elder-pith, xxxvi Embryonal tissue, 28 Enamel, 62 Endocardium, 47 — ■ fibres of Purkinje, 48 End-bulbs of penis, 1 19 End-plates, 45 Endomysium, 37 Endothelium, 13 Eosin, xiv Epidermis, 90 Epididymis, 117 Epiglottis, 18 Epineurium, 44 Epithelium, single layer, 13 — squamous stratified, 8 — ciliated, 1 1 — columnar, 10 — transitional, 12 — secretor)', 12 Examining a simple object, xxiv Eye, 104 Eye-lids, 1 10 Eyepiece, xxii FALLOPIAN TUBE, 121 Farrant's solution, xlviii Fat-cells, 27 — membrane of, 27 — development of, 27 Feeding blood-corpuscles, 4 Ferrein, pyramid of, 85 Fibrilte of muscle, 39 Fibrils, intra-cellular plexus of, 9 — intra-nuclear plexus of, 9 Fibrin in blood, 5 Fibro-cartilage, 14 Fibrous tissue, white, 21 yellow, 20 Filiform papilUi;, 60 Forceps, xix Formic acid, xlv Freezing microtomes, xxxviii — mixture, xxxviii — process, xxxviii Fresh tissues, examination of, xxx Fungiform papillse, 60 G ANGLIA, of bladder, 89 — lung, 59 HYD Ganglia, of tongue, 61 — Gasserian, 102 — spinal, 102 — sympathetic, 103 Gas-chamber, xxix Gases, application of, xxix Gasserian ganglion, 103 Gastric follicles, 67 — method of isolating, 67 Gauge for cover-glasses, xxv Gelatine injections, li Germinal vesicle, 120 Giant-cells, 35 Glass slides, xix Glandular epithelium, 12 Glisson's capsule, T>, Glycerine, xlviii Glycerine jelly, xlviii Goblet-cells, 10 Gold chloride, xlv — for Auerbach's plexus, 71 • — cornea, 105 — nerve-terminations, 45 — skin, 95 — and formic acid, xlv Graaffian follicle, 120 Granular layer of cerebellum, 100 • muscle, 40 retina, 109 Granules, free in blood, 5 Grey matter of cerebrum, !0[ of cord, 97 Gum for imbedding, xxxviii HEMACYTOMETER, 7 Hasmaglobinometer, 7 Hjematoxylin, xlii Haimin, 7 Haemoglobin crystals, 6 Hairs, 94 Hair-follicle, 93 Hand sections, 94 Hardening, methods of, xxx Hartnack's microscope, xxi Hassall's corpuscles, 8t Haversian canals, 31 Heart, muscular substance of, 47 — frog and mammal, 47 Heat, methods of applying, xxviii Henle, looped tubules of, 84 Hensen, cells of, 115 Hepatic cells, 73 — vein, -j^, High powers, xxii Hollow ground razor, xix Hot stage, xxviii Hyaline cartilage, 14 Hydrochloric acid for bone, xxxiii — — blood, 1 128 INDEX. HYD Hydrochloric acid for uriniferous tubules, 87 Hypophysis cerebri, 82 T LLUMINATION, methods of, xxiii -L Imbedding, methods of, xxxvi Incisures of a nerve-fibre, 42 Infundibulum of lung, 57 Injection, method of, i — apparatus, water pressure, Iv — apparatus, mercury pressure, liv — of an entire animal, Iv — of blood vessels, Iv — of lymphatics, Iv — mass, method of preparing, li Intervertebral disc, 18 Intramembranous bone, 35 Intercellular substance, 13 Intestine, 68 Intra-cellular network, 9 Intra-nuclear network, 9 Involuntary muscle, 36 Iodine-green, xlvii Iodine solution for cartilage, 14 Iodised serum, xxxiv Irrigation, 2 KIDNEY, structure of, 84 Kleinenberg's logwood, xliii — picric acid, xxxii Krause's membrane, 39 LABELS, xxvi J Lachrymal gland, no Lamp, xxiii Large intestine, 70 — slides, xix Larynx, 53 Lateral discs, 39 Lens, 107 Lieberkiihn's follicle, 70 Ligamentum nuchas, 20 Lip, 60 List of apparatus, xix staining reagents, xlvii histological requisites, xix Liver, 71 Living tissues, study of, xxvii Logwood staining-fluids, xlii Lung, 53 Lymphatics, injection of, Iv — of diaphragm, 76 Lymph-glands, simple, 78 compound, 77 MAGENTA, xliv — for elastic fibres, 20 — for blood-corpuscles, xliv Malassez, 7 NER Malpighian bodies, 79 — capsules, 84 — tufts, 84 Mammary glands, 122 Marrow, red, 35 — giant-cells of, 35 Measuring cover-glasses, xxv Medulla oblongata, 99 Medullated nerve-fibres, 42 Membrana granulosa, 120 Meissner's plexus, 71 Mesentery of newt, 38 Mesocolon of rabbit, 2 1 Methyl-aniline, xliv Microscope, xxi — compound, xxi — dissecting, xxxv — preparation of, xxi — testing of, xxi — Hartnack's, xxi — Swift's, xxi Microtomes, xxxvii ■ — Ranvier's hand, xxxvii — A. B. Stirling's, xxxvii — Rutherford's freezing, xxxviii — Williams' freezing, xxxix — B. Lewis's ether, xl Milk, 122 Modiolus, 114 Moist chambers, xxvii Mounting of specimens, 1 — solutions, xlviii Mouth, 60 Mucigen, 64 Mucin, 8 Muco-salivary glands, 64 Mucous glands of tongue, 61 Mucous tissue, 28 Miiller's fibres, 109 — fluid, xxxii Multipolar nerve-cells, 98 Muscle, non-striped, 36 — striped, 38 — and tendon, union of 41 — of crab, 39 — of heart, 47 — nerves of, 45 Muscularis mucosEe, 66 NAILS, 94 Nasal mucous membrane 1 1 Needles mounted, xix Nerve-cells of ganglia, 102 spinal cord, 98 frog, 103 Nerve-fibres, medullated, 42 — non-medullated, 45 — of spinal cord, 44 — with osmic acid, 43 INDEX. 129 NER Nerve-fibres, silver nitrate, 43 — of cornea, 105 muscle, 45 skin, 95 — terminal organs, 45 Nerve-fibrils, 105 Neurilemma, 44 Neurogleia, 97 Neutral tint-reflector, xxvii Nodes of Ranvier, 43 Non-striped muscle, 36 Nosepiece, xxii OBJECTIVES, xxi — dry and immersion, xxii Ocular, xxii Odontoblasts, 63 Gisophagus, 65 Oil of cloves, XX Oil immersion lenses, xxii Olfactory cells, 1 1 1 Olivary body, 99 Omentum, 26 Opaque injections, 74 Osmic acid, xlvi Ossification, intra-cartilaginous, 34 — intra-membranous, 35 Osteoblasts, 33 Osteoclasts, 35 Outline plates, how to fill in, xvii Ovary, 120 Ova, development of, 121 PACINIAN corpuscles, 46 Pancreas, 75 Papillje of tongue, 60 Paraffine for imbedding, xxxvi Parotid gland, 64 Penis, 119 Peptic glands, 67 Peptic cells, 66 Pericardium, 47 Perimysium, 37 Perineurium, 44 Periosteum, 31 Peripheral terminations of nerves, 45 Peyer's glands, 69 Photophore, xxxv Pia mater, vessels of, 49 Picric acid, xxxii Kleinenberg's, xxxii for bone, 30 Picrocarminate of ammonia, xliii Pigment-cells of choroid, 107 Pith for imbedding, xxxvi Placenta, 123 Plasma cells, 22 Pleura, 56 SER Potassic bichromate, xxxi Potassic hydrate, xxxiv Pressure bottle, injection apparatus, liv Prickle cells, 60, 91 Prism for drawing, xxvii Prostate, 119 Prussian blue, soluble, li Pulmonary vessels, injection of, 58 Pulp of tooth, 62 spleen, 79 Pure soluble blue, li Purkinje's fibres of heart, 48 ■ — cells of cerebellum, 100 Purpurine, xliv Pyloric end of stomach, 67 RANVIER'S microtome, xxxvii — nodes, 43 — photophore, xxxv — picrocarmine, xliii Razor, xix Reagents, bottles for, xx — list required, xx Reissner, membrane of, 114 Respiratory organs, 53 Rete Malpighii, 90 Retina, 108 Rodded epithelium of kidney, 86, 87 Rods of retina, 109 Rolando substantia gelatinosa, 97 Rosanilin nitrate, xliv Rosein, xlv SALINE solution, xxx Saliva, 8 Salivary corpuscles, 8 — glands, 64 Salt solution, xxx Sarcolemma, 38 Sarcous elements, 39 Scala vestibuli, 114 Scalp, 93 Schafer's heating apparatus, xxviii Schneiderian mucous membrane, 1 1 1 Scissors, xix Schwann, sheath of, 42 — white substance of, 43 Sclerotic, 107 Sealing preparations, 1 Sebaceous glands, 93 Sections, making of, xxxvi — mounting of, xlix — how to float on to a slide, xxvi Semicircular canals, 113 Seminiferous tubules, 116 Septum cysterniE lymphaticoe maguK, 76 Serous membranes, 13 — glands, 61 I30 SHA Sharpey's fibres, 32 Silver nitrate, xlv — lines, 13 — process, xlv Skin, 90 — blood-vessels of, 94 — nerves of, 95 Slides, xix Small intestine, 67 Softening fluids, xxxiii Solitary glands, 70 Spermatoblasts, 117 Spermatozoa, 1 1 8 Spinal cord, 97 isolation of cells of, 98 Spinal ganglia, 102 Spiral groove, 114 — lamina, 1 14 — ligament, 114 Spleen, 78 Squamous epithelium, 8 Stage of microscope, xxii — warm, xxviii Staining fluids, xlvii — methods of, xlii Stirling, W., method of artificial digestion, 92 Stomach, 66 Stomata, 76 Stratified epithelium, g Stratum corneum, 90 — lucidum, 90 — granulosum, 90 — Malpighii, 90 Strieker's warm stage, xxviii Striped muscle, 38 Sub-arachnoid trabecule, 23 Sub-lingual gland, 64 Sub-maxillary gland, 64 Supra-renal bodies, 82 Sweat-glands, 91 Sympathetic nerves, 45 Syringe for injecting, Hi — subcutaneous, 22 TACTILE corpuscles, 92 Tadpole, tail of, 50 Tail of a rat, 25 Tannic acid, effect of, on blood, 2 Taste-bulbs, 62 Teasing, method of, xxxv Tectorial membrane, 1 14 Tendon, 23 — cells, 25 Test objects, xxi Testis, 116 — of newt, 118 Thin slides, xix INDEX. ZON Thymus gland, 81 Thyroid gland, 82 Tongue, 60 — mucous glands of, 61 — serous glands of, 61 Tonsils, 65 Tooth, 62 Trachea, 54 Transitional epithelium, 12 Treble-staining, xlvii Trypsin digestion, xxxiv Tubuli seminiferi, 84 Tunica adventitia, 49 — adnata, 1 16 — albuginea, 1 16 — granulosa, 1 20 Turntable, xlix UMBILICAL cord, 124 , Unstriped muscle, 36 Ureter, 88 Urinary bladder, 89 Uriniferous tubules, 84 Uterus, 121 VAS deferens, 117 Vasa efterentia of testis, 31 Vasa recta, kidney, 88 testis, 116 Veins, 51 Villi of small intestine, 68 — of placenta, 123 Vitelline membrane, 120 Vitreous humour, 28 Vocal cords, 53 Voluntary muscle, 38 WAGNER'S corpuscles, 92 Warm stages, xxviii Water, action of, on blood-corpuscles, 2 Wharton's jelly, 28 White blood-corpuscles, 3 method of feeding, 4 White fibro-cartilage, 18 White fibrous tissue, 21 White matter of cerebellum, 100 of cerebrum, loi of spinal cord, 97 White substance of Schwann, 42 'WELLOW fibro-cartilage, 18 ZEISS, camera lucida, xxvi — oil immersion lenses, xxi Zinc cement, 1 Zona pellucida, 120 Spottuiroode .)■ Co., Printers, Nm-street Square, lAmdon. SMITH, ELDE R, & CO.'S P UBLICATIONS. ATLAS OF HISTOLOGY. By E. KLEIN M.D., F.R.S. LECTURER O.N HISTOLOGY AT ST. BARTHOLOMEW'S HOSPITAL MEDICAL SCHOOL, By NOBLE AND SMITH, P.R.C.S. Edin. &c. FORMERLY SENIOR HOUSE-SURGEON TO ST. MAKY's HOSPITAL, AND RESIDENT MEDICAL OFFICER TO THE CHILDREN'S HOSPITAL AT BRISTOL, AND THE LOCK HOSPITAL, LONDON. A complete Representation of the Microscopic Structure of Simple and Compound Tissues of Man and the higher Animals, in carefully executed Coloured Engravings, with E.xplanatory Text of the Figures, and a concise Account of the hitherto ascertained facts in Histology. Royal i,to. with 48 Coloured Plates, bound in half-leatJier, fn'ee ^£4. 4s.; or in Thirteen Farts, priee 6s. each OPINIONS OF THE PRESS. ' We are much pleased with these plates. Tliey give very faithful delineations of the objects described in the text, and they are not overcharged. They should be on the table of every practitioner wlio desires to make himself familiar with the pro- gress of Histolog)'.' — Lancet, April 26, 1879. ' We have great pleasure in drawing attention to this very handsome work, which is certainly the most richly illustrated treatise on Histolog)' which has ever been published. . . . Mr. Noble Smith has executed drawings from the choicest preparations made by Dr. Klein, which in colour and form present to the eye precisely the same effect as the preparations themselves when accurately focussed. . . . The medical student will find this work a surer guide to the knowledge of modern His- tology than the older books, even those of high authority and reputation.' — The Quarterly Journal of Microscopical Science, October, 1S80. ' This admirably executed Atlas has throughout maintained the high promise with which it started, and fulfils all that could be expected from the joint work of our best histologist and the mojt accomplished of British medical draughtsmen. The illustrations of the last two parts, especially devoted to the display and brief description of the intimate structure of the nervous centres and nerve ganglia, are admirable for the per- fection and beauty of the sections and the choice of specimens, as they are for the delicate skill and intimate knowledge of the draughtsman. The text is only too sh-jrt for it is full of accurate, recent, and lucid information. It is long since any work of more entirely sntisfactory conception and execution has issued from the English medical press. This is very high praise ; but the rare merits of this standard production deserve no less.' British Medical Journal, October 4, 1879. \Vith 208 Illustrations, 8vo. T2j. COMPENDIUM OF HISTOLOGY. TWENTY FOUR LECTURES. By HEINRIOH FREY, Professor. Translated from the German, by permission of the Author, by GEORGE R. CUTTER, M.D. \\'ith numerous Illustrations. Crown 8vo. 10^. td. A COURSE OF PRACTICAL HISTOLOGY. By EDWARD ALBERT SCHAFER, assistant professor of phvsiologv, university college. We are very much pleased with Mr. .Schafer's book. It is clearly written, well and originally illustrated, and possesses the merit of being both concise and complete. In conclusion, we may say that any student who has gone fairly over the ground covered by this treatise, wdl find that he has not only gained a mastery over the modern methods of microscopical investigation. but a sound knowledge of the several structures of which the body is composed.' — Lancet. 'Concerning Mr. Schafer's " Practical Histology" we have nothing to say except in praise. 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