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 COLLEGE OF PHYSICIANS 
 AND SURGEONS 
 
 Reference Library 
 
 Given by 
 
OUTLINES 
 
 OF 
 
 PRACTICAL HISTOLOGY 
 
OUTLINES 
 
 OF 
 
 PRACTICAL HISTOLOGY 
 
 BY 
 
 WILLIAM RUTHERFORD, M.D., 
 
 F.R.SS. L. AND E. 
 
 PROFESSOR OF THE INSTITUTES OF MEDICINE 
 j IN THE UNIVERSITY OF EDINBURGH, EXAMINER IN PHYSIOLOGY 
 IN THE UNIVERSITY OF LONDON 
 
 SECOND EDITION 
 
 WITH SIXTY-THREE WOOD ENGRAVINGS 
 
 LONDON 
 J. & A. CHURCHILL, NEW BURLINGTON STREET 
 
 PHILADELPHIA : LINDSAY AND BLAKISTON 
 1876 
 
 [All rights reserved.] 
 
PREFACE 
 
 TO 
 
 THE SECOND EDITION. 
 
 No subject more than Practical Histology requires 
 for its successful pursuit that guidance which oral 
 instruction alone can give. If the beginner attempts 
 to study it without a teacher, he is baffled in learn- 
 ing how to use the microscope, how to manipulate 
 the tissues, and how to apprehend rightly the phe- 
 nomena that come under his observation. Much 
 valuable time is thus lost, and the final result is 
 generally failure and disappointment. 
 
 These Outlines are therefore primarily intended 
 to assist beginners who are working under the 
 guidance of a teacher in a laboratory furnished with 
 the usual appliances, together with a full set of those 
 microscopical specimens that cannot be readily made. 
 They are at the same time, however, designed to assist 
 those who may already have learned the rudiments of 
 
vi PREFACE. 
 
 the subject at a time when its details were not taught 
 as they now are. Care has been taken to render all 
 instructions as definite as possible, and the advanced 
 student will find in Part I. such directions for the 
 preliminary preparation of the tissues as will enable 
 him to work through the Histological Demonstra- 
 tions in Part III. without embarrassment. The 
 paragraphs in small type are especially intended for 
 the advanced student. 
 
 These Outlines are essentially the Notes of the 
 course of Practical Histology given by me in King's 
 College, London, and in the University of Edin- 
 burgh. That course is conducted on the principle 
 that, inasmuch as the time at the disposal of most 
 students of medicine is very limited, the only 
 practicable method of teaching large numbers is 
 what may be termed the regimental system, whereby 
 the students all work at the same moment, at one 
 subject, and are addressed collectively. In this 
 way classes, under thirty in number, can be con- 
 ducted through the leading points of Practical His- 
 tology in about thirty-six lessons, extending from 
 an hour to an hour and a half. As it is impossible in 
 this short time to prepare every desirable microscopi- 
 cal specimen, special demonstrations are made by the 
 teacher in order that the introduction to the subject 
 may be as comprehensive as possible. The special 
 
PREFACE. Vii 
 
 working out of many complicated processes cannot 
 be attempted in a general course of this sort, but 
 must be done by the advanced student working in 
 the laboratory for several hours daily. The ex- 
 ceptionally high magnifying powers sometimes men- 
 tioned in the following pages, may be used by him, 
 but in the case of the beginner it is understood that 
 they are intended to be used for special demonstra- 
 tion by his teacher. 
 
 In the course of the demonstrations (Part III.), 
 the student is desired to read the general considera- 
 tions regarding methods, in Part IV. ; and when he 
 finds no statement regarding the preservation of a 
 specimen, it is to be inferred that it is needless to 
 preserve it. 
 
 In this edition, the book has been considerably 
 extended by an account of the microscope, and 
 fuller descriptions of the tissues. All figures of the 
 latter have been omitted, however, for they are to be 
 found in the systematic text books already in the 
 hands of everyone. Their repetition here would 
 therefore have entailed needless addition to the 
 expense of the work. It is hoped that the demon- 
 strations of the tissues, if made as directed, will 
 supply the place of pictures, and that the descrip- 
 tions will, together with hints from the teacher, prove 
 a sufficient guide to them. 
 
viii PREFACE. 
 
 The blank paper at the end is intended for 
 drawings and notes. 
 
 The author is glad of the indication of the 
 usefulness of the book, afforded by the rapidity with 
 which the first edition was exhausted. 
 
 University of Edinburgh, 
 September 1876. 
 
TABLE OF CONTENTS. 
 
 PART I. 
 
 INTRODUCTION. 
 
 Apparatus required . 
 Preliminary Preparation of Tissues 
 
 PAGE 
 
 i 
 
 PART II 
 THE MICROSCOPE. 
 
 Fundamental Principles 
 
 Lenses . . 
 
 Theory of the Simple Microscope . 
 
 Theory of the Compound Microscope 
 
 Faults of a Lens 
 
 Optical Parts of the Compound Microscope 
 
 Mechanical Parts of the Compound Microscope 
 
 Different Forms of Microscopes 
 
 Accessory Apparatus for Illumination 
 
 Method of Testing the Microscope 
 
 Method of Working with the Microscope 
 
 Preparation of a Simple Object 
 
 Description of the Object . 
 
 Delineation of the Object . . 
 
 Estimation of the Magnifying Power of the Microscope 
 
 Estimation of the Size of an Object 
 
 8 
 ii 
 16 
 17 
 19 
 22 
 
 30 
 33 
 36 
 
 39 
 
 40 
 
 4i 
 4i 
 
 43 
 46 
 48 
 
CONTENTS. 
 
 PART III. 
 
 HISTOLOGICAL DEMONSTRATIONS. 
 
 Brownian Movement. Tradescantia 
 
 Yeast . . . -»•■•. 
 
 Penicillium. Starch. Cotton Fibres 
 
 Linen Fibres. Disc-Bearing Tissue. Bacteria 
 
 Blood of Newt 
 
 Blood of Bird, Fish, and Man 
 
 Mucus. Epithelium . 
 
 Connective Tissues . 
 
 Cartilage 
 
 Bone 
 
 Tooth 
 
 Muscle . 
 
 Nerve Tissue . 
 
 Blood-vessels . 
 
 Circulation of the Blood 
 
 Lymphatics 
 
 Blood Glands . 
 
 Lung 
 
 Tongue . 
 
 Stomach 
 
 Intestine 
 
 Liver 
 
 Salivary Glands and Pancreas 
 
 Kidney . 
 
 Urinary Deposits 
 
 Skin 
 
 Eye 
 
 Cochlea . 
 
CONTENTS. 
 
 XI 
 
 schneiderian membrane 
 
 Spinal Cord and Medulla Oblongata 
 
 Cerebellum and Cerebrum . 
 
 Male Generative Organs 
 
 Female Generative Organs . 
 
 PAGE 
 
 136 
 
 137 
 
 139 
 141 
 
 143 
 
 PART IV. 
 GENERAL CONSIDERATIONS REGARDING HISTO- 
 
 LOGICAL METHODS. 
 
 Methods of Applying Re-agents to the Tissues 
 
 Application of Fluids and Gases . 
 
 Application of Heat .... 
 
 Application of Electricity . 
 
 Methods of Studying Living Tissues 
 
 Methods of Hardening the Tissues 
 
 Methods of Softening the Tissues . 
 
 Mechanical Dissociation of Tissue Elements 
 
 Methods of Making Sections 
 
 Methods of Increasing the Transparency of the 
 
 Methods of Staining the Tissues . 
 
 Methods of Injection. . . , 
 
 Methods of Preserving the Tissues 
 
 Method of Keeping Preparations . 
 
 Index ..... 
 
 .. 144 
 
 . 144 
 
 ; 145 
 . 148 
 
 i 149 
 
 152 
 
 . 157 
 
 • 159 
 
 • iS9 
 Tissues 169 
 
 . 171 
 
 . 179 
 
 . 185 
 
 190 
 
 191 
 
ABBREVIATIONS. 
 
 H. means high power; a combination of lenses such as the 
 \ inch objective of an English microscope, with an eye- 
 piece of medium power, or the No. 7 objective and No. 3 
 eye-piece of Hartnack's microscope — with the tube of the 
 microscope drawn out — magnifying about 300 diameters 
 linear. 
 
 L. means low power ; a combination of lenses such as the 
 1 inch objective of an English microscope, with an eye- 
 piece of medium power, or the No. 3 objective and No. 3 
 eye-piece of Hartnack's microscope, magnifying about 50 
 diameters. 
 
 V. S., L. S., and T. S., respectively mean vertical, longitudinal, 
 and transverse section. 
 
Fig. i.— Hartnack's No. IIIa Microscope. A, Eyepiece. D, Coarse adjust- 
 ment. C, Condenser. H, Fine adjustment y, Objective. L, Stage. M, 
 Diaphragm. N, Mirror. 
 
OUTLINES: 
 
 OF 
 
 PRACTICAL HISTOLOGY. 
 
 ♦ 
 
 PART I. 
 INTRODUCTION. 
 
 APPARATUS REQUIRED. 
 
 i. The student who is entering upon the study of Practical 
 Histology must be provided with a compound micro- 
 scope capable of giving a low magnifying power of about 
 50 diameters, and a high power of about 300 diameters ; a 
 thin-bladed razor of good steel (§ 294); a pair of strong 
 needles in handles (§ 290); scalpels, forceps, and scissors, 
 such as are found in an ordinary dissecting case, the bent 
 scissors shown in Fig. 5 5 are, however, to be preferred ; 
 three or four camel-hair brushes of medium size ; five or 
 six dozen ground glass slides, 3x1 inch ; about a hundred 
 extra thin circular cover-glasses, j inch in diameter ; a box 
 to hold about fifty preparations. The student will thus be 
 enabled to preserve the principal specimens made during 
 the course of study indicated in the .following pages. 
 
 2. He should also be provided with the following set 
 of the ordinary reagents and mounting fluids. These may 
 be conveniently placed in one-ounce bottles, with ordinary 
 corks (Fig. 2).* 
 
 * Corks of vulcanised indiarubber are not suitable. 
 B 
 
PRACTICAL HISTOLOGY. 
 
 8. 
 
 9- 
 10. 
 
 ii. 
 
 12. 
 
 y 
 
 In bottles, with 
 a glass rod 
 fixed in the 
 cork. 
 
 i. £■ per cent Salt Solution (§269) 1 
 
 2. Magenta Solution (§ 324 a) 
 
 3. Picrocarmine Solution (§ 322) 
 
 4. Logwood Solution (§ 323) 
 
 5. Dilute Acetic Acid (§ 313) 
 
 6. Dilute Alcohol (§ 284) 
 
 7. Glycerine 
 Farrants' Solution (§ 349) 
 Dammar mounting fluid (§ 347^) 
 Clove Oil. In a bottle, with a 
 
 brush fixed to a stick (Fig. 2). 
 
 Size Cement I bottleS) ^ camel . 
 
 hair brushes. 
 
 small sable hair 
 
 wide - mouthed 
 
 White Zinc Cement 
 
 It is convenient to place these bottles in a -wooden 
 framework similar to that used for test-tubes, and to cover 
 
 • the whole with a paper or 
 glass shade, to protect it from 
 dust. 
 
 The advanced student 
 will find it advantageous to 
 place the first eight of the 
 above-mentioned reagents in 
 the bottles with capillary- 
 stoppers (Fig. 3). In these 
 the reagent is completely 
 protected from the entrance 
 of dust by a glass cap. These 
 bottles, however, though ex- 
 cellent for very fine work, 
 are not to be recommended 
 for ordinary purposes, be- 
 cause of the serious length 
 of time required to obtain a 
 
 Fig. 2.— Corked Fig. 3.— Capillary drop of the reagent the 
 
 reagent bottle. reagent bottle. , *1 . . ° . 
 
 bottle requiring to be in- 
 verted, and held in the hand until its heat expands the air 
 and expels the fluid. 
 
INTRODUCTION. 3 
 
 3. The advanced student who may be desirous of 
 working through the following course of instruction pri- 
 vately — without the resources of a laboratory — requires, in 
 addition to the foregoing apparatus and reagents, a magni- 
 fying power of about 800 or 1000 diam. ; a stage and an 
 eye-piece micrometer (§§ 59, 60) ; a freezing microtome 
 (§ 302) ; a camera lucida (§ 58, a or b) ; a lamp with a chim- 
 ney of pale blue glass for night work (§ 51) ; a warm stage 
 (§ 261); a small and a large injection syringe (Fig. 60) , 
 and perhaps a pressure bottle injection apparatus (Fig. 61) ; 
 a turn-table (Fig. 6$). A polarising apparatus may be 
 added, but other accessories are of very little use. Various 
 other reagents are required, but these need not be particu- 
 larised here. 
 
 PREPARATION OF TISSUES PREVIOUS TO THEIR 
 EXAMINATION.* 
 
 4. The following fluids require to be prepared : — 
 
 I. f- per cent Salt Solution. Dissolve 7*5 grammes 
 of dried ordinary NaCl in 1000CC distilled water. 
 
 II. One per cent Chromic Acid Solution. Dissolve 
 10 grammes chromic acid in 1000CC water. This can be 
 readily diluted when required. 
 
 III. Chromic Acid and Spirit Solution. Dissolve 1 
 gramme chromic acid in 20CC water, and slowly add it to 
 180CC rectified spirit. 
 
 IV. Chromic and Bichromate Solution. Dissolve 1 
 gramme chromic acid, 2 grammes potassium bichromate 
 in 1200CC water. 
 
 V. Chromic and Nitric Fluid. Chromic acid 1 gramme, 
 water 200CC, then add 2CC nitric acid. 
 
 VI. Miiller's Fluid. Dissolve 25 grammes potass, 
 bichrom. and 10 grammes sodium sulphate in 1000CC 
 water. 
 
 VII. One per cent Bichromate Solution. Dissolve 10 
 grammes potass, bichromate in 1000CC water. 
 
 * This section is designed to assist the advanced student when he 
 undertakes histological work in private. 
 
4 PRACTICAL HISTOLOGY. 
 
 5. Most of the requisite tissues and organs may be ob- 
 tained from the cat and guinea-pig. Feed the cat, and an 
 hour or so afterwards place it in a bag ; drop chloroform 
 over its nose until insensibility is produced. Open the 
 chest by a linear incision through the sternum ; speedily 
 open the right ventricle, and allow the animal to bleed to 
 death. 
 
 6. Divide the trachea immediately below the cricoid 
 cartilage, and inject it with £ per cent chromic acid fluid ; 
 tie it to prevent the escape of the fluid, and place the dis- 
 tended lungs in the same fluid, and cover them with cotton 
 wool. Change the fluid at the end of eighteen hours. 
 Allow them to remain in this fluid for a month, then cut 
 into small pieces and transfer to rectified spirit* until 
 required. 
 
 7. Open by a linear incision the stomach, small and 
 large intestine, and wash their inner surface with -f- per cent 
 salt solution. Place the tongue, divided transversely into 
 a number of pieces, and a portion of the small intestine in 
 chromic and bichromate fluid (Solution 4). Change the 
 fluid at the end of eighteen hours ; and at the end of a 
 fortnight transfer them to rectified spirit until required. 
 Place the tongue, divided transversely into five or six 
 pieces, together with a portion of the stomach and large 
 intestine, in \ per cent chromic acid solution. Change the 
 fluid at the end of eighteen hours ; and at the end of a 
 month transfer them to rectified spirit till required. 
 
 8. Open the bladder, wash it with salt solution, and 
 place it in a 1 per cent solution of bichromate of potash 
 (Solution 7) for two or three days. 
 
 9. Remove the kidneys ; divide one transversely, 
 the other longitudinally, and place them in Miiller's fluid. 
 Change the fluid at the end of eighteen hours ; and at the 
 end of four weeks transfer them to rectified spirit until 
 required. They will be ready after having been a fortnight 
 in the spirit. It is also advantageous to have the kidneys 
 
 * In this edition rectified spirit has been substituted for methylated 
 spirit in the preparation of the tissues, for undoubtedly the tissue ele- 
 ments are better prepared in it. (Its sp. gr. is 0*838.) 
 
INTRODUCTION. 5 
 
 of a rabbit prepared as follows : — Divide the kidneys longi- 
 tudinally ; place them for a fortnight in a i per cent solu- 
 tion of chromic acid, then in rectified spirit till required. 
 
 i o. Cut half of the liver into small pieces, and place them 
 in Muller's fluid. Change the fluid at the end of eighteen 
 hours ; and at the end of four weeks transfer them to 
 rectified spirit till required. 
 
 ii. Place the spleen, uterus, some thin muscles from 
 the limbs or abdomen, in \ per cent chromic acid. Change 
 the fluid at the end of eighteen hours ; and at the end of a 
 month transfer them to rectified spirit. 
 
 12. Through several points in the tunica albuginea of 
 testis of cat, inject a 1 per cent solution of osmic acid, 
 and then place it in rectified spirit ; and finally — a day or 
 two before making sections — in absolute alcohol. 
 
 i2A. Place the ovaries of cat or dog in Muller's fluid. 
 Change the fluid after the first day, and after three weeks 
 transfer to a mixture of equal parts of absolute alcohol and 
 water for thirty-six or forty-eight hours, then in absolute 
 alcohol for a day, after which they will be ready for section. 
 
 13. Remove both eyes. Divide them transversely behind 
 the crystalline lens. Remove the vitreous. Place the pos- 
 terior halves in the chromic and spirit (Solution 3). Change 
 the fluid at the end of eighteen hours. Transfer to recti- 
 fied spirit at the end of ten days. Place the crystalline 
 lens in chromic acid and spirit for two weeks, and then in 
 rectified spirit. Place the cornea in J per cent chromic 
 acid for a month, and then in rectified spirit. 
 
 14. Cautiously open the cranial and spinal cavities. 
 Remove brain and spinal cord, and strip off arachnoid. 
 Partially divide spinal cord into pieces half an inch long. 
 Partially divide the brain transversely into a number of 
 pieces. Place them in a cool place in rectified spirit for 
 eighteen hours. Transfer the spinal cord to \ per cent 
 chromic acid for six or seven weeks. Change the fluid at 
 the end of eighteen hours. Prepare the sciatic nerve in 
 the same manner. Place the brain in chromic and bi- 
 chromate fluid. Change at the end of eighteen hours, and 
 then once a week, until the brain is hard and tough. If it 
 
6 PRACTICAL HISTOLOGY. 
 
 be not tough and leathery at the end of six weeks, place it 
 in £ per cent chromic acid for a fortnight, and then in rec- 
 tified spirit. If the brain be placed in a too strong chromic 
 acid solution, or if it be too long exposed to the action of 
 even a moderately strong solution, it becomes friable and 
 useless. Procure a portion of human cerebrum [parietal 
 lobe), as fresh as possible, and treat it in the same manner 
 as the cat's brain. A portion of human cerebellum may 
 be prepared in the same manner. At the end of six or 
 seven weeks transfer all the tissues to rectified spirit to com- 
 plete the hardening. The brain and spinal cord should 
 always be placed on cotton wool in their hardening fluid, 
 in order that the fluid may reach every part equally. 
 
 15. Remove the muscles, but not the periosteum, from 
 the bones of the upper or lower limb, and remove both from 
 the lower jaw. Divide the jaw and the long bones trans- 
 versely in two or three places, and put them into the 
 chromic and nitric fluid (Solution 5). Change the fluid re- 
 peatedly until the bone is just sufficiently softened, and then 
 transfer it to rectified spirit. If the softening is not com- 
 pleted in a month, double the quantity of nitric acid in the 
 fluid, or place them simply in dilute nitric acid (2 per cent). 
 The softening is most rapidly accomplished by suspending 
 the bones in the fluid. For this purpose they may be 
 placed in a muslin bag. The bone must not, however, 
 remain in the softening fluid after all calcareous matter has 
 been removed. 
 
 16. Place a piece of human scalp, skin from palmar 
 surface of finger, in chromic and spirit fluid (Solu- 
 tion 3). Change the fluid at the end of eighteen hours. 
 At the end of a month transfer to rectified spirit. 
 
 1 7. Remove the petrous portion of the temporal bone, 
 open the tympanum, pull the stapes from the oval fene- 
 stra, and place the cochlea in chromic and spirit fluid. 
 Change it at the end of eighteen hours, and again on the 
 sixth or seventh day. But if a copious brown precipi- 
 tate fall, change the fluid every third day. On the tenth 
 or twelfth day transfer to chromic and nitric fluid. Change 
 the fluid frequently until the bone is soft. Then place 
 
INTRODUCTION. 7 
 
 it in rectified spirit. In the guinea-pig the cochlea forms 
 a very evident projection into the tympanum, and is 
 therefore very convenient for enabling the student to see 
 how the cone is to be sliced when sections are to be made. 
 Kill a young guinea-pig, sever the head from the body, 
 disarticulate the lower jaw, open the tympanic bulla, remove 
 the stapes, and prepare the cochlea as above. In a young 
 animal the chromic and spirit fluid alone may suffice to 
 soften the bone, and the chromic and nitric fluid may 
 therefore not be required, or at any rate only for a short 
 time. 
 
 It is very important to place all the above tissues in a 
 cool place. A cellar is best in summer. Protoplasm rapidly 
 undergoes change in even a medium temperature. 
 
PART IL 
 THE MICROSCOPE. 
 
 FUNDAMENTAL PRINCIPLES. 
 
 1 8. The Visual Angle. — We form an opinion of the 
 magnitude of an object from the size of the image which 
 it produces on the retina. The lateral rays from any 
 object [intersect behind the crystalline lens. The size of 
 the retinal image depends on the size of the visual angle 
 — that is, the angle formed by the intersection of the rays 
 
 H iti^rjj?. %,V*4?fc v* *■ si A3WVK0' B»^B 
 
 Fig. 4. — Visual angle. 
 
 from the extreme points of the object. All objects which 
 have the same visual angle appear of the same size. Thus 
 the objects ABC (Fig. 4), although actually of different 
 sizes, have — when placed at proper relative distances from 
 the eye — the same apparent size ; because the visual angle 
 is the same for all, and the magnitude of the image on 
 the retina (N) consequently the same. 
 
THE MICROSCOPE. 9 
 
 Moreover, an object of the same actual size appears of 
 different sizes at different distances from the eye, — the 
 magnitude appearing less the greater the distance. This 
 depends on the size of the visual angle being inversely as 
 the distance of the object from the eye. Thus, if we 
 look at a line in the position A (Fig. 5), it produces the 
 
 Fig. 5. — Visual angle. 
 
 image a a on the retina : in the position B it appears 
 larger ; because its image b b excites a greater retinal area ; 
 in the latter the visual angle is evidently greater than in the 
 former. 
 
 1 9. Theory of the Microscope.— The eye can see the 
 same object clearly when it is successively placed at various 
 distances, because of an alteration of the curvature of the 
 crystalline lens, whereby parallel rays from an object at a 
 great distance, or divergent rays from a near object, may be 
 successively focalised accurately upon the retina. The 
 more minute the object, the nearer one brings it to the eye 
 in order to increase the visual angle, and get as large an 
 image as possible. But there is a limit to this. Within 
 eight or ten inches from a normal eye of an adult, the 
 object cannot be seen without an unpleasant effort to render 
 the crystalline lens sufficiently convex to focalise the diver- 
 gent rays on the retina ; and within two inches or so the 
 eye completely fails to accomplish the task ; the image is 
 therefore ill denned. But if a convex lens (Fig. 1 9) be in- 
 terposed between the near object and the eye, it acts in the 
 same sense as the crystalline lens, and assists it focussing 
 the divergent rays, and thus the object apparently magni- 
 fied can be clearly seen. \ * 
 
IO 
 
 PRACTICAL HISTOLOGY. 
 
 20. Refraction and Reflection of Light. — The 
 
 action of a lens depends on the following principles. As 
 long as a luminous ray travels in a uniform medium it 
 pursues a straight line. If it pass at a right angle from 
 one medium to another, it does not deviate from its 
 course, however different the refractive powers of the two 
 media may be. But if the ray traverse the plane sepa- 
 rating two media of different refractive powers at any 
 
 other than a right angle, it 
 is bent. Thus, the ray R 
 (Fig. 6), passing through air 
 and striking the surface of a 
 glass plate obliquely at B, 
 is bent in two directions ; a 
 portion (r) is reflected, while 
 the remainder is refracted 
 towards the perpendicular 
 (J>). The refracted ray again 
 strikes the plane between the 
 two media obliquely at C ; a 
 portion (r) is reflected, while 
 the remainder is refracted 
 from the perpendicular as it re-enters the air. The emergent 
 ray is of less intensity after traversing the glass, in conse- 
 quence of its partial reflection at the 
 two surfaces ; there is therefore a loss 
 of light at every refraction ; a very im- 
 portant fact in microscopical optics. 
 
 21. Index of Refraction. — 
 The refractive power of any medium 
 is expressed by its index of refrac- 
 tion. This is ascertained by measur- 
 ing the amount of refraction which 
 a ray undergoes in passing from air 
 into another medium ; thus, let r 
 (Fig. 7) be a ray passing through air, 
 incident upon water at /, and refracted in the direction r . 
 The sines of the angles of incidence r i p, and of refraction 
 r i p' , are obtained by describing a circle with the point 
 
 Fig. 6. — Refraction and reflection by 
 parallel plate. 
 
 ..1 
 
 ft - - 
 
 
 
 Fig. 7. — Law of refraction. 
 (Lommel.) 
 
THE MICROSCOPE. n 
 
 i as the centre, and drawing lines from the perpendicular 
 at right angles with it to the points at which the circle 
 intersects the incident and refracted rays. Thus a b and 
 c d are the sines of the angles of incidence and refraction 
 respectively. In the case of air and water the sines are 
 as 4:3. The refractive index of air is so small, that for 
 practical purposes its refractive power is discarded, and 
 therefore -£=i*333 the refractive index of water. The 
 following table shows the indices of refraction for various 
 substances, many of which are used in practical histology : — 
 
 Indices of Refraction. 
 
 Diamond 
 
 2-44 
 
 Flint glass 
 
 1*642 
 
 Crown glass 
 
 I'll* 
 
 Canada balsam 
 
 i"54P 
 
 Clove oil 
 
 • i'535 
 
 Oil of turpentine 
 
 1-478 
 
 Glycerine 
 
 i'475 
 
 Alcohol 
 
 ■ 1-372 
 
 Aqueous humour 
 
 1*337 
 
 Pure water 
 
 1-336 
 
 Air 
 
 1*000294 
 
 As will afterwards be seen, such substances as Canada 
 balsam, clove oil, turpentine, and glycerine, having a com- 
 paratively high refractive index, are constantly employed 
 in histology, for the purpose of increasing the transparency 
 of tissues. 
 
 22. Lenses. — If a ray pass obliquely through a medium 
 with parallel faces (Fig. 6), the excident assumes the same 
 direction as the incident ray, but, if the faces be not par- 
 allel, the excident ray assumes a new direction. Thus, 
 let r (Fig. 8) be a ray impinging on a triangular prism at a, 
 it bends towards the perpendicular fi, and on emerging at 
 b, bends from it. The excident ray r thus takes up a 
 direction entirely different from r. If two prisms be joined 
 by their bases (Fig. 9), the rays A B, being refracted as in- 
 dicated in Fig. 8, are rendered convergent and brought to 
 
12 PRACTICAL HISTOLOGY. 
 
 a focus (C). On the other hand, if the angles of the 
 
 Fig. 8. — Refraction by prism. 
 
 Fig. 9. — Convergence of rays by prisms. 
 
 prisms be turned towards each other (Fig. 10), the rays A 
 B, are rendered diver- 
 gent. The converging 
 lenses A B and C in 
 Fig. 11 act after the 
 manner of the prisms in 
 Fig. 9, while the diver- 
 ging lenses D E and F 
 (Fig. 11) act like the 
 prisms arranged as in 
 Fig. 10. 
 
 Fig. 10. — Divergence of rays by prisms. 
 
 Fig. 
 
 -Lenses. 
 
 23. Focal Length. — Take the simple case of a plano- 
 convex lens (Fig. 1 2). It may be regarded as composed of 
 two rectangular prisms joined by their bases, with the oblique 
 surfaces curved so as to form the segment of a sphere whose 
 radius is C S. The rays A R R all strike the plane surface 
 of the lens at right angles, and therefore undergo no refrac- 
 tion until the spherical surface is reached. The ray A S 
 passing through the centres of both surfaces of the lens is 
 
THE MICROSCOPE. 13 
 
 termed the axial ray. Being perpendicular to both sur- 
 faces, it undergoes no refraction. The rays R S, on 
 emerging from the lens, are bent from the perpendicular 
 C S, and meet in the focus F. In a plano-convex lens the 
 
 FtG. 12. — Effect of plano-convex lens. (Hannover.} 
 
 optical centre is placed at the point where the axial ray- 
 meets the convex surface ; in a bi-convex lens it is placed 
 within the lens, at its very centre, if the convexity of the 
 surfaces be equal. The distance of the focal point F from 
 the optical centre is the focal distance of the lens. In a 
 plano-convex lens of crown glass the focal distance is almost 
 exactly twice the radius of curvature (C S), while in a bi- 
 convex lens of the same substance having equally curved 
 surfaces; the focal length is very nearly the same as the 
 radius of curvature. But the focal length depends not 
 only upon the degree of curvature of the lens, it also de- 
 pends on the refractive power of its material ; thus, if the 
 focal lengths of two lenses — the one of flint, the other of 
 crown glass — of equal curvatures be compared, the flint 
 lens is found to have a shorter focus than the other, be- 
 cause of its higher refractive power (§ 21). 
 
 24. Principal Focus and Conjugate Foci. — The 
 principal focus of a convex lens (A, Fig. 13) is the point 
 (F) to which rays that were parallel before entering the 
 lens are brought. (The same is shown in Fig. 1 4, where 
 P P are the parallel rays, and F the principal focus.) If 
 the rays be divergent (D D, Fig. 14) before entering the 
 lens, they are focalised outside the principal focus (D') : 
 while, if they be convergent (C C), they are focalised within 
 
'4 
 
 PRACTICAL HISTOLOGY. 
 
 it (C). It is evident that if a cone of rays proceeded from 
 a luminous point at F, the divergent pencil would be con- 
 verted into a cylinder of parallel rays. If the luminous 
 
 Fig. 13. — Principal focus. {Deschanel.) 
 
 point were placed within the principal focus — say at C', the 
 more divergent cone would not be rendered parallel by the 
 lens, but would still continue to diverge, though to a smaller 
 degree, after passing through it ; but if the luminous pencil 
 
 Fig. 14. — Principal focus (F) and secondary foci (C D 7 ). (Hannover.} 
 
 proceeded from a point outside the principal focus, say at D', 
 the less divergent rays would be rendered convergent 
 (D D) by the lens, and would come to a focus somewhere 
 on the other side of it. In Fig. 15a cone of rays is seen 
 proceeding from a point S', outside the principal focus F, 
 and brought by a lens to a real focus at S. At S a real 
 image of the point S' would be produced. Conversely, if 
 the rays proceeded from S, they would come to a real focus 
 at S', and if S were placed nearer the lens, but outside the 
 
THE MICROSCOPE. 
 
 15 
 
 principal focus, S' would recede from the lens. The points 
 S and S' are therefore termed real conjugate foci ; that is, 
 
 : 
 
 Fig. 15. — Conjugate foci, both real. (Deschanel.) 
 
 points so correlated that rays diverging from the one con- 
 verge to the other. In Fig. 15 the luminous point is 
 placed in the principal axis of the lens ; if it were placed 
 above or below the axis, the rays would be focalised on 
 the opposite side of the axis (Fig. 17). 
 
 25. Virtual Focus. — Let Fig. 16 represent a luminous 
 point T placed within the principal focus F of the lens 
 A B, the rays are never ^ 
 brought to a focus, but 
 are only rendered less 
 divergent by the lens. 
 The rays after travers- 
 ing the lens proceed 
 just as if they eman- , 
 ated from the point V, 
 to which they would 
 pass if prolonged backwards, without the intervention of 
 the lens. To an observer a luminous object at T would 
 appear to be at V. V would therefore be a virtual but not 
 a real image of the object T, or, otherwise expressed, T is 
 a real, and V a virtual focus. 
 
 26. Real Image. — If a luminous object be placed at 
 twice the focal distance from a convex lens, a real image of 
 the same size is produced at the same focal distance ; but 
 if the object (a b, Fig. 17) be placed at less than twice the 
 
 Fig. 16. — Real and virtual foci. [Lotnntel.) 
 
i6 
 
 PRACTICAL HISTOLOGY. 
 
 focal distance, but outside the principal focal point (F), a 
 real magnified image of the object (A B) is produced. The 
 divergent luminous pencil proceeding from a is brought to 
 a real focus at A. The position of A is determined by the 
 point at which the refracted ray a g intersects the secondary 
 
 Fig. 17. — Production of a real magnified image. (Lommel.) 
 
 axial ray a O A : — that is, the ray passing from a through 
 the optical centre of the lens. Luminous pencils, of 
 course, proceed from every point of the object a b, and are 
 focalised at corresponding points of AB; these, however, 
 are for the sake of clearness omitted from the figure. 
 
 Conversely, it will be readily seen that if A B were the 
 object placed at more tha?i twice the focal distance, a real 
 diminished image would be produced at a b. The real 
 images produced by a convex lens are always inverted. 
 
 27. Virtual Image, with the Simple Microscope. — 
 If an object A B (Fig. 18) be placed within the focal dis- 
 tance of a convex lens, 
 no real image can be 
 produced, because the 
 rays proceeding from 
 A issue divergingly 
 from the lens as if they 
 came from the virtual 
 point a. Although in 
 this case the lens cannot 
 focalise the rays proceeding from A B, the crystalline lens 
 of the eye of an observer can do this, and thereby produce a 
 real image of A B on the retina, provided that A B is 
 placed only a little way within the focal distance of the 
 
 Fig. 18. — Virtual magnified image. {Lommel.) 
 
THE MICROSCOPE. 
 
 17 
 
 lens (L). To him the rays from A would appear as if they 
 proceeded from a. He would therefore see an enlarged 
 virtual image {a b) of the object. This is the principle of 
 the simple microscope. Fig. 19 explains the theory of en- 
 largement by the simple microscope more clearly. Let A 
 
 a 
 
 Fig. 19. — The simple microscope. 
 
 B be an object placed within but near the principal focus 
 of the lens L, the rays from A B are rendered less divergent 
 by the lens, and are focalised by the crystalline lens of the 
 eye upon the retina. To the observer they appear as if 
 they proceeded from an object in the position a b at the 
 distance of ten inches from the eye. 
 
 The simple microscope is often used for dissection (§ 50) 
 and other purposes, where a high magnifying power is not 
 required. 
 
 28. Compound Microscope. — The compound micro- 
 scope is always employed where high magnifying powers 
 are needed. Its simplest form (Fig. 20) consists of two 
 convex lenses, one — the objective (O) placed near the 
 object, the other — the ocular (O'), placed next the eye. 
 The object (a b) is placed slightly beyond the focal length 
 of the objective O. A real magnified image is produced 
 at a! b' c, slightly within the focal distance of the ocular O', 
 by which it is again magnified, so that the observer sees 
 the virtual magnified image ABC. The compound 
 microscope is therefore really just a conjunction of two 
 
 c 
 
i8 
 
 PRACTICAL HISTOLOGY. 
 
 simple microscopes. The complete form of the compound 
 microscope (Fig. 21) has an objective (O) consisting of 
 
 Fig. 20.— Compound microscope- 
 Sitnplest form. (Desckanel.) 
 
 Fig. 21.— Compound microscope- 
 Complete form. {Carpenter. ) 
 
 several lenses superimposed, while the ocular is always 
 composed of two converging lenses, the lower one, termed, 
 the field-glass (F F), collecting the rays from O. so as to 
 
THE MICROSCOPE. 19 
 
 form at B B a real image of a dimension capable of being 
 seen through the upper lens, termed the eye-glass, E E. 
 A diaphragm or stop B B, consisting of a blackened metal 
 disc with a hole in the centre, cuts off the extraneous rays, 
 and thus gives sharpness to the image. 
 
 29. The Faults of a Lens. — There are two faults in 
 every lens which must be overcome before a satisfactory 
 image of an object can be produced. These faults are of 
 trivial importance with very low magnifying powers, but in 
 the case of high powers they become so serious, if not 
 corrected, that the microscope is useless. The faults are : — 
 1. Spherical, 2. Chromatic aberration. 
 
 30. Spherical aberration is due to the manner in which 
 rays are refracted by a spherical surface. The axial ray A 
 (Fig. 22), striking the lens at a right angle, is not refracted. 
 The rays C C around the axis are bent to a slight extent 
 
 Fig. 22. — Spherical aberration. 
 
 and brought to a focus at c ; the lateral rays R R, striking 
 the lens much more obliquely, are focalised at r ; conse- 
 quently the image of an object is rendered indistinct, its 
 margins r r are misty and ill-defined, owing to the lateral 
 rays having already passed their focal point. 
 
 31. Spherical aberration is to a large extent remedied 
 by intercepting the lateral rays with a diaphragm. This 
 method is often had recourse to in simple microscopes, 
 but it has the disadvantage of diminishing the aperture 
 of the lens, and thereby causing a loss of light. In the 
 eyepiece of the compound microscope, however, where 
 the lenses are large, a diaphragm is used (B B, Fig. 21). 
 
20 
 
 PRACTICAL HISTOLOGY. 
 
 Other methods of preventing spherical aberration are also 
 had recourse to ; thus, in the eyepiece of the microscope, 
 two lenses (E F, Fig. 31) are so arranged that the rays 
 which are internal in passing through the one become ex- 
 ternal in passing through the other, so that the spherical 
 fault of the one corrects that of the other. The manner 
 in which spherical aberration is corrected in the objective 
 would require an explanation entirely beyond the limits 
 of this work. It must, therefore, be simply stated that it 
 is by a combination of converging with diverging lenses, of 
 such curvature and arranged at such distances, that the 
 positive spherical aberration of the one set is neutralised 
 by the negative spherical aberration of the other. 
 
 32. Chromatic aberration is due to the fact that the 
 different coloured rays which compose white light are 
 unequally refrangible ; those of the longest wave length — 
 the red — being least, and those of shortest wave — the violet 
 
 Fig. 23. — Chromatic aberration. 
 
 — being most easily bent. In passing through a lens, 
 therefore, white light is dispersed, the violet rays are foca- 
 lised (V, Fig. 23) nearer the lens than the red (R), while 
 the foci of the other colours are intermediate between 
 these. The image of a colourless object therefore appears 
 coloured. The coloration is most marked at V and R, 
 least so at F K ; for in that plane. there is an intersection 
 of the red and violet rays. 
 
 33. To understand the manner in which chromatic 
 aberration is remedied, it is necessary to carefully distin- 
 guish between the deflection and the dispersion of light. 
 
THE MICROSCOPE. 
 
 21 
 
 The indices of refraction, already alluded to (§ 21), are 
 really the indices of deflection ; they indicate the amount of 
 deflection that a ray of homogeneous light, e.g. pure red, 
 would undergo when transmitted through various sub- 
 stances. The table of indices (§21) shows that in flint 
 glass the deflective power is 1*642, while in crown glass it is 
 1*53. It would, however, be easy to have a flint prism 
 that would give the same deflection as a crown prism ; the 
 former only requires to be made of a smaller angle than 
 the latter; thus a prism of flint glass with a refracting 
 angle of 52 would have the same deflective power as a 
 6o° prism of crown glass ; a ray of pure red light would be 
 deflected to the same extent by both. But if a ray of 
 white light were transmitted through each prism, the 
 spectrum produced by the flint would be nearly twice as long 
 as that produced by the crown glass ; that is to say, the dis- 
 persive power of flint is nearly double (17 in exact figures) 
 that of crown glass. 
 
 34. The construction of an achromatic lens then is 
 essentially this. If we combine in opposite positions two 
 prisms, one of crown 
 glass of about 6o° (c, 
 Fig. 24), the other (/) 
 of flint glass of about 
 30 , a ray (r) passing 
 into the prism c is de- 
 flected and dispersed. 
 On passing into/ it is deflected and dispersed in an op- 
 posite direction. But, ow- 
 ing to the smaller angle 
 of/ the outward {upward 
 in the Fig.) deflection of 
 the ray does not overcome 
 its inward deflection in c, 
 therefore, it emerges with 
 a part of the deflection 
 given to it in c still re- 
 tained. But the dispersive power of flint being nearly 
 double that of crown glass, the outward dispersion in / 
 
 SQ 
 
 Fig. 24. — Achromatic prisms. (Lommel.) 
 
 Fig. 25. — Achromatic lens. 
 
22 PRACTICAL HISTOLOGY. 
 
 is greater than the outward deflection ; and so, by a careful 
 adjustment of the angles of the prisms, the dispersive 
 power of / can exactly reverse that of c, while the deflec- 
 tion is not overcome, hence the ray r emerges from / 
 deflected but not dispersed. The system is achromatic. An 
 achromatic object-glass may consist (Fig. 25) of a bi-convex 
 lens of crown, and a plano-concave lens of flint glass 
 cemented together with Canada balsam. The effect is the 
 same as that of the prisms in Fig. 24. Luminous rays, 
 whether travelling to or from the point F, leave the lens 
 achromatic. 
 
 THE OPTICAL PARTS OF THE COMPOUND 
 MICROSCOPE. 
 
 35. The Objective. — The value of a compound 
 microscope depending principally on the quality of the 
 objective, it is usually termed the lens. It consists of one 
 or more compound lenses (O, Fig. 21). In objectives of 
 medium power, e.g. |~inch focus, it has been generally the 
 fashion to construct them of three compound plano-con- 
 vex achromatic glasses. Recently, however, Mr. Wenham 
 (Proc. Roy. Soc. vol. xxi. p. in) has devised an objective, 
 in which only one concave lens of dense flint glass is used 
 to correct four convex lenses of crown glass. By this 
 means the cost of the production of lenses of high quality 
 has been much lessened. 
 
 36. The powers of continental lenses are usually dis- 
 tinguished by the numbers 1, 2, 3, and so on; in this 
 country, their powers are indicated by their focal length. 
 The lenses most useful for the beginner are the i-inch and 
 ^-inch ;* giving, with a suitable ocular, a magnifying power 
 of about 50 and 300 diameters. Much higher powers in 
 addition to these are required by the advanced student, 
 such as T V, tV» anc * yV or ~th- A -^ has been constructed, 
 but for ordinary purposes, the Y V is a s high a power as need 
 be employed. The lenses made by Hartnack and by 
 Zeiss, on the Continent, are commendable on account of 
 
 * These have a power similar to objectives 3 and 7 of Hartnack's 
 microscope. 
 
THE MICROSCOPE. 
 
 23 
 
 their* comparatively small cost and good quality. The 
 lenses made in England, especially those by Ross, R. and 
 J. Beck, Powell and Lealand, are of great excellence : Wen- 
 ham's \ made by Ross, the new -J- by Powell and Lealand, 
 and the T V by R. and J. Beck, are all lenses of such 
 beauty as has, in the opinion of competent authorities, not 
 been equalled elsewhere. 
 
 37. Angle of Aperture. — The angle of aperture of 
 an objective is the angle formed between the most external 
 rays that can penetrate its entire system of lenses from a 
 luminous point placed in the focus; thus, in Fig. 26, rays 
 are seen diverging from a luminous point 
 in the focus/: some of them (a a) enter the 
 first or front lens, too near its periphery to get 
 through into the second. The rays b b' 
 are the most peripheral rays that are capable 
 of passing through all the lenses of the ob- 
 jective, and therefore the angle between b 
 and b' is the angle of aperture : from this it 
 appears that the actual aperture of a lens is 
 a very different thing from its angular aper- 
 ture. There has been much discussion re- 
 garding the relative merits of lenses of small 
 and of large angular aperture. With a large 
 angle more of the oblique rays are admitted, 
 and therefore for the resolution (definition) of 
 very fine lines, such as those on diatom 
 scales, and the rods and cones of the retina, 
 the larger angle is preferable ; but the more the angle is 
 increased, the greater is the loss of penetrati?ig power ; by 
 penetrating power is meant the power of seeing with toler- 
 able distinctness points lying a little above and below the 
 exact focus. This is a matter of very great importance in 
 studying the structure of tissues and organs. It may be 
 readily understood from Fig. 26 that the smaller the angle, 
 the more can the objective be raised or lowered without 
 throwing the object much out of focus. At the same time, 
 however, lenses of large angle give a more brilliantly illu- 
 minated field, and are on that account more agreeable to 
 
 Fig. 26. — Angle of 
 aperture of the 
 objective. {Dip- 
 pel.) 
 
24 
 
 PRACTICAL HISTOLOGY. 
 
 work with. It may be safely stated that for ordinary h*isto- 
 logical purposes, lenses of "small," or rather of medium, 
 are preferable to those of " large " angle. In such a lens 
 as -J- inch, 70 is considered a "small," and 130 a "large " 
 angle. 
 
 38. Adjustment of the Objective for covered and 
 uncovered Objects. — If rays pass from a luminous 
 point (S, Fig. 27) to the eye obliquely through a parallel 
 
 glass plate, there is an 
 apparent displacement 
 of the point to the 
 position S'. The dis- 
 tortion of an oar when 
 dipped in the water is 
 due to a similar cause. 
 If, therefore, a plate of 
 glass be interposed be- 
 tween the lens and an 
 object, the latter is dis- 
 torted, and the amount 
 of distortion increases 
 with the thickness of the glass. Thus, in Fig. 28, we have 
 rays diverging from the point a and passing through a 
 cover-glass c. The 
 ray r, close to the 
 perpendicular /,would 
 appear to an observer 
 to proceed from an ob- 
 ject very nearly in the 
 position of a. — the ray 
 r from an object at 
 a, and r" from an 
 object at a". The 
 single luminous point 
 a would thus be spread 
 out and rendered indistinct ; and the more so the thicker the 
 cover-glass ; hence the importance of employing thin cover-glass. 
 It is evident from Fig. 28 that the divergence of the rays is 
 increased by the cover-glass ; hence, if the objective be ad- 
 
 Fig. 27. — Apparent displacement of a luminous; 
 pj point by a parallel plate. (JDeschanel. ) 
 
 
 Fig. 28. — Effect of the cover-glass. 
 
THE MICROSCOPE. 
 
 justed so as to clearly define an object without a cover-glass, 
 it will fail to do so if it have a cover. Mr. Ross first pointed 
 this out, and made the front lens of the objective movable, so 
 that the observer might adjust it for covered or uncovered 
 objects (Fig. 29). The front lens is fixed in the case a — 
 the inner lenses, in &, e, and <?', are 
 two lines engraved on the outer 
 case a, and d a line on a portion 
 of the inner case projecting through 
 a slit in a. For a covered object 
 the lenses are approximated by a 
 screw until the line e is opposite d, 
 while, for an uncovered object, 
 they are separated until the line 
 e is opposite d. For ordinary Fig. 29.— Objective with adjust- 
 lenses, however, this adjustment SSSJobjS. eMd and un " 
 is unnecessary, for one really 
 
 scarcely ever looks at an uncovered object, and, moreover, 
 the adjustment is liable to shift and thereby throw things 
 into confusion. For very high powers, however, it is essen- 
 tial, because cover-glasses vary a good deal in thickness, 
 and if the cover be not of the very thickness with which the 
 optician corrected the lens, definition is impaired. In 
 that case, therefore, it is important to have an adjusting 
 screw on the lens. 
 
 39. Dry and Immersion Lenses. — With a dry lens, 
 the rays pass through air between it and the cover-glass. 
 
 With an immersion lens, some 
 fluid, such as water or glycerine, 
 is interposed. Very high powers 
 are now almost always con- 
 structed on the immersion sys- 
 tem, for one of its great advan- 
 tages is the conservation of 
 light. Fig. 30 explains this ; 
 in A the oblique ray r is seen 
 passing from the cover-glass past 
 the lens. In B it passes from the 
 cover-glass into water, the refractive index of which being 
 
 iyBS 
 
 A. 
 
 B 
 
 I^H 
 
 »J3 
 
 5Sl 
 
 H H 
 
 Fig. 30.- 
 
 -A.JDry. B. Immersion 
 lens. 
 
26 PRACTICAL HISTOLOGY. 
 
 higher than that of air, the ray is not so much deflected 
 from the perpendicular at the upper surface of c in B, 
 as in A, and therefore it enters the lens. In the case of 
 very high powers, the full illumination of the object is a 
 most essential consideration ; hence the impoitance of the 
 immersion principle. For the same reason that water is 
 better than air, glycerine is better than water ; care, how- 
 ever, must be taken to employ pure glycerine. Glycerine 
 is, however, not so convenient as water, and for ordinary 
 purposes the latter is all that is required ; only distilled 
 water should be used, however, for the film upon the lens, 
 which ordinary water leaves when it dries, is by no means 
 unimportant. The immersion system is also advantageous 
 in another respect. If Fig. 28 be compared with B, Fig. 
 30, it will be evident that, by diminishing the obliquity of 
 the rays, the water under the immersion lens must contri- 
 bute to the better definition of the object. The immersion 
 system should, however, be employed only for very high 
 powers, it being inconvenient in doing ordinary work. By 
 Ross and some other opticians, lenses are now made that 
 may be used either dry or immersed by altering an 
 adjustment. 
 
 , 40. The Eye -Piece or Ocular. — The eye-piece 
 commonly employed is that invented by Huyghens. It 
 consists of two plano-convex lenses placed as in Fig. 31 
 (E F). The rays from the objective would, if uninterrupted, 
 form an image at v v and r r. With the field-glass (F) 
 they are focalised at v v and r r , and so produce a 
 smaller image capable of being embraced by the eye-glass 
 (E). Both lenses produce chromatic and spherical aberra- 
 tion, but these are reduced to a minimum as follows : — If 
 the objective were perfectly achromatic, the light would be 
 decomposed by F, and the violet rays focalised nearer to F 
 than the red rays (Fig. 23). The focal length of E being, 
 as in the case of F, shorter for the violet than for the red, 
 an achromatic image would be impossible. This difficulty, 
 however, is overcome by over-correcting chromatic aberration 
 in the objective, so that the coloured light issuing from it 
 is focalised by the converging lens F in a manner the 
 
THE MICROSCOPE. 
 
 27 
 
 reverse of that indicated in Fig. 23 ; that is to say, the 
 violet rays V V are focalised at a greater distance from F 
 than the red rays R R. The 
 focus of E being, of course, 
 shorter for the violet than 
 for the red, these different 
 rays are focussed by E on 
 the same points of the retina, 
 and thus an image, colour- 
 less or nearly so, is seen. 
 The manner in which spheri- 
 cal aberration in the eye- 
 piece is prevented by adjust- 
 ing its lenses, so that the 
 spherical fault of the one 
 corrects that of the other, 
 has been already explained 
 
 (§3i)- 
 
 With most microscopes 
 
 eye-pieces of different powers 
 are supplied. Those of Eng- 
 lish microscopes are distin- 
 guished by letters A, B, 
 etc., while those of foreign 
 makers are distinguished by numbers 1, 2, 3, and so on; 
 the A of the former and the 1 of the latter having the 
 lowest magnifying power. The shorter the eye-piece, the 
 higher is its power. By many English writers a short is 
 termed a " deep " eye-piece ; the reader has therefore to 
 remember that when " deep eye-piecing" is recommended by 
 some writers, the employment of an ocular of high magni- 
 fying power is indicated. As the confusion is perplexing 
 and altogether needless, the term "deep" ought to be 
 abandoned. 
 
 Seeing that the eye-piece merely magnifies an image 
 produced by the objective, any fault in the image produced 
 by the latter is exaggerated. On this account, only very 
 perfect lenses can bear a shallow eye-piece, and therefore 
 its employment is of much service in testing the quality of 
 
 Fig. 31. — The eye-piece of Huyghens. 
 {Dippel, slightly altered.) 
 
28 PRACTICAL HISTOLOGY. 
 
 an objective. Generally speaking, it is well to keep to eye- 
 pieces of medium power, such as the B or C of English 
 makers and the No. 3 of Hartnack. 
 
 41. Distance of the Ocular from the Objective. 
 — The magnifying power of the compound microscope may 
 be varied by altering the distance between the objective 
 and the ocular. Some microscopes are so constructed that 
 this is impossible. Nevertheless it is extremely convenient 
 to have the tube between the lens and the ocular made 
 like that of a telescope (Fig. 1). One not unimportant 
 advantage is, that such a microscope can be packed in a 
 comparatively small box. The great advantage, however, 
 is, that by varying the length of the tube very fine gradations 
 of power can be obtained with the same lens and ocular. 
 In measuring an object with an eye-piece micrometer it 
 will afterwards be seen how important this is. The tube 
 when fully elongated should not, however, reach more than 
 ten inches above the stage, for it is often important in 
 drawing and in measuring the magnifying power of the 
 microscope to have the surface of the stage in the focus of 
 the eye that is not looking through the microscope. 
 
 42. The Bull's Eye Condenser. — Opaque objects are 
 seen by reflected light. The light is concentrated upon them 
 with an ordinary condenser consisting of a plano-convex 
 lens (c, Fig. 1). This may be fixed to a separate support, 
 but in an ordinary microscope there is no place for it so 
 convenient as that shown in Fig. 1. Considerable experi- 
 ence is required in the use of this apparently simple 
 apparatus. It is important to bear in mind that the 
 condenser should always, if possible, be placed at right 
 angles to the direction of the light, and that, with a covered 
 object, the light should fall on the cover-glass as directly 
 as possible, for if it be very oblique it is to a large extent 
 lost by reflection from the cover. 
 
 43. The Mirror. — Transparent objects are illuminated 
 by transmitted light reflected from a mirror (N, Fig. 1). 
 The mirror is usually slightly concave, and its distance 
 from the object should be capable of alteration by a sliding 
 or jointed movement, so that when working with a low 
 
THE MICROSCOPE. 29 
 
 power the mirror may be placed at such a distance from 
 the object as is necessary to illuminate the entire field, while 
 with a high power the rays should be brought to a focus in 
 or near to the object. The light is exactly focalised en 
 the object, when a window bar, that may be brought into 
 the field, can be seen sharp and distinct, or when the shape 
 of a lamp flame can be distinctly seen. With artificial 
 light it is often inconvenient to have the flame thus exactly 
 focalised on the object, and therefore the lamp should be 
 drawn a little nearer to the mirror to avoid this. The 
 mirror should in general be so placed that the cone of rays 
 is reflected from it at a right angle. One generally arranges 
 the mirror so as to send the light directly through the object, 
 but sometimes for the resolution of fine structure it is im- 
 portant to throw the light into the object obliquely. By 
 such means one side of the object is thrown into shadow. 
 In the more expensive microscopes the mirror has two 
 surfaces, one concave, the other flat. The flat surface 
 is used when the light is to be focalised on the object 
 with an achromatic condenser (§ 52). 
 
 44. The Diaphragm usually consists of a blackened 
 brass disc (M, Fig. 1) placed below the stage. It has 
 apertures of various sizes, which can be successively brought 
 under the aperture in the stage (L, Fig. 1). It is very 
 important to attend carefully to the use of the diaphragm, 
 and so regulate the amount of light transmitted through 
 the object ; for if a glare of light be allowed to pass through 
 the object, the finer shadows are lost, and definition is thus 
 rendered indistinct. With a high power a small aperture is 
 employed. The diaphragm is, for convenience, usually 
 placed about half-an-inch below the object. For lenses of 
 very short focus, however, -^ inch and upwards, it is very 
 important to have the diaphragm close to the slide. In 
 Hartnack's more expensive microscopes this is effected by 
 a movable tube having one end partially closed by a dia- 
 phragm. Every objective has its appropriate diaphragm, 
 the aperture being smallest for the lenses of shortest focus. 
 The advantage of this arrangement is that the diaphragm 
 can be pushed close up to the slide as it lies on the stage, 
 
30 PRACTICAL HISTOLOGY. 
 
 or withdrawn to any desirable distance. Nevertheless, it 
 entails loss of time to shift the tubes, and for ordinary 
 work nothing is so convenient as the circular plate. Its 
 margin should be notched like a mill-headed screw, and 
 there should always be a spring catch for arresting the rota- 
 tion of the plate when any one of its apertures comes 
 exactly under that of the stage. 
 
 45. Employment of Daylight. — The best light for 
 microscopical work is that reflected from a white cloud. 
 The light from the blue sky is not so good, and the light 
 from gray clouds is about the worst. The window for 
 microscopical work should, if possible, face that part of the 
 sky opposite the sun at the time the microscope is used. 
 On this account a north light is that which is most useful. 
 If the microscopist is compelled to work at a window into 
 which the sun shines, he should cover it with a white* calico 
 blind, for direct sunlight must never be employed. Good 
 daylight is almost always to be preferred to artificial light ; 
 it often happens, however, that the latter must be employed. 
 The mode of obtaining it will be considered in § 51. 
 
 It is of great importance to protect the eye from 
 extraneous light. For this purpose a black paper or card- 
 board shade should be placed in front of the eye, so that 
 as little light as possible may reach it, save through the 
 microscope. With such a simple precaution one can work 
 much longer without fatigue, and the sharpness of the 
 microscopic image is much improved. 
 
 THE MECHANICAL PARTS OF THE COMPOUND 
 MICROSCOPE. 
 
 46. The Supports. — The pedestal of the microscope 
 may be of a tripod or of a horse-shoe form, it matters 
 little, provided that perfect steadiness be obtained. 
 
 The stage (L, Fig. 1) should be a little broader than the 
 length of a slide (3 inches), and it is very important that it 
 be not too high. It ought to allow the forearm to rest on 
 the table while the thumb and forefinger are moving the 
 slide to and fro upon the stage. It is singular that English 
 
THE MICROSCOPE. 31 
 
 opticians should have, for so long a time, sacrificed this 
 primary consideration to the far less important one of occa- 
 sionally fitting a large achromatic condenser below the stage. 
 When the pillar of the microscope is bent, the glass slip 
 must be prevented from sliding down the stage. Nothing 
 does this so simply and so efficiently as a pair of clips made 
 of brass, below which the ends of the slip are placed. A 
 movable brass ledge sometimes is placed across the stage to 
 serve instead of these clips ; but a simpler contrivance for 
 wasting one's time could scarcely have been devised. Movable 
 stages are lauded by some ; those with screws are scarcely 
 worthy of serious attention, but the stage moved by a lever 
 with a ball and socket joint, devised by R. and J. Beck, is 
 — if a movable stage be desired — a good arrangement. At 
 the same time, however, it is, I believe, much better to learn 
 to do without any movable stage unless for very exceptional 
 work under very high powers ; in that case it is useful to 
 those whose fingers have not acquired the necessary amount 
 of dexterity to move the glass slip carefully. Nothing is so 
 convenient as the motion communicated directly to the glass 
 slip by the fingers. If the stage be kept smooth and clean — 
 and a polished glass surface to the stage is on this account 
 the best — the slide can be readily moved in any direction, 
 and when working with very high powers it is only necessary 
 to put the slip under the clips to keep it from moving too 
 easily. With a little practice it is remarkable what a 
 delicate movement may be given by the fingers. 
 
 The pillar of the microscope should be flexible, in order 
 that the head may be kept in the erect posture, a condition 
 that greatly diminishes the fatigue of a day's work. When 
 working with fluids, the stage has of course to be made 
 level. It is important to see that the hinge of the pillar be 
 thoroughly well made, otherwise the stage will be unsteady. 
 
 The telescope tube holding the ocular and the objective 
 has been already alluded to (§ 41). 
 
 A nose-piece (Fig. 32) is an excellent device for saving 
 time in changing from one objective to another. In this 
 simple contrivance, devised by Mr. Brooke, the low and 
 high lenses are screwed to a framework which can be 
 
32 
 
 PRACTICAL HISTOLOGY. 
 
 Fig. 32. — Nose-piece, made by Swift. 
 
 rotated round a vertical axis, so as to bring the one or the 
 other lens under the tube. In the original form of this 
 
 apparatus both lenses re- 
 mained vertical. Powell 
 and Lealand, however, im- 
 proved it by causing the 
 lens not in use to project 
 free from the stage. The 
 form shown in Fig. 32 is 
 a still further improve- 
 ment made by Swift, in 
 which the objective is 
 readily centred accurately 
 under the tube. The cap 
 over the lens not in use 
 should always fit closely 
 to prevent the entrance 
 of dust. The nose-piece may be made to carry three or 
 even four lenses. 
 
 47. The Adjustments. — These are two in number, 
 the coarse and the^<?. The former moves the tube with 
 the lenses rapidly, the latter moves it slowly up or down, 
 so that the objective may be quickly brought to its focus 
 with the former, and then very accurately by the latter. 
 The fine adjustment is almost always a fine screw (H, Fig. 
 1) ; the fingers are constantly kept upon it during the 
 examination of an object, in order to slightly change the 
 focus and examine every stratum of it thoroughly. If, 
 therefore, the screw be not sufficiently low in position to 
 allow the arm to rest on the table, the fatigue becomes very 
 inconvenient. The arrangement in Fig. 1 serves the pur- 
 pose fairly well. 
 
 In such a microscope as that shown in Fig. 1 the coarse 
 adjustment is effected by seizing the lower milled head on 
 the tube (D), and giving it a rapid spiral motion. The 
 only disadvantage of this is, that the tube is sometimes apt 
 to jerk, and so to bring the lens suddenly down on the 
 object. Moreover, unless the tube be kept quite clean, 
 verdigris accumulates and it becomes stiff. Still, the pre- 
 
THE MICROSCOPE. 33 
 
 caution of cleaning it with a cloth when one begins to work 
 is easily attended to. And the difficulty can also be over- 
 come by having the split tube that clasps the sliding body 
 of the microscope lined with leather or cloth. If a nose- 
 piece is not employed, this form of coarse adjustment is 
 the best, because the tube can be readily pulled out of its 
 split sheath when the lens is to be changed : for nothing 
 can be more inconvenient than attempting to screw or unscrew 
 a lens with the tube fixed over the stage; the hand being 
 placed in an unusual position, the lens is apt to fall, and 
 certainly much time is lost in getting the thread of the 
 screw to bite when the hand is thus placed. When a nose- 
 piece is employed, however, a coarse adjustment, consisting of 
 a rack and pinion finely made, is the best arrangement, for 
 then all jerking is avoided, and the spiral motion of the 
 tube, which is disadvantageous with a nose-piece, is unne- 
 cessary. 
 
 FORMS OF MICROSCOPES. 
 
 48. Monocular Compound Microscopes. — Excel- 
 lent microscopes are made in this country by Ross, R. and 
 J. Beck, Powell and Lealand, Swift, Crouch, Collins, Baker, 
 Browning, and others : in Paris by Hartnack, and Verick : in 
 Jena by Zeiss. Their forms are well-nigh endless, and can- 
 not be considered here.* The main points required in 
 any compound microscope are (1), good lenses ; (2), per- 
 fect steadiness ; (3), power of easy adjustment ; (4), porta- 
 bility; (5), moderate cost. These qualities are wonder- 
 fully well united in the form of microscope (Fig. 1) which 
 is very similar to that originally made at the request of the 
 late Professor Bennett, by Oberhauser, and now by his suc- 
 cessor Hartnack, and also by Verick of Paris. English 
 opticians have been too much given to the manufacture of 
 machines of imposing appearance. Now, however, they 
 are wisely endeavouring to rival the cheap continental 
 microscopes, and it is by no means improbable that ere 
 long they will surpass continental makers in this direc- 
 
 * The reader is referred to Dr. Carpenter's excellent work on 
 "The Microscope," 5th ed. 1875, for full information on this subject. 
 
 D 
 
34 PRACTICAL HISTOLOGY. 
 
 tion. What is wanted for ordinary microscopic work 
 undoubtedly is an instrument resembling that shown in 
 Fig. i ; it matters not who makes it, provided it be well 
 made and at moderate cost. In the Edinburgh school, 
 Hartnack's microscope has always been recommended 
 hitherto, because it has met the requirements of the student 
 better than other forms of the instrument. Quite recently, 
 however, Mr. Swift of London sent for my inspection a 
 student's microscope somewhat resembling Hartnack's. I 
 was struck by the excellence of many of its parts. The 
 objectives are ■%■ inch and i inch, the former is of low angle, 
 and the lens sent to me as a sample was certainly a better 
 lens than the majority of Hartnack's No. 7. Its power was 
 considerably higher. Swift's lens, however, wants a thin 
 brass ring round its margin to give it more protection. His 
 medium eye-piece (C), equal to Hartnack's No. 3, is excel- 
 lent, but the shallow eye-piece (D), though better than 
 Hartnack's No. 4, is of little use. With the -^-inch objective 
 and the C eye-piece it is possible by simply altering the 
 length of the telescope tube to get a power ranging from 
 250 to 450 diam., a great convenience, and in this respect 
 surpassing Hartnack. The pedestal, the hinge, the pillar, 
 and the support of the body of the microscope, are all 
 steadier than in Hartnack's III.A microscope* (Fig. 1). 
 The fine and coarse adjustments are certainly better made. 
 But the instrument is not yet perfect. The diaphragm is 
 let into the stage from above, so that the disc rotates just 
 under the slide ; a faulty arrangement, with which it would 
 be impossible to keep the stage clean. Acids getting be- 
 tween the diaphragm and the stage would spoil everything. 
 The object of the arrangement is to have a rotating dia- 
 phragm with its apertures close to the slide, but until a 
 
 * Hartnack's No. III.A microscope, with objectives 3 (1 inch), 
 and 7 (not quite \ inch), and oculars 3 and 4, may be obtained from 
 Bryson, 60 Princes Street, Edinburgh, for £7 : ios. ; also from Tisley 
 and Spiller, 172 Brompton Road, London, S.W. ; and Baker, 244 
 High Holborn, London, W.C. Mr. Swift's (43 University Street, Tot- 
 tenham Court Road, London) microscope above alluded to, with 
 £-in. and I -in. objectives, and C and D ocular, similar to Hartnack's, 
 posts £6 : 12s. 
 
THE MICROSCOPE. 35 
 
 more convenient stage and diaphragm are added, Hart- 
 nack's microscope will still be preferred. What is wanted 
 is a stage of blackened glass. The clips upon Mr. Swift's 
 microscope are faulty, and should be improved, and the 
 condenser would be more convenient if fixed to the body 
 of the instrument, as in Hartnack's microscope, and not 
 placed on a separate stand, which always gets in one's way 
 when working. The microscope, however, costs less than 
 Hartnack's, and surely it will not be difficult to remedy its 
 slight defects. When that is done, it will be a better micro- 
 scope than Hartnack's. A nose-piece, and in that case a 
 rack-and-pinion coarse adjustment, would render it a very 
 perfect microscope for all ordinary work. 
 
 If a more expensive microscope stand be desirable, one 
 similar to Hartnack's No. VII. stand should be procured. 
 It is perfectly steady. The stage is covered with blackened 
 glass. It has an easily worked rack-and-pinion coarse 
 adjustment. The stage is not too high, and it — together 
 with the tube and lenses — can be turned round a vertical 
 axis, for the variation of the illumination of the object. 
 Any English optician could make such a stand ; meanwhile 
 Hartnack's may be safely recommended. 
 
 49. Binocular Microscope If the rays transmitted from an 
 
 object through each lateral half of the objective be focalised on the corre- 
 sponding retina, so that two slightly dissimilar pictures are produced, 
 the effect is stereoscopic ; the object stands out en bas relief. Various 
 methods have been invented for effecting this, most of which imply 
 a permanent division of the tube of the microscope into two. Hart- 
 nack's arrangement is the best, for it is entirely confined to a special 
 double-tubed eye-piece, which may at any time be placed in the 
 ordinary monocular microscope. In this eye-piece there are four rec- 
 tangular prisms (Fig. 33), in which the rays undergo total reflection. 
 In two of these (A B) the rays from the objective are divided into two 
 lateral bundles, that are again reflected by the prisms C and D, and 
 focalised by the ordinary lenses of two eye-pieces at E and F. Such 
 an arrangement may be satisfactorily employed with low powers, but 
 with very high powers the loss of light entailed by the rays encounter- 
 ing so many glass surfaces, even at right angles, is a serious drawback, 
 and definition is apt to be impaired. Therefore, although it is less 
 fatiguing for the eyes to use the binocular, the monocular is still the 
 microscope for ordinary work. - j 
 
36 
 
 PRACTICAL HISTOLOGY. 
 
 Fig. 33. — Course of the rays through Hartnack's 
 binocular eye-piece. [Ranvier.) 
 
 50. Dissecting Microscopes. — The simple dissecting micro- 
 scope devised by Darwin (Fig. 34) answers very well for ordinary pur- 
 poses. Many, however, will prefer the binocular dissecting microscope 
 made by R. and J. Beck, or Lawson's binocular dissecting microscope 
 made by Collins. 
 
 A Briicke's lens (Fig. 35) is a very convenient form of simple 
 microscope for assisting in dissecting and preparing microscopical 
 specimens, and in making fine drawings. It consists of two plano- 
 convex lenses, with their convex surfaces facing each other, and a con- 
 cave lens above all. This arrangement increases the working distance 
 between the microscope and the object. It has been adopted by R. 
 and J. Beck in their binocular dissecting microscope. 
 
 ACCESSARY APPARATUS FOR ILLUMINATION. 
 
 51. Lamp. — If good daylight cannot be obtained, a 
 lamp is necessary. Artificial light is far more fatiguing to 
 the eyes than daylight, on account of the predominance of 
 yellow rays. These may, however, be removed by trans- 
 
THE MICROSCOPE. 
 
 37 
 
 mitting the light through pale blue glass. A plate of it 
 may be placed on the stage of the microscope, or a blue 
 chimney may be put on the lamp. Although the light is 
 softened thereby, it is, however, at the expense of illumi- 
 
 Fig. 34. — Darwin's dissecting microscope. 
 
 very 
 
 high 
 
 nating power, and therefore sometimes, with 
 powers, the yellow flame has to be employed. 
 
 An Argand gas-burner may be used, and answers re- 
 markably well if covered with a blue chimney. The light 
 from a colza oil or a belmontine lamp is, however, softer. 
 An ordinary colza oil reading lamp does extremely well ; 
 but if a special lamp has to be procured, a lamp for burn- 
 
3 8 PRACTICAL HISTOLOGY. 
 
 ing paraffin, or better still, the purified form of it named 
 belmontine, is to be preferred. The simple form of lamp, 
 devised by How of London (Fig. 36) serves very well for 
 
 Fig. 35. — Briicke's simple microscope, made by Hartnack. 
 
 ordinary purposes. It is made with an ordinary bottle that 
 can therefore be easily replaced, if broken. A white por- 
 celain cylinder serves as a reflector, and also for cutting off 
 extraneous light from the eye. It should have a chimney 
 of white, and one of pale blue glass. 
 
 For very high powers, especially when an achromatic condenser is 
 used, the diverging rays from the lamp must be rendered parallel by a 
 large condenser. A lamp with this arrangement is made by R. and 
 J. Beck, Ross, and others. 
 
 The best lamp, however, for work with very high powers is that 
 devised by Mr. Dallinger, and made by Mr. G. S. Wood of Liverpool. 
 (See Monthly Microscopical Journal, vol. xv. p. 165.) The lamp is 
 expensive, but very perfect. 
 
 52. Achromatic Condenser. — This apparatus is of much 
 service in illuminating objects when very high powers are employed. 
 It may be simply made by fixing a half or quarter inch objective 
 in a tube under the stage. 
 
 The flat surface of the mirror is used to reflect the light through 
 the object, which must be arranged so that the light is focalised on 
 the object. Special forms of this instrument, with diaphragm, having 
 apertures of various sizes and shapes for giving different illuminating 
 
THE MICROSCOPE. 
 
 39 
 
 effects, are admirably made by English opticians. As they are designed 
 for their own microscopes, however, a special small condenser would 
 have to be made for such a micro- 
 scope as Hartnack's. (I cannot 
 recommend Hartnack's achro- 
 matic condenser.) Mr. Dallin- 
 ger's article il On a New Arrange- 
 ment for Illuminating and Cen- 
 tering with High Powers," the 
 reference to which is given in 
 § 51, is well worthy of careful 
 perusal in this connection. Mr. 
 Dallinger and Dr. Drysdale have, 
 by their remarkable researches on 
 bacteria, shown how very tho- 
 roughly they have studied the 
 points to be attended to for the 
 proper illumination of microscopic 
 objects under high powers. 
 
 53. Polarising Apparatus. 
 — This commonly consists of two 
 Nicol's prisms. One, termed the 
 polariser, is placed below the 
 stage ; the other, termed the ana- 
 lyser, is placed either immediately 
 above the objective, or above the 
 eye-piece. Hartnack's polari- 
 scope is perhaps the best. The 
 analyser is placed in a special 
 eye-piece, with a graduated circle 
 and indicator to show the num- 
 ber of degrees through which the 
 prism is rotated. The ray, polar- 
 ised by the lower prism, can pass /Fig. 36.— How's lamp for the microscope, 
 through the upper prism, when 
 
 the principal sections of the two are parallel ; but when they are placed 
 at right angles, the polarised ray is arrested in the upper prism, and all 
 is darkness. 
 
 If an object be placed on the stage and the Nicols be crossed, it is 
 invisible if it refract light singly, but it appears luminous if it refract 
 light doubly ; the reason being that the object so twists the polarised 
 ray that it can pass through the upper prism. Polarised light is used 
 in the examination of striped muscle and other tissues, and various 
 crystals. 
 
 THE METHOD OF TESTING THE MICROSCOPE. 
 54. Test Objects. — Before purchasing a microscope 
 
40 PRACTICAL HISTOLOGY. 
 
 it should invariably be tested by a practised observer. Clear 
 daylight — from a white cloud if possible — should always be 
 employed, and its regulation by the diaphragm carefully 
 attended to. The cover-glass should be extra thin and 
 perfectly clean. The salivary corpuscle is a good test- 
 object for -j^-inch lens. If the margin of the nucleus, and 
 of the corpuscle, and the Brownian motion of the granules 
 (§ 86, b), cannot be sharply seen, the definition is imper- 
 fect. If the corpuscle, when exactly in focus, shows any 
 colour, the microscope is not achromatic. A drop of blood 
 should also be used. It should form a very thin layer 
 under the cover-glass. If, when the tube of the microscope 
 is fully elongated, the corpuscles in the centre of the field 
 are sharp, while those near the periphery are misty, the field 
 is not flat. The adjustments must work smoothly. The 
 stand of the microscope must be perfectly steady, and the 
 objective must on no account move from side to side when 
 the fine adjustment is used. A microscope should be rejected 
 without hesitation for any one of these faults. The fine 
 markings upon the scales of Lepisma saccharina, Podura 
 plumbea, various diatoms, and Nobert's test-object con- 
 sisting of parallel lines of various degrees of approximation 
 ruled upon glass, are all recommended as test-objects. 
 Doubtless, they are good tests of defining power, but they 
 are well-nigh useless as tests of achromatism, because of 
 the dispersion which they themselves occasion. 
 
 THE METHOD OF WORKING WITH THE MICROSCOPE. 
 
 55. Preparation of the Microscope. — a. Select the 
 objective and ocular required, and place them on the 
 microscope. In the present case the -j^-inch lens (No. 7 of 
 Hartnack) and an eye-piece of medium power (No. 3 of 
 Hartnack) will be needed. See that their outer surfaces 
 are clean, if not, rub them gently with soft wash-leather. 
 If they are difficult to clean use dilute ammonia. 
 
 b. If the objective be the -J-inch (No. 7 of Hartnack), 
 place it halfan-inch above the level of the stage ; if it be 
 the 1 -inch (No. 3 of Hartnack), place it two inches above the 
 stage. In these positions they are of course outside the focus. 
 
THE MICROSCOPE. 41 
 
 c. Arrange the mirror and illuminate the field of vision ; 
 use a small aperture of the diaphragm in the case of a high 
 power. If any specks be visible on looking through the 
 microscope, they are upon either the upper or lower glass 
 of the eyepiece (probably on the upper surface of the 
 latter), and must be removed. Muscse volitantes must not 
 be mistaken for such specks. The latter move as the eye 
 changes its position. Dirt upon the objective is indicated 
 by no definite forms on looking through the microscope, but 
 by a dimness of the object which may be under examination. 
 
 d. Clean one or more cover-glasses. If the glass be 
 new, use dilute alcohol. The cover-glass ought never to be 
 laid flat upon the table, but should always be tilted on edge 
 against some suitable support until required ; otherwise, the 
 surface to be applied to the object will be contaminated 
 with various particles. 
 
 56. Preparation of a Simple Object. — Place a small 
 drop of milk upon the centre of a slide. Hold the cover- 
 glass by the margin, between the thumb and forefinger; 
 rest the free margin nearest the finger-tips upon the slide, 
 and allow the glass to fall, in this oblique manner, gently 
 upon the object. If the cover-glass be perfectly clean the 
 fluid will meet its surface evenly, if not, unmoistened 
 islands will be left, and air thus entangled. By gently 
 raising the margin of the cover-glass with a needle once or 
 twice the air-bubbles may be got rid of. If too much fluid 
 has been taken, remove it from the margin of the cover 
 with bibulous paper. 
 
 In mounting speci- r-^^x 
 
 mens it is very important /1^^J^^-^\ 
 
 to allow the cover-glass ^^^S ffgl ^- "%>—*_ 
 
 to fall evenly, and with- /-— ^— ^^^^B^^^^. -. "\}Xk 
 
 out shifting its place, on < ^ a ^^= — ^rffe^J tip. 
 
 the slide. This is best ^HJJ' 1 
 
 done with a pair Of for- Fig. 37.— Application of the cover-glass. 
 
 ceps (Fig. 37). 
 
 57. Description of the Object — It is very important 
 that the student should be educated in the method of 
 describing microscopic objects. To facilitate description, 
 he should attend to the following : — 
 
42 PRACTICAL HISTOLOGY. 
 
 a. Shape. Are the milk corpuscles globules, or are they 
 circular discs like coins? To determine this, touch the 
 edge of the cover-glass with a needle, and cause the cor- 
 puscles to roll over and over, or effect this by inclining the 
 stage. Farther, attend to this : when the focus is altered 
 a globule comes into and goes out of focus gradually, while 
 a flat corpuscle passes into and out of focus suddenly. 
 
 b. Size. Attend in this case to the relative, but omit 
 the absolute size. 
 
 c. Border. Is it smooth and regular, or notched ? 
 
 d. Upper and under surface. Elevate the objective, then 
 slowly lower it, and carefully watch the upper surface as it 
 comes into view. Is it smooth or rough ? The character 
 of the lower surface is unimportant in this case, but when 
 a membrane such as the mesentery is under observation, it 
 is important. 
 
 e. Colour. When exactly in focus, are the corpuscles 
 coloured or colourless? 
 
 f. Trafisparency. Bodies may be opaque, transparent, 
 or semi-transparent. The first transmits no light ; through 
 the second the outlines of subjacent objects are visible ; the 
 third transmits light, but the outlines of subjacent objects 
 cannot be distinctly seen. 
 
 g. Cofitents. Does the corpuscle appear to be homo- 
 geneous, or can included particles be seen on carefully 
 focussing through its whole depth ? 
 
 h. Effects of re-agents. Add acetic acid and observe its 
 effects on the corpuscles. For this purpose place a drop 
 of the acid on the slide close to the edge of the cover-glass. 
 If it fail to pass under the cover, dip a strip of bibulous 
 paper into the milk at the margin of the cover-glass 
 opposite the acid. The acid will then speedily penetrate 
 and irrigate the corpuscles (see § 257). After it has 
 affected them, those that lie near each other no longer 
 remain distinct, but coalesce and form irregular masses, 
 from which it is inferred that each milk globule has an 
 envelope that is soluble in acetic acid. From the results 
 of the chemical analysis of the milk, this envelope is 
 believed to consist of casein, a substance soluble in an 
 excess of acetic acid. 
 
THE MICROSCOPE. 43 
 
 i. A knowledge of the effects of re-agents implies nothing 
 less than an acquaintance with the majority of histological 
 methods. In the course of the following demonstrations 
 the student will more correctly appreciate the effects of 
 re-agents by bearing in mind that they are applied to the 
 tissues for the purpose of ascertaining — 1. Their chemical 
 composition, e.g. fat is blackened by osmic acid, and 
 dissolved by ether ; starch is coloured blue by iodine ; 
 earthy salts are dissolved by mineral and various other 
 acids ; albuminous tissues are rendered transparent, and 
 may be partially dissolved by acetic acid. 2. Their 
 structural coinposition. Many textures are so transparent 
 and their outlines so faint, that great assistance in deter- 
 mining their structure is obtained by stainitig them with 
 various substances : — e.g. a solution of nitrate of silver, by 
 darkening the outlines of epithelial cells, brings into view 
 what is otherwise invisible. Chloride of gold, by rendering 
 nerve fibrils of a violet colour, often reveals them where 
 they cannot otherwise be seen ; carmine, magenta, and 
 logwood, by staining nucleated protoplasts, are of , the 
 greatest service in showing their boundary lines. Also 
 such substances as acetic acid, glycerine, and oil of cloves, 
 by rendei-ing transparent some parts of the tissues, permit 
 of others being seen. 3. Some re-agents are applied for 
 the purpose of studying their effects on vital phenomena. 
 With this view heat, electricity, and various chemicals, are 
 applied to corpuscles that exhibit movement — e.g. white 
 blood corpuscles, cilia, etc. 
 
 58. Delineation of the Object. — It is very important 
 that the student should embrace every opportunity of 
 making drawings of microscopical objects, for drawing 
 necessitates a thorough inspection of an object, and it 
 impresses its features deeply upon the memory. The 
 smooth but not too highly glazed "antique note paper" 
 made by Pirie and Son, Aberdeen, is an excellent paper 
 for drawing. The pencils used are H.B., 4 H., and 6 H. 
 The shading should not be produced by lines, as in 
 ordinary drawing and wood-engraving, but it should be a 
 continuous shade produced by a rapid to and fro move- 
 
44 PRACTICAL HISTOLOGY. 
 
 ment of the pencil. This continuous smooth shading, so 
 necessary for giving a fine effect, may be readily produced 
 by rubbing the pencil drawing with an artist's " stump," 
 consisting of a firm cone of soft paper on the end of a 
 stick. The student should draw the object just as he sees 
 it, without altering its apparent size. In making drawings 
 of complicated objects, for the purpose of publication, it 
 is often necessary to reduce the size, and to fill in the 
 details of one drawing from many specimens, but with this 
 the beginner has nothing to do. The only practice advisable 
 in his case is to draw things just as he sees them. It is 
 important to practise even the drawing of outlines without 
 the aid of a camera. The details of objects must indeed 
 always be filled in without such aid, nevertheless it is often 
 difficult to get the exact size and the general form without 
 a camera. To all drawings a statement of the magnifying 
 power should be added, e.g. milk corpuscles x 300; and 
 in the case of things very difficult to see it is well to state 
 the objective and ocular employed, not only their numbers, 
 but also the names of their makers. 
 
 a. Chevalier's camera lucida is an exceedingly good one 
 (Fig. 38). The eye-piece of the microscope is removed, 
 and the camera inserted in its place, and fixed in any 
 position with a screw (s). The rays penetrate a prism (/), 
 undergo total internal reflection, emerge, are collected by a 
 special eye-piece, and penetrate a second prism (fl), where 
 they are reflected to the eye of the observer, to whom the 
 object appears to lie upon the table. The outlines of the 
 object may be traced with a pencil, as shown in the figure. 
 It must be remembered, however, that the pencil cannot 
 possibly be seen through the prism. The rays from the 
 pencil have to pass its margin, and so reach the pupil. 
 One-half of the pupil has therefore to be devoted to the 
 rays from the prism, and the other to rays from the pencil 
 and the paper. With Chevalier's camera the stage of the 
 microscope is kept horizontal, so that it is perfectly service- 
 able in the case of fluids. The paper and the point of the 
 pencil should be eight or ten inches from the eye. With 
 a microscope having a tube ten inches long, the paper 
 should be placed on a box on a level with the stage. 
 
THE MICROSCOPE. 
 
 45 
 
 b. Single prism. Mr. Bryson of Edinburgh has recently 
 fixed a small rectangular prism in a simple support, that 
 can be readily clipped on the eye-piece of the microscope. 
 
 Fig. 38.— Chevalier's camera lucida. with Hartnack's microscope. 
 
 It acts like p in Fig. 38, gives as sharp an image as 
 Chevalier's, and is far less expensive. The observer keeps 
 his head in a vertical position, and makes the drawing on a 
 suitable support placed vertically or slightly inclined ten 
 inches in front of the eye. The observer should, therefore, 
 sit at the left side of the microscope when it is directed 
 towards the window, so that he may not obstruct the illumi- 
 nation of the mirror, nor get the direct light from the 
 window into the eye that looks at the paper. The position 
 of the hand in drawing is not quite so convenient as when 
 it rests on the table, but really a camera is not very often 
 employed, and the cheapness of the apparatus is a com- 
 pensation. 
 
 c. Parallel plate reflector. A slip of thin glass placed above the 
 ocular at an angle of 45 (r, Fig. 39), has long been recommended in 
 
4 6 
 
 PRACTICAL HISTOLOGY. 
 
 place of a prism. It may be fitted to a support that can be clasped to 
 the eye-piece. A cover-glass answers the purpose. Dr. Beale, how* 
 ever, recommended that the glass should be of neutral tint, to diminish 
 the glare from the paper, an advantage certainly. This is, doubtless, 
 the cheapest apparatus for drawing, but it is certainly not so good as 
 the prism ; it cannot be so good, because in the prism the rays from 
 the object undergo total reflection, whereas they are only partially re- 
 flected from the surface of the plate ; moreover, the microscope must 
 be inclined. 
 
 Fig. 39. — Drawing with parallel plate reflector, r. 
 
 Success in using #, b, or c will not, however, be attained 
 unless the following be carefully attended to. The object 
 must not be too brightly illuminated ; a black pasteboard 
 shade should be placed between the window and the eye to 
 cut off extraneous light, and the paper on which the image 
 appears to lie should be rather dimly lighted, otherwise the 
 point of the pencil will not be clearly seen. The beginner 
 should always close one eye. 
 
 d. Squared glass. A plate of glass with fine. squared 
 lines (Fig. 43) placed in the eye-piece is of much service for 
 enabling one to draw objects of their exact size and form 
 without a camera. The same piece of glass should be 
 engraved, so as to serve as an eye-piece micrometer. 
 
 59. Estimation of the Magnifying Power of the 
 Microscope. — A stage micrometer is necessary for the 
 determination of the magnifying power of any combination 
 
THE MICROSCOPE. 47 
 
 of objectives and oculars that may be placed on the micro- 
 scope. Such a micrometer consists of a glass slide with a 
 very fine scale engraved on it with the aid of a machine. 
 In the English micrometer the divisions are 1 \ ths and 
 y-j^j-ths of an inch. In the French micrometer they are 
 Tthr tns °f a millimeter. Measurements are easiest with 
 the latter. 
 
 Let the -|-in. objective and the ordinary ocular be on the 
 microscope (the No. 7 objective and No. 3 ocular in the 
 case of Hartnack's microscope), with the tube fully elon- 
 gated, what is the magnifying power? Place a millimeter 
 micrometer on the stage, and focalise five of its larger 
 divisions, the space included is 0*05 millimeter. The 
 magnified image requires in some way or other to be com- 
 pared with an ordinary hand millimeter scale to determine 
 how much the 0*05 MM has been magnified. Every one 
 should try to do this as follows : — Hold the points of a pair 
 of compasses ten inches from the eye (on a level with the 
 stage of Hartnack's microscope), keep both eyes open, look 
 at the micrometer lines with one, and at the points of the 
 compasses with the other eye, open or close the points 
 until they include exactly the five divisions of the micro- 
 meter, then apply the compasses to a millimeter scale. 
 Suppose they cover exactly 15 millimeters, the magnifying 
 power is ascertained by dividing the apparent size (15 MM) 
 by the real size (0*05 MM). 
 
 Apparent Size. Real Size. Magnifying Power. 
 
 15MM -T- o*o5MM = 300 diameters. 
 
 If an English micrometer be used, bring into focus a 
 10 1 00 th inch division. Suppose it magnified to 0*3 inch, 
 then ; 
 
 o*3 inch -ro'ooi inch = 300 diameters. 
 The use of compasses in this process is not essential ; 
 the millimeter, or inch scale itself, may be held at the side 
 of the microscope on a level with the stage. The process, 
 however, is much easier with compasses, because their 
 points are more readily seen, and, unlike the lines of the 
 scale, they are not apt to produce confusion. 
 
48 
 
 PRACTICAL HISTOLOGY. 
 
 By the above method the magnifying power can be 
 ascertained with perfect accuracy; there is, however, an 
 easier way, which may be had recourse to by those who 
 possess a simple prism or a parallel plate reflector (§ 58, 
 b and c). (Chevalier's camera is not here admissible, as it 
 involves an increase of magnifying power by lengthening 
 the tube.) If a scale be held ten inches in front of the 
 prism, or the same distance below the parallel plate, the 
 extent to which the micrometer divisions are magnified 
 may be readily ascertained. 
 
 Having measured the power with the tube elongated, 
 shorten it and estimate the power again. It will be found 
 that the shorter the tube, the lower is the power. In this 
 manner the powers of all the possible combinations of 
 objectives and oculars belonging to a microscope should 
 be ascertained and tabulated as follows.* 
 
 
 Ocular. 
 
 No. 3. 
 
 No. 4. 
 
 Objective. 
 
 No. 3 
 No. 7 
 
 Diameters. 
 300 
 
 Diameters. 
 
 65 
 450 
 
 ) Tube 
 ) elongated. 
 
 The maker of the microscope generally supplies a table 
 such as the above ; but it is essential that the student should 
 know how it is constructed, in order that he may test its 
 accuracy, and measure the power of any lens that he may 
 add to his microscope. 
 
 60. Estimation of the Size of a Microscopic Ob- 
 ject. — This is done most easily and most accurately with 
 an eye-piece micrometer (Fig. 40). This consists of a 
 piece of glass with a fine scale engraved on it. It is most 
 convenient to have a special eye-piece containing a micro- 
 meter, but it is also possible to get a micrometer adapted 
 to the ordinary eye-piece. The ocular micrometer may be 
 
 * This table applies to a Hartnack's microscope, but it serves to 
 indicate how the powers of any form of microscope may be tabulated. 
 
THE MICROSCOPE. 49 
 
 in the form of a circular piece of glass, that is dropped into 
 the eye-piece when required, or the glass slip may, as re- 
 commended by Mr. Jackson, be mounted in a brass plate 
 capable of being pushed into the eye-piece through a slit 
 in its side. The former is preferable, but it will not neces- 
 sarily follow that, if the micrometer be dropped into the 
 eye-piece, its lines will be clearly seen. They require to 
 be exactly in the focus of the upper lens of the eye-piece, 
 and to bring this about, an optician may have to fit the 
 micrometer into a tube at such a height, that when it is 
 dropped into the ocular, and rests on the stop, the lines 
 will appear sharp and distinct. The micrometer lines 
 usually form a series like that represented in Fig. 40, and 
 for the purpose of ordinary measurement this is doubtless 
 all that is necessary. It is, however, 
 of great advantage to have the micro- 
 meter plate divided into squares, each 
 square being the breadth of ten of the 
 
 finest divisions Of the SCale (Fig. 43). FlG ; 40— Ordinary form 
 . , A • ■ i- of eye-piece micrometer, 
 
 A micrometer so constructed* is indis- with a human blood cor- 
 pensable for counting the blood-cor- d?am. e ,'seen through i 3 t?° 
 puscles (Fig. 43), and also serves as the 
 square of glass for drawing, alluded to in § 58 d. In 
 measuring the breadth of the human coloured blood cor- 
 puscles, use the J-in. objective, elongate the tube, 
 and substitute the micrometer in the ocular. Bring a 
 single coloured corpuscle into focus. It is seen through 
 the micrometer lines (Fig. 40). Suppose its broad diameter 
 covers four of the smallest divisions : push in the tube ; the 
 corpuscle does not now cover so many spaces ; hence the value 
 of the divisions of the ocular micrometer varies with the mag- 
 nifying power, and it is necessary to determine their value 
 for all the powers employed. Therefore substitute a stage 
 micrometer for the blood. If an English one be used, 
 focalise a 10 \ inch division. Elongate or push in the 
 tube until it covers just ten divisions of the ocular micro- 
 meter, then evidently each division must, with this magni- 
 
 * Such micrometers are made by Mr. Bryson, 60 Princes Street, 
 Edinburgh. 
 
 E 
 
50 PRACTICAL HISTOLOGY. 
 
 fying power, be equivalent to t^j-Jits" inch. This ascer- 
 tained, substitute the blood for the stage micrometer. 
 Suppose that the broad diameter of a coloured corpuscle 
 covers three divisions of the ocular micrometer (Fig. 40),' 
 then 10 g 00 = -g^Vs mcn - The corpuscles vary slightly 
 in size ; the average size being 3 ^ 0() inch. 
 
 61. It is very desirable that a uniform system of 
 measurement should be adopted by histologists. We era^ 
 ploy the metric system for fluid measures and weights, and 
 it seems very desirable also to use it for measures of length. 
 The expression of lengths in fractions of an inch is always 
 clumsy, and apt to lead to confusion. The micromillimeter 
 is the best histological unit ; and it is very conveniently 
 written by the Greek /a, and in speaking, shortened to 
 micro. A micromillimeter is the 1 oooth of a millimeter 
 (0*0000397 inch). 
 
 In using the millimeter instead of the English stage 
 micrometer for the above observation, its divisions (o*o 1 MM) 
 will be seen through the lines of the ocular micrometer. 
 Probably, with the magnifying power employed (300 diam.), 
 it will be found that, with the tube somewhat shortened, 
 three ocular micrometer divisions cover one division of the 
 stage micrometer. If 3 oc. mic. divisions = o'oiMM; 
 evidently 1 oc. mic. division must = 0*0033 millimeter; that 
 is, 3*3 micromillimeters. With the tube of the microscope 
 thus shortened, a red blood corpuscle would be found to 
 cover very nearly 2*5 oc. mic. divisions ; hence 3*3 x 2*5 = 
 8*25 p. The average size is 8 /£. By remembering this 
 figure for the blood corpuscle, it is easy to have an idea of 
 the size of an object when expressed in micromillimeters. 
 
PART III. 
 
 HISTOLOGICAL DEMONSTRATIONS. 
 
 62. Granules and Brownian Movement. (H.) — Rub 
 a piece of gamboge in a drop of water on a slide until the 
 fluid is faintly tinged ; cover, and examine. The term 
 fine granule is applied to a minute particle of any shape 
 that has no light centre. The term coarse granule is ap- 
 plied to a particle of irregular shape having a light centre 
 surrounded by a dark border. The difference between the 
 optical characters of the former and those of the latter is 
 entirely owing to size. Brownian motion is exhibited by 
 all minute particles floating in a fluid. The motion is pro- 
 bably owing to thermal currents in the fluid ; the solid par- 
 ticles merely indicating the directions of these currents. 
 
 63. Hairs of Stamens of Tradeseantia.* — a. With 
 a pair of sharp-pointed forceps pull off a few hairs from a 
 stamen of a flower (a white flower if possible) that is just 
 opening. Place them in a drop of water on a slide ; cover, 
 and examine. 
 
 (L.) The hair is a chain of cells rounded at its apex, 
 elongated towards its base. The youngest cell is at the 
 apex. 
 
 (H.) Observe the envelopes, protoplasm, and nucleus of 
 each cell. In the youngest cells the protoplasm fills the 
 whole cavity. In the elongated cells there are irregular 
 spaces (vacuoles) in the protoplasm, between " which it 
 stretches in the form of threads across the cavity of the 
 cell, towards the nucleus. It also forms a more or less 
 complete layer (primordial utricle) inside the envelope. 
 The protoplasm is a colourless finely granular jelly. Ob- 
 
 * The Tradeseantia Virginica is in flower from the middle of June 
 until the middle of October. 
 
52 PRACTICAL HISTOLOGY. 
 
 serve its movements : in the threads, it streams in some 
 towards, in others from, the nucleus. The whole mass of 
 the protoplasm, clear substance as well as granules, moves. 
 
 b. Apply heat with the aid of a warm stage. The 
 simple tin-plate described, § 259, Fig. 47, will answer the 
 present purpose. A gentle elevation of the temperature 
 hastens the protoplasmic movement, probably by accelerat- 
 ing chemical changes on which the movement in some way 
 or other most likely depends. If the temperature be raised 
 too high, however, the movement entirely ceases, owing to 
 death of the protoplasm. 
 
 64. Germinating Yeast.* (H.) — a. Examine torulae 
 of various sizes. The vacuole seen amidst the protoplasm 
 'in the larger torulae must not be mistaken for a nucleus. 
 
 b. Place a drop of magenta solution at the margin of 
 the cover-glass. It stains the buds most rapidly, The 
 protoplasm in many of the torulae becomes stained, while 
 the envelope through which the staining agent must neces- 
 sarily pass to reach the protoplast remains comparatively 
 colourless. A nucleus may be seen in some. The nucleus 
 is generally the part of the cell which is most deeply stained 
 by such agents as carmine, logwood, and magenta,. Some 
 torulae never become stained. These are probably the 
 oldest ones. Observe that the envelope is not equally 
 thick in all the torulae. In the youngest (smallest) bud no 
 envelope can be seen. This arises either from the absence 
 or from the excessive tenuity of the envelope. As the bud 
 increases in size, the envelope becomes evident, and grows 
 thicker with advancing age. The envelope is apparently 
 
 * If beer yeast ( Toriria cerevisice) cannot be obtained, procure Ger- 
 man yeast, and sow it in sugar and water, or in the following modifica- 
 tion of Pasteur's fluid : — 
 
 Magnesium sulphate o*2 gramme. 
 
 Calcium phosphate 0*2 ,, 
 
 Potassium ,, o*2 ,, 
 
 Ammonium tartrate 10 ,, 
 
 Cane sugar . 130 ,, 
 
 Water . . 800CC. 
 
 Keep the yeasty fluid gently warmed near the fire, or in a hot 
 chamber for a few hours. 
 
HISTOLOGICAL DEMONSTRATIONS. 53 
 
 produced by the protoplasm. It is not a deposit from the 
 surrounding fluid ; for it is known that the envelope of the 
 vegetable cell consists of cellulose, and there is certainly no 
 cellulose in Pasteur's fluid. 
 
 c. Rupture the torulae. For this purpose make a pad 
 of blotting paper, place it on the cover-glass, and beat it 
 with the handle of a needle. Colourless ruptured envelopes 
 will then be found scattered throughout the field, while the 
 coloured protoplasm lies in masses here and there. Some- 
 times it is only partially outside the ruptured bag. 
 
 65. Penicillium.* (H.) — Examine in water. Observe 
 that the hyphae (stems and rootlets) consist of chains of 
 elongated cells. Each aerial hypha (stem) produces at its 
 summit four or five rows of small germ-cells {conidid) of a 
 greenish colour. Even after staining with magenta no 
 nucleus is rendered visible in any of the cells. (This 
 fungus closely resembles the Achorion Schonleinii met with 
 in the hair follicles in Favus.) 
 
 66. Starch. (H.) — a. Gently scrape the cut surface of 
 a potato with a scalpel ; place the scraping on a slide ; add 
 a drop of water ; diffuse the scraping in the water with the 
 point of a needle ; remove all coarse particles ; apply the 
 cover-glass ; and examine the starch corpuscles. 
 
 Observe their lines arranged concentrically around a 
 bright refracting spot — the so-called " nucleus " of the cor- 
 puscle. This " nucleus " appears merely to be the first part 
 of the concretion that is produced, the various layers being 
 deposited concentrically around it. That the concretions 
 are produced within the protoplasm of the cell, may be readily 
 seen in a very thin section, with a power of about 800 diam. 
 
 b. Allow a drop of iodine solution (tincture of iodine 1 
 part, water 1 2 parts) to flow under cover-glass, and observe 
 the blue coloration of the starch. 
 
 67. Cotton Fibres. (H.) — a. Place a few fibres of 
 cotton wool on a slide in a drop of the above iodine solu- 
 tion ; cover, and examine. Notice the twist that is charac- 
 
 * The green dust of common mould should be blown upon the 
 surface of modified Pasteur's fluid ten days or so beforehand. Fungi 
 grow rapidly in this fluid. 
 
54 PRACTICAL HISTOLOGY. 
 
 teristic of these fibres. The fibres consist of cellulose. 
 Unlike starch, it is not rendered blue by iodine, but acquires 
 a brownish - yellow tinge like that of the iodine solution 
 itself. 
 
 b. Allow a drop of dilute sulphuric acid (acid and 
 water equal parts) to flow under the cover-glass. The fibres 
 soon become swollen, and acquire a blue colour. If the 
 blue colour be slow in appearing, add a small drop of strong 
 sulphuric acid to that already on the slide. The reaction 
 takes place equally well if the acid be added before the 
 iodine. 
 
 68. Linen Fibres. (H.) — Examine in water. Unlike 
 cotton fibres, they have no twist. They are for the most 
 part solid, with here and there a crevice running along their 
 interior. 
 
 , 69. Disc-bearing Tissue. (H.) — Make, with the aid 
 of a scalpel, an excessively thin vertical section of an ordi- 
 nary cedar pencil, or of a piece of common fir. Examine 
 in water, and observe the disc-like depressions on the fibres. 
 
 70. Bacteria. (H.) — These may be readily developed 
 in an infusion of some albuminoid matter — e.g., muscle. 
 
 a. Mince some lean meat, and allow it to soak in cold 
 water for an hour ; squeeze it in the water with the hand ; 
 filter through calico, and then through paper. Place the 
 filtrate in a hot chamber, or near the fire, to keep it at 
 summer heat, for thirty-six hours or so. A pellicle forms 
 on the fluid. 
 
 b. Place a particle of the pellicle with a small drop of 
 the surrounding fluid on a slide ; cover, and examine. 
 
 The pellicle consists of very minute, clear, motionless 
 granules. These are young bacteria. They are imbedded 
 in a clear jelly-like matter, that is, however, difficult to 
 see unless a dye be used. In the fluid around the pellicle, 
 the bacteria are in active motion. The minute granular 
 bacteria dance about in directions that are indefinite, like 
 the granules of gamboge already examined. The elongated 
 bacteria exhibit, as long as they are alive, movements in a 
 definite direction. They shoot across the field with great 
 rapidity. 
 
HISTOLOGICAL DEMONSTRATIONS. 55 
 
 c. Add magenta. The fluid stains and kills them. The 
 latter effect is probably due to the alcohol contained in the 
 solution. When dead, they exhibit only Brownian move- 
 ment. The direction of this movement is indefinite. The 
 movement of the elongated bacteria in a definite direc- 
 tion results from contractility. 
 
 Examine the stained pellicle. The young granular bac- 
 teria are brightly tinged, while the jelly that surrounds them 
 is very faintly so. 
 
 If the elongated bacteria, after staining with magenta, be carefully 
 examined with a i-25th inch lens, it may be seen that, as in the stained 
 torula already examined, there is a brightly-stained matter in the centre, 
 enclosed in a substance that remains colourless. The enclosed matter 
 is doubtless protoplasm, and the enveloping substance is pi - obably cel- 
 lulose, because, like cellulose and unlike any soft albuminoid matter, 
 it is unaffected by a solution of caustic potash. Dallinger and Drys- 
 dale {Monthly Microscop. Jonrn. vol. xiv. p. 105) have detected a 
 minute flagelhtm or cilium at the end of the bacterium, which — acting 
 like the tail of a fish — propels it through the fluid. 
 
 67, 68, 69, and 70 are examined because of the fre- 
 quency of their occurrence in the urine and other fluids 
 examined by the medical microscopist. 
 
 BLOOD. 
 
 71. Blood of Newt. (H.) — Stun the animal, dry the 
 tail, snip a piece from its extremity, and then express a small 
 drop of blood upon a slide. Place the cover-glass upon it 
 immediately. 
 
 a. The coloured corpuscles. — Shape when seen in front 
 face and in profile. Size. Border. Colour. Transpar- 
 ency. Contents. Observe that the corpuscles at first appear 
 to be homogeneous, but that, after a time, a light oval spot 
 appears in the centre of many \ this is the position of the 
 nucleus. 
 
 b. The white corpuscles. — There are several varieties of 
 these. Some are very finely granular nucleated masses of 
 protoplasm, as large, or even twice as large, as the coloured 
 corpuscles. These are the common white corpuscles. 
 Others are coarsely granular. These are. not nearly so 
 
56 PRACTICAL HISTOLOGY. 
 
 numerous as the first. A third set consists of nucleated 
 protoplasts, finely granular, and much smaller than the 
 others. The nuclei can seldom be distinctly seen in the 
 white corpuscles without the addition of re-agents. 
 
 c. The amoeboid movements of the white corpuscles. — 
 Select one of the large finely-granular corpuscles of irregu- 
 lar shape, and watch it carefully for some minutes. Usually, 
 even at the ordinary temperature, it changes its shape with 
 considerable rapidity, and it is instructive to draw the out- 
 line of the corpuscle at the end of every two minutes or so, 
 in order to be fully convinced of the changes that occur. 
 The direction of the motion is indefinite ; a protrusion may 
 take place at any part of the protoplasm, and be afterwards 
 withdrawn, or the protoplasmic process may adhere to the 
 slide or cover-glass, and the mass of the corpuscle be drawn 
 after it, thus leading to a locomotion of the corpuscle. 
 Sometimes neighbouring processes coalesce, and the readi- 
 ness with which they do so is opposed to the idea that the 
 protoplasm is enclosed in a membrane, as some are inclined 
 to suppose. 
 
 d. Effect of acetic acid. — Place a small drop of dilute 
 acetic acid at the margin of the cover-glass, and gently 
 raise the cover with the point of a needle, to break the 
 coagulum, and so allow the acid to penetrate. The 
 coloured corpuscle suddenly becomes enlarged, but still 
 retains its oval form. This probably results from softening 
 of the envelope of the corpuscle, together with endosmose ; 
 the former permitting and the latter producing the enlarge- 
 ment. At the same time, the corpuscle loses colour, and 
 the nucleus becomes evident. After a time it becomes 
 very difficult to see the envelope of the corpuscle ; the 
 nucleus, however, remains perfectly distinct. It is usually 
 somewhat corrugated. It sometimes happens that the 
 nucleus becomes of a yellow colour, owing to the acid solu- 
 tion of the blood pigment tinging it, after the manner of 
 such a dye as magenta. Until the acid is added, the nu- 
 cleus is not permeated by the blood pigment. This lies 
 entirely outside the nucleus, between it and the envelope 
 of the corpuscle. The general mass of each white corpuscle 
 
HISTOLOGICAL DEMONSTRATIONS. 57 
 
 becomes transparent, and the nuclei very distinct. From 
 three to five nuclei are usually revealed in the large cor- 
 puscles. The amoeboid motion is arrested by the acid. 
 
 72. Effect of Dilute Alcohol. — Dilute alcohol (rectified spirit 
 I part, 'water 2 parts) is a useful re-agent for the blood corpuscles 
 (Ranvier). Under its influence the frog or newt's coloured corpuscle, 
 and also its nucleus, become swollen, and a nucleolus is revealed. The 
 nuclei of the colourless corpuscles become evident, and one or more 
 very delicate clear blebs grow from the periphery of the white cor- 
 puscle, apparently consisting of some colloid substance, into which 
 endosmose takes place rapidly. 
 
 73. Effect of Water. — Make a preparation of the 
 same blood, and add water as described in § 71, d. The 
 coloured corpuscles become more or less globular, owing 
 to endosmose, and they lose colour owing to diffusion of 
 their pigment into the surrounding fluid. 
 
 74. Effect of Strong Syrup. — Place a drop of the 
 same blood on a slide, add a drop of strong syrup, and 
 quickly mingle the two with the point of a needle, then 
 apply the cover-glass. The coloured corpuscles shrivel, 
 owing to exosmose. It is to be observed that some of the 
 coloured corpuscles shrivel rapidly, while others undergo 
 no change for a considerable time. Possibly the latter are 
 older corpuscles and have thicker envelopes. 
 
 75. Effect of Magenta. — To a drop of the same 
 blood add a drop of the special magenta fluid used for 
 staining blood corpuscles (§ 324, b). Mix the two with the 
 point of a needle, and then cover. The nuclei of coloured 
 and colourless corpuscles become brilliantly stained. The 
 magenta fluid here employed contains a quantity of glycerine 
 sufficient to render its specific gravity similar to that of the 
 blood, with a view to prevent the swelling of the coloured 
 corpuscles. If the coloration take place slowly, however, 
 a drop of the ordinary watery magenta fluid may be added 
 to facilitate the penetration of the corpuscle by the dye. 
 
 76. Effect of Tannic Acid. — Dissolve 5 decigrammes 
 tannic acid in 113CC hot water, and allow it to become 
 cold. Place a drop of the same blood on a slide, and 
 cover. Bring into focus the corpuscles near the right 
 margin of the cover-glass, and place as quickly as possible 
 
58 PRACTICAL HISTOLOGY. 
 
 a drop of the tannic acid solution near this point. The 
 coloured corpuscles often become globular, and after a time 
 the haemoglobin becomes slowly or suddenly extravasated 
 from the corpuscle in the form of one or more buds adherent 
 to the wall, or in the form of fine granules scattered irregu- 
 larly around the wall. With 1-2 5th inch objective it may 
 be seen, after the action of tannic acid, that the coloured 
 corpuscle has a definite envelope, and this envelope may 
 sometimes be found ruptured where the bud has formed. 
 With such a power as this one may see the pigment rolling 
 away from the envelope like a cloud. It is not always 
 extravasated, but may collect around the nucleus in a 
 stellate mass. This effect of tannic acid was discovered by 
 Dr. Roberts of Manchester. A 2 per cent solution of 
 boracic acid (Briicke) has a somewhat analogous action. 
 For §§ 73, 74, and 75, frog's blood does perfectly, but for 
 §76 newt's blood is preferable. 
 
 77. Blood of Bird (H.) — a. Observe the pointed, oval, 
 biconvex shape of the coloured corpuscles, and their much 
 smaller size as compared with those of the amphibian. A 
 colourless corpuscle may be seen here and there. 
 
 b. Add acetic acid. A nucleus is revealed in the 
 coloured corpuscle. 
 
 78. Blood of Fish (H.) — This blood must be ex- 
 amined in a perfectly fresh condition. The corpuscles 
 resemble those of the bird. The coloured corpuscles, how- 
 ever, are not quite so pointed. 
 
 79. Human Blood (H.) — Take a small drop of blood 
 from the finger, cover it immediately, and find the focus as 
 quickly as possible. 
 
 a. The coloured corpuscles. — Observe them running to- 
 gether to form rouleaux. Study an isolated corpuscle. 
 Shape ; when seen in front face and in profile. Size. 
 Border. Colour. Transparency. Observe in a corpuscle 
 — seen in front face — the dark spot in the centre sur- 
 rounded by a light margin, and that when the focus is 
 changed, the centre becomes light and the margin dark. 
 This results from the biconcavity of the corpuscle. 
 
 b. The white corpuscles. — Few in number j found singly 
 
HISTOLOGICAL DEMONSTRATIONS. 59 
 
 or in groups of two or three here and there amidst the 
 rouleaux. Their shape is irregular, and amoeboid movement 
 may possibly be seen. Generally, however, the blood 
 requires to be raised to its normal temperature (3 8° C.) with 
 the warm stage (Fig. 48 or 49) before the movements can 
 be clearly seen. The size of the white corpuscles varies. 
 Some are twice as large as the coloured corpuscles, others 
 are about the same size. 
 
 c. Fibrin. — Very delicate colourless homogeneous 
 threads of fibrin may possibly be found stretching across 
 the spaces between the corpuscles. 
 
 d. Effect of acetic acid. — Add acetic acid at margin of 
 cover-glass, and if necessary gently raise the cover with the 
 point of a needle to break the coagulum. Observe that 
 the coloured corpuscles become globular, and lose their 
 colour, while the white corpuscles become very transparent 
 and most of them exhibit three nuclei. Sometimes, how- 
 ever, though rarely, there is only a single nucleus, and 
 sometimes there are as many as five. 
 
 80. Crenation of the Corpuscles. — Allow a drop of 
 the same blood to remain exposed to the air for twenty 
 seconds or so before applying the cover-glass in order to 
 produce crenation of the coloured corpuscles. A corpuscle 
 thus changed is covered with delicate spinous projections, 
 like a thorn-apple. In most bloods only a few of the cor- 
 puscles become crenated, while in other specimens nearly 
 all the corpuscles undergo this change. Possibly those 
 corpuscles which are prone to crenate have thinner enve- 
 lopes than the others, and on this account more readily 
 permit of the slight exosmose to which the crenation is 
 commonly ascribed. There is reason for suspecting, how- 
 ever, that crenation may be due to some other cause as yet 
 unknown ; for it is regularly produced in such an animal as 
 a dog when poisoned with Calabar bean (Fraser), and in 
 that case it seems unreasonable to ascribe it to any change 
 in the specific gravity of the blood plasma or corpuscles. 
 
 81. Effect of Magenta. — Add to the same blood, 
 before applying the cover-glass, a drop of the special 
 magenta fluid for staining blood corpuscles (§ 324, b); mix 
 
60 PRACTICAL HISTOLOGY. 
 
 the two with the point of a needle, and cover. No nucleus 
 is revealed in the coloured corpuscle, but in the white 
 corpuscle three nuclei usually appear. 
 
 82. Effect of Tannic Acid. — Add to the same blood 
 a drop of tannic acid solution (§ 76). Mingle the two with 
 the point of a needle; cover, and examine immediately. 
 The coloured corpuscles undergo a change similar to those 
 noticed in the newt's blood. Frequently, however, a deli- 
 cate envelope may be seen outside the bud of extravasated 
 haemoglobin. 
 
 83. Structure of the Blood Corpuscles. — In all 
 animals the white blood corpuscles are nucleated masses of 
 protoplasm without any apparent envelope. The coloured 
 corpuscles are nucleated in fishes, amphibians, and birds, 
 but not in mammals ; and it is to be observed that the 
 mammalian corpuscle comports itself towards re-agents not 
 like the nucleus, but like the perinuclear portion of the 
 nucleated coloured corpuscles. Notwithstanding the con- 
 trary opinion of Rollett (Strieker's Histology, i. p. 408), we 
 maintain with Hensen (lib. cit., p. 409) that the coloured 
 corpuscles have an envelope. The membrane can be 
 distinctly seen with a power of about 1000 diam. in the 
 newt's corpuscle after the addition of magenta and tannic 
 acid. In the mammalian corpuscles it is difficult to form 
 a decided opinion from direct observation, but the be- 
 haviour of the mammalian corpuscle towards re-agents is so 
 similar to that of the newt's corpuscle, that the existence of 
 a membrane in the former may be safely inferred. The 
 haemoglobin lies between the nucleus and the envelope. 
 All that is to us perfectly clear, but the remainder is 
 doubtful. Rollett maintains that the pigment fills the 
 interstices of a colourless sponge-like stroma. Hensen 
 (quoted by Rollett) states that he has seen a system of 
 extremely delicate filaments stretching from the nucleus of 
 the newt's corpuscle outwards to the envelope. Whether 
 or not this sponge-like or thread-like stroma is present it 
 would be difficult to say, but its existence would assist us 
 in comprehending how the non-nucleated human corpuscle 
 so definitely and regularly maintains its biconcave shape. 
 
HISTOLOGICAL DEMONSTRATIONS. 61 
 
 83 a. Effect of Gases. — The influence of a stream of carbonic 
 acid upon the coloured corpuscles may be studied by Strieker's method. 
 A drop of water is placed on the floor of the chamber in his hot stage 
 (Fig. 48), and a cover-glass, on which a drop of newt's blood has been 
 previously placed, is inverted over the chamber. A ring of oil is 
 painted round the margin of the cover to prevent evaporation. The 
 stage is then gently heated until the water in its moist chamber evapo- 
 rates, and the corpuscles at the periphery of the drop become affected 
 by the moisture. When the red corpuscles are just about to lose their 
 colour, the further addition of heat is discontinued, and a stream of 
 carbonic acid is driven through the chamber by the apparatus described 
 in § 258 (Fig. 46). If the coloured corpuscles have been sufficiently 
 acted on by water, the carbonic acid produces a cloudiness within 
 them, — which, according to Schmidt and others, is due to the precipi- 
 tation of paraglobulin. The precipitate disappears if a stream of 
 oxygen or common air be substituted for the carbonic acid. 
 
 83 b. Effect of Electricity. — Make a preparation of human 
 blood on a slide with gold leaf electrodes (Fig. 51), and transmit shocks 
 through it from a Du Bois Reymond's induction apparatus, in connection 
 with a voltaic cell as powerful as a small Grove's element. The second- 
 ary should be pushed close up over the primary coil of the apparatus. 
 The electricity causes the white corpuscles to withdraw their processes, 
 and to become more or less globular. The coloured corpuscles become 
 successively crenated, globular, and finally colourless, owing to a diffu- 
 sion of their pigment into the surrounding stroma. A similar effect is 
 produced by discharges from a Leyden jar. These effects, first shown 
 by Rollett and Neumann, are not entirely explained, but in all proba- 
 bility they chiefly result from electrolysis ; although induced is not 
 nearly so powerful as voltaic electricity in giving rise to electrolytic 
 effects. 
 
 83c. Inclusion of Solid Particles by the Colourless 
 Corpuscles. — As in the case of an amoeba, a white blood corpuscle 
 can entangle and envelope solid particles. This is effected by protru- 
 sions of the protoplasm growing around the particles. To demonstrate 
 this phenomenon, human or newt's blood may be employed ; the latter 
 is, however, to be preferred, ,on account of the larger size and greater 
 number of the white corpuscles. Fine granules of carmine, vermilion, 
 indigo, or aniline blue that has been precipitated by alcohol from a 
 watery solution, may be employed. (Schultze, Schultzis Archiv, vol. 
 i. p. 1 . ) These substances are reduced to a very fine state of division, 
 mixed with aqueous humour, and introduced into the blood, which may 
 be previously diluted with an equal bulk of aqueous humour. In the 
 absence of aqueous humour, dilute albumin (white of egg I part, f 
 per cent salt solution 2 parts, strained through muslin) may be em- 
 ployed. The blood with the pigment is placed on a slide and covered. 
 Evaporation is prevented by a ring of oil painted round the cover- 
 glass, and the preparation is kept on the warm stage (Fig. 49), at 30 
 C. in the case of newt's blood, and at 3 8° C. if the blood be human. 
 
62 
 
 PRACTICAL HISTOLOGY. 
 
 Cohnheim proved the practical importance of feeding the white blood 
 corpuscles with these pigments, by injecting for several days in succes- 
 sion 2 or 3CC of water with fine particles of aniline blue, into a sub- 
 cutaneous lymph sac of the frog. The lymph corpuscles enveloped the 
 blue granules and entered the circulation. After some days he in- 
 duced inflammation of the cornea, and found among the pus-cells at the 
 seat of irritation, some containing the blue granules, proving that they 
 had emigrated from the blood-vessels into the corneal tissue. 
 
 83D. Enumeration of the Blood Corpuscles.— Malassez 
 and Potain have devised a method for estimating the number of coloured 
 corpuscles in a given quantity of blood {Archives de Physiologie, 1874, 
 p. 32), a matter of much importance both in physio- 
 logy and pathology. 
 
 The blood is diluted with an artificial serum in 
 order to prevent coagulation, and to facilitate the 
 counting of the corpuscles. The artificial serum 
 consists of 1 vol. watery solution of gum arabic, 
 having a Sp. G. of 1020, 3 vols, watery solution of 
 equal parts of sodium sulphate and sodium chloride, 
 having a Sp. G. of 1020. The mixture of the 
 blood with the above serum may be made in an 
 ordinary CC measure, but if the quantity of blood 
 be small, the pipette shown in Fig. 41 is essential. 
 This is a glass pipette with a capillary tube, *and a 
 bulb containing a glass bead for mixing the blood 
 and serum. An elastic tube, about a foot in length, 
 is attached to one extremity. In estimating the 
 number of coloured corpuscles in human blood, the 
 finger is pricked, and a large drop of blood ex- 
 pressed, the elastic tube is placed in the mouth, the 
 pointed end of the pipette in the drop of blood, and 
 the fluid is sucked into the tube until it exactly 
 reaches the line marked 1. The point of the pipette 
 is then placed in the serum, and the aspiration con- 
 tinued until the fluid exactly reach the line c. 
 During the entrance pf the serum, the pipette is 
 shaken, so that the glass bead may cause its thorough 
 admixture with the blood. The instrument is so 
 graduated that the cavity of the pipette below the 
 line 1 contains exactly T -^ part of the cavity of the 
 bulb from the line 1 to the line c. The mixture of 
 blood and serum, therefore, contains one per cent 
 of blood. In cases where the blood corpuscles are 
 very numerous, it is advantageous to have only half a per cent of blood 
 in the mixture. In such a case the aspiration of the blood into the 
 pipette is arrested when the line marked ^ is reached, and the serum is 
 added as before. One must be careful to remember that after the 
 mixture is made, the fluid in the long stem of the pipette is merely 
 serum, and must be expelled as useless. 
 
 Fig. 41.— Pipette for 
 diluting blood with 
 artificial serum. 
 {After Malassez.) 
 
HISTOLOGICAL DEMONSTRATIONS. 
 
 63 
 
 The diluted blood is now introduced into a capillary glass tube (Fig. 
 42) flattened and fixed to a glass slide. Near the upper surface of this 
 
 Fig. 42. — Capillary tube for enumeration of blood corpuscles. {After MaZassez.) 
 
 tube there is an exceedingly fine elliptical cavity, the capacity of 
 various lengths of which is carefully ascertained by the maker. The 
 different lengths are tabulated in micromillimeters (§ 61) in the left 
 column of numbers engraved on the slide (Fig. 42), while the capacities 
 of these various lengths are expressed in fractions of a cubic millimeter 
 in the right column ; e.g. 500 fi contain yit-g- of a cubic millimeter. 
 
 The capillary is filled with the diluted blood by placing a drop at the 
 extremity, and allowing it to enter by capillary attraction. It may be 
 necessary, however, to hasten its entrance by aspiration. As soon as 
 the glass tube is filled, the remainder of the drop must be carefully 
 removed by bibulous paper, otherwise the corpuscles will not come to 
 rest within the tube. 
 
 Suppose we desire to count the corpuscles in 500 micromillimeters 
 of the capillary, we proceed thus : — A millimeter stage micrometer 
 is focalised by a power of about 100 diameters. A squared micro- 
 meter (Fig. 43) is placed in the eye-piece, and the tube of the 
 microscope is shortened or lengthened until the whole breadth — viz. 
 ten squares — of the ocular micrometer exactly include 500 micromilli- 
 meters of the stage micrometer. It avoids future trouble if a circular 
 line be drawn on the tube of the microscope to indicate its position 
 when the ten squares of the ocular micrometer have this value, with a 
 given combination of lenses. 
 
 The capillary tube with the blood is now substituted for the stage 
 micrometer, care being taken not to alter the length of the tube of the 
 microscope. Fig. 43 represents the capillary seen through the squared 
 micrometer. The coloured corpuscles within the whole of the 500 
 micromillimeters of the capillary have now to be counted. The squares 
 in the eye-piece facilitate the process, and the number found in each 
 square should be tabulated to avoid error. 
 
 Suppose that 334 coloured corpuscles are counted in 5 00 micro- 
 millimeters of the tube, this would, in a tube of the calibre of that 
 under consideration, be the number in r \- (5 part of a cubic millimeter 
 
6 4 
 
 PRACTICAL HISTOLOGY. 
 
 of the diluted blood. Evidently then, 334 x 150 = 50, 100, the number 
 of corpuscles in one cubic millimeter. But as this diluted blood con- 
 tains only xfo part of blood, therefore 50, 100 x 100 = 5,010,000, the 
 number of coloured corpuscles in one cubic millimeter of the blood 
 drawn from the finger. 
 
 To secure an accurate result, it is, of course, essential — I. that the 
 capillary be of uniform capacity throughout its entire length ; 2. that 
 
 Fig. 43. — Capillary tube with diluted blood, seen through a squared ocular 
 micrometer, X 100 diam. {After Malassez). 
 
 the blood be thoroughly mixed with the serum; 3. that the enumera- 
 tion be accurately performed. 
 
 The average number of coloured corpuscles in a cubic millimeter 
 of healthy human blood is 5,000,000. The number may be as high 
 as 6,000,000, and in diseased conditions it may fall as low as 800,000 
 (Malassez, lib. cit.) 
 
 The method for counting the white corpuscles is essentially the 
 same as the above ; but, owing to their smaller number, the mixture 
 of blood and serum should contain two instead of one per cent of 
 blood. This is readily effected as follows : — Fill the pipette with 
 blood to the line 1, then aspirate air until the blood enters the bulb. 
 Aspirate the same quantity of blood a second time, and then mix the 
 blood with serum as before. The average number of white corpuscles 
 in a cubic millimeter of human blood is about 9000, that is about 
 ■y^ of the number of the coloured corpuscles. 
 
HISTOLOGICAL DEMONSTRATIONS. 65 
 
 84. Blood Crystals (H.) — a. Hcemoglobin may be 
 readily prepared as follows : — Kill a rat or mouse by the 
 inhalation of ether. Place a drop of its blood upon a 
 slide, and add twice its volume of water. Mix the two 
 with the point of a needle, and allow slight evaporation. 
 Prismatic crystals, either isolated or in rosettes, will be 
 found. These may be preserved by allowing the blood 
 completely to dry before the application of the cover-glass, 
 and then applying a drop of dammar solution and covering. 
 
 b. HcBinin crystals are most readily prepared in con- 
 siderable quantity as follows : — Place some finely-powdered 
 dried blood in a test tube ; add about one-twentieth of 
 chloride of sodium, and about ten times its bulk of glacial 
 acetic acid. Heat, and keep it a temperature not higher 
 than 50 C. for three or four minutes (the solution should 
 not be boiled). Filter and evaporate slowly. 
 
 MUCUS. 
 
 85. (H.) — Mucous corpuscles may be obtained by 
 gently scraping the interior of the trachea of an animal 
 recently killed, or by hawking up some mucus from the 
 back of the throat. They have usually a single globular 
 nucleus ; sometimes, however, there are two or three. 
 They exhibit amoeboid movement, especially if they be 
 warmed to 3 8° C. on the hot stage (Fig. 49). Mucous 
 corpuscles may also be obtained from the urine by allowing 
 it to stand in a conical glass for six or seven hours until 
 the corpuscles are deposited. If the greater part of the 
 supernatant fluid be poured off, they can then be readily 
 obtained with a pipette (Fig. 45). Those from the urine 
 exhibit no amoeboid movement, even if they be warmed ; 
 probably because of the paralysing influence of the water 
 or other urinary constituent. 
 
 EPITHELIUM. 
 
 86. Stratified Squamous Epithelium (H.) — Move 
 the tip of the tongue roughly over the gums and interior of 
 
 F 
 
66 PRACTICAL HISTOLOGY. 
 
 the lips to detach some epithelial scales ; then place a large 
 drop of saliva on a slide, skim off the air-bubbles with a 
 needle ; cover, and examine. 
 
 a. Squamous epithelial cells; single or cohering together 
 in • groups, will be found scattered throughout the field. 
 Observe their shape, large size (30-50 //,), the nucleus 
 usually single and oval. 
 
 The epithelium of the mouth is stratified. The cells 
 under examination belong to the most superficial layers. 
 
 b. Salivary corpuscles will be found singly or in clusters. 
 Size, rather larger than that of a mucous corpuscle. Observe 
 the Brownian motion of the granules between the nucleus 
 and the envelope. There is no amoeboid movement. 
 
 c. Mucin. — Place a drop of magenta at the margin of 
 the cover-glass, and elevate it slightly to allow the dye to 
 penetrate the viscous fluid. The nuclei of the epithelium 
 and salivary corpuscles become brilliantly stained, and 
 very delicate films or fibrils of precipitated mucin will 
 probably be found stretching here and there across the 
 field. The mucin is precipitated by the alcohol in the 
 magenta fluid, and as it is slightly stained by the dye it 
 may be found without much difficulty. Acetic acid may 
 be used to precipitate the mucin, but the films being 
 colourless are, in that case, much more difficult to 
 find. 
 
 87. V. S. skin" of palmar surface of finger hardened 
 first in chromic acid and spirit (§ 16). The skin may be im- 
 bedded in paraffin, as described in § 300, and held in the hand 
 during section ; or it may be imbedded in paraffin and cut 
 in a microtome, as described in § 303 ; or, best of all, it 
 may be frozen, and cut in the freezing microtome, as de- 
 scribed in § 305. However obtained, the section must be 
 extremely thin. If the slice be large and difficult to spread 
 • out on the slide, put in a tumbler or beaker full of water. 
 Before it has had time to sink to the bottom thrust the 
 slide into the water, and with the aid of a needle draw the 
 section upon the centre of the slide, with the epidermic 
 surface facing the broad margin of the slide. Dry the 
 slide with a cloth or bibulous paper close up to the tissue. 
 
HISTOLOGICAL DEMONSTRATIONS. 67 
 
 Apply a drop of Farrants' solution or glycerine ; cover, and 
 examine. 
 
 (L.) The epidermis consisting of squamous cells 
 roughly divided into two layers, the horny layer above and 
 the rete mucosum below. The cutis vera with its papillae. 
 Select an extremely thin piece of the rete mucosum close 
 to the cutis vera ; place it exactly in the centre of the field, 
 and fix the slide in that position with clips. 
 
 (H.) The epidermic cells are produced by fission in the 
 lowest layer of the rete mucosum. Observe the small size 
 of the cells there, as compared with the upper part of the 
 rete, where they are growing but not dividing. The margins 
 of all the cells are jagged. This can be clearly seen in the 
 rete mucosum, and in the lower part of the horny layer. 
 Towards the upper part of the horny layer the cells become 
 scaly, and the nucleus often disappears. In the lowest 
 layers the cells appear to be nucleated protoplasts. As 
 they grow old they become horny, owing to the production 
 of keratin, a substance into which the protoplasm is 
 probably transformed. 
 
 The history of the epithelium lining the mouth is 
 similar to that of the epidermis. As it is more difficult to 
 see the jagged margins of the cells in the former, the latter 
 is taken here. (This preparation will be required again 
 when we study skin?) 
 
 87A. The evidence of the fission of the cells in the deepest layers of the 
 epidermis can only be clearly seen with a power of about 800 diam. 
 The nuclei of the cells are generally elongated, and mostly contain 
 more than one nucleolus. Fission of the nucleus may sometimes be 
 seen. It cannot be doubted that the perinuclear plasm also divides, but 
 it is astonishingly difficult to see a cell in the act of division, probably 
 because they are so closely packed together. 
 
 87B. The serrated margins of the cells are well seen in skin of human 
 finger, treated by a method which we owe to Ranvier. Inject \ per 
 cent osmic acid (§281) into the lower part of the epidermis by thrusting 
 the nozzle of Wood's syringe (§ 344) obliquely into the cutis vera, and 
 driving the fluid wherever it will go. Harden in rectified spirit, 
 make V. S., and preserve in glycerine. The lowest epidermic cells are 
 slightly darkened, and a power of about 800 diam. shows that the 
 so-called "prickles" are not the teeth of neighbouring cells "dove- 
 tailed one with another," but that, as Dr. Martyn [Monthly Microscop. 
 Journ., xiv., p. 59) has shown — they are bridges between the cells 
 
68 PRACTICAL HISTOLOGY. 
 
 with intervening spaces. One may correctly imitate the arrangement by 
 expanding the fingers of both hands and apposing their tips. Possibly 
 the spaces constitute a system of lymph channels. 
 
 87c. The " prickle " cells may be isolated by teasing sections of the 
 skin after prolonged maceration in ^ per cent potassium bichromate, to 
 which one or two drops of carbolic acid have been added to prevent 
 putrefaction (§ 289). They may also be readily obtained (Lister) by 
 placing one's foot in hot water for half-an-hour, and then scraping off 
 the epidermis, and macerating it in the above bichromate solution. 
 
 87D. Simple Squamous Epithelium. — Omentum of 
 full-grown cat or guinea-pig, previously silvered by the 
 method described in § 328. Excise a small piece of the 
 membrane with scissors, and extend it on a slide by the 
 method described in § 87. Apply a drop of glycerine; 
 cover, and examine. 
 
 (L.) The membrane is at some parts areolar, at others 
 continuous. In the non-areolar portions, chains of fat cells 
 may be recognised, and it will be just possible to see a fine 
 network of dark lines, the silver lines that surround the 
 epithelial cells. The omentum is a thin serous membrane 
 with a simple layer of squamous epithelium on both sur- 
 faces. Between the epithelial coverings there is fibrous 
 tissue, and at the trabecular there are, in addition, fat cells, 
 bloodvessels, and nerves. 
 
 (H.) Examine the silver lines upon the trabecular 
 The silver blackens what appears to be an interstitial sub- 
 stance that cements the cells together. If, however, the 
 tissue have been for more than three or four minutes in the 
 silver solution, the nucleus, and, to a less extent, the peri- 
 nuclear part of the scale, become darkened. Over the 
 bands of tissue between the spaces in the areolar portion 
 of the membrane, the silver lines are often imperfect. 
 Profile views of the nuclei of the epithelial plates that clasp 
 the fibrous bundles in this situation will probably be readily 
 seen. 
 
 Seal up the preparation. All glycerine projecting 
 beyond the margin of the cover-glass must be removed 
 with a pipette (Fig. 45), and, if necessary, the slide must 
 be washed with bibulous paper dipped in water. Paint a 
 ring of gold size around the margin of the cover-glass, and 
 
HISTOLOGICAL DEMONSTRATIONS. 69 
 
 next day, when this has dried, cover it with white zinc 
 cement (see § 358). 
 
 (This preparation will be required for fat cells and 
 areolar tissue on a future day.) 
 
 88. Ciliated Epithelium. — Ciliary Motion may be 
 readily studied in the gill of the mussel. The salt-water 
 mussel is much to be preferred to the fresh-water one. In 
 opening the shell, collect the salt water in a watch glass 
 for future use. The gill consists of parallel bars, the free 
 ends of which are at its free margin. 
 
 a. With scissors and forceps remove a small piece of 
 the gill, including its free margin, place it on a slide, and 
 apply a drop of sea water. As the gill in each half of the 
 animal consists of a double membrane, separate the two 
 portions with a pair of needles (a dissecting microscope (Fig. 
 34) is here of service), and then very gently pull asunder a 
 few of the bars of one of the membranes ; apply the cover- 
 glass; place the preparation on the tin hot stage (Fig. 47), 
 and examine. 
 
 (L.) Observe the bars with their free rounded ends 
 covered with cilia moving " like a field of corn before the 
 wind." Particles in the surrounding fluid are propelled 
 in a definite direction, the same as that in which the cilia 
 bend. 
 
 (H.) The cilium tapers from base to tip. (When 
 motionless, and examined with a power of about 1000 
 diam., it is seen to be a filament, flat, perfectly trans- 
 parent, and apparently homogeneous.) The cells on 
 which the cilia are placed cannot be clearly recognised in 
 this preparation, but the clear band at the margin of the 
 cells, from which the cilia spring, may be distinctly seen. 
 The movement of the cilium has a definite direction, con- 
 sisting in its becoming alternately bent and straight. The 
 propulsion of the surrounding fluid in the direction of the 
 bend may be accounted for by supposing that the velocity 
 with which the cilium bends is greater than that with which 
 it regains its erect position. 
 
 b. Effect of heat. — The hot stage is already under the 
 slide, apply heat as in the experiment with Tradescantia 
 
2d PRACTICAL HISTOLOGY. 
 
 (§ 63, b). A slight increase of temperature accelerates the 
 ciliary motion, probably by hastening the chemical changes 
 on which it depends. A too high temperature arrests the 
 movement, doubtless by destroying the delicate molecular 
 machinery of the contractile substance. 
 
 89. Effect of Chloroform (H.) — To study the effect of 
 chloroform vapour on ciliary motion, first shown by Mr. Lis- 
 ter, take a slide with a glass cell (Fig. 6 2, b) or make a cell with 
 a ring of putty, and place it on a slide. Place a small piece 
 of the mussel's gill in a drop of sea water on a cover-glass. 
 Put a small drop of chloroform in the cell ; invert the cover- 
 glass over it, so that the preparation will hang into the cham- 
 ber. Find the focus quickly, and observe that the ciliary 
 movement gradually comes to a standstill. The chloroform 
 molecules evidently penetrate the contractile substance of 
 the cilia, and inhibit or restrain those chemical or other 
 changes in its molecular machinery that are essential for 
 the contractile movement. If — when the movement is 
 nearly at or has just come to a standstill — the cover-glass 
 be removed, the cell washed out, and the preparation 
 exposed for some time to the air, the chloroform molecules 
 escape, and the movement is resumed. This experiment 
 may be more easily performed with the aid of a gas- 
 chamber (Fig. 49). 
 
 90. Effect of Carbonic Acid and Oxygen — With the aid 
 of a gas-chamber (Fig. 49), and cilia from the mussel's gill or frog's 
 pharynx, arranged as in the last experiment, the effects of various gases 
 may be studied. 
 
 A stream of carbonic acid (supplied by the usual apparatus, Fig. 46) 
 at first accelerates, but soon retards, and finally arrests the movement. 
 On the substitution of a stream of oxygen or air the movement is after 
 a time resumed. — {Kiihne.) 
 
 91. Effect of Alkalies — The addition of a very dilute solution 
 of caustic potash accelerates the movements of cilia when these are 
 becoming sluggish previous to their death ; it may even recall the 
 movement after it has ceased for some time. — (Virchcrw.) 
 
 For other experiments on ciliary motion see Kiihne, Schidtze's 
 Archiv, ii. 372; Engelmann, Journ. of Anat. and Physiol., iii. 420. 
 
 92. Structure of Ciliated Epithelium (H.) — Ciliary 
 
HISTOLOGICAL DEMONSTRATIONS. 71 
 
 motion and the structure of ciliated epithelium may be studied 
 in cells obtained from the frog's pharynx, or from the trachea 
 of rabbit or cat. If the latter animals be employed, they 
 should be killed by a blow on the head, and not by ether 
 or chloroform, otherwise the cilia will be paralysed. The 
 cells may be examined fresh, and then stained with 
 magenta to bring out the nucleus and the granular pro- 
 toplasm ; or they may be placed for twenty-four hours 
 in -A- per cent osmic acid, and then examined. The cells 
 may be rendered easily separable by a twenty-four hours' 
 immersion in dilute alcohol (§ 284), and then scraping 
 the epithelial surface. Magenta then stains the cells very 
 readily. 
 
 93. Scrape off some cells from the tracheal mucous mem- 
 brane of a cat, hardened in osmic acid as above mentioned ; 
 add glycerine ; tease with needles ; cover, and examine. 
 Observe the nucleus and nucleolus, the granular protoplasm 
 sometimes with vacuoles, the cilia and the clear band under 
 them. The clear band, however, may not now appear 
 homogeneous, as in the fresh condition, but striated verti- 
 cally. 
 
 With a power of about 1000 diam., it may be seen that the striae 
 are apparently continuations of the cilia into the granular protoplasm. 
 They may be seen passing a very short way below the clear band ; but 
 we have never seen them — even after various methods of staining — 
 passing to the nucleus, as has been alleged. 
 
 A preparation of ciliated epithelium for preservation 
 will be obtained in the section of hardened trachea to be 
 afterwards examined (§11 5). The cells hardened in osmic 
 acid may be preserved in potassium acetate (§351) or 
 Farrants' solution. 
 
 94. Columnar Epithelium and " Chalice Cells " 
 (H.) — Remove a portion of the wall of the large intestine 
 from a cat recently killed. Wash the mucous membrane 
 with } per cent salt solution. Scrape off the epithelium 
 and diffuse it with the point of a needle in a drop of 
 magenta solution. Columnar cells and chalice cells will be 
 found. 
 
72 PRACTICAL HISTOLOGY. 
 
 a. Columnar cells. — These resemble ciliated cells with- 
 out their cilia. At the free border of the cell there is a 
 clear band which, unlike the granular substance of the cell, 
 does not stain readily with magenta. Fine vertical striae 
 may possibly be seen in the band; these, however, will 
 probably be more evident in the next preparation. 
 
 b. Chalice cells. — These cells are found amidst the 
 ciliated epithelium of the respiratory passages, and the 
 columnar epithelium of the intestine more especially. They 
 are most readily found in the large intestine of the cat, and 
 after staining with magenta, they may be easily seen in a 
 preparation made as above. 
 
 They are generally about twice as broad as the ordinary 
 columnar cells ; some, however, are about the same 
 breadth. The free end of the cell is open, with a margin 
 that may be regular or irregular. The mouth of the cell 
 leads into a large vacuole. A nucleus with a small quantity 
 of protoplasm is usually found near the attached end of the 
 cell. 
 
 95. Scrape the mucous membrane of small intestine of 
 cat, hardened in chromic acid and potassium bichromate 
 (§ 7). Diffuse the scraping in water with the point of a 
 needle ; cover, and examine (H). 
 
 a. Profile View of the Cells. — The ordinary columnar 
 cells may be formed singly or in clusters. The fine vertical 
 lines, in their striated borders, are most readily seen with a 
 power of 1000 diam., nevertheless, they are discernible 
 with a power of 300, in specimens that have been macerated 
 a sufficient time. They are probably fine canals (Kolliker). 
 A chalice cell may perhaps he found here and there be- 
 tween the columnar cells. 
 
 b. View of the free ends of the cells. — A superficial view 
 of the free ends of the cells has the appearance of a mosaic. 
 The polygonal shape of the cells results from their collateral 
 pressure during their growth. The rounded opening of a 
 chalice cell may be seen. This is generally smaller than 
 the ends of the neighbouring columnar cells. 
 
 96. The mosaic and the openings of the chalice cells may be readily 
 seen if a portion of perfectly fresh small intestine of the cat or frog be 
 
HISTOLOGICAL DEMONSTRATIONS. 73 
 
 laid open, washed in distilled water, and silvered (§ 328), the out- 
 lines of the cells are rendered dark and distinct. In the silver process 
 in this case, use ^ per cent solution of silver nitrate, and agitate the in- 
 testine in the solution for a minute or so. 
 
 97. The existence of mucin in the ordinary columnar cells may be 
 thus shown. Scrape the mucous membrane of the frog's small in- 
 testine. Diffuse the scraping with a needle in a drop of water. Cover, 
 and examine at once. From the striated ends of the cells very deli- 
 cate hyaline blebs rapidly grow and become detached, their place being 
 taken by others. These hyaline corpuscles apparently result from 
 endosmose into the mucin within the cells. 
 
 The precise significance of chalice cells must still be 
 regarded as doubtful. Whether they are to be regarded as 
 unicellular glands (F. E. Schultze) for the special purpose 
 of secreting mucus, or whether they are merely old cells 
 undergoing mucin transformation and breaking down, is not 
 fully shown. The fact that cells resembling them may be 
 produced in large numbers by prolonged maceration of the 
 intestine in chromic acid favours the latter view. 
 
 98. Transitional Epithelium (H.) — Examine a pre- 
 paration made by scraping the mucous membrane of the 
 bladder of cat or rabbit prepared as directed in (§ 8), and 
 preserved in Farrants' solution. In this variety of epithe- 
 lium the cells may be squamous, columnar, or quite indefi- 
 nite in shape. 
 
 The various forms of secreting epithelium will be exa- 
 mined in the different glands, and other points regarding 
 epithelium will be conveniently studied with the various 
 organs. 
 
 CONNECTIVE TISSUES. 
 
 99. Elastic Tissue (H.) — Cut the ligamentum michce 
 of a calf or ox vertically, and strip off a few fibres from the 
 cut surface with forceps. Place the tissue on a slide, add 
 a drop of acetic acid, and tease it thoroughly with a pair of 
 needles (§ 290, a). The acetic acid softens the white 
 fibrous tissue of the ligament, and so facilitates the separa- 
 tion of the elastic fibres, upon which it appears to have 
 no action. Cover, and examine. 
 
74 PRACTICAL HISTOLOGY. 
 
 The fibres refract the light strongly, and therefore have 
 a distinct dark border. They branch and form an anasto- 
 mosing network. Their ends may be curled or straight. 
 The fibres under examination are a very coarse variety, 
 being about the breadth of a blood corpuscle. In the 
 vocal cords and in areolar tissue generally, they are much 
 finer. This preparation may be preserved in Fan-ants' 
 solution or glycerine ; in the last, however, the borders of 
 the fibres are not nearly so dark, owing to the high refrac- 
 tive index of the medium. 
 
 ioo. Areolar Tissue (H.) — a. Rapidly spread out 
 with needles on a dry slide a small piece of subcutaneous 
 tissue from a cat or rabbit. A dry slide is taken in order 
 that the tissue may adhere to it, and so be spread out 
 readily. The tissue must not, however, be allowed to dry. 
 Add salt solution and examine. Notice that the white 
 fibres consist of bundles of fibrils. The borders of the 
 white fibres are faint and ill defined. An elastic fibre may 
 be found here and there in the field ; unlike the white, fibre, 
 its margin is sharply defined, owing to its substance being 
 strongly refractile. 
 
 b. Remove the cover-glass and add acetic acid. It 
 causes no change in the elastic fibres, but the white fibres 
 swell up, the fibrils disappear, and nothing marks their posi- 
 tion excepting a clear jelly. The nuclei of the connective 
 tissue corpuscles are brought into view. Areolar tissue may 
 be preserved in potassium acetate or weak spirit. Glycerine 
 causes the white fibres to swell up and become transparent 
 or hyaline. This preparation need not, however, be pre- 
 served, as specimens will be obtained in sections of the 
 alimentary canal. 
 
 The splitting of the white fibres into fibrils is very easily effected 
 after maceration for a fortnight or so in a io per cent solution of 
 sodium chloride, often changed, or in lime or baryta water. (Rollett.) 
 These agents appear to soften or dissolve a cement which unites the 
 fibrils. 
 
 White fibrous tissue may be dissolved by boiling in water (§ 285) or 
 dilute sulphuric acid (§ 286), and may be softened by maceration in 
 dilute alcohol (§ 284). 
 
 1 01. (H.) — Examine a preparation of intermuscular 
 
HISTOLOGICAL DEMONSTRATIONS. 75 
 
 fascia from the leg, or of sub-peritoneal tissue from the lum- 
 bar region of a frog recently killed. A small portion of the 
 tissue is excised with scissors, and gently spread it out on a 
 slide in a drop of aqueous humour (§ 265). Observe the 
 stationary and amoeboid connective tissue corpuscles. The 
 latter, often termed the wandering cells of connective tissue, 
 appear to be identical with white blood corpuscles. The 
 former are the true connective tissue corpuscles. Endeavour 
 to see the locomotion of the wandering cells. The changes 
 of shape, but without locomotion, of the proper connective 
 tissue corpuscles may perhaps also be seen. 
 
 102. Connective tissue corpuscles may also be advan- 
 tageously studied in the loose subcutaneous tissue of the 
 groin of a young rabbit, cat, or guinea-pig, recently killed. 
 The tissue should be gently spread out on the dry slide, as 
 in § 100, and magenta added before the tissue becomes 
 dry. This dye reveals the corpuscles with great readiness. 
 The connective tissue corpuscles proper are usually flattened. 
 When seen in profile they are often fusiform. Some are 
 continuous with elastic fibres, others have no distinct pro- 
 cesses. 
 
 103. (H.) — Examine the preparation of omentum 
 already made (87D), and observe the connective tissue cor- 
 puscles amidst the fibres, and the epithelium here and there 
 clasping the bundles of fibres. Amoeboid cells (no longer 
 moving, of course) may sometimes be seen singly or in groups 
 here and there on the surface of the bundles. 
 
 1 04. Tendon. — Sections of fresh tendon may be readily 
 made with the freezing microtome (§ 302) ; or the tissue 
 may be hardened in \ per cent chromic acid, or hardened 
 by drying, and then cut ; the tendon being either simply 
 held in the hand or imbedded in paraffin and cut in the 
 microtome (§ 303). 
 
 Dry a tendon of an animal such as a sheep or a rabbit, 
 until it is just dense enough to be readily sliced. Make an 
 excessively thin transverse section with a scalpel or a razor. 
 The former is preferable, as the tendon is often over-dried, 
 and in that case apt to spoil a blade so thin as that of a 
 razor. The knife should be wetted with a spirit, for water 
 
76 PRACTICAL HISTOLOGY. 
 
 does not adhere to the blade (see § 295). Probably several 
 sections may have to be made before a sufficiently thin one 
 is obtained. It is not necessary to have one embracing the 
 whole thickness of the tendon, but care must be taken to 
 have it exactly transverse and not oblique. Examine the 
 section in water. 
 
 (L.) A tendon consists for the most part of bundles of 
 fibres running longitudinally; a few transverse fibres encircle 
 the longitudinal bundles, forming a sheath for the whole 
 tendon. This sheath sends inwards branching trabeculae 
 between the bundles, so as to divide the tendon into com- 
 partments. These trabeculae and the cut ends of the en- 
 closed longitudinal bundles are readily seen with L. 
 
 (H.) Observe the compressed branching spaces between 
 the transverse sections of the longitudinal fibres. Some 
 of the spaces contain nucleated corpuscles. (This prepara- 
 tion need not be preserved.) 
 
 If such a section be placed in glycerine, the fibres swell up and be- 
 come hyaline. The cut ends of elastic fibres may then be seen within 
 the longitudinal bundles and the nuclei of the cells in the branching, 
 spaces may also be seen. 
 
 105. Examine T. S. tendon hardened in chromic or 
 picric acid, and preserved in glycerine. In this, the spaces 
 between the bundles are not so compressed as in the dried 
 tendon. 
 
 106. Examine a tendon from the tail of a rat' prepared 
 as follows. Divide the skin about the middle of the tail of 
 a young rat or mouse just killed. Forcibly detach the ex- 
 tremity of the tail, so as to rupture the caudal vertebras, and 
 pull out the bundles of fine tendons with which these are 
 surrounded. Two methods may then be adopted — 1. They 
 may be placed in ^ per cent solution of chloride of gold for 
 two or three minutes, and then exposed to the light in water 
 acidulated with a few drops of acetic acid (see § 330). The 
 tendons are then teased with needles, and mounted in 
 Farrants' solution or in glycerine. 2. The tendons may be 
 placed for a minute or two in water slightly acidulated with 
 acetic acid, to cause them to swell up and become trans- 
 parent. They are then washed in water stained with log- 
 
HISTOLOGICAL DEMONSTRATIONS. 77 
 
 wood (§ 323) and mounted in Farrants^ solution, or in 
 glycerine. 
 
 Rows of flattened cells (Ranvier's cells) will be seen 
 lying between the longitudinal bundles of fibres. The cells 
 are somewhat quadrangular plates consisting of a distinct 
 nucleus surrounded by granular protoplasm. Their relation 
 to the fibres has been the subject of considerable dispute. 
 In a piece of tendon stained with gold and successfully 
 teased with needles, one may see, as Boll first pointed out 
 (Schultzis Archiv, 1871, p. 281), that the cells clasp the 
 bundles and apparently extend about half-way round them. 
 A longitudinal stripe in the row of cells was pointed out by 
 Boll, and termed [by him the " elastic stripe." More than 
 one stripe may be seen in each row. A view in full face of 
 the margin of a cell has the appearance resembling Boll's 
 stripe. Ranvier has shown, however (Traiti Technique 
 d 'Histologic, p. 352), that Boll's stripes are due to ridges on 
 the cells ; such a ridge as that seen on a bent tile or plate 
 of zinc on the top of a house. 
 
 107. For the demonstration of these ridges and the isolation of 
 tendon cells, Ranvier (loc. cit. ) recommends the following : — Place a 
 tendon for twenty-four hours in I per cent osmic acid, and then for the 
 same period in pi cro- carmine. Endeavour to isolate the tendon cells by 
 teasing with needles, and examine in glycerine containing 1 per cent 
 formic acid. 
 
 1 08. Adipose Tissue (H). — Examine the preparation 
 of omentum of guinea-pig or cat already made (§ 87D). 
 Observe the shapes of the fat cells when they are isolated, and 
 when they are pressed one against another. The nucleus of 
 the fat cell may have been stained by the silver, and if so, it 
 will be readily seen, especially if a profile view be obtained. 
 The nucleus is also rendered evident by carmine or logwood 
 staining. Each cell consists of a thin membrane more or 
 less filled by a drop of fat, having a nucleus with a small 
 quantity of protoplasm in contact with the membrane at 
 one part of the cell. Fat cells when silvered and mounted 
 in Farrants' solution or in glycerine are very perfectly pre- 
 served. 
 
 109. The envelope of the fat cell may be readily shown by the 
 
78 PRACTICAL HISTOLOGY. 
 
 old method of placing in ether for twenty-four hours a piece of adipose 
 tissue from an animal just killed. The fat is thus abstracted more or 
 less completely from some of the cells, and the collapsed membranes 
 are rendered visible. Perhaps the best method, however, is the 
 following : — Digest a piece of skin as described in § 223, b ; make 
 vertical sections, stain with carmine, and preserve in glycerine. In 
 many of the fat cells there is a wide space between the fat and the 
 envelope, and the stained nucleus is readily seen on the interior of the 
 latter. 
 
 109A. Examine a preparation of fat cells showing crystals 
 of margarine. These sometimes appear after death. They 
 are almost always to be found in fat taken from an animal 
 recently killed, and mounted for some time in glycerine. 
 They are also often found in the fat cells of organs, e.g., 
 skin hardened in chromic acid and mounted in glycerine. 
 
 no. Adenoid Tissue (H.) — With the aid of the 
 freezing microtome make an extremely thin section of a 
 fresh mesenteric gland of a fasting animal recently killed. 
 Detach the lymph corpuscles from the adenoid tissue by 
 gently shaking the slice in a test-tube with j per cent salt 
 solution ; mount the section in Farrants' solution, and 
 examine. 
 
 Adenoid tissue consists of a reticulum of nucleated 
 
 connective tissue corpuscles, the processes of which have 
 
 been transformed into fibres. Lymph corpuscles will be 
 
 found in the interspaces. — (This section will be wanted on a 
 
 future day.) 
 
 in. Examine a section of a lymph gland prepared as 
 follows : — Into the mesenteric gland of a fasting cat or dog 
 recently killed, thrust the nozzle of a Wood's syringe through 
 the capsule of the gland, and. inject \ per cent silver nitrate 
 solution. Make sections with the freezing microtome, wash 
 away the superfluous silver with distilled water, expose in 
 the water to diffuse daylight for a few hours, stain with 
 logwood. The adenoid tissue is coloured brown, while the 
 lymph corpuscles are rendered violet. The latter may be 
 detached by shaking in a test-tube with ordinary water. 
 The sections may be preserved in Farrants' solution, or in 
 glycerine, or glycerine jelly. 
 
 Other preparations of adenoid tissue will be obtained 
 
HISTOLOGICAL DEMONSTRATIONS. 79 
 
 on a future occasion in sections of the lung, intestine, and 
 spleen. 
 
 112. Mucous Tissue (H.) — Examine a preparation 
 of vitreous humour, made by mounting a small piece of the 
 vitreous humour of the kitten before birth, in Fan-ants' 
 solution. Connective tissue corpuscles are seen in a jelly- 
 like matrix. 
 
 CARTILAGE. 
 
 113. White Fibro- Cartilage (H.) — Make a section 
 with scissors of fresh white fibro-cartilage from the peri- 
 pheral part of the intervertebral disc of an ox or sheep 
 after hardening for forty-eight hours in a saturated solution 
 of picric acid. Tease with needles, and preserve in 
 Farrants' solution or in glycerine. 
 
 The matrix consists of very delicate fibrils like those of 
 white fibrous tissue. Sometimes, however, it is so difficult 
 to see the fibrillation that one is apt to suppose that the 
 matrix is homogeneous. The cells consist of a nucleated 
 protoplast enclosed in a capsule, resembling that found 
 around the protoplasts of hyaline cartilage. The cells may 
 be found in various stages of fission. Their characters are 
 much more clearly seen with a power of 800. 
 
 114. Yellow Fibro- Cartilage (H.) — Place the epi- 
 glottis of. a sheep for a day in rectified spirit : nothing 
 else hardens this so well. Make sections with a razor. 
 Examine, and preserve in Farrants' solution or in glycerine. 
 
 This tissue consists of nucleated cells surrounded by a 
 matrix, which, in the greater part of the cartilage, consists 
 of a spongy material apparently of the same nature as 
 elastic tissue. Towards the periphery of the cartilage there 
 are distinct yellow elastic fibres in the matrix. But the 
 greater part consists of a yellow spongy substance, the fine 
 spicules of which when divided transversely resemble 
 granules ; hence the erroneous statement that the matrix 
 of this tissue consists of elastic fibres imbedded in a granular 
 material. It is advantageous to examine this tissue with a 
 power of about 800. 
 
 115. Hyaline Cartilage, Trachea — T. S. tracheal 
 
80 PRACTICAL HISTOLOGY. 
 
 rings of cat, hardened in J per cent chromic acid (§ 6). 
 The specimen will be a valuable one if the section be made 
 with the freezing microtome ; for in that case the ciliated 
 epithelium lining the trachea will not be destroyed, as is 
 apt to be the case when such a tissue is imbedded in 
 paraffin. Mount the section in Farrants' solution or in 
 glycerine, and examine. 
 
 (L.) The position of the cartilage, epithelium, and the 
 tracheal glands. 
 
 (H.) a. The cartilage, consisting of cells in a hyaline 
 matrix. Observe the nucleus and granular protoplasm of 
 which the cells consist ; the distribution of the cells in the 
 matrix ; the groups result from recent proliferation of the 
 cells. 
 
 b. The perichondrium, consisting of white fibrous tissue. 
 Notice the gradual transition between the matrix of the 
 cartilage and the fibres of the perichondrium on the one 
 hand, and between the cells of the cartilage and those of 
 the perichondrium on the other. The latter, however, are 
 difficult to discern unless they are stained. 
 
 c. The ciliated epithelium. — The characters of the ciliated 
 cell have been already studied. The cells are here seen in 
 situ. There are several layers of them, but only the upper 
 layer (those next the cavity of the trachea) are ciliated, 
 The others are of irregular shape, and are apparently young 
 cells that are destined to develop into those with cilia. 
 
 d. The glands, cut in all directions, will be found inside 
 the cartilage. They are lined by epithelium, and their 
 ducts may perhaps be traced to the inner aspect of the 
 membrane on which they pour their mucous secretion. 
 
 1 1 6. Costal Cartilage of human adult kept for 
 twenty-four or forty-eight hours in a saturated solution of 
 picric acid. With a razor make a thin T. S. through a 
 point of incipient ossification. Mount in Farrants' solution 
 or in glycerine, and examine. 
 
 (H.) The diffuse yellow colour resulting from the picric 
 acid might be removed by immersion in water or in alcohol, 
 but it is advantageous to retain the colour in this case. 
 At the point of incipient ossification groups of proliferating 
 
HISTOLOGICAL DEMONSTRATIONS. 81 
 
 cartilage cells will be found, and a clear capsule will be 
 seen around most of the protoplasts — thickest in the largest 
 ones. The appearance reminds one of the capsule enclosing 
 each protoplast in white nbro-cartilage. The diffuse picric 
 acid staining is of much service in rendering these capsules 
 evident. The matrix of hyaline cartilage apparently consists 
 of these capsules pushed outwards and matted together by 
 new ones that are secreted by the protoplasts proliferating 
 by fission within the old ones. Side by side with the 
 proliferation of the cartilage cells, observe the fibrillation of 
 the matrix. This consists in the appearance of great 
 numbers of calcified fibrils running parallel one with 
 another, and usually with the long axis of the groups of 
 proliferating cells. The proliferation of the cells and the 
 calcified fibrillation of the matrix are the two earliest stages 
 of intra-cartilaginous ossification, which will be further 
 studied in another preparation (§125). If this preparation 
 happen to be the costal cartilage of an old subject, cartilage 
 cells in a state of fatty degeneration may be found here 
 and there. The protoplasm becomes replaced by fatty 
 particles. 
 
 117. Staining Agents for Hyaline Cartilage. — a. Osmic 
 acid. The fatty particles that occur sparingly in most cartilage cells, 
 and often so abundantly in those of an old subject, may be readily 
 demonstrated by placing the slices in \ per cent osmic acid for 12 to 
 24 hours. The fat is blackened by the acid. 
 
 b. Chloride of gold is very useful for staining the cells. It renders 
 them violet, and it has the great advantage of not causing any retrac- 
 tion of the protoplasm from the matrix. Vertical or longitudinal sec- 
 tions of the articular cartilage of the femur of a frog, recently killed, 
 when stained with gold give readily the characteristic appearance. It 
 should be kept in the gold solution twenty minutes, and otherwise 
 treated as described in § 330. 
 
 c. Silver Nitrate stains the matrix of hyaline cartilage, and is there- 
 fore of value in tracing its relation to the protoplasts. Ranvier's 
 method is to wash the head of the frog's femur in distilled water for a 
 few seconds ; place it in \ per cent solution of silver nitrate until it 
 becomes opaque ; wash in distilled water for some minutes : remove 
 thin slices from the articular surface; place them in glycerine on a 
 slide, and expose to the light till the matrix has become sufficiently 
 dark. Or the articular surface may be shaved off, and the sections 
 placed at once in the silver solution. 
 
 d. The value of picric acid has been alluded to in § 116. 
 
 G 
 
82 PRACTICAL HISTOLOGY. 
 
 BONE. 
 
 1 1 8. Method of preparing Unsoftened Bone. — The bone 
 is cut into tolerably small pieces, and freed from its soft parts by 
 maceration for months in water changed from time to time. The 
 bone must not be allowed to become dry previous to maceration, 
 otherwise the bony tissue may become indelibly marked with greasy 
 stains. In the case of a long bone, much of the fat may be rapidly 
 detached from the medullary canal and cancellated tissue by connecting 
 it to a water-tap by an elastic tube. The bone is then dried, and if it 
 contain much spongy tissue, it is impregnated with a strong solution of 
 gum, hardened for a day in methylated spirit, dried, and then cut with 
 a fine saw in any desirable direction. The spongy tissue is supported 
 by the gum, and thus rendered less liable to be fractured by the saw. 
 The sections are ground sufficiently thin by rubbing them on an ordi- 
 nary hone, moistened with methylated spirit to retain the gum in situ. 
 They are then placed in water to remove the gum, thoroughly washed 
 in a stream of water, and finally dried. — {Ranvier.) They are com- 
 monly mounted dry, that is to say, in air. They may also be mounted 
 in dammar (§ 347, c) ; this is apt to render them too transparent. 
 
 119. Method of preparing Softened Bone. — The 
 
 soft parts of bone may be successfully hardened and pre- 
 served, while the calcareous matter is removed, by macera- 
 tion in J per cent chromic acid, with the addition of 1 per 
 cent nitric acid, after the method described in § 15. When 
 the decalcified bone is transferred to rectified spirit, it 
 becomes of a greenish tint, owing to the production of 
 chromium sesquioxide (chromic oxide). The softened 
 bone may be readily sliced with a knife, or torn into shreds 
 for the demonstration of its lamellae. 
 
 Ranvier recommends for the softening of bone a 
 saturated solution of picric acid, and this, for foetal bone, 
 is perhaps the best agent. The bone should be divided 
 into very small pieces and placed in a large quantity of the 
 fluid, kept saturated by the presence of an excess of the 
 crystals. When softened, the picric acid may be partially 
 or completely removed by immersion in water or spirit. It 
 should be wholly removed only when the preparations are 
 to be stained, e.g., in picro-carmine. 
 
 120. Staining Agents for Softened Bone. — a. The 
 greenish coloration of the matrix by chromic oxide has 
 been alluded to in § 119. 
 
HISTOLOGICAL DEMONSTRATIONS. 83 
 
 b. Picro-carmine (§ 322) gives fair results. It stains the 
 bone corpuscles, and to a less extent the matrix. Before 
 staining with this agent all chromic or picric acid should be 
 removed by washing the sections in water. 
 
 c. Purpurine is strongly recommended by Ranvier as a 
 staining agent for the bone corpuscles, on account of its 
 staining them only. Chromic or picric acid is previously 
 washed out of the sections, and they are then placed in a 
 solution of purpurine (§ 326) for twenty-four to forty-eight 
 hours. It does not, however, appear to show more than 
 picro-carmine does. 
 
 Sections of softened bone unstained should be mounted 
 in rectified spirit or glycerine jelly (§ 350) ; glycerine 
 renders them too transparent. If stained, they may be 
 mounted in Fan-ants' solution, glycerine, or glycerine jelly. 
 
 121. Structure of Bone. — Examine a T. S. un- 
 softened bone prepared as directed in § 118. 
 
 (L.) The compact and spongy bone. The Haversian 
 systems in the former, with a Haversian canal cut trans- 
 versely in or near the centre of each system. The lacunae 
 with their canaliculi. 
 
 (H.) The lacunae usually more or less flattened. The 
 canaliculi dividing and anastomosing with those of neigh- 
 bouring lacunae, so as to form what is probably a system 
 of lymphatic channels. The canaliculi of the lacunae 
 nearest the Haversian canal open into it, and thus the 
 lymph can readily pass from the blood capillaries in the 
 canal into the canaliculi. The contents of the Haversian 
 canals and lacunae will be found in preparations of decalci- 
 fied bone. 
 
 122. Examine a V. S. unsoftened long bone. (L.) The 
 Haversian canals mostly cut vertically. They anastomose, 
 and some may be found opening on the periosteal surface. 
 Others may be found passing into the spaces in the spongy 
 bone. The Haversian systems being divided vertically, the 
 concentric arrangement of their lacunae is of course not 
 here seen. 
 
 123. T. S. softened long bone of cat. If the section is 
 unstained, examine it in dilute alcohol (§ 284) — for the 
 
84 PRACTICAL HISTOLOGY. 
 
 cover-glass requires to be removed — and finally mount in 
 glycerine jelly ; glycerine renders the bone corpuscles too 
 transparent. If, however, the section be stained with 
 picro-carmine, examine, and preserve it in Farrants' solu- 
 tion, glycerine, or glycerine jelly. 
 
 (L.) The compact and spongy bone. — If the section be 
 through the central part of the shaft of the bone, little cancel- 
 lated tissue will be seen. The Haversian systems — each 
 consisting of a Haversian canal, with a concentric series of 
 lamellae — are seen divided transversely. The systems are 
 of somewhat irregular shape, and the Haversian canal is 
 often placed eccentrically. This results from the substance 
 of the system growing from the Haversian canal outwards, 
 most rapidly in the direction of least resistance. Under 
 the periosteum the lamellae have a direction parallel with 
 that membrane. Between the Haversian systems there are 
 small sets of lamellae, that are generally more or less 
 parallel with the direction of the sub-periosteal lamellae. In 
 the development of the bone the lamellae produced around 
 the Haversian canal grow outwards, and cause the par- 
 tial, and in some places the complete, absorption of those 
 lamellae parallel with the periosteum, and originally pro- 
 duced from it. The lamellae and the bone corpuscles will, 
 however, be more clearly seen with H. 
 
 (H.) The bone corpuscles. — Nucleated masses of proto- 
 plasm in branching spaces, the lacunae and canaliculi. The 
 last are difficult to see in softened bone, unless it be stained 
 with picro-carmine. The wall of the lacunae and canaliculi 
 consists of a homogeneous calcified membrane that may be 
 detached from the lamellae, although with difficulty — if 
 softened bone be teased with needles. (Virchow.) 
 
 b. The lamellce. — Examine them in a Haversian system. 
 Five or six lamellae usually intervene between two neigh- 
 bouring lacunae. The elliptical shape of the lacunae 
 apparently results from compression between the lamellae. 
 
 c. Sharpens fibres may be found perforating the sub- 
 periosteal lamellae. They are calcified fibres that run from 
 the periosteum inwards between the Haversian systems. 
 They are not found within the latter. Remove the cover- 
 
HISTOLOGICAL DEMONSTRATIONS. 85 
 
 glass, and tear off a few of the peripheric lamellae. After 
 this some of Sharpey's fibres may be found isolated, by 
 being torn out of their sockets. 
 
 d. The periosteum. — In the adult animal the periosteum 
 consists simply of fibrous tissue, with a connective tissue 
 corpuscle here and there imbedded amidst the fibres, or 
 in contact with the bone, but they are not readily seen 
 unless the section be exceedingly thin and stained. In 
 another preparation it will be seen that the periosteum of 
 the young bone has a very different appearance. The 
 contents of the cancelli and of the Haversian canals will 
 also be better seen in other preparations. 
 
 124. Development of Bone. — Young periosteum. 
 (H.) — Examine a T. S. long bone from kitten at birth, 
 softened in the chromic and nitric fluid, or in picric acid, 
 and stained with picro-carmine. A layer of nucleated cells 
 (osteoblasts) is found under the fibrous tissue of the perios- 
 teum close to the bone. On carefully examining the whole 
 thickness of the periosteum, it may be seen that these cells 
 are proliferations from connective tissue corpuscles. They 
 may be traced into the Haversian canals, and through these 
 into the cancelli. Everywhere they are in contact with the 
 bone. These osteoblasts ultimately become the bone cells ; 
 the matrix of the bone grows around and encloses them. 
 The manner in which the canaliculi are formed is disputed ; 
 some maintaining that they are mere spaces left in the bony 
 matrix during its growth. It may be seen, however, with 
 (H.), and still more clearly with a power of 1000 diam., 
 that the osteoblasts have often very distinct processes 
 before they are included in the bone. This preparation 
 shows a very delicate fibrous tissue in the cancelli and in 
 the Haversian canals, through which it is continuous with 
 the periosteum, capillaries and larger vessels may also be 
 found in the cancelli, Haversian canals, and in the perios- 
 teum, especially in its inner layer. 
 
 125. Intra- cartilaginous Formation of Bone — 
 V. S. bone and articular cartilage from softened bone of adult 
 cat, prepared as directed in § 15. Treat in the same 
 manner as the softened bone (§ 123). 
 
86 PRACTICAL HISTOLOGY. 
 
 (L.) Examine cancelli, articular cartilage, and a layer 
 of calcified cartilage close to the bone. 
 
 (H.) a. Osteoblasts. — In the cancelli osteoblasts will 
 be found close to the bone. Observe their irregular shape, 
 some with distinct processes, and all of them having a bright 
 refractive appearance. The other cells found in marrow 
 will be examined in a preparation from fresh bone. 
 
 b. Articular cartilage. — Near the articular surface the 
 cells are flattened ; about the middle of the cartilage they 
 are in irregular groups ; near, and in the calcified layer, 
 they are arranged in rows, owing to repeated proliferation 
 of the cartilage cell by cleavage at right angles to the long 
 axis of the row. The lime has of course been removed 
 from the matrix in the calcified zone, but in specimens not 
 fully decalcified, a fibrillation of the matrix similar to that 
 seen in the rib (§ 1 1 6) may be recognised. On very care- 
 fully examining the line of junction between the cartilage 
 and the bone, one can here and there see a cartilage pro- 
 toplast becoming surrounded by the matrix of the bone 
 that grows up around it. The production of a bftne cell 
 from a cartilage cell may be thus demonstrated ; but this 
 mode of bone formation seen above the epiphysis in an 
 adult animal must be regarded as exceptional, and differing 
 decidedly from the mode discernible under the epiphysis in 
 a young animal. 
 
 126. The intra-cartilaginous development of bone may 
 be very clearly studied in a V. S. of a long bone or scapula 
 of a kitten at birth, and in a V. S. of the phalanx of a 
 human foetus at the fourth month (doubtless the phalanx 
 of a kitten would show the same, but this we happen not 
 to have examined). The bones should be softened in 
 picric acid, and the former may be unstained while the 
 latter is stained with picro-carmine. It is advantageous to 
 inject the blood-vessels with soluble Prussian blue (§ 333) 
 previous to softening. 
 
 127. The vertical section of the phalanx, prepared as 
 above, is exceedingly instructive. 
 
 (L.) The ossification begins in the middle of the shaft, 
 and then extends towards the ends. A wedge-shaped piece 
 
HISTOLOGICAL DEMONSTRATIONS. 87 
 
 of bone may be seen growing inwards from the perichon- 
 drium to meet its fellow from the opposite side. Between 
 the two there are irregular medullary spaces rilled with cells. 
 Blood-vessels extend into them from the perichondrium 
 through canals in the bone. Rows of cells may be seen in 
 the cartilage near the medullary spaces. 
 
 (H.) The proliferation of the cartilage cells close to the 
 medullary spaces may be readily seen. A single cavity 
 contains a number of cells, and these cavities finally be- 
 come continuous with the medullary spaces into which 
 blood-vessels have grown from the perichondrium. The 
 cartilage matrix is entirely absorbed, as has long been 
 known, but whether the cartilage cells become marrow 
 cells or osteoblasts, or what becomes of them, it would be 
 difficult to say, for one may detect something very like the 
 diapedesis of white blood corpuscles through the walls of 
 the capillaries, and the emigrated blood corpuscles may play 
 some part in the process of forming osteoblasts, as has been 
 suggested by Gegenbaur and others. 
 
 128. (H.) — A V. S. of a somewhat older bone of a 
 kitten, prepared as above, shows the rows of cartilage cells, 
 the medullary spaces with their capillaries, and cells around 
 them. The fate of the plates of cartilage matrix between 
 the medullary spaces may be clearly traced. 
 
 The cartilage matrix becomes encrusted with the matrix 
 of the future bone, and slowly disappears. Osteoblasts are 
 gradually enclosed in the bony matrix, and thus become 
 bone cells. It would be difficult to say what is the precise 
 genetic relation between the osteoblasts and the bony matrix, 
 for a thin layer of the matrix may be seen extending up 
 towards the cartilaginous ends of the medullary spaces, 
 without any appearance of osteoblasts in it, in the first in- 
 stance. The exact mode of origin of the osteoblasts in in- 
 tra-cartilaginous formation of bone, must therefore still be 
 held as doubtful. 
 
 129. Marrow (H.) — As yellow marrow consists chiefly 
 of fat cells, it need not here be alluded to. Red marrow, 
 however, is worthy of careful study. Take a rib from a 
 rabbit, cat, or dog, not fully grown ; divide it transversely, 
 
88 PRACTICAL HISTOLOGY. 
 
 and then with a common knife remove a slice of the spongy- 
 tissue. Place it on a slide in a drop of salt solution. With 
 needles detach the bony spicules from the marrow and 
 throw them away. Cover, and examine. 
 
 Most of the cells in the preparation are the proper 
 marrow cells of Kolliker. These somewhat resemble white 
 blood corpuscles, but when acted on by dilute alcohol or 
 acetic acid, they usually exhibit a single nucleus, and often 
 also yellowish particles. Large multi-nucleated masses of 
 protoplasm, commonly termed giant cells (the myeloplaxes of 
 Robin), may be found here and there. They vary in size 
 from five to twenty times that of a blood corpuscle. In a 
 section of the cancelli of decalcified bone of a somewhat 
 young subject, a giant cell may be found in contact with 
 the osseous tissue here and there amongst the osteoblasts. 
 Believing that they are concerned in the absorption of the 
 neighbouring bone — which often appears as if scooped out 
 — Kolliker has named them osteoclasts. 
 
 Neumann and Bizzozero have advanced the opinion {Ceniralblait, 
 1868, pp. 689, 855, and 1869, p. 149), that on account of the pre- 
 sence of nucleated red blood corpuscles, similar to those of the embryo, 
 red marrow is to be regarded as a blood gland. We have entirely 
 failed to find such corpuscles ; but, on the other hand, one can some- 
 times see a group of red corpuscles enveloped in a mass of clear colour- 
 less colloid material, similar to the bodies found in the spleen, and 
 which are there regarded as evidence of the disintegration of red cor- 
 puscles. 
 
 TOOTH. 
 
 130. Sections of unsoftened tooth are prepared accord- 
 ing to the same method as bone (§ 118). Owing to the 
 absence of fat from the tooth pulp, however, the maceration 
 is soon accomplished. The method for softening tooth is 
 the same as that adopted for bone (§11 9). The enamel, 
 however, disappears, owing to its very large percentage of 
 calcareous matter. 
 
 131. Structure of Unsoftened Tooth. V. S. Unsoft- 
 ened Tooth. (L.) — Observe the pulp cavity, the dentine, 
 the enamel outside the dentine in the crown, and the crusta 
 petrosa outside the dentine in the fang of the tooth. The 
 
HISTOLOGICAL DEMONSTRATIONS. 89 
 
 wavy lines running from within outwards in the dentine are 
 the dentinal tubules. The arched incremental lines which 
 may sometimes be seen crossing the dentinal tubules in the 
 crown of the tooth indicate a lamination of the dentine 
 apparently due to incomplete calcification of its matrix 
 during development. The enamel consists of prisms set 
 with their ends on the dentine. Lines more or less con- 
 centric, and of a brown colour, may often be seen crossing 
 the enamel prisms. Their cause is uncertain, but they look 
 as if they resulted from the deposit of a brown pigment. 
 
 (H.) The enamel prisms are arranged in bundles that 
 often decussate at their inner ends near the dentine. In 
 the preparation there may be some of these bundles cut 
 transversely. These show the fibres to be more or less 
 hexagonal. The dentinal tubules have primary dichotomous 
 divisions, and give off great numbers of fine lateral branches. 
 The latter are usually very evident in the outer part of the 
 dentine of the fang of the tooth. In the crown, very pro- 
 bably, minute rounded apertures in the transverse sections 
 of the dentinal tubules may be found. These are, how- 
 ever, most easily seen in a V. S. of dentine made at one 
 side of the pulp cavity. Interglobular spaces may perhaps 
 be seen in the outer part of the dentine. These are irre- 
 gular cavities, which, like the incremental lines that fre- 
 quently open into them, apparently result from defective 
 calcification. In such a preparation as this they have a 
 black appearance, resulting from their being filled with 
 debris during the grinding of the tooth. The crusta petrosa 
 is merely bone. 
 
 132. Structure of Softened Tooth. V. S. jaw of 
 cat softened in dilute chromic and nitric acid, as directed 
 in § 15. Mount in Farrants' solution or in glycerine jelly. 
 The latter is preferable. 
 
 (L.) The tooth in the alveolus. The periodontal mem- 
 brane serving as a periosteum for the crusta petrosa and 
 the interior of the alveolus. If the section be one of the 
 lower jaw, the canal containing the inferior dental nerve 
 and blood-vessels will be cut across, and good sections of 
 these structures thus obtained. All the parts of the tooth 
 
90 PRACTICAL HISTOLOGY. 
 
 are present except the enamel, and probably also much of 
 the pulp will be wanting. 
 
 (H.) The dentinal tubules will be readily seen. 
 Carefully examine the pulp close to the dentine for odonto- 
 blasts. These are nucleated cells that send one or more 
 somewhat stiff processes from their outer aspects into the 
 dentinal tubules, constituting the dentinal fibres, or fibres 
 of Tomes. The odontoblasts are, however, very liable to 
 be lost during the softening of the tooth. Nerves and blood- 
 vessels and a delicate connective tissue are also found in 
 the pulp, but these need not be looked for in this prepara- 
 tion. 
 
 133. The structure of the pulp of the full-grown tooth can only be 
 investigated by breaking the tooth across with a hammer. The broken 
 tooth should then be hardened for twenty-four hours in -| per cent osmic 
 acid. 
 
 134. The odontoblasts and fibres of Tomes maybe seen in sections 
 of the teeth in situ, when they are very young and still within the den- 
 tal sacs. 
 
 135. The dentinal tubules may be isolated by boiling a softened 
 tooth for about ten minutes in 1 per cent sulphuric acid. The* tubules 
 appear to be calcified membranes, imbedded in a calcified tissue some- 
 what similar to that of bone. The latter disappears when boiled in the 
 dilute acid. 
 
 MUSCLE. 
 
 136. Non-Striped Muscle. — Methods of Preparation. 
 — a. Kolliker's method for the isolation of the fibres con- 
 sists in placing a piece of fresh intestine or stomach in 
 dilute nitric acid (acid one part, water four parts) for 
 twenty-four or thirty-six hours. The muscular fibres are 
 hardened and assume a yellow colour, while the fibrous 
 tissue that binds them together is softened and dissolved. 
 The acid is then removed by washing in water. The nu- 
 cleus in each fibre may be clearly brought into view by 
 logwood staining. The fibres are separated by teasing. 
 
 b. The fibres may also be separated by teasing after 
 maceration for two days or so in dilute alcohol (§ 284). 
 
 c. The fibres may also be prepared by the gold method. 
 For this purpose portions of the muscular coat, from the 
 small intestine of a rabbit or other animal recently killed, 
 
HISTOLOGICAL DEMONSTRATIONS. 91 
 
 are placed in gold chloride solution, and treated as directed 
 in § 330. The nucleus of the fibre is stained. 
 
 d. The nuclei of the fibres may also be well stained 
 with picro-carmine. A piece of frog's bladder when treated 
 with this dye (§ 322) gives beautiful preparations. 
 
 137. Structure of Non-striped Muscle. — Strip off a 
 small piece of the muscular coat of the intestine, hardened 
 in dilute nitric acid as above described. Tease it with 
 needles in a drop of Farrants' solution ; cover, and examine. 
 
 Each fibre is a spindle consisting of a nucleated band 
 of sarcous substance, without an envelope. The whole 
 fibre is evidently an elongated cell, the protoplasm of which 
 has been transformed into a substance, that — like the sub- 
 stance of a cilium — contracts in a definite direction. 
 
 138. Examine a preparation of frog's bladder, carmin- 
 ised. Notice the spindle-shaped and the triradiate fibres. 
 The latter might, by the inexperienced observer, be mistaken 
 
 for nerve cells. 
 
 Excellent preparations of the fasciculi of non-striped 
 muscle, divided transversely and longitudinally, will be 
 found in the sections of stomach and intestine at a future 
 time. 
 
 139. Striped Muscle. — The Sarcolemma (H). — a. 
 Decapitate a frog, and isolate one of its muscles; the 
 sartorius is to be preferred, because its fibres are parallel 
 and easily separated. Make a vertical section of the muscle 
 with scissors, and tease the fibres in a drop of water, in 
 order to study the sarcolemma. Look for the sarcolemma 
 raised from the sarcous substance, by imbibition, in the form 
 of a vesicle, or stretched between the ends of fibres broken 
 across, or prolonged as a funnel-shaped process from the 
 end of a ruptured fibre. 
 
 b. Effect of acetic acid. — Remove the preparation from 
 under the lens, raise the cover and add a drop of dilute 
 acetic acid. It renders the sarcous substance very trans- 
 parent, and reveals the nuclei of the sarcolemma. In 
 mammals the nuclei are only found under the sarcolemma. 
 In the frog's muscle they are also found amidst the sarcous 
 substance. 
 
92 PRACTICAL HISTOLOGY. 
 
 Each striped fibre of the voluntary muscles is an elon- 
 gated spindle developed from a cell. The sarcolemma may- 
 be regarded as the cell membrane ; the nuclei inside the 
 sarcolemma are proliferations from the original nucleus, and 
 the sarcous substance is a modification of the original pro- 
 toplasm. 
 
 140. The Sarcous Substance (H.) — To soften the 
 connective tissue between the fibres, and permit of their 
 ready dissociation with needles, prepare muscle as follows. 
 Take a narrow strip of the sartorius of a rabbit just killed ; 
 tie a thread round both ends ; fix the threads to a piece of 
 wood with the muscle gently stretched ; then place in dilute 
 alcohol (§ 284) for two days or so. Tease a small piece 
 with needles in Farrants' solution; cover, examine, and pre- 
 serve. 
 
 Each fibre is seen to be crossed by alternate light and 
 dim stripes. By altering the focus it may be seen that the 
 stripes are not superficial, but extend through the whole 
 thickness of the fibre. 
 
 141. Cleavage of the Sarcous Substance (H.) — a. 
 With a razor make a thin V. S. of striped muscle of cat or 
 rabbit hardened in \ per cent chromic acid (§ 11) to 
 facilitate the cleavage of the sarcous substance. Tease 
 with needles in a drop of Farrants' solution; cover, and 
 examine. 
 
 The fibre cleaves most readily in a longitudinal direc- 
 tion. The result of such cleavage is a bundle of fibrils, 
 each marked with a light and dim transverse stripe like the 
 original fibre. A very fine dark line may — with a power 
 of about 1000 — be seen crossing the light stripe trans- 
 versely, and dividing it into two equal portions. If the 
 fibre or fibrils cleave transversely, the rupture takes place 
 through this faint dark line in the clear stripe. 
 
 b. Remove the slide from under the lens, and forcibly 
 press down the cover-glass with the handle of a knife or 
 needle, and examine again. The pressure often causes the 
 fibres and fibrils to cleave transversely. When a fibre 
 cleaves transversely, it gives rise to a number of discs. 
 The transverse cleavage of a fibril produces a number of 
 
HISTOLOGICAL DEMONSTRATIONS. 93 
 
 oblong particles, which have been conveniently named sar- 
 cous prisms. & fibril consists of these prisms united end to 
 end ; a disc is formed by their transverse union. 
 
 If striped muscle be partially digested in such a digestive 
 fluid as that mentioned in § 223 I?, it, as has long been 
 known, becomes very apt to split into discs. 
 
 142. Examine, with a power of 800 or 1000 diam., 
 a preparation of the muscle of a bee, hardened in $• per 
 cent chromic acid, and mounted in glycerine. The sarcous 
 prisms are beautifully seen. The house-fly and the water- 
 beetle serve equally well for making such specimens. 
 
 143. Amputate the leg of a water-beetle (JDytiscus mar- 
 ginalis). With a knife slit open the chitinous case ; care- 
 fully pick out the muscle ; make a vertical section with 
 scissors ; add a drop of salt solution, or, still better, aqueous 
 humour; gently separate the fibres with needles; cover, and 
 examine without delay. (H.) Observe the contraction 
 waves sometimes implicating the whole thickness, sometimes 
 only a part of the fibre. The stripes are easily seen. Faint 
 dark lines are seen crossing the dim stripe at right angles 
 to it. These are the lines through which longitudinal 
 cleavage of the fibre takes place. 
 
 When magnified 1000 diam. or so, the fine longitudinal lines 
 crossing the broad dim stripe at right angles may be traced into the 
 clear stripe on each side, near the middle of which each line becomes 
 enlarged and forms a knob-like extremity. The whole has been com- 
 pared by Mr. Schaefer to a dumbbell with a very slender shaft. He 
 has termed these structures "muscle rods." The heads of the rods 
 form the faint dark line in the dim stripe. The discussion regarding 
 the cause of the stripes of muscle cannot be entered into here ; but it 
 may be safely said that the whole question is still very obscure. 
 
 144. Examine (H.) striped muscle stained with carmine. 
 The nuclei are stained, but not the sarcous substance or 
 the sarcolemma. 
 
 145. Examine (H.) a T. S. of striped muscle, hardened 
 in chromic acid. Such a section as this will be obtained 
 in a T. S. of the tongue (§ 185). The fibres are seen to be 
 polygonal, owing to collateral pressure. In the living 
 muscle they are rounded. 
 
94 PRACTICAL HISTOLOGY. 
 
 Transverse sections of fresh muscle may be readily 
 made with the freezing microtome. 
 
 146. Examine (L.) an L. S. of muscle with its blood- 
 vessels injected. The capillaries are seen to run mostly 
 parallel with the fibres. These longitudinal vessels are united 
 by short transverse branches. 
 
 147. With regard to the examination of muscle by polarised light 
 (§ 53), the memoirs by Brticke (Strieker's Histology, vol. i. p. 235) and 
 Schaefer ("Minute structure of the Leg-Muscles of the Water- Beetle," 
 Philosophical Trans., 1873) may be consulted. 
 
 NERVE TISSUE. 
 NERVE FIBRES. 
 
 148. White Nerve Fibres (H.) — Place the lumbar or 
 sciatic nerves of a frog in \ per cent osmic acid for two 
 days. Wash in distilled water for a day. Tease with 
 needles, and preserve in Farrants' solution. 
 
 Observe in each fibre a central somewhat clear band — the 
 axial cylinder. At the broken end of a fibre this band 
 may be found projecting. It is apparently homogeneous, 
 but in the spinal cord it may be seen that at the junction of 
 the axial cylinder with a nerve cell it is fibrillated. Therefore 
 the axis is believed to be a bundle of nerve fibrils held to- 
 gether by an interstitial cement. Outside the axis there is the 
 medullary sheath, or white substance of Schwann darkened by 
 the osmic acid (for convenience we term this the white sheath). 
 In the cerebro-spinal nerves — but not in the brain and 
 spinal cord — the medullary sheath is enclosed in a delicate 
 homogeneous membrane, the primitive sheath of Schwann 
 (for convenience we term this the gray sheath). It is difficult 
 to see this, but it may perhaps be found prolonged from 
 the broken end of a fibre. On tracing the fibres lengthwise, 
 Ranvier's nodes will readily be found. Each node is a 
 slight constriction, at which the white sheath is deficient. 
 The axial cylinder, however, extends through the node, so 
 that the continuity of this — the essential part of a nerve 
 fibre — is intact. These nodes — which are only to be found 
 
HISTOLOGICAL DEMONSTRATIONS. 95 
 
 in the cerebrospinal nerves, and not in the centres — indi- 
 cate the division of the nerve fibre lengthwise into a number 
 of segments, each segment being apparently a transformed 
 cell. The gray sheath represents the cell membrane, in 
 which a single nucleus is to be found in each segment, 
 while the axial cylinder and white substance may be 
 regarded as modified cell contents. 
 
 149. The nuclei of the gray sheath are best brought into 
 view by silver staining. Place the sciatic nerve of a frog, 
 or the intercostal nerves of a rabbit, in \ per cent solution 
 of silver nitrate for two or three minutes, then treat in the 
 ordinary way (§32 8). The nuclei are coloured brown ; 
 another very characteristic appearance, however, is pro- 
 duced, viz. that of small brown crosses in the fibres. Each 
 cross results from the silver penetrating the axial cylinder 
 and staining it, together with the cement that covers it, at 
 the node where the medullary sheath is wanting. In such 
 a preparation, also, the simple layer of squamous epithelium 
 that covers the trunk of every cerebro-spinal nerve may be 
 seen, the epithelial outlines being silvered. 
 
 150. Remove a portion of a lumbar or sciatic nerve of 
 a frog just killed. Fray out one end of the nerve on a 
 slide with the point of a needle in a drop of salt solution 
 or aqueous humour. Cover the frayed portion only, and 
 examine. (H.) There is a double coiitour, that is to say, 
 a pair of lines on either side of the centre. The axial 
 cylinder is placed between the two pairs of lines, while the 
 interval between the outer and inner line of each pair is 
 occupied by the white and the gray sheaths. The gray 
 sheath may sometimes be found uniting the two ruptured 
 portions of a fibre, or stretching as an infundibuliform 
 process from a torn extremity. A double contour is not 
 perceptible in a living medullated fibre. Its appearance is 
 probably due to coagulation. 
 
 If the fresh nerve be frayed out in water, the white 
 substance coagulates, and often assumes a fibrillated 
 appearance. 
 
 151. Make a thin L. S. with scissors of the white 
 matter of fresh spinal cord of sheep or other animal An 
 
96 PRACTICAL HISTOLOGY. 
 
 extremely small piece must be taken. Place it on a slide, 
 add no fluid, gently press down the cover-glass, and examine 
 the fibres. As the white fibres in the brain and spinal cord 
 are not surrounded by a gray sheath, they readily yield to 
 the influence of pressure, becoming ampullated, and even 
 broken into pieces. The fragments assume a rounded 
 form, each one being marked like the original fibre by a 
 double contour. This serves to distinguish them from nerve 
 cells, for which they are apt to be mistaken by the inex- 
 perienced observer. 
 
 152. T. S. sciatic nerve of rabbit or cat hardened in 
 chromic acid (§ 14). Stain with logwood (§ 323) by placing 
 the section in a large drop of logwood solution on a slide. 
 After staining and washing in water, remove the water 
 by immersing first in methylated spirit in a watch glass, 
 and then in absolute alcohol for two or three minutes. 
 Lay the section on a slide, allow it to partially dry (let it 
 just become glazed and sodden), then insinuate a drop of 
 clove oil under the section with a sable-hair brush (camel- 
 hair is not stiff enough). Allow the oil to rise through the 
 section, and so drive away the spirit. The principle of 
 this method is explained in § 3 47 a. 
 
 (L.) Watch the clarifying effect of the oil (the section 
 being still uncovered, of course). Finish the clarification 
 by touching the upper surface of the section with the oil. 
 Lastly, cover with a drop of dammar or Canada balsam 
 solution, put on the cover-glass, and gently press it dowm 
 Water and spirit are not miscible with dammar. The spirit 
 replaces the water, the clove oil replaces the spirit, increases 
 the transparency of the tissue, and is miscible with dammar 
 or Canada balsam dissolved in benzole or chloroform. 
 
 (H.) In the section prepared and mounted as above, 
 divided nerve fibres of various sizes are seen, each with 
 a violet centre — the axial cylinder — surrounded by the 
 medullary substance unstained. Observe the connective 
 tissue {neurilemma, pei'ineuriuni) forming a sheath to the 
 nerve, and sending processes between the fasciculi of the 
 nerve fibres. 
 
 153. Gray nerve fibres. (H.) — Examine a preparation 
 
HISTOLOGICAL DEMONSTRATIONS. 97 
 
 of these made as follows: — Place the cervical sympathetic of a 
 rabbit in J per cent chromic acid for two or three days. Tease 
 a small piece with needles, stain with logwood, and mount in 
 glycerine. A spindle-shaped nucleus brightly stained can be 
 seen here and there in the course of a fibre. The nuclei ap- 
 pear to belong to the gray sheath enclosing the nerve fibrils. 
 
 154. Examine (H.) a preparation of cornea, showing 
 primitive nerve fibrils stained with gold, as directed in 
 § 232, b. A network of fibrils is seen. The fibrils have 
 usually a beaded appearance. A power of 800 or 1000 
 diam. is advantageous for their examination. 
 
 CENTRAL NERVE TERMINATIONS. 
 
 155. Nerve Cells (H.) — Make a section with scissors 
 of the Gasserian ganglion of a sheep or other animal 
 recently killed. Tease the section gently in a drop of salt 
 solution ; cover, and examine. The nucleus, nucleolus, and 
 general protoplasm of the nerve cells can be readily seen. 
 The processes of the cells are usually broken off. 
 
 If such a preparation be teased in a drop of magenta instead of salt 
 solution, the cells are readily stained, and the axial cylinders of the white 
 nerve fibres may often be found torn out of the medullary sheath and 
 stained. 
 
 The membranous capsule, with its nuclei around each cell, may be 
 demonstrated by freezing a fresh Gasserian ganglion, and staining the 
 sections with magenta. 
 
 156. Examine (H.) multipolar nerve cells from the 
 hardened spinal cord prepared thus : — Cut the fresh spinal 
 cord of a calf into pieces about a quarter of an inch in 
 length. Place them for a month in one per cent potass, 
 bichrom. solution. Remove a thin slice of the gray matter 
 of the anterior horn with scissors, tease with needles, stain 
 with carmine, and mount in glycerine. Or freeze the fresh 
 cord of the calf; make transverse sections. Place them in 
 4- per cent osmic acid for twenty-four hours ; wash in water, 
 and examine in potassium acetate. Or place them for a 
 day in iodised serum, and then examine. 
 
 Each cell has a nucleus and a nucleolus and several 
 processes. With a power of about 1000 diam. the 
 
 H 
 
98 PRACTICAL HISTOLOGY. 
 
 nerve fibrils may be seen in the processes, especially at the 
 junction of these with the cell, into the granular protoplasm 
 of which they may be traced. If the cells and their pro- 
 cesses have been very successfully isolated, it may be seen, 
 as Deiters pointed out, that each cell has one process — the 
 " axial cylinder process " —which undergoes no division, 
 and at some distance from the cell becomes enclosed in 
 the medullary sheath, and thus becomes a white fibre. All 
 the other processes. remain gray; they repeatedly divide, and 
 form a reticulum in the gray matter of the cord. 
 
 The nerve cells of the brain will be examined with that 
 organ. 
 
 157. Examine a preparation of the pear-shaped cells 
 of the ganglia of the frog's heart with a power of about 800 
 diam. Such a preparation may be made as follows (Beale) : 
 — Carminise the auricular septum of the frog's heart. Ma- 
 cerate for some days in glycerine (1 oz.), and glacial acetic 
 acid (5 drops); isolate the ganglia by teasing with needles 
 under a dissecting microscope. Preserve in glycerine. 
 
 As pointed out by Beale each cell has two processes : 
 a straight fibre resembling in position the stalk of a pear ? 
 and a spiral fibre coiled round it for some distance. 
 
 PERIPHERAL NERVE TERMINATIONS. 
 
 The methods for demonstrating these in the various 
 sense organs will be conveniently studied with these organs 
 themselves. 
 
 158. Plexus of Fibrils.— This has been already seen 
 in the cornea stained with gold, § 154. 
 
 A fine plexus of nerve fibrils may also be beautifully shown in the 
 tail of the tadpole of the green tree frog {Hyla arbo7'ea), prepared as 
 recommended by Klein. Place the tail of a tadpole in which the pos- 
 terior extremities are just appearing in ^ per cent solution of chloride 
 of gold for from thirty to forty minutes ; wash in distilled water, and 
 leave it in the water exposed to diffuse daylight for twenty-four hours 
 or so. Then strip off the ' epidermis from one side with a pair of 
 forceps, and mount in glycerine with the inner surface of the epidermis 
 uppermost. The removal of the epidermis is facilitated by immersion 
 in absolute alcohol for fifteen minutes or so. If this preparation be 
 successful, a plexus of nerve fibrils will be found close to the epidermis; 
 
HISTOLOGICAL DEMONSTRATIONS. 99 
 
 159. — Pacini's Corpuscles. — These may be obtained 
 by making a V. S. of the pad of the cat's foot, hardened in 
 chromic acid. Stain with carmine or logwood, and mount 
 in dammar. They may, however, be found with far greater 
 readiness in the mesentery of the cat, where they are evi- 
 dent to the unaided eye as small, clear, oval, bead-like bodies. 
 Examine a corpuscle removed with scissors from the cat's 
 mesentery, and placed in a drop of glycerine. . 
 
 (L.) The general appearance : nerve fibre entering at 
 one extremity, concentric layers of tissue covering it. 
 
 (H.) The axial cylinder of the nerve fibre in the centre 
 of the corpuscle. The medullary sheath disappears as the 
 nerve enters. The axial cylinder is surrounded by a layer 
 of finely granular substance, outside which there are a num- 
 ber of membranes that form a series of concentric tunics. 
 The fresh specimen may be fairly well preserved in 
 glycerine, but they are more perfectly preserved when 
 hardened in chloride of gold or chromic acid. 
 
 Each tunic of the capsule of the corpuscle consists of an outer 
 and an inner layer of epithelial scales," with white and elastic fibres 
 surrounded by an albuminous fluid interposed (Key and Retzius). The 
 fibres may be demonstrated by teasing the corpuscle with needles after 
 maceration for a few days in -g- per cent chromic acid (Schaefer, Quart. 
 Mic. jfourn., xv. 139). 
 
 160. Nerve Terminations in Striped Muscle. — 
 These may be most readily found in the muscles of the 
 thigh or back of the lizard {Lacerta viridis or Lacerta agilis), 
 and in the recti muscles of the eyeball of a rabbit or other 
 animal. 
 
 Examine (H.) a preparation made as follows : — With 
 scissors make a thin longitudinal section of a rectus oculi 
 of a rabbit just killed ; place it in very dilute acetic acid 
 (two drops of pyroligneous acid in half an ounce of water) ; 
 and, after a minute or two, gently dissociate the fibres with 
 needles. On carefully looking along the fibres, profile 
 and superficial views of the nerve " end organs " may be 
 obtained. The nerve fibre penetrates the sarcolemma, 
 loses its medullary substance, and appears to end in a plate 
 of nucleated granular protoplasm that forms an eminence 
 
ioo PRACTICAL HISTOLOGY. 
 
 under the sarcolemma (Doyere's eminence) ; but it is 
 exceedingly doubtful that this plate is the real termination 
 of the nerve, for, after staining with gold, Gerlach has found 
 a plexus of fine varicose fibrils — like nerve fibrils — through- 
 out the muscular substance. (Sitzungsberichte der Phys. Med. 
 Soc. ; Erlangen, 1873.) Kuhne's article on this subject in 
 Strieker's Histology, vol. i. p. 202, may be advantageously 
 consulted by the advanced student. 
 
 BLOOD-VESSELS. 
 
 161. Structure of Blood-Vessels (H.) — Blood-vessels 
 may be conveniently prepared from the pia mater ; that of 
 a sheep does perfectly. With scissors remove a slice about 
 a quarter of an inch in depth from the surface of a cerebral 
 convolution. Lay it upon a slide, with the pia mater 
 touching the glass. Fix one side of the slice with forceps, 
 and with the back of a scalpel gently scrape away the brain 
 matter. With a camel-hair brush and water, "pencil" 
 away the remainder. Tease with needles; cover, and 
 examine in water. 
 
 a. Capillaries. — Observe the nuclei in the capillaries. 
 They appear to be placed in a homogeneous membrane, 
 but in a silvered preparation it will be seen that the 
 membrane is mapped out by silver lines into polygonal 
 areas, each one enclosing a nucleus. These are, therefore, 
 the nuclei of epithelial squames, with which the whole 
 vascular system is lined. Whether or not a very delicate 
 homogeneous membrane lies outside the epithelium of the 
 capillary is doubtful. 
 
 If the capillary be traced up to a large vessel, the first 
 obvious change consists in the addition here and there of 
 a non-striped muscular fibre coiled round the vessel ; then 
 the other tissues found in the larger vessels make their 
 appearance. 
 
 b. Small arteries and veins. — In small arteries and 
 veins three coats may in general be recognised. The inner 
 coat consists of an elastic membrane lined by a single layer 
 of squamous epithelium. The middle coat is composed of 
 
HISTOLOGICAL DEMONSTRATIONS. 101 
 
 non-striped muscular fibres mostly circular in their arrange- 
 ments, with elastic and white fibrous tissue superadded in 
 vessels of considerable size. In very large vessels, such as 
 the aorta, there is a great preponderance of elastic elements 
 in the form of elastic membranes and fibres. The outer 
 coat is made up of elastic and white fibrous tissue. In 
 arteries and veins the same structural elements are gene- 
 rally present, the veins having only thinner walls than the 
 arteries. 
 
 a. Search for vessels that appear about an inch in 
 breadth when seen with the power we are at present 
 employing (300 diam.) In the outer coat the wavy fibrous 
 tissue will be readily recognised. Inside the outer tunic an 
 appearance of rounded cells will be seen, provided the 
 vessel under examination be an artery; if not, shift the 
 preparation and search for a vessel with this characteristic. 
 This appearance of rounded cells is due to the muscular 
 fibres seen through their long axes as they curl round the 
 vessel. The nucleus appears as a bright refracting spot, if 
 the part of the fibre seen through happen to contain it. 
 With careful focussing the margins of these rounded bodies 
 at the periphery are seen stretching across the vessel as the 
 outlines of the muscular fibres. In the smallest vessels 
 there is only a single layer of muscular fibres, but in those 
 of larger size two and even three layers may be found. 
 The elastic membrane will be more readily seen when the 
 outer and middle coats have been rendered transparent by 
 acetic acid. 
 
 b. Add acetic acid. The tunica adventitia swells up, 
 becomes transparent, and the nuclei of its connective tissue 
 corpuscles appear. The muscular fibres become very 
 transparent, and show their more or less staff-shaped nuclei 
 crossing the vessel. With deeper focussing other nuclei 
 may be seen, usually of an oval shape, with their long axes 
 generally parallel with that of the vessel. These belong to 
 the epithelial lining. 
 
 The elastic membrane is composed of a transparent 
 highly refractile elastic substance. It may be of uniform 
 extension or fenestrated. It is generally marked by ir- 
 
102 PRACTICAL HISTOLOGY. 
 
 regular lines that often look like a network of elastic fibres. 
 The vessel under observation has a distinct double contour. 
 The sharp inner line is due to the elastic membrane, and 
 the long somewhat irregular lines seen here and there 
 running along the vessel, generally result from the foldings 
 of this membrane on account of the collapsed state of 
 the vessel. Possibly at the end of a vessel the elastic 
 membrane may be found isolated from the other tissues. 
 
 162. Examine (H.) a preparation of. the elastic mem- 
 brane isolated as follows : — Remove the basilar or other 
 artery from the base of the brain, e.g., of a sheep. Take a 
 short piece of the vessel, lay it open, add a drop of acetic 
 acid, tease with needles, and mount in glycerine. 
 
 163. Examine (H.) a preparation of blood-vessels of 
 pia mater stained with carmine. It brings out the nuclei 
 of the muscular fibres. 
 
 164. Examine (H.) a T. S. blood-vessel of medium 
 size stained with carmine. The muscular fibres, the elastic 
 membrane, and the nuclei of the epithelial lining will be 
 seen. Such a section as this will be found in the prepara- 
 tion of spinal cord yet to be obtained (§ 245), and also in 
 the V. S. tongue (§ 185). 
 
 Transverse sections of blood-vessels in their fresh con- 
 dition may be readily made with the freezing microtome. 
 
 1 64A. Examine capillaries and larger vessels with epithe- 
 lium silvered according to the method described in § 329. 
 Such a preparation is preserved in glycerine. 
 
 Numerous preparations of blood-vessels for preservation 
 will be found in the sections of organs yet to be made. 
 
 CIRCULATION OF THE BLOOD. 
 
 165. The circulation may be studied in the tail of the 
 fish and tadpole ; in the web, mesentery, tongue, and lung 
 of the frog ; and amongst warm-blooded animals in the 
 wing of the bat and the omentum of the guinea-pig. The 
 last requires to be kept carefully warmed on a special hot 
 stage devised by Strieker and Sanderson (see Quarterly 
 Microscop. Journ. Oct. 1870). 
 
HISTOLOGICAL DEMONSTRATIONS. 103 
 
 166. The Frog's Web.— Inject one, two, or three 
 minims — according to size of frog — of a watery solution of 
 curara, each minim of which contains 0*032 milligramme of 
 the poison. This dose is small, and will take from ten to 
 twenty minutes to produce paralysis. It is well, however, 
 not to give a larger dose, otherwise the vasomotor nerves 
 will be paralysed. 
 
 When paralysis has supervened, tie a soft woollen thread 
 gently round the longest toe, close to its tip, and another 
 round the toe next to, and on one or the other side of it. 
 Lay the frog on its back, and stretch the web gently over a 
 triangular window in a piece of cardboard (A, Fig. 44) by 
 drawing the threads attached to the toes gently through the 
 slits a and a . The web must be kept wet by the repeated 
 addition of water. 
 
 (L.) Artery, vein, capillaries, and pigment cells. The 
 velocity of the stream in the artery is greater than in the 
 vein. This results from the calibre of the artery being less 
 than that of the vein, and from the greater pressure of the 
 blood in the former. 
 
 (H.) (Tube of microscope shortened.) Examine the 
 manner in which the coloured and colourless corpuscles 
 move along the capillaries — the former with comparative 
 rapidity, owing to their polished surfaces ; the latter tardily, 
 owing to an apparent tendency to adhesion between the 
 wall of the vessel and the corpuscle. In the arteries and 
 veins it may often be seen that the coloured corpuscles 
 move principally in the centre of the stream, while the 
 colourless corpuscles may often be found adhering to the 
 wall of the vessel, or moving tardily along it. The peri- 
 pheral part of the stream is. often devoid of coloured 
 corpuscles (the " lymph space " of older observers). The 
 smaller velocity of the peripheral, as compared with the 
 central part of the stream, is owing to the friction between 
 the vessel and the blood, and the adhesion here and there 
 of the white corpuscles. 
 
 167. The Frog's Mesentery. — The student is not 
 permitted to do the following experiment, but it may be 
 shown to him by his teacher thus: — The curarised frog is 
 
104 PRACTICAL HISTOLOGY. 
 
 first stunned by a blow on the head, so that no pain can 
 possibly be produced, and then a vertical incision is made 
 on the left side of the abdomen ; a vein immediately below 
 the axilla must be avoided, and also the stomach and 
 duodenum. The small intestine is gently drawn out with 
 forceps, and fixed with small pins to a crescent of cork close 
 to a window (B, Fig. 44) in the cardboard. If the ex- 
 
 Fig. 44. — Frog-plate of cardboard. The slits c and d are only used when the 
 frog is not curarised and requires to be secured in a bag. 
 
 amination is to extend over a considerable time, a thick 
 cover-glass should be fixed with sealing-wax to the glass on 
 the cork, leaving, however, the outer half of the latter exposed. 
 The mesentery is spread out on the glass ; a small and very 
 thin cover-glass is placed over it, and the membrane is kept 
 moist with aqueous humour. The circulation in the 
 mesentery is extremely beautiful ; for the vessels are only 
 covered by a layer of very transparent epithelial scales. 
 
 168. Tail of the Tadpole. — The circulation may be 
 readily studied in the tadpole's tail. It is only necessary 
 to lay the animal on a slide in a drop of water under a 
 cover-glass. The tadpole is apt to move, however, and 
 therefore it is advantageous to give curara by putting the 
 animal in a watch-glassful of water containing two or three 
 minims of the curara solution (§ 166). It is removed to a 
 slide and covered as before when the palsy is complete. 
 The phenomena of the circulation are similar to those 
 already described in the frog's web, but there is this in> 
 portant difference — the diapedesis or emigration of the white 
 blood corpuscles through the walls of the capillaries and 
 small veins, so rarely seen in the frog's web or mesentery 
 unless inflammation be induced, is almost always to be seen 
 in the tadpole's tail under ordinary circumstances : but if a 
 
HISTOLOGICAL DEMONSTRATIONS. 105 
 
 number of tadpoles be kept for some days in unchanged 
 water, a remarkable tendency to diapedesis manifests itself; 
 the white corpuscles can be found in all stages of emigra- 
 tion. The diapedesis is, however, a very slow process ; a 
 corpuscle may take from one to three hours to pass through 
 the vascular wall, thin though it be. 
 
 When a small drop of ammonia is placed on the tail, a 
 great emigration of white corpuscles ensues after a time. 
 Red as well as white corpuscles often pass out. The emi- 
 gration of the former probably results from pressure, for 
 they have no contractile power. 
 
 Study the method of injecting blood-vessels (§§ 333-343). 
 
 LYMPHATICS. 
 
 169. Stomata of Lymph Sac (H.) — Examine a 
 silvered preparation of the septum cysterticz lymfihaticce 
 magna of the frog prepared thus : — 
 
 Open the abdomen of the animal just killed, and remove the 
 viscera. The great lymph sac is placed on either side of the spinal 
 column, immediately behind the stomach. Wash away the blood with 
 distilled water, pour a half per cent silver nitrate solution over it, and 
 allow to remain for three or four minutes ; wash thoroughly in dis- 
 tilled water, and expose to the light either in distilled water -or in 
 glycerine until a brownish colour appear. Excise a portion of the sac 
 and mount it in glycerine. 
 
 The silvered outlines of the transparent epithelial plates 
 that form the wall of the septum will be readily recognised. 
 Alter the focus, and observe that there are two layers of 
 these, the cells of the one — the peritoneal — being more or 
 less elongated, those of the other — the layer belonging to 
 the sac — being more rounded in form. In both, the out- 
 lines of the cells are somewhat sinuous. The stomata, or 
 apertures leading from the peritoneal into the lymph sac 
 are bounded by a special layer of very finely granular poly- 
 hedral cells, that are readily stained throughout by the 
 silver. These cells in diseased states often proliferate, 
 hence they have been designated "germinating." {Klein.) 
 
 Clusters of cells of this germinal serous epithelium may 
 often be found on the mediastinal pleura of the dog, and 
 
106 PRACTICAL HISTOLOGY. 
 
 on the peritoneal surface of the centrum tendineum of the 
 guinea-pig and rabbit, in the chronic peritonitis induced by 
 tubercle-inoculation. [Klein.) 
 
 Stomata essentially similar to these may be demon- 
 strated by the above process in other situations, such as 
 the pleura and the surface of the centrum tendineum. 
 Their existence was first made known by Recklinghausen 
 in the latter situation. 
 
 170. Lymphatics of Diaphragm. — Examine a pre- 
 paration made as follows : — 
 
 Kill a rabbit — a young one is to be preferred — and remove the 
 anterior wall of the chest. Ligature the inferior vena cava near its 
 cardiac end ; separate the pleurae from the diaphragm, and remove the 
 heart and lungs. Remove the investing serous epithelium by pencilling 
 the upper surface of the diaphragm with a camel-hair brush dipped in 
 the serum found in the chest. Wash the pencilled surface with dis- 
 tilled water ; pour over it a half per cent nitrate of silver solution ; 
 allow to remain for three or four minutes, and then wash thoroughly 
 with distilled water. The diaphragm with its osseous and cartilaginous 
 attachments is then cut out and placed in a vessel with distilled, water, 
 until the silvered surface becomes of a brownish colour. The centrum 
 tendineum may then be cut into pieces and mounted in glycerine. The 
 lymphatics near the under surface of the diaphragm may be demon- 
 strated in a similar manner. 
 
 (L.) The lymphatics form an anastomosing system of 
 irregular channels of various sizes. The silvered outlines 
 of their epithelial wall are evident. Alternate dilatations 
 and constrictions are often seen on the smaller vessels. 
 The appearance resembles a series of Florence flasks set 
 one above another. There are no valves at the swellings, 
 such as are found in large lymphatics. 
 
 (H.) In the smallest lymphatics the epithelial outlines 
 are more sinuous than in the larger vessels, and in the 
 latter the outlines of the cells are more or less parallel with 
 the long axis of the vessel. In favourable specimens, the 
 smallest lymphatics may be seen opening into connective 
 tissue spaces, as described by Recklinghausen. This mode 
 of origin may be beautifully seen in the superficial lymph- 
 atics of the intestinal villi (§ 193). 
 
 The smallest lymphatics consist entirely of a simple 
 
HISTOLOGICAL DEMONSTRATIONS. 107 
 
 layer of squamous epithelium. The large vessels, e.g. the 
 thoracic duct, have a structure similar to veins. 
 
 171. Study the puncture method of injecting lymph- 
 atics described in § 344. 
 
 Klein's memoir on The Anatomy of the Lymphatic System, part i., 
 London, 1873, may be advantageously consulted by the advanced 
 student. 
 
 BLOOD GLANDS. 
 
 172. Lymph G-land. — The methods of preparing 
 lymphatic glands in the fresh condition, and after injection 
 with silver nitrate, have already been described (§§ no, in). 
 
 Examine the section of lymphatic gland already pre- 
 pared (§ no). 
 
 (L.) The fibrous capsule sends into the interior numerous 
 trabecular that are membranous in the cortex, and cord- 
 like in the medulla. These trabecular may be seen cut 
 longitudinally and transversely. Contiguous with the capsule 
 and the trabecular, there is everywhere a somewhat clear 
 space, the lymph sinus. From the capsule and trabecular 
 a loose adenoid tissue extends across the sinus. The spaces 
 amidst the tissue elements constitute the lymph path 
 continuous with the afferent lymphatics through the cortex, 
 and with the efferent lymphatics through the hilus of the 
 gland. The lymph sinus everywhere encloses a ramifying 
 mass of " follicular tissue," consisting of a somewhat con- 
 densed adenoid tissue closely packed with lymph corpuscles. 
 
 (H.) The fibres of the trabecular. The loose adenoid 
 tissue of the lymph sinus, with probably here and there a 
 lymph corpuscle not shaken out of the spaces (§11 o). 
 The follicular tissue, with numerous lymph corpuscles. 
 The corpuscles are identical with the smaller white blood 
 corpuscles. They appear to be produced in the follicular 
 tissue, and to pass from thence into the lymph stream. 
 
 Examine also (L. and H.) a section of silvered lymphatic 
 gland prepared as stated in § in. 
 
 173. Spleen. — Methods. — a. The spleen may be pre- 
 pared by hardening in chromic acid and alcohol as described 
 in § 11. Sections may be readily made with the -hand, or 
 
108 PRACTICAL HISTOLOGY. 
 
 in the microtome imbedded in paraffin. The sections are 
 mounted in Farrants' solution or in glycerine. They may 
 be unstained or stained with logwood. This dye is especi- 
 ally useful for mapping out the splenic corpuscles. 
 
 b. For the study of the splenic pulp, it is advantageous 
 to place small portions of perfectly fresh spleen in one per 
 cent potassium bichromate solution for five or six days, and 
 then to make sections in the freezing microtome. It is 
 difficult to get good sections of the spleen by freezing when 
 it has not been previously hardened by other methods, but 
 the difficulty is entirely overcome by impregnating the tissue 
 with a thick solution of gum previous to freezing (§ 309). 
 The blood corpuscles may be removed from the adenoid 
 tissue of the pulp by gently shaking the sections with salt 
 solution in a test-tube, as recommended for lymphatic 
 gland (§ no). 
 
 c. The cell elements of the spleen may be examined in 
 the fresh condition in aqueous humour. 
 
 174. Structure. — Examine, without the microscope, the 
 cut surface of the fresh spleen of ox. The tough fibrous 
 capsule is evident. Scrape away the pulp, and observe the 
 branching cord-like trabecular continuous with the capsule, 
 and running from one surface to the other of the organ. 
 Look at a cut surface that has not been scraped, and en- 
 deavour to see the splenic corpuscles — minute things like 
 millet seeds, or like very small grains of unboiled sago. 
 The cut ends of the trabecular must not be mistaken for 
 them. Endeavour to pick out a splenic corpuscle with the 
 point of a scalpel. They are generally attached to the 
 tunica adventitia of the arteries. It is sometimes possible 
 to pull out a small artery, and see the corpuscles adhering 
 to it like small i beads. The structure of the spleen is 
 somewhat analogous to that of a lymph gland. The cap- 
 sule and trabecular are similar. The splenic corpuscles 
 apparently correspond to the follicular tissue of the latter, 
 while the splenic pulp may be compared to the lymph 
 sinus. In the spleen, however, blood-vessels open into the 
 spaces amidst the adenoid tissue of the pulp, to which they 
 appear to be related in a manner similar to that- of the 
 
HISTOLOGICAL DEMONSTRATIONS. 109 
 
 lymphatics to the sinus of the lymph gland ; the arteries 
 pass into capillaries, some of which penetrate the splenic 
 corpuscles (W. Sanders). They all eventually open into 
 the spaces in the pulp, from which the venous radicles are 
 derived (W. Miiller). There are lymphatics in the spleen 
 around the blood-vessels, and in the trabecular ; their mode 
 of origin, however, has not been fully investigated. 
 
 175. Section of chromic acid spleen of cat stained 
 with logwood. Mount in Fan-ants' solution or in glycerine. 
 
 (L.) Capsule. Trabecular. Splenic corpuscles much 
 larger here than they are in the human spleen. The pulp. 
 
 (H.) The trabecular, chiefly consisting of elastic fibres ; 
 non-striped muscle is also present in them and in the cap- 
 sule. The splenic corpuscles, containing great numbers of 
 lymph corpuscles. The envelope consists of a delicate 
 condensed adenoid tissue. The pulp, consisting of adenoid 
 tissue, with numerous blood-corpuscles in its spaces. Lymph 
 corpuscles are probably produced in the splenic corpuscles, 
 and find their way into the pulp, and from thence by the 
 veins. 
 
 176. If the pulp of the fresh spleen be examined in aqueous 
 humour, clear colloid masses containing coloured blood-corpuscles, 
 variously transformed, may be found. These are supposed to be cor- 
 puscles breaking up. Similar bodies may be found in the red marrow 
 of bone (§ 129). 
 
 177. Examine (L.) a preparation of injected spleen. 
 
 178. Thyroid Gland. — (L. and H.) Examine sec- 
 tions of human thyroid gland injected and uninjected. 
 
 The uninjected gland is best hardened in Mtiller's fluid for a month, 
 and then in methylated spirit for a fortnight. Preserve in Farrants' 
 solution, or in glycerine, or stain with carmine or logwood, and mount 
 in dammar. The injected gland may be hardened in j per cent 
 chromic acid for a fortnight, and then cut by the freezing method, 
 stained with logwood or carmine, and mounted in dammar. 
 
 The gland consists of sacs imbedded in fibrous tissue. 
 The sacs are lined by a somewhat cubical epithelium. 
 They contain lymph corpuscles. They are not penetrated 
 by capillaries, as is the case with the splenic corpuscles. 
 In the human subject they are often found enlarged, and 
 filled with a colloid material. 
 
no PRACTICAL HISTOLOGY. 
 
 179. Suprarenal Body (L. and H.) — Examine V. S. 
 suprarenal body prepared as follows : — 'Harden in Miiller's 
 fluid for a month, and then in methylated spirit for a fort- 
 night. Mount the sections in glycerine or dammar. Groups 
 of nucleated brownish cells are seen with (H.) They are 
 chiefly placed in the outer part of the organ, arranged in 
 rows, or in irregular clusters, in spaces amidst the fibrous 
 tissue. 
 
 LUNG. 
 
 A section of the trachea has already been examined 
 
 (§"5)- 
 
 180. Methods. — a. The lung may be extremely well 
 
 prepared by hardening in chromic acid, and then in alcohol, 
 as directed in § 6. 
 
 b. The outlines of the epithelial cells in the alveoli may be silvered 
 as follows :^— Allow the lungs to collapse by opening the chest of an 
 animal just killed. Inject a half per cent solution of silver nitrate into 
 a bronchus, and fully distend the lung, Make sections at once in the 
 freezing microtome, using distilled water instead of the gum. "Wash 
 the sections in distilled water, and expose them to the light in it. 
 Preserve in Farrants' solution, or in glycerine. Some sections may be 
 good, but the staining is apt to fail. 
 
 c. Sections of lung, either fresh or hardened in chromic 
 acid and spirit, are best made with the freezing microtome. 
 The spirit must of course be previously removed by im- 
 mersing the piece of lung in water for twelve hours or so. 
 Good sections may, however, also be made without a 
 machine, by imbedding the lung in gum, as described in 
 § 301, b. The gum is easily removed from the sections by 
 placing them in water. 
 
 d. The sections may be examined unstained or stained 
 with logwood. If unstained, they are mounted in Farrants' 
 solution or glycerine ; if stained, they may be preserved in 
 the same fluids, or in dammar (§ 152). 
 
 e. The blood-vessels of the lung may be injected, e.g. with carmine 
 and gelatine (§ 343). The lung is then hardened in alcohol, as de- 
 scribed in § 337, and sections made as above mentioned. 
 
 f. The ganglia of the lung may be examined in sections of the 
 lung, hardened in chromic acid in the usual way. They are found 
 
HISTOLOGICAL DEMONSTRATIONS. in 
 
 close to the pulmonary vessels and bronchi. The ganglia may also be 
 isolated by cutting out a portion of the wall of a bronchus of a lung 
 prepared as above, and, with the aid of a dissecting microscope, the 
 small ganglia lying outside the cartilages may easily be isolated. ( W. 
 Stirling. ) 
 
 1 8 1. Structure. — An unstained section of the lung of 
 cat across a minute bronchus, made as above described. 
 Mount in Farrants' solution, or in glycerine, and examine. 
 
 (L.) Notice the general appearance of the section ; — the 
 T. S. bronchus with two or three plates of cartilage cut 
 across ; — the larger vessels, the pulmonary artery having a 
 thicker wall than the vein; — the air vesicles somewhat 
 collapsed. 
 
 (H.) The air vesicles. — The somewhat indefinite fibril- 
 lated tissue with numerous elastic fibres, of which the 
 alveolar walls principally consist, will be easily recognised, 
 and also the rounded nuclei of the simple layer of squamous 
 epithelium, with which the alveolus is lined. Capillaries may 
 be found cut across here and there between the alveoli. 
 
 The bronchus. — Internally, ciliated epithelium ; and ex- 
 ternally, plates of hyaline cartilage imbedded in a fibrous 
 membrane. The bronchial glands lie chiefly between the 
 two, but often the sacs of the glands pass between the car- 
 tilaginous plates, and occupy a position external to them. 
 Circular non-striped muscular fibres, difficult to recognise 
 as such, lie near to the epithelium. Around the glands 
 there is either fibrous or adenoid tissue. The latter is 
 abundantly found in the bronchial mucous membrane, 
 either diffuse or in the form of cords, which, according to 
 Klein, have a longitudinal direction. The cut ends of the 
 elastic fibres that run longitudinally along the bronchi close 
 to the epithelium may also be seen. They are most readily 
 detected in a section stained with logwood. With L. they 
 are seen as bundles causing ridges on the mucous membrane. 
 
 The sections of the pulmonary arterioles and veinlets 
 may also be examined. 
 
 The internal ganglia of the lung may also be found in 
 this preparation. As Remak pointed out, they are placed 
 on the nerves that accompany the bronchi. The cells are 
 large and multipolar. ( W. Stirling?) 
 
H2 PRACTICAL HISTOLOGY. 
 
 182. It is advantageous to stain a section of cat's lung 
 with logwood for comparison with the foregoing. 
 
 It is also advantageous to have a section of the lung of 
 a child, hardened as described in § 6, cut in the freezing 
 microtome, and stained with logwood. Mount in Farrants' 
 solution. The important point of difference between the 
 cat's lung and this, is the small amount of interalveolar 
 fibrous tissue in the normal human lung. The logwood 
 brings out the nuclei of the alveolar epithelium clearly. 
 
 183. Examine (L.) a section of lung with its blood- 
 vessels injected with gelatine and carmine and mounted in 
 dammar (§ 180 e) ; also (H.) a section of silvered lung 
 showing the outlines of the alveolar epithelia. 
 
 Regarding the lymphatics of the lung, Klein's memoir, Anatomy of 
 the Lymphatic System, Part ii., London, 1875, may be advantageously 
 consulted by the advanced student. 
 
 ALIMENTARY CANAL. 
 TONGUE. 
 
 184. Method. — The tongue, when cut into small pieces, 
 may be hardened in chromic acid and then in alcohol, as 
 mentioned in § 7. Sections are best made in the freezing 
 microtome, but they may also be made from tongue 
 imbedded in paraffin. The sections may be preserved 
 unstained in Farrants' solution or glycerine, or they may be 
 stained with logwood and preserved in the same fluids or 
 in dammar. 
 
 185. Structure. — V. S. unstained chromic acid tongue, 
 made as above. Mount in Farrants' solution or glycerine, 
 and examine. 
 
 (L.) The papilla. — Unlike the filiform papillae of the 
 human tongue, those that correspond to them in the cat are 
 short thick stumps. The muscular fibres are seen divided 
 longitudinally and transversely. The fasciculi of the latter 
 are well seen, also the general arrangement of the fibres — 
 some passing transversely, others vertically, and others 
 longitudinally. 
 
HISTOLOGICAL DEMONSTRATIONS. 113 
 
 (H.) Stratified squamous epithelium. — The individual 
 cells and their nuclei may be clearly seen in the lower and 
 middle layers ; but near the free surface, where it is raised 
 into papillae, the scales are so compressed that in profile 
 they are not unlike fibres. The cells are jagged in the 
 deeper layers, but this appearance is not so well marked 
 here as in the skin. 
 
 186. Examine (L.) V. S. human tongue, showing the 
 fungiform and filiform papillae ; a V. S. of the same, showing 
 a circumvallate papilla ; a V. S. injected tongue, showing the 
 capillaries passing into the fibrous tissue at the base of each 
 papilla. 
 
 187. Taste Bulbs. — Examine (L. and H.) V. S. gustatory 
 disc of rabbit's tongue hardened in absolute alcohol. The 
 sections are stained with logwood, and mounted in glycerine 
 or dammar. The gustatory disc {papilla foliata) is found 
 on either side of the dorsum of the tongue near its root. 
 
 Each taste bulb consists of a single layer of investing 
 ordinary epithelial cells surrounding a cluster of modified 
 epithelial cells, at the free end of which there is a short 
 and fine hair-like process, and at the other extremity delicate 
 protoplasmic processes that are probably connected with 
 nerve filaments. The bunch of hair-like processes projects 
 into an opening {gustatory pore) at the exposed extremity of 
 the taste bulb. In the human tongue the taste bulbs 
 chiefly occur on the outer aspect of the central projection 
 of the circumvallate papillae. 
 
 Engelmann's article in Strieker's Histology, vol. iii. p. I, may be 
 consulted by the advanced student for other methods of preparation. 
 
 STOMACH. 
 
 188. Methods. — a. The stomach may be well hardened 
 in chromic acid and alcohol as described in § 7. Sections 
 may be made in the freezing microtome, or by imbedding 
 in paraffin. The latter is, however, not nearly so good, for 
 the paraffin is very apt to enter the mouths of the glands. 
 The sections unstained are best mounted in Farrants' 
 solution or in glycerine. 
 
 1 
 
H4 PRACTICAL HISTOLOGY. 
 
 b. The cells of the peptic glands are remarkably well seen in a 
 stomach hardened in osmic acid. A portion of the mucous membrane 
 is separated from the muscular coat of the stomach of a cat just killed. 
 The mucus is washed away with a stream of salt solution. One or 
 two pieces about an eighth of an inch square are placed in \ per cent 
 osmic acid for twenty-four hours. It may then be readily cut in the 
 freezing microtome, and the sections mounted in glycerine or Farrants' 
 solution. The nuclei of the cells may be well brought into view by 
 slight staining with logwood. 
 
 c. Sections of the fresh stomach may be readily made in the freezing 
 microtome, and stained with magenta or a watery solution of anilin blue. 
 
 189. Structure. — V. S. chromic acid stomach of cat ; 
 mount in glycerine. 
 
 (L.) The relations of the mucous, submucous, and 
 muscular coats. 
 
 (H.) The mucous coat. — The peptic follicles cut vertically. 
 The columnar epithelium at their open ends, and the 
 epithelial cells of irregular shape throughout the greater part 
 of the follicle. The outer or " parietal," and the inner or 
 "chief," varieties of these may be recognised, but these 
 will be better seen in the osmic acid stomach. 
 
 Bring into view the muscularis mucosa outside the closed 
 ends of the follicles. It consists of two layers of non-striped 
 muscular fibres, bundles from both of which may be traced 
 upwards between the follicles, and may in very thin sections 
 be traced nearly to the open ends of the glands, where, 
 according to Klein, they form a transverse network between 
 the glands. 
 
 The submucous coat consists of areolar tissue ; blood- 
 vessels may be found here and there. 
 
 The. muscular coat. — The fasciculi of non-striped fibres 
 can be seen cut longitudinally and transversely. 
 
 190. Examine (H.) a V. S. mucous membrane of cat's 
 stomach hardened in osmic acid as above described. If a 
 peptic follicle can be traced throughout its entire length, it 
 will be found that near and at the orifice the epithelium is 
 columnar, with the nuclei placed near the attached ends of 
 the cells. In about the half of the follicle nearest its closed 
 extremity two sorts of cells may be seen. A large, some- 
 what spheroidal, nucleated cell here and there placed exter- 
 nally close to the basement membrane, and within this, and 
 
HISTOLOGICAL DEMONSTRATIONS. 115 
 
 occupying the intervals above and below them, smaller 
 cells of irregular shape. The former have long been 
 termed " peptic " cells. By Heidenhain they have been 
 named " parietal," while the others have been termed by 
 him the "chief" cells, from his supposing that they are 
 especially concerned in the secretion of gastric juice. 
 About the middle of the follicle the " parietal " cells form 
 an almost continuous layer. 
 
 In a V. S. of stomach, near its pyloric orifice, glands, 
 lined throughout by columnar epithelium, may be seen. 
 These are commonly termed mucous glands. They occur, 
 though in relatively small numbers, throughout the re- 
 mainder of the stomach. 
 
 THE INTESTINE. 
 
 191. Methods. — a. The intestine may be hardened 
 in a solution of chromic acid and potassium bichromate, 
 followed by alcohol, as described in § 7. Sections of the 
 small intestine are best made in the freezing microtome, for 
 the villi are apt to be spoiled by paraffin or any similar im- 
 bedding agent. The sections unstained are mounted in 
 Farrants' solution or in glycerine, or they may be stained 
 with logwood or carmine, and mounted in the same fluids or 
 in dammar. 
 
 b. A method devised by Klein gives good results. Place portions 
 of the intestine in a mixture of two parts of \ per cent solution of 
 chromic acid, and one part of methylated alcohol. Change the fluid 
 two or three times, and at the end of seven days transfer to dilute 
 spirit (methylated spirit I, water 2 parts). After two or three days 
 place them in methylated spirit, and when they are sufficiently hard 
 make sections. Place the sections for a few minutes in I per cent 
 sodium bicarbonate solution, and then stain them with logwood. 
 
 c. Auerbach's plexus between the two layers of the muscular coat 
 may be thus prepared. {Klein.) Take the small intestine of a rabbit 
 just killed, and, after washing out its contents with a stream of salt 
 solution, inflate it with air. With broad-pointed forceps strip off the 
 longitudinal layer of muscular fibres in as membranous a form as pos- 
 sible. Place the membranes thus detached in \ per cent chloride of 
 gold solution for twenty-five minutes ; wash in distilled water, and after- 
 wards treat as stated in § 330. 
 
 d. The openings of the chalice cells may be rendered very distinct 
 by the silver process already described in § 96. 
 
n6 PRACTICAL HISTOLOGY. 
 
 e. The simple layer of epithelial squames that forms a basement 
 membrane under the columnar epithelium as described by Debove 
 {Archives de Physiologie, 1 8 74, p. 19) may be shown without much 
 difficulty thus : — Open the small intestine of a rabbit or cat just killed. 
 Isolate a small portion and wash the mucous membrane with distilled 
 water. Pour \ per cent silver nitrate solution over the interior, and 
 leave it till it becomes opalescent. Wash in distilled water. Then 
 pencil away the epithelium, and silver the subjacent tissue in the usual 
 way (§ 328). If the villi are then snipped off and mounted in glycerine, 
 the silver lines of a squamous epithelial membrane, somewhat like that 
 of a serous membrane, may be seen. 
 
 f. The same process as that just described also serves to show very 
 beautifully the superficial lacteals of the villi and their communications 
 with the spaces in the reticulum of adenoid tissue. 
 
 192. Structure of Small Intestine, T. S. or V. S. — 
 
 Small intestine of cat prepared as stated in § 191 a. Mount 
 in Farrants' solution or in glycerine. (L.) The mucous 
 coat showing the villi, follicles of Lieberkiihn, and mus- 
 cularis mucosa. The submucous coat. The muscular 
 coat with its two layers of fibres. 
 
 (H.) The columnar epithelium of the villi with their 
 striated border, and with a chalice cell here and there. 
 
 The follicles of Lieberkiihn, lined throughout by colum- 
 nar epithelium, the nuclei of which are placed near to the 
 attached ends of the cells. In very thin sections the epi- 
 thelium may be found detached from the basement mem- 
 brane. (The membrane consists of simple epithelial 
 squames (Watney), but this can only be seen in silvered 
 specimens.) 
 
 The muscularis mucosa forming here as in the stomach 
 two layers. Endeavour to trace bundles of fibres from it 
 up into the lateral part of the villus, where they run nearly 
 to its tip. 
 
 The ade?ioid tissue ; the only connective tissue found 
 in the villi, and also largely found between the follicles of 
 Lieberktihn. 
 
 There is nothing remarkable about the submucous and 
 muscular coats. 
 
 193. Lacteals. — Examine (H.) villi silvered after removal 
 of the columnar epithelium (§ 191,/). The clear lines 
 parallel with the villus are the superficial lacteals which 
 
HISTOLOGICAL DEMONSTRATIONS. 117 
 
 may be seen communicating with the spaces in the adenoid 
 tissue. 
 
 194. Blood-vessels. — Examine (L.) T. S. small intestine 
 with blood-vessels of villi injected. 
 
 195. Brunner's glands. — Examine (L.) T. S. duo- 
 denum stained with carmine, showing Brunner's glands. 
 They are compound saccular glands. 
 
 196. Beyer's glands. — a. Examine (L.) T. S. or V. S. 
 small intestine, showing Peyer's sacs. These structurally 
 resemble the splenic corpuscles. 
 
 b. (L.) A similar section with the capillaries in Peyer's 
 sacs injected. 
 
 c. (H.) A similar section showing the lymph corpuscles 
 within the sac. 
 
 The solitary glands found here and there in the small 
 and large intestine structurally resemble a single sac of a 
 Peyer's patch. 
 
 197. Structure of Large Intestine. — Unstained, T. S. 
 or V. S. large intestine of cat prepared as directed in 
 § 191, a. Mount in Farrants' solution or in glycerine. (L.) 
 The follicles of Lieberkiihn in the mucosa. The sub- 
 mucous and muscular coats. 
 
 (H.) Lieberkuhn's follicles similar to those of the small 
 intestine. 
 
 198. Examine (H.) T. S. follicles of Lieberkiihn in 
 order to be able to recognise tubular glands such as those 
 when divided transversely. 
 
 LIVER. 
 
 199. Methods. — a. The liver may be very successfully 
 hardened in Miiller's fluid, and then in rectified spirit, as 
 directed in § 10. A one per cent solution of potassium 
 bichromate for a fortnight or so, followed by rectified spirit, 
 also gives very fair results. 
 
 Sections of the hardened liver, imbedded in paraffin, 
 may readily be made. The sections should be examined 
 unstained and also stained with logwood, a dye peculiarly 
 suitable for the liver and kidney. In either case they 
 should be mounted in Farrants' solution, or in glycerine. 
 
u8 PRACTICAL HISTOLOGY. 
 
 b. The blood-vessels and bile ducts may be injected as 
 directed in §§343, 346. 
 
 200. Structure. — Cells (H.) — a. Scrape the cut sur- 
 face of the fresh liver of rabbit or cat ; diffuse the scraping 
 in a drop of salt solution ; cover and examine. The cells 
 have one — rarely two — nuclei ; the protoplasm is distinctly 
 granular, and there is no envelope. 
 
 b. Prepare in the same manner the cells of the liver of 
 the ox. Particles of fat are usually to be seen in these. 
 
 201. Thin section of liver of cat hardened in Miiller's 
 fluid and spirit (§ 199, a). The section should be made 
 across one of the larger bile ducts. Stain with logwood, 
 by placing the section on a slide in a few drops of strong 
 logwood solution for two or three minutes. Wash in water ; 
 and mount in Farrants' solution, or in glycerine. 
 
 (L.) The lobules. -r-ObsQTve. the network of the gland 
 cells in each. If the section be across the long axis of a 
 lobule, branching columns of cells may be seen radiating 
 from the centre. A network of capillaries occupies the 
 spaces between the cells. The T. S. of one of the larger 
 bile ducts may perhaps be seen, with a section of the 
 hepatic artery and portal vein near it. If there be a T. S. 
 of the bile duct in the preparation, it is convenient to place 
 it exactly in the centre of the field, otherwise it will not be 
 readily found with H. 
 
 (H.) The bile duct lined by a layer of columnar epithe- 
 lium. There is nothing remarkable about the sections of 
 the hepatic artery and portal vein. 
 
 The capsule of Glisson. — A fine fibrous tissue that sur- 
 rounds all the vessels and runs between the lobules. The 
 whole liver is enclosed in a thin fibrous capsule. The tissue 
 now seen is a prolongation of the capsule around the vessels 
 that enter at the portal fissure. The capsule also sends fine 
 trabecular between the lobules from many points of the sur- 
 face. Look for these near the margin of the section. 
 (They are most readily seen, however, when the cells have 
 been pencilled away.) 
 
 The columns of cells are easily seen, and in the intervals 
 between the columns, collapsed capillaries with blood cor- 
 puscles. 
 
HISTOLOGICAL DEMONSTRATIONS. 119 
 
 Mount an unstained section of liver in Farrants' solu- 
 tion, and compare it with the preceding. 
 
 202. Blood-vessels (L.) — Section of liver of cat, injected 
 from the portal vein with Prussian blue and gelatine, and 
 hardened in rectified spirit. Mount in dammar, and examine. 
 The radicles of the hepatic vein lie in the centre, the ter- 
 minations of the portal vein between the lobules and capil- 
 laries connect the two venous systems. 
 
 203. Bile ducts (H.) — Examine a section of rabbit's 
 liver having the bile ducts injected with soluble Prussian 
 blue (§ 346), and the venous system injected with carmine 
 and gelatine, mounted in dammar. The larger (inter- 
 lobular) bile ducts are filled with injection, around which 
 the columnar epithelium may be seen. Lateral branches 
 may be readily traced into the lobules. The columnar epi- 
 thelium becomes shorter and shorter, and soon disappears. 
 The branches ramify, and soon open into a network of fine 
 ducts — the bile capillaries between the individual hepatic 
 cells. These capillaries appear to be mere rounded spaces 
 between the cells. If, after having seen this injection, the 
 student carefully look with H. at the uninjected preparation 
 already made (§ 200), he may see here and there fine pores 
 between the cells — the empty bile capillaries — much smaller 
 than the spaces that contain the blood capillaries. 
 
 204. Glands of the Bile Ducts. — The large bile ducts 
 have numerous mucous glands which may be seen in T. S. 
 of the duct, most readily after they have been filled with 
 injection from the common bile duct. Examine (L.) a 
 preparation showing these. 
 
 SALIVARY GLANDS AND PANCREAS. 
 
 205. Methods. — a. These organs if cut into sufficiently small 
 pieces may be well hardened in a saturated solution of picric acid, as 
 recommended by Ranvier. About forty-eight hours are required. 
 Sections may then be readily made with the freezing microtome. They 
 may be mounted unstained in Farrants' solution, or in glycerine ; or 
 they may be stained with picro-carmine, and mounted in the same 
 fluids. 
 
 b. The glands may also be hardened in alcohol, and sections made 
 with the hand. The sections may be stained with Beale's carmine fluid 
 (§318, a), diluted four or five times with water. 
 
120 PRACTICAL HISTOLOGY. 
 
 c. Osmic acid is also a good hardening agent. Small portions of 
 the glands are hardened for two days in \ per cent solution. They 
 may then be cut by freezing, or may be farther hardened in alcohol and 
 cut by the hand. This method of hardening is especially suitable when 
 it is desired to dissociate the gland elements with needles. The sec- 
 tions are mounted in Farrants' solution, or in potassium acetate solu- 
 tion. 
 
 206. Structure. — The salivary glands and pancreas 
 have an essentially similar structure. They are all racemose 
 glands ; the ducts lined by columnar epithelium, and the 
 acini containing polyhedral epithelial cells, with channels 
 for the secreted fluid occurring here and there between 
 them. The secreting cells of the submaxillary gland differ 
 from those of the parotid and pancreas in containing 
 mucin. 
 
 Examine (L.) a section of submaxillary gland, prepared 
 stated in § 205, a. The acini. 
 
 (H.) The cells of the acini and those of the ducts. 
 
 KIDNEY. 
 
 207. Methods. — (a.) The epithelium of the fresh 
 kidney may be examined by merely scraping the surface of 
 a Malpighian pyramid cut vertically, and examining the 
 scraping in salt solution. 
 
 b. Sections of the fresh kidney may be made with a 
 Valentin's knife (Fig. 56), but far better with the freezing 
 microtome. Sections of the unhardened kidney can scarcely 
 be preserved. 
 
 c. The best ordinary methods for preparing the kidney 
 are precisely the same as in the case of the liver. There- 
 fore all that is stated in § 199, a applies equally to both. 
 
 d. Small pieces of kidney may also be hardened in a 
 solution of chromic acid. The kidney of the rabbit may 
 be very successfully hardened in 1 per cent solution, as 
 directed in § 9. 
 
 e. The renal tubules may be isolated (Ludwig) by making thin 
 vertical sections of the Malpighian pyramids of the fresh kidney, and 
 putting them in a flask containing one part of hydrochloric acid to four 
 parts of rectified spirit. A cork, with a long glass tube passing through 
 it, is fitted to the flask, and it is boiled over a sandbath for two or three 
 
HISTOLOGICAL DEMONSTRATIONS. 121 
 
 hours, and then the mixture is replaced by distilled water, in which 
 they are left for a day. The tubules are then isolated by shaking a 
 slice in a test tube, with a little water. 
 
 / The epithelium lining the Malpighian capsules is best demon- 
 strated in the kidney of the child at birth. Cut it into small pieces. 
 Place them in I per cent potassium bichromate solution for a week, 
 then in £ per cent chromic acid for a day, and make sections in the 
 freezing microtome. 
 
 g. The blood-vessels may be readily injected with gelatine and 
 carmine, etc., as described in § 342. 
 
 208. Structure. — Examine V. S. Malpighian pyramid 
 without the aid of the microscope ; the whitish compact 
 medullary substance near the apex of the pyramid ; the 
 softer reddish-brown cortex at the base. The longitudinal 
 striae in the medullary substance are due to the straight 
 tubules. The pyramids of Ferrein — bundles of the straight 
 tubules — are easily seen where the medullary passes into 
 the cortical substance. The red lines between these pyra- 
 mids are due to the vasa recta. In the cortex it is just pos- 
 sible to see fine granulations on the cut surface, produced 
 by the glomeruli. Tear off the fibrous capsule, and in so 
 doing notice the fine filaments that appear to be torn 
 across ; these are capillaries that pass between the cortex 
 and the capsule. 
 
 209. V. S. Malpighian pyramid of cat's kidney hardened 
 in Miiller's fluid, as described in § 9, cut in the freezing 
 microtome, and stained with logwood. Mount in Fan-ants' 
 solution or in glycerine. 
 
 (L.) Trace the tubules from the apex of the Malpi- 
 ghian pyramid into the cortex. The tubules are straight in 
 the medulla, and form bundles — the pyramids of Ferrein — 
 towards its outer part. These pass nearly to the outer part 
 of the cortex. In the cortex the straight tubules give off 
 lateral branches that become the convoluted tubules, and 
 finally end in the capsules enclosing the glomeruli. These 
 lie amidst the convoluted tubules, and can be readily re- 
 cognised. 
 
 (H.) The glomerulus — or Malpighian body — a cluster 
 of capillaries enclosed in a capsule formed by expansion of 
 the basement membrane of a convoluted tubule lined by 
 
122 PRACTICAL HISTOLOGY. 
 
 squamous epithelium. Profile views of the nuclei of the 
 epithelial cells will probably be obtained. The squamous 
 epithelium of the capsule is reflected over the capillaries 
 of the glomerulus, but it is scarcely possible to see this, 
 save in the young child. Examine the epithelium of the 
 convoluted and that of the straight tubules. 
 
 210. Mount an unstained V. S. Malpighian pyramid, 
 and compare it with the above. Especially look at the ap- 
 pearance of the unstained epithelium of the tubules. 
 
 211. Examine (H.) V. S. Malpighian pyramid of rabbit's 
 kidney hardened in chromic acid (§ 207, d). The columnar 
 epithelium of the larger straight tubules can be remarkably 
 well seen near the apex of the pyramid. The epithelium 
 of the convoluted tubules is not so good as in the Miiller's 
 fluid kidney. The divisions of the straight tubules may be 
 seen ; and the small looped tubules of Henle may often be 
 recognised. 
 
 212. (H.) Scrape the cut surface of the cortex of fresh 
 kidney of sheep to get isolated glomeruli, epithelium, and 
 basement membranes. Examine in salt solution, then add 
 acetic acid and notice its effect on the glomerulus. It 
 renders the capillary walls extremely transparent, and re- 
 veals their nuclei. 
 
 213. (H.) T. S. Malpighian pyramid of Miiller's fluid 
 kidney near its apex. Examine and preserve in Farrants' 
 solution or in glycerine. (The section is best made from a 
 frozen kidney, but may also be made with a Valentin's knife.) 
 Study the T. S. tubules, and the matrix of connective tissue 
 between them. The connective tissue is evident in the 
 medullary part of the kidney, but it is very scanty in the 
 cortex. 
 
 214. V. S. Malpighian pyramid of injected kidney. 
 Mount in dammar, and examine. 
 
 (L. and H.) See the interlobular arteries in the cortex ; 
 the glomerulus, each with its afferent vessel derived from 
 the interlobular artery and the efferent vessel breaking up 
 into a second set of capillaries around the convoluted 
 tubules. See also the vasa recta between the pyramids of 
 Ferrein. 
 
HISTOLOGICAL DEMONSTRATIONS. 123 
 
 URINARY DEPOSITS. 
 
 The various forms of these are studied in the chemical 
 division of the class of Practical Physiology. It is con- 
 venient, however, to study here the methods of mounting 
 them, in order that the student may prepare specimens 
 similar to those which he will afterwards be shown. 
 
 215. Collection and Examination. — Allow the urine 
 to deposit in a conical glass, and remove, with the aid of a 
 long pipette, a portion of the sediment for microscopical 
 examination, or remove the supernatant fluid with a syphon, 
 in order that the deposit may not be diffused through the 
 fluid by the disturbing influence of the pipette. For the 
 ordinary examination of the deposit it is generally con- 
 venient to place a drop of the urine on a slide with a 
 shallow cell. The best cell for this purpose consists of a 
 rounded shallow cavity ground in the centre of the surface 
 of the slide. A cell made of a thin ring of dammar varnish, 
 allowed to dry, also answers the purpose. The cell pre- 
 vents the diffusion of the deposit under the weight of the 
 cover-glass. A magnifying power of 250 is most suitable. 
 Those who employ Hartnack's microscope, with the No. 7 
 objective and No. 3 ocular, should shorten the tube, fully, 
 as the power is a little too high with the tube elongated — 
 that is to say, the lens requires, in the latter case, to come 
 so near the cover-glass that the whole depth of the cell is 
 not visible. 
 
 216. Mounting. — The deposit should be washed at 
 least twice with the fluid in which it is to be mounted, or in 
 rectified spirit in the case of such deposits as uric acid, etc., 
 when they are to be mounted in dammar or Canada balsam. 
 For this purpose allow the urine to deposit. Remove the 
 supernatant fluid with a syphon, or decant it ; pour on the 
 preservative fluid ; allow deposit to form ; remove fluid with 
 a syphon or a pipette, again pour on preservative fluid, and 
 keep the mixture until wanted. When deposits are to be 
 mounted in fluid media they should always be placed in a shal- 
 low dammar or balsam cell, made by painting a ring of fluid 
 dammar, or Canada balsam, on a slide. With the aid of a 
 
124 PRACTICAL HISTOLOGY. 
 
 pipette, place a particle of the deposit jn such a cell, add a 
 drop of the preservative fluid, and seal up. 
 
 217. Urates. — Mount in weak spirit or glycerine solu- 
 tion (see § 221). 
 
 218. Cystine. — Mount in dilute acetic acid (ordinary 
 pyroligneous acid 1 part, water 20 parts). 
 
 219. Triple Phosphate. — Mount in strong ammonia 
 1 part, water 6 parts. 
 
 220. Calcic Phosphate is usually amorphous, but 
 sometimes occurs in rosettes of crystals. These may be 
 readily obtained by simply adding to urine a small piece of 
 calcium chloride. The precipitate is allowed to form in a 
 conical glass, and may be mounted and preserved in the 
 mother liquor. 
 
 221. Uric Acid and Oxalate of Lime. — Mount in 
 equal parts of glycerine and camphor water, (Camphor water 
 is thus prepared : wrap 3 grammes of camphor in a piece of 
 muslin, place it in a bottle containing 1500CC of water, 
 shake repeatedly. The fluid is ready for use in three or 
 four days). Or they may be mounted in glycerine jelly as 
 follows. Wash the deposit in weak spirit. Heat the 
 glycerine jelly in a water bath until it becomes fluid. Place 
 a drop on a warm slide, add the urinary deposit, and after 
 a few seconds apply a warm cover-glass. When the jelly is 
 cold, scrape it away from the edge of the slide, and seal 
 up with zinc cement, to prevent the hygroscopic jelly from 
 attracting moisture from the air. As the jelly answers 
 admirably, the use of Canada balsam or dammar is un- 
 necessary ; and this is fortunate, for the use of these 
 media in this connection is attended with considerable 
 difficulty. 
 
 222. Casts and Epithelium. — Pus and mucus may be 
 preserved in naphtha and creosote fluid prepared as fol- 
 lows : — {Beale) A, creosote, 35CC; B, wood naphtha, 
 28CC; C, precipitated chalk, 130 grammes; D, water, 
 600CC. Mix thoroughly A and B and C in a mortar; 
 very gradually add D, keeping the mixture in motion with 
 the pestle all the while. Place the whole, together with a 
 few pieces of camphor, in a lightly covered vessel for two 
 
HISTOLOGICAL DEMONSTRATIONS. 125 
 
 or three weeks, occasionally shaking or stirring the mixture, 
 then filter, and preserve in corked or stoppered bottles. 
 (Beak) 
 
 SKIN. 
 
 223. Methods. — a. Very good preparations may be 
 made of the skin hardened in the mixture of chromic 
 acid and spirit, as described in § 16. The sections may 
 be advantageously stained with picro-carmine. They are 
 best mounted — stained or unstained — in Farrants' solution 
 or in glycerine. Stained sections may, however, also be 
 mounted in dammar. 
 
 b. The method of digestion may be applied to the skin with 
 advantage [IV. Stirling) for the purpose of studying the arrangements 
 of the muscular and elastic fibres. Mix iCC pure hydrochloric acid 
 with 500CC water, and add 1 gramme pepsine.* After keeping the 
 mixture at 38 C. for three hours, shake it thoroughly. Stretch a piece 
 of skin of man or dog, as fresh as possible, over the mouth of a glass 
 dialysing jar, and tie it firmly round the jar to keep it stretched. 
 Digest the skin in the above fluid at 3 8° C. for a period varying from 
 two to eight hours, according to the size and age of the skin. Young 
 skin digests more quickly than that which is old. It is advantageous 
 to use only about 100CC of the digestive fluid at one time, and to 
 change the fluid every second hour if the piece of skin be large, in 
 order to remove the peptones, and thereby facilitate the digestive 
 process. After partial digestion, place the skin in water for twenty- 
 four hours. In this it becomes swollen and transparent. It can then 
 be hardened in the ordinary fluids, and stained with logwood or carmine. 
 By the above process the white fibrous tissue swells up and becomes 
 extremely transparent, thus permitting of a clear view of the other 
 tissues. 
 
 c. The outlines of the cells of the rete mtuosum may be well shown 
 by treatment with osmic acid, as described in § 87B. 
 
 d. The epidermic cells may be rendered prone to separate by 
 maceration in potassium bichromate solution, as described in § 87c. 
 
 e. Staining with gold and osmic acid is serviceable for the demon- 
 stration of the structure of the tactile corpuscles and their connection 
 with nerves. (See Thin, Joum. of Anat. and Phys., vol. viii. 30.) 
 
 224. Structure of the Skin. — Examine the V. S. 
 human skin already prepared (§ 87). 
 
 (L.) In the epidermis observe the rete mucosum below, 
 
 * The ^epsina porci of the British Pharmacopoeia may be conveniently 
 employed. 
 
126 PRACTICAL HISTOLOGY. 
 
 the horny layer above. The lower stratum of the latter is 
 clear and almost homogeneous in appearance. 
 
 In the cutis vera observe the papillae, the areolar tissue, 
 sudoriferous ducts ; probably the coils of the ducts and 
 fat cells will be found in the deeper stratum. 
 
 (H.) The epidermic cells have already been examined 
 (§ 87), out it will be well to complete the subject by looking 
 at the clear layer at the junction of the horny layer with 
 the rete mucosum, — sometimes spoken of as the layer of 
 Langerhans. It consists of epidermic cells so condensed 
 that it is scarcely possible to see their outlines, unless the 
 section be stained with such a dye as picro-carmine. 
 
 The papillce consist of a compact fibrous tissue. Deeper 
 in the cutis the tissue becomes distinctly areolar. 
 
 The sweat glands are simple tubules that will probably 
 be found cut in various directions. In the cutis there is a 
 basement membrane lined by the gland cells. In the 
 cuticle the sweat canal is an irregular somewhat twisted 
 channel amidst the epidermic cells. 
 
 A touch corpuscle may be found in a papilla here and 
 there, somewhat oval in shape, and having a fibrous appear- 
 ance. A nerve fibre may, perhaps, be seen in connection 
 with it. (The nerve fibre passes to the summit, and then 
 ends within the corpuscle in a soft core that is enclosed in 
 a tunic of fibrous tissue. This, however, can only be seen 
 when the corpuscle has been rendered very transparent, 
 e.g., by acetic acid.) 
 
 Pacinian corpuscles may possibly be found in the lower 
 part of the cutis (seeing that the skin under examination 
 is from the palmar surface of the finger). The structure of 
 these has been already considered (§ 159). 
 
 225. Examine (L.) V. S. skin of human heel. The 
 sudoriferous glands may be very distinctly seen in this 
 situation. 
 
 226. Examine (600 diam.) a tactile corpuscle in an 
 extremely thin V. S. of skin from palmar surface of point 
 of human finger. 
 
 227. Human Hair. (H.) — Pluck out a hair of the 
 head — with its bulb, if possible ; place a drop of dammar 
 
HISTOLOGICAL DEMONSTRATIONS. 127 
 
 upon it, cover and examine. In a hair three parts may 
 usually be distinguished: — 1. The cuticle, consisting of a 
 layer of epidermic scales arranged in an imbricated fashion ; 
 2. The cortex, composed of a substance which often appears 
 almost homogeneous, with a few longitudinal lines and gra- 
 nules scattered through it, but which, on bruising a hair that 
 has been boiled in sulphuric acid, is found to consist of epider- 
 mic scales elongated and resembling fibres ; 3. The pith, 
 consisting of irregular cavities amidst the epidermic fibres. 
 These cavities are often absent. The pigment of the hair 
 is either in the form of masses of granules scattered through 
 the cortex, or is diffused throughout the epidermic matter. 
 
 228. Hair of Sheep. (H.) — Mount in dammar. Ob- 
 serve the strongly marked borders of the cuticular cells, 
 readily seen in profile. 
 
 229. Hair of Rabbit (H.) — Mount in dammar. Ob- 
 serve the single or double row of cavities in the pith, and 
 the relatively small amount of fibrous cortex. 
 
 230. The Hair Follicle. — The hair in its follicle is 
 enclosed in envelopes derived from the dermis and epider- 
 mis. From without inwards, the envelopes are — 
 
 I. Dermic Coverings. 
 
 1. A layer of white fibrous tissue with its bundles 
 arranged longitudinally. 
 
 2. A fibrous layer of somewhat indeterminate character, 
 the connective tissue corpuscles and the indistinct fibres 
 around them being arranged transversely. 
 
 3. A hyaline layer of doubtful structure, resembling a 
 basement membrane. 
 
 II. Epidermic Coverings. 
 
 4. The outer root sheath, consisting of a mass of cells 
 irregularly disposed, continuous with the rete mucosum of 
 the adjoining skin. 
 
 5 . The in?ier root sheath, composed of two layers : — 
 
 a. Henle's layer (fenestrated layer), made up of a 
 
128 PRACTICAL HISTOLOGY. 
 
 stratum of flat non-nucleated cells with irregular 
 spaces between them. 
 b. Huxley's layer, composed of a stratum of flat 
 
 nucleated cells without fenestrae. 
 The structure of layers a and b can only be clearly 
 seen where they have been isolated by dissec- 
 tion. 
 6. The cuticle of the root sheath, formed of a layer of 
 imbricated cells in contact with the cuticle of the hair. 
 
 The inner root sheath and its cuticle seldom pass 
 higher than the openings of the sebaceous glands. 
 
 231. Examine the following preparations: — a. (H.) 
 T. S. hair follicle from scalp, preserved in glycerine or 
 dammar. Layers 1, 2, 4, 5 will be readily seen. 3 and 6 
 will probably be difficult to recognise. 
 
 b. V. S. hair follicle of scalp. 
 
 (L.) The follicle, usually somewhat oblique in position, 
 with the sebaceous gland or glands opening into the upper 
 part. 
 
 (H.) The fibrous coverings of the follicle and the outer 
 root sheath may be readily seen, but it will probably be 
 difficult to see the others clearly. 
 
 c. (L.) V. S. skin of dog digested (§ 223, b) and pre- 
 served in glycerine. Examine the erector muscle of the 
 hair follicle, composed of non-striped fibres passing from 
 the lower part of the hair follicle to end in elastic fibres 
 close to the surface of the cutis vera, at a little distance 
 from the follicle, but on that side of it towards which the 
 hair slopes. It is rare to see the whole length of the 
 muscle in a single preparation. 
 
 EYE. 
 
 232. Cornea. — Methods. — a. The cornea may be readily 
 hardened in chromic acid, as directed in § 13. This method 
 serves very well for the demonstration of the epithelium, 
 the elastic lamina, and the fibrous tissue. It may be 
 readily cut if imbedded in paraffin, and the sections un- 
 stained or stained with logwood, mounted in Farrants' 
 solution or in glycerine. 
 
HISTOLOGICAL DEMONSTRATIONS. 129 
 
 b. The nerves and connective corpuscles of the cornea may be 
 stained with gold chloride, as described in § 330, a. 
 
 c. The cell spaces amidst the corneal fibres may be readily demon- 
 stated as follows : * — The epithelium is scraped away from the anterior 
 surface of the cornea of a living frog anaesthetised by chloroform, or 
 stunned to prevent all pain, and one or two drops of a 2 per cent 
 solution of nitrate of silver are introduced within the conjunctiva. At 
 the end of fifteen minutes the cornea is excised, placed in glycerine, and 
 exposed to diffuse daylight till it is of a brown colour. The cornea 
 may be split into layers with scissors and forceps. This will be found 
 advantageous, for the transparency of the cornea is greatly diminished 
 by silvering, and the outer layers are rendered useless by the process. 
 The preparations are mounted in glycerine. Such preparations may 
 also be made from the cornea of the rabbit, anaesthetised with ether. 
 
 233. Structure. — V. S. chromic acid cornea of cat, 
 prepared as stated in § 232, a. Mount in Farrants' solution, 
 and examine. 
 
 (L.) Anterior epithelium. — Fibrous tissue. Elastic lamina 
 with posterior epithelium. Unless the section be made by 
 freezing, the posterior epithelium is very apt to be torn off. 
 
 (H.) Several layers of the anterior epithelium, the lower 
 cells resting immediately upon the fibrous tissue without the 
 intervention of a thin elastic lamina — Bowman's membrane 
 — as in the case of the human cornea. 
 
 The single layer of epithelium placed posteriorly on the 
 membrane of Descemet — a thick elastic lamina. 
 
 The fibrous tissue with the nuclei of connective tissue 
 corpuscles (corneal corpuscles), 
 
 234. Examine (H.) a cornea silvered by the method 
 described in § 232, c. The fibrous tissue is darkened by the 
 silver, and the connective tissue spaces are readily seen 
 forming an anastomosing system of channels (" lymph 
 canalicular system") in which the corneal corpuscles are 
 placed, and through which the lymph from the marginal 
 capillaries can readily permeate. 
 
 235. Examine (600 diam.) the nerves and corneal 
 corpuscles stained with gold by the method described in § 
 330, b. Plexuses of nerve fibrils lie between the layers of 
 fibres, and may be exposed by stripping off the fibrous layers 
 from say the anterior surface of the cornea. Nerve fibres 
 
 * The student is not permitted to do this. 
 K 
 
130 PRACTICAL HISTOLOGY. 
 
 are seen dividing into fibrils, and these forming a network 
 without — even when examined with very high powers — any 
 apparent connection with surrounding tissue. The nerve 
 fibrils have almost always a beaded appearance. 
 
 236. Crystalline Lens (H.) — Fibres of lens of cod 
 fish boiled for ten minutes in dilute sulphuric acid (1 per 
 cent). Dissociate with needles and preserve in Farrants' 
 solution. The serrations of the fibres are very distinctly 
 seen. The lens of the ox, cat, or rabbit may be prepared 
 in the same manner, but the serrations are not nearly so 
 well marked. 
 
 237. Examine a. (L.) T. S. lens hardened in absolute 
 alcohol. The laminae of fibres are readily seen. 
 
 b. (H.) T. S. fibres of lens hardened in chromic acid 
 and spirit (§ 13), showing their hexagonal shape. 
 
 238. Hexagonal Pigment Cells (H.) — With the point 
 of a scalpel scrape off some of the pigmentary layer inside 
 the choroid of an eye hardened in chromic acid and spirit, 
 § 13, and place the scraping in a drop of Farrants' splution. 
 Cover, and examine. The hexagonal cells, each with a clear 
 nucleus, will be readily seen. 
 
 239. Retina. — Methods. — a. The retina may be well 
 prepared for section in the chromic acid and spirit fluid, as 
 mentioned in § 13. The sections may be unstained or 
 stained with dilute carmine (§ 318, a) and preserved in 
 Farrants' solution or in glycerine. 
 
 b. The retina may also be well hardened by osmic 
 acid. It is placed in a \ per cent solution for thirty-six 
 hours, and then washed in distilled water for twenty-four 
 hours or so. Sections are preserved in Farrants' solution, 
 or in a saturated solution of potassium acetate. Osmic 
 acid is the best agent for showing the general form and 
 connections of the rods and cones. These may be isolated 
 by teasing sections made by freezing, as described below, 
 or more simply by means of scissors. 
 
 c. The gold method is very useful (W. Stirling) for 
 facilitating the cleavage of the outer segments of the rods 
 transversely into their plates, as described by Max Schultze. 
 The posterior half of the eyeball of a frog is placed for an 
 
HISTOLOGICAL DEMONSTRATIONS. 131 
 
 hour or so in a J per cent solution of gold chloride, and 
 after thorough washing in water, and exposure to diffuse 
 light for two days in water slightly acidulated with acetic 
 acid, it will be found that on teasing out a small piece of 
 the retina the outer segments of the rods readily tend to 
 cleave transversely. 
 
 d. To make good sections of the retina has hitherto 
 been a matter of the greatest difficulty, for any imbedding 
 agent that may be used is so apt to spoil the bacillary layer. 
 The freezing microtome has, however, overcome the difficulty 
 completely, and it is easy to obtain any number of perfect 
 sections with its aid. 
 
 240. Structure (H.) — V. S. retina of cat prepared as 
 stated in § 239, a. Place the ends of the section towards 
 the ends of the slide, add a drop of Farrants' solution. 
 Cover, and examine. 
 
 The general scheme of the structure of the retina is this 
 (Schultze) : — Delicate nervous elements are supported by 
 a framework of connective tissue. The rods and cones are 
 the terminations of the fibres of the optic nerve with which 
 their connection is probably the following. A nerve fibril 
 passes from the inner segment of each rod, and has one or 
 two nuclear swellings upon its course inwards. From the 
 inner segment of each cone there passes a bundle of fibrils, 
 which has also at least one nuclear swelling upon its course 
 inwards. Both the rod and cone fibrils join multipolar 
 nerve cells at the inner part of the retina, which in turn 
 are connected with the fibres of the optic nerve. The 
 actual continuity of the rod and cone fibrils with the 
 multipolar nerve cells has not, however, been traced as yet. 
 
 The connective tissue elements principally consist of a 
 membrane {inner limiting membrane) on the inner aspect of 
 the retina, and another (outer limiting membrane) inside the 
 rods and cones, with trabecular (fibres of Mi'tller) passing 
 between the two. Nine or even ten layers may be dis- 
 tinguished in the retina which we have now to examine in 
 detail in the preparation. 
 
 1. Inner limiting membrane, with the fibres of Miiller 
 extending from it outwards. 
 
132 PRACTICAL HISTOLOGY. 
 
 2. Layer of nerve fibres consists of the axial cylinders ot 
 the optic nerve fibres. 
 
 3. Layer of nei-ve cells is composed of multipolar cells, 
 the central process of each being an undivided axial cylinder 
 process continuous with an optic nerve fibre, and the external 
 processes, several in number, dividing again and again, and 
 thus giving rise to smaller bundles of fibrils that pass out- 
 wards into the next layer, through which, however, they 
 have not as yet been traced. 
 
 4. Lnner granular layer ("inner molecular layer"). 
 With this magnifying power (300) it appears to be nothing 
 but minute granules, but when very highly magnified a clear 
 matrix containing fine granules may be seen. The fibres 
 of Miiller pass through it, and probably so do the nerve 
 fibrils. 
 
 5. Lnner nuclear layer contains distinct nuclei, each with 
 a nucleolus. A few of them belong to the fibres of Miiller ; 
 the majority, however, belong to the nerve fibrils passing 
 outwards. (The nerve fibrils cannot be distinctly seen 
 without a much higher power.) 
 
 6. Outer granular layer is much thinner, but is in other 
 respects similar to the inner granular layer ; its limits are 
 much more easily seen in a retina stained with carmine, 
 for then the inner and outer nuclear layers are stained, while 
 the granular layers are not. 
 
 7. Outer nuclear layer contains many nuclei, the majority 
 of which belong to the rod nerve fibrils, one nucleus upon 
 each fibril placed at various parts of their course. The 
 fibrils, however, can only be seen with a very high power, 
 and best in the osmic acid retina. Transverse striation 
 of these nuclei first pointed out by Henle is very evident ; 
 the general substance of the nucleus is highly refractile, 
 and is crossed by one, sometimes two stripes of a less 
 refractile substance. There is no nucleolus. It is much 
 more difficult to find the single nucleus with nucleolus upon 
 the cone fibre placed just within the outer limiting membrane. 
 It has no stripes, and is exactly like the nuclei of the inner 
 nuclear layer. It is best seen in the osmic acid retina of 
 the frog. 
 
HISTOLOGICAL DEMONSTRATIONS. 133 
 
 Cannot be 
 clearly seen 
 in this pre- 
 paration. 
 
 8. Outer limiting membrane. 
 
 9. Rods and eones (Jacob's membrane, or 
 the bacillary layer). 
 
 10. Pigmentary layer. 
 The pigmentary layer consists of the hexagonal pigment 
 
 cells already examined (§ 238), hitherto commonly regarded 
 as the inner layer of the choroid, but included by Max 
 Schultze as one of the layers of the retina, because of 
 delicate connective tissue fibrils, apparently connected with 
 the cells, passing inwards between the outer segments of 
 the rods and cones. 
 
 240A. Examine the following preparations : — a. V. S. 
 osmic acid retina of frog prepared as directed in § 239, b, 
 and cut by freezing. A power of 300 diam. will do, but 
 600 is much to be preferred. The rods being far larger 
 than in the cat's retina are easily seen, each consisting of 
 an outer and an inner segment. The former will probably 
 be much blackened by the osmic acid. It is normally as 
 clear as glass. The inner segment is seen perforating the 
 extremely thin outer limiting membrane. A cone is usually 
 seen with difficulty. Its outer segment is shorter than 
 that of the rod. 
 
 b. (H.) Rods of frog's retina hardened in gold (§ 239, c). 
 Observe the outer segments of the rods cleaving transversely 
 into discs. The outer segments of a cone cleave in like 
 manner. According to Schultze the inner segments of rods 
 and cones are the true nerve terminations, while the outer 
 segments consist of columns of plates that play the part of a 
 reflecting apparatus. 
 
 c. (L.) V. S. optic nerve entering eyeball, tinged with 
 carmine, showing the prominence {papilla) of the nerve, 
 its central blood-vessels, and the fibres of the lamina 
 cribrosa of the sclerotic, through the spaces between which 
 the nerve fibres enter the eyeball. 
 
 d. (L.) V. S. from before backwards, of ciliary muscle, 
 iris, sclerotic, cornea, choroid and ciliary processes. The 
 anterior attachment of the muscle to the cornea is seen, and 
 its fibres are seen to spread out posteriorly on the choroid. 
 
 e. (L.) Inner aspect of ciliary processes injected — - 
 opaque or transparent. 
 
i 3 4 PRACTICAL HISTOLOGY. 
 
 e. Branched pigment cells of the substance of the 
 choroid. These may be seen in any thin section of the 
 tunics of the eyeball. 
 
 COCHLEA. 
 
 241. Methods. — a. The structure of Corti's organ may be in- 
 vestigated in the fresh condition, but, except in very young animals, 
 little can be learned by such a mode of examination regarding the 
 relations of the various soft tissues. For this purpose, Corti's organ 
 must be hardened, its bony surroundings softened, and the cochlea 
 sliced vertically. It is of the greatest importance that the organ be 
 placed in preservative fluid as soon after death as possible. If the 
 human cochlea be prepared, it should be taken not later than twelve 
 hours after death ; and in order to preserve it as thoroughly as possible, 
 it is well, immediately after death, to inject rectified spirit into the 
 tympanic cavity with an ordinary subcutaneous syringe thrust through 
 the membrana tympani. 
 
 b. Hardening. — Osmic acid and chromic acid are the best hardening 
 agents. The latter is to be preferred when sections are to be made of 
 the surrounding bone. The best chromic fluid is the mixture of chromic 
 acid and spirit (§ 4, Solution 3). This fluid is used in the manner 
 indicated in § 17. 
 
 c. Softening the bone. — In the case of foetal cochleae, the bone may 
 be softened by chromic acid alone, but for older cochlea? a stronger 
 acid should be added. German authors recommend for this purpose 
 hydrochloric acid diluted with water ten times, but far better results 
 are obtained by using the bone-softening fluid already mentioned. 
 (See § 4, Solution 5.) A large quantity of this fluid is necessary, and 
 in the case of the larger cochleae, they should be kept in motion by 
 some such means as an ordinaiy meat-jack. With the aid of this 
 device, even a large cochlea may be softened in a few days. 
 
 d. Section. — When sections are to be made of the softened cochlea, 
 the lamina spiralis and organ of Corti require support to maintain them 
 in situ. Melted wax and a hot solution of gelatine have both been 
 used for this purpose with some success, but the method of imbedding 
 in gum is decidedly preferable. Remove the cochlea from the softening 
 fluid, and place it in a small cone of bibulous paper, containing a strong 
 solution of gum arabic for four or five hours, then immerse the cone in 
 methylated spirit for forty-eight hours or so, until the gum has become 
 hard and tough ; then remove the superfluous gum, and imbed the 
 whole in the ordinary paraffin mixture in a microtome (§ 303), with the 
 modiolus of the cochlea placed at right angles to the axis of the tube 
 of the microtome, so that Corti's organ will be sliced vertically. Or 
 transfer the cochlea from the softening fluid to a solution of gum placed 
 in the well of the freezing microtome, and, after letting it soak in the 
 gum for some hours, freeze and make sections. After either the one or 
 
HISTOLOGICAL DEMONSTRATIONS. 135 
 
 other mode of section, the slices should be immediately placed in 
 rectified spirit. 
 
 e. Staining, — The sections may be stained with logwood, silver, and 
 carmine. The last two yield the best results. Carmine may be success- 
 fully used in the following way. Mix equal parts of water, methylated 
 spirit, and Beale's carmine fluid, with all the ammonia retained (see 
 § 3 1 8a). (The spirit is added to the mixture in order to prevent the 
 rapid swelling up and solution of the supporting gum. ) Transfer the 
 sections from the rectified spirit to the above carmine fluid, and let 
 them remain in this for about twelve hours, until a deep diffuse staining 
 is obtained. Then pour away the carmine and wash the sections very 
 gently with dilute alcohol (rectified spirit I part, distilled water 3 parts). 
 Next cover them with an acid washing fluid, consisting of rectified 
 spirit 60 parts, water 39 parts, hydrochloric acid I part. After wash- 
 ing in this fluid for six or eight hours, until all diffuse staining is 
 removed, cautiously pour off the fluid and preserve the sections in ordi- 
 nary rectified spirit until required. Silver staining is useful for the 
 fresh tissue. It reveals the outlines of the epithelium of the cochlea. 
 The process of staining is the ordinary silver method (§ 328). 
 
 f. Mounting. — Great care is necessary in mounting the sections, lest 
 important parts be injured. If the sections have not been stained, 
 the gum must be carefully removed or softened by the above, but if 
 glycerine jelly be used for mounting, this precaution is unnecessary. 
 The best pi'eservative media are glycerine, glycerine jelly, and dammar. 
 The last is only resorted to when the sections have been stained. For 
 unstained, and even for stained sections, glycerine and glycerine jelly 
 are good preservative media. In mounting vertical sections of the 
 cochlea, the modiolus should always be placed across the glass slip to 
 facilitate subsequent microscopical study. 
 
 These methods give good results, and they were to a large extent 
 devised by my late assistant, Dr. Pritchard, to whom I gave this as the 
 subject for a thesis for the University of Edinburgh. 
 
 242. Structure. — Examine a series of preparations 
 
 hardened in chromic acid and unstained or stained with 
 
 carmine. 
 
 a. (L.) V. S. cochlea showing modiolus. Lamina 
 spiralis ossea and membranacea, with the organ of Corti on 
 the latter. 
 
 b. (H.) V. S. cochlea showing inner and outer rods of 
 Corti, both planted on the basilar membrane. The head 
 of the inner rod resembles somewhat the head of the ulna. 
 The head of the outer rod is articulated with it. A short 
 process passes outward from the head of each rod, to which 
 the membrana reticularis here seen in section is connected. 
 The outer rods are longer than the inner ones. 
 
136 PRACTICAL HISTOLOGY. 
 
 The cells of Corti will be easily seen external to the 
 outer rods. There are three rows in the cat and some 
 other animals, four in man. Each cell has short stiff hair- 
 like processes on its upper extremity projecting through the 
 membrana reticularis. A process passes from the lower end 
 of the cell to be attached to the basilar membrane. Out- 
 side Corti's cells the epithelium is columnar, but without 
 hairs. It gradually becomes short and cubical, and so 
 passes gradually into the epithelium lining the outer part of 
 the scala media. 
 
 The membrana basilaris may be seen stretching from 
 the lamina spiralis ossea to a fan-shaped (in V. S.) expan- 
 sion — the ligamentum spirale of Kolliker (the cochlear 
 muscle of Bowman). The tissue of the ligament is 
 peculiar, and it is difficult to say what is precisely its 
 nature. 
 
 The membrana tectoria may be seen extending from the 
 limbics lamina spiralis over the heads of the rods and the 
 hair cells. In the normal condition it appears to hang 
 free above these, but very often in prepared cochleae it lies 
 closely upon the hair cells. According to Waldeyer it con- 
 sists in the unhardened cochlea of a soft pulpy material. 
 
 The membrane of Reissner may perhaps be found. It 
 consists of a single layer of flattened epithelial cells separat- 
 ing the scala media from the scala vestibuli. 
 
 The spiral ganglion may be seen in carminised sections 
 — within the bone of the spiral lamina. 
 
 c. (H.) A preparation showing a superficial view 
 of Corti's organ ; the heads of the rods resembling a row 
 of pianoforte keys ; the ' membrana reticularis consisting 
 of plates that have the form of rings and phalanges. The 
 hairs of Corti's cells project through the rings. 
 
 SCHNEIDERIAN MEMBRANE. 
 
 243. Examine (H.) a preparation of olfactory cells from 
 the nose of the newt, prepared thus : — Harden the head in 
 Miiller's fluid for four or five days. Snip out the upper part 
 of the nostril. Scrape the mucous membrane of the detached 
 portion with the point of a scalpel. Dissociate with needles, 
 
HISTOLOGICAL DEMONSTRATIONS. 137 
 
 and preserve in Fan-ants' solution. The olfactory cells are 
 long andj slender, with a large nucleus about the centre. 
 The ordinary epithelial cells are much broader. In the 
 newt and frog the former have delicate hairs on their peri- 
 pheral extremities, but these can rarely be seen. 
 
 SPINAL CORD AND MEDULLA OBLONGATA. 
 
 244. Methods. — a. The spinal cord and medulla 
 oblongata may be hardened by immersion in' rectified spirit 
 followed by a \ per cent solution of chromic acid {Lock- 
 hart Clarke) as directed in § 14. Sections are made in a 
 microtome with the cord imbedded in carrot or paraffin. 
 The sections may be stained with carmine or logwood. 
 The former gives good results if the staining be very 
 slowly done. The dilution of Beale's carmine fluid with all 
 the ammonia retained (§ 318, a) produces excellent results. 
 Twenty-four hours or more may be required. Carmine 
 may, however, completely fail if the tissue have been 
 over-hardened by chromic acid. In that case the carmine 
 staining may be readily effected after the previous action of 
 palladium chloride, as described in § 3 1 9, or the sections 
 may be stained with logwood. 
 
 b. The sections, whether stained or unstained, are 
 usually mounted in dammar. 
 
 c. A method for isolating the nerve cells of the cord 
 has been already described in § 156. 
 
 245. Structure of the Cord. — a. T. S. spinal cord of 
 cat carminised. Mount in dammar (§ 152). 
 
 (L.) The anterior and posterior median fissures. The 
 gray matter brightly stained with carmine. The nerve roots 
 may perhaps be seen; the anterior forming two or three 
 bundles, the posterior only a single bundle. The anterior, 
 lateral, and posterior white columns in each half of the cord. 
 The gray (posterior) and the white (anterior) commissures 
 uniting the two halves of the cord. The central canal in 
 the gray commissure. Leave the canal exactly in the centre 
 of the field, and change the lens. 
 
 (H.) The central canal lined by a single layer of ciliated 
 
138 PRACTICAL HISTOLOGY. 
 
 columnar epithelium ; the cilia, however, have been destroyed 
 in the hardening process. 
 
 The gray matter with its multipolar cells relatively larger 
 in the anterior horn. The nerve fibres in the white columns 
 mostly cut transversely with the axial cylinders stained. 
 The pia mater sending in here and there well-marked pro- 
 cesses continuous with a fine connective tissue (neuroglia) 
 lying between the nerve elements. The processes contain 
 an elastic tissue which may be shown by pencilling a sec- 
 tion of the hardened cord. In the spinal cord, medulla, 
 and perhaps in other parts at the base of the brain, the 
 neuroglia is a nucleated connective tissue, the matrix of 
 which is fibrillated. In the cerebrum and cerebellum the 
 matrix appears to be a sort of jelly, but it is difficult to de- 
 fine it exactly. 
 
 b. Mount an unstained T. S. cord in dammar for com- 
 parison with the preceding. 
 
 246. Examine the following preparations : — 
 
 a. (L.) V. S. white and gray columns of spinal cord 
 carminised. 
 
 b. (L.) T. S. spinal cord injected. The gray is far 
 more vascular than the white matter. 
 
 246A. Structure of Medulla Oblongata. — Examine 
 the following preparations : — 
 
 a. (L.) T. S. carminised medulla oblongata through 
 the decussation of the anterior pyramids. Observe the 
 decussation; the enlarged tips of the posterior horns of 
 gray matter, now termed the tubercles of Rolando ; the 
 posterior pyramids. 
 
 b. (L.) T. S. carminised medulla through the olivary 
 bodies, and the lower part of the fourth ventricle. The 
 following parts may be recognised from the anterior median 
 line outwards in either half of the medulla. The anterior 
 pyramid without any gray matter. Fibres of the hypoglossal 
 nerve passing to the gray matter at the floor of the fourth 
 ventricle. The olivary fasciculus with its ganglion — the 
 corpus dentatiwi — a crumpled sheet of gray matter. Fibres 
 of the vagus nerve passing to the gray matter at the floor 
 of the ventricle. The restiform tract and its gray matter. 
 
HISTOLOGICAL DEMONSTRATIONS. 139 
 
 The posterior pyramids are now difficult to distinguish from 
 these. They contain gray matter. The floor of the fourth 
 ventricle with its gray matter is evident. 
 
 CEREBELLUM AND CEREBRUM. 
 
 247. Methods. — a. The methods of preparation are 
 similar to those recommended for the spinal cord and me- 
 dulla. The brain, however, cannot bear a solution of 
 chromic acid so strong as that recommended for the cord. 
 The dilute solution of chromic acid and potass, bichrom. 
 (Solution 4, § 4), used as directed in § 14, gives good results. 
 The sections are treated as in the case of the spinal cord. 
 
 b. The brain may also be hardened first in rectified 
 spirit, and then in a 2 per cent solution of potass, bichrom. 
 
 c. The cells of the cerebrum may be prepared for examination in 
 their fresh condition by a method devised by Mr. Lewis of Wakefield 
 {Monthly Microscop. Journal, vol. xvi. p. 105). The pia mater is 
 stripped from a convolution of the human cerebrum as fresh as possible. 
 Thin vertical sections of the gray matter are then made with a broad- 
 bladed and perfectly sharp razor, the surface of which is deluged with 
 spirit. As thin a section as possible is floated from the razor upon a 
 slide, and Midler's fluid is added, to make a pool of fluid above and 
 below the section, so that the tendency of the nervous matter to adhere 
 to the glass during the next process may be diminished. The cover-glass 
 is then applied and cautiously pressed down with the point of a 'needle 
 applied to its centre, until the nervous matter becomes a thin trans- 
 parent film. The slide is then placed in a'flat dish and covered with 
 methylated spirit. After about forty seconds the slide is removed from 
 the dish, one edge of the cover-glass is steadied with the finger, while 
 the other is slowly raised with the point of a scalpel. The spirit faci- 
 litates the removal of the cover-glass, and the nerve film is left floating 
 on the slide, or adhering to the cover-glass. To stain the nerve cells 
 a drop of a 1 per cent solution of aniline black (§ 325) is placed on the 
 film. When the colour is sufficiently deep the slide is lowered under- 
 water in a shallow vessel, and washed by gently moving the water with 
 a brush. Previous to mounting, the slide, with the film uncovered, is 
 placed under a bell glass with sulphuric acid, and when perfectly dry 
 a few drops of clove oil, or still better, chloroform, are placed on the 
 film, then a drop of dammar, and then the cover-glass. Staining agents 
 other than the above mentioned may be used. 
 
 248. Structure of Cerebellum. — V. S. cerebellum of 
 man or of cat, carminised. Mount in dammar. 
 
 (L.) The primary and secondary convolutions. The 
 
140 PRACTICAL HISTOLOGY. 
 
 white matter within and two layers of gray matter outside, 
 the inner of the two being the more intensely coloured, and 
 having a granular appearance with this magnifying power. 
 The cells of Purkinje may just be detected lying at the 
 inner part of the outer gray layer. 
 
 (H.) Outer gray layer. — The cells of Purkinje have a 
 single central process that passes into the inner gray layer. 
 It is often very difficult to see this, but there is no difficulty 
 in seeing one or two processes passing from the outer 
 aspect and dividing again and again, and thus forming a 
 network in the outer gray layer as the branching processes 
 of the cells of the spinal cord do in its gray matter. The 
 nuclei scattered here and there belong to the neuroglia. 
 
 In the inner gray layer great numbers of nuclei are 
 easily seen. They closely resemble the nuclei of the neu- 
 roglia. 
 
 248A. Structure of Cerebrum. — a. V. S. convolu- 
 tions of carminised human cerebrum. Mount in dammar. 
 
 (L.) The convolutions are simple in character — without 
 secondary folds as in the cerebellum. In very favourable 
 specimens a lamination of the gray matter may be seen. 
 This indeed in the unhardened brain is evident to the eye, 
 assisted by a simple lens. There are alternate white and 
 gray layers in the gray matter, but little heed need be 
 given to an examination of these with a low power, for one 
 of the layers that has a whitish appearance contains most 
 ' of the pyramidal cells that are so characteristic of the cere- 
 bral ganglion. 
 
 (H.) It is really very difficult to succeed in getting a 
 specimen just thin enough, and just sufficiently well stained, 
 to show the five layers described in the gray matter by 
 Lockhart Clarke and Meynert. The student may very 
 possibly see little more than the pyramidal cells ; even these 
 will not be sufficiently seen if the section be not perfectly 
 vertical. The pyramidal cells occur about the middle of 
 the gray master, of which they are the largest cells. Their 
 apices are directed outwards, numerous processes come off 
 from the base, and usually one from the apex. According 
 to Meynert, all the processes ramify excepting one — the 
 
HISTOLOGICAL DEMONSTRATIONS. 141 
 
 axial cylinder process — connected with the centre of the 
 base of the pyramid. 
 
 From without inwards the layers of the gray matter, as seen in very 
 good specimens, are : — 
 
 1. A thin layer, consisting almost entirely of neuroglia. 
 
 2. A thin layer of minute closely placed nerve cells of irregular 
 shape, with branching processes. 
 
 3. A layer three or four times the breadth of either 1 or 2, con- 
 taining the pyramidal cells, scattered throughout its whole thickness. 
 
 4. A thin layer of closely set small granule-like corpuscles of 
 irregular shape. 
 
 5. A layer twice as broad as the last, consisting of cells, for the 
 most part of fusiform shape. This layer gradually merges into the 
 central white matter of the convolution. 
 
 Fine fasciculi of medullated fibres pass outwards in the gray matter. 
 Other fasciculi of medullated {Meynert) fibres run at right angles to 
 these, and leave interstices that are occupied by the nerve cells and the 
 reticulum formed by their branching processes {Gerlach). 
 
 b. Examine a V. S. cerebral convolutions injected, and 
 observe the greater vascularity of the gray as compared with 
 the white matter. 
 
 GENERATIVE ORGANS. 
 
 MALE ORGANS. 
 
 249. Testis. — Methods. — Mihalkovics (Ludwig's Arbei- 
 ten, 1874, p. 19) recommends the following method of 
 hardening the testis for the purpose of section, as preferable 
 to all others : — Inject, with a sharp-pointed syringe, 1 
 per cent osmic acid at several points through the tunica 
 albuginea of the testis of a cat ; place it in rectified spirit 
 for several days, and then in absolute alcohol for a day or 
 two, previous to making sections. Stain with carmine or 
 logwood, and mount in glycerine. 
 
 b. Hardening in Miiller's fluid, and then in alcohol, is 
 also a fairly good method for studying the cells in the 
 tubuli seminiferi in teased preparations. 
 
 c. For the isolation of the tubules, Sappey long ago recommended 
 the maceration of small portions of the organ in. dilute hydrochloric 
 acid (acid I, water 2 parts) for one or two days. 
 
i 4 2 PRACTICAL HISTOLOGY. 
 
 d. The lymphatics of the testis may be readily injected by the 
 method described in § 344. 
 
 250. Structure. — a. T. S. testis of cat hardened in 
 osmic acid and alcohol ; mount in glycerine. 
 
 (L.) Tunica albuginea ; mediastinum and septa passing 
 from it to the tunica. Tubuli seminiferi. 
 
 (H.) Nucleated cells — the spermatoblasts, and perhaps 
 also spermatozoids, within the tubuli seminiferi. The mem- 
 brane of the tubules is thick, and, according to Mihal- 
 kovics, consists of several layers of flattened cells, that are 
 unaffected, and may, indeed, be demonstrated by a twelve 
 hours' immersion in strong hydrochloric acid. 
 
 b. Examine (L.) a. V. S. testis of cat, prepared as above, 
 showing tunica albuginea, tubuli seminiferi, tubuli recti, 
 rete testis, and perhaps also the vasa efferentia, passing to 
 the epididymis. 
 
 c. Examine (H.) a preparation of spermatozoids of ox 
 or sheep, preserved by drying. 
 
 251. Prostate. — This may be hardened in a 5 per cent chromic 
 acid, followed by alcohol. 
 
 FEMALE ORGANS. 
 
 252. Uterus. — This may be readily hardened in a 
 \ per cent chromic acid followed by alcohol (§ n). T. S. 
 uterus of cat, unstained ; mount in glycerine. (L.) Mucous 
 membrane and muscle. (H.) The uterine glands ; tubular, 
 and lined by columnar epithelium. 
 
 253. Ovary. — Methods. The ovary maybe exceedingly 
 well prepared by hardening in Muller's fluid and alcohol, as 
 directed in § 12 a. The sections are best stained with 
 logwood, and may be mounted in glycerine or in dammar. 
 
 254. Structure, — Examine a section of the ovary pre- 
 pared as directed above. 
 
 (L.) Graafian follicle surrounded by a fibrous stroma. 
 
 (H.) The following parts may be recognised from within 
 outwards in a mature Graafian follicle : — 1. The germinal 
 spot ; a nucleolus. 2. The germinal vesicle ; a nucleus. 
 3. Granular protoplasm; the vitellus. 4. The zona pellu- 
 
HISTOLOGICAL DEMONSTRATIONS. 143 
 
 cida ; the envelope of the cell. 5. The tunica granulosa 
 covering the zona. 6. The space that contains the liquor 
 folliculi placed outside the tunica. 7. The tnembrana 
 granulosa lining the ovisac. 8. The cumulus proligerus 
 uniting the tunica with the membrana. 5, 7, and 8 consist 
 of epithelial cells. 9. The ovisac composed of a fibro-vas- 
 cular tissue. 
 
 255. Examine (H.) the section of an ovary of a young 
 kitten, showing here and there one of the epithelial cells of 
 the columnar epithelium on the peritoneal aspect of the 
 ovary enlarged. This, as described by Foulis, becomes the 
 nucleated protoplast of the future ovum. It becomes en- 
 closed by connective tissue corpuscles growing from the 
 stroma up around it. These eventually produce the cells 
 of the tunica and membrana granulosa, which for a long 
 time form one mass, but eventually become separated by 
 the appearance of the liquor folliculi. The zona pellucida 
 is an after formation, possibly a sort of excretion derived 
 from the protoplasm of the ovum, or from the cells of the 
 tunica granulosa. 
 
 The tubes which have been described in the ovary 
 appear to be nothing more than sulci produced by the 
 unequal outward growth of the ovary {Foulis). Young ova 
 may be found growing on the surfaces of the sulci in the 
 same manner as on the free surface of the ovary. 
 
 DEVELOPMENT. 
 
 256. Examine a series of models of the human embryo, 
 and preparations of the embryo chick. A description of the 
 structure of the latter, and of the details of the methods 
 of preparation, would carry us beyond the limits of these 
 outlines. The advanced student will, however, find a com- 
 plete account of the subject in the Elements of Embryology, 
 by Foster and Balfour. (London: Macmillan, 1874.) 
 
PART IV. 
 
 GENERAL CONSIDERATIONS REGARDING 
 HISTOLOGICAL METHODS. 
 
 METHODS OF APPLYING RE-AGENTS TO THE 
 TISSUES. 
 
 257. Application of Fluids. — Irrigation. When a 
 microscopic object is to be irrigated, a drop of any desirable 
 fluid is placed on the slide close to the margin of the cover- 
 glass, and if necessary a strip of bibulous paper is dipped 
 in the fluid at the opposite side, to remove that which is to 
 be replaced. For the removal of thick fluids such as 
 glycerine, it is necessary to use a pipette (Fig. 45), which 
 may be readily made by blowing a bulb on a glass tube, 
 and drawing one extremity of the tube to a sufficiently fine 
 point (Read § 57, /.) 
 
 258. Application of Vapours and Gases. — An ex- 
 ample of a simple method for applying a vapour to a micro- 
 scopic object is given in § 89, where the chloroform vapour 
 is applied to cilia. A drop of the fluid is placed on the 
 floor of a glass cell, and the tissue is placed on the 
 under surface of a cover-glass inverted over it. 
 
 For the application of gases, it is necessary to have 
 two tubes in connection with the cavity of a cell : one for 
 the entrance, the other for the exit of the gas. The warm 
 stage, to be presently described (Figs. 48, 49), is so 
 arranged that it may serve as a gas chamber for the attain- 
 ment of this object. Various gases, such as carbonic acid, 
 oxygen, etc., may be employed. 
 
 For the production of carbonic acid an apparatus such 
 as that shown in Fig. 46 is convenient. Dilute hydro- 
 chloric acid is placed in a bottle A, and pieces of broken 
 
HISTOLOGICAL METHODS. 
 
 145 
 
 marble in another bottle C. When the gas is desired, A 
 is elevated upon some support. The gas is passed ^-» 
 through a wash bottle (C) containing water, and 
 thence to a T tube (D). The microscopic gas 
 chamber is joined to the tube E j and when the 
 transmission of gas through it is desired, a clamp 
 (G) is placed upon the waste-tube F. When it is 
 
 Fig. 46. — Carbonic acid apparatus. 
 
 necessary to rapidly arrest the transmission of the 
 gas, a clamp is placed on H. The gas accumu- 
 lates in B, and drives the acid back into A. A 
 stream of air may be pumped through the gas 
 cell by connecting an elastic pump to F, and 
 placing the clamp G upon H. 
 
 259. Application of Heat. 
 stage. — A stage suitable for roughly heating a 
 
 I I 
 
 Fig. 45. 
 c j. 7 14. Pipette for 
 Kiimple hOt removing 
 
 fluid from 
 "S"-' »««-wvw«' --"■ w "6"v —„—.»—.£, — microsco- 
 
 microscopic object may be made of a plate of tin p ic ob - 
 9 x 2 J inches, with an aperture about a quarter 
 of an inch in diameter in the centre (Fig. 47). The plate 
 should be long enough to project some inches on each 
 side of the stage of the microscope. The slide, with the 
 object to be heated, is laid on the plate, the object is focal- 
 ised, and a spirit lamp is applied to one end of the plate. 
 It is convenient to apply the lamp to the right extremity, 
 and to keep the forefinger of the left hand on the plate 
 
 L 
 
146 
 
 PRACTICAL HISTOLOGY. 
 
 close to the slide, so that the lamp may be removed the 
 moment the temperature becomes unpleasant to the touch. 
 
 Simple hot stage. 
 
 260. Strieker's hot stage. — Max Schultze was the first to in- 
 vent an apparatus for keeping a microscopic object at a constant 
 temperature. His instrument, however, was not very convenient, and 
 modifications of it have therefore been devised. Strieker's hot stage 
 (Fig. 48) is one of these. It consists of a rectangular piece of ebonite 
 fixed on a brass plate that rests on the stage of the microscope. On 
 the upper surface of the ebonite there is a copper disc, with an aperture 
 (c) leading into a short metal tube closed inferiorly by a piece of glass. 
 The object to be heated is placed between two cover-glasses and laid 
 on the disc, a ring of olive or almond oil is then painted around the 
 cover-glasses to prevent evaporation. For heating the object, a thick 
 copper wire (w), coiled at one end, is stuck upon the copper tube (a), 
 
 Strieker's hot stage and gas chamber. 
 
 by the wall of which the heat is conducted to the copper disc. The 
 wire is about six inches long, and is heated by a spirit lamp or gas- 
 burner placed on the table in front of the microscope. The temperature 
 depends on the distance of the point of application of the flame from 
 the stage, and on the proximity of the flame to the wire. The tempera- 
 
|| :•!:!! 
 
 HISTOLOGICAL METHODS. 147 
 
 ture of the copper disc is indicated by a thermometer (/). The tempera- 
 ture which it shows, however, is only approximately that of the object. 
 This method of heating such a stage is convenient, because of the 
 rapidity with which it may be used. When, however, a constant 
 temperature is to be maintained for any considerable length of time, 
 the stage is best heated by a stream of hot water, as suggested by 
 Strieker and Sanderson (Fig. 49). Before passing to it, however, it 
 should be stated that the tubes a and a (Fig. 48) are inlet and outlet 
 tubes for the conveyance of gases through the cell. The wall of the 
 tube a is used as a heat-conductor merely for convenience. 
 
 261. Strieker and Sanderson's Hot Stage and Gas 
 Chamber is the best for maintaining a constant tempera- 
 ture. It (Fig. 49) is a hollow flattened brass box through 
 which hot water flows by the 
 tubes i and 0. There is a 
 central cell similar to c in Fig. 
 48, with a thermometer (/) 
 coiled round it. Two tubes, 
 
 a and b, for gases, COmmuni- Fig. 49.— Strieker and Sanderson's 
 
 cate with the cell. Recently ot stage * 
 
 Dr. Sanderson has adapted to this stage the wire-heating 
 apparatus shown in Fig. 48, so that the same stage may be 
 heated by the wire or by hot water — a great convenience.* 
 
 262. The simplest arrangement for supplying a stream 
 of hot water at a constant temperature is that (Fig. 50) de- 
 vised by Mr. Schaefer (Quarterly Microscop. Journ., vol. xiv. 
 p. 349). a represents a vertical section of the cell in the 
 stage, e e is an indiarubber tube for the circulation of 
 water through the stage from the reservoir B. The water 
 is heated by a gas-jet, the size of which is regulated by the 
 expansion of mercury in the apparatus A. When the stage 
 has attained the desired temperature, the mercury A is 
 elevated by the screw S until it touches the metal tube,/! 
 The gas can then only issue through the lateral slit in the 
 tube, so that the jet is reduced to a very small size. It 
 readily enlarges, however, the moment the mercury falls.f 
 
 These forms of the hot stage are only adapted for keep- 
 
 * This form of the stage is made by Hawksley, Oxford Street, 
 London. 
 
 + This apparatus is made by Casella, 147 Holborn Bars, London. 
 
148 
 
 PRACTICAL HISTOLOGY. 
 
 ing small objects at a constant temperature. Strieker 
 and Sanderson have described a larger stage, whereby such 
 an object as the omentum of the guinea-pig may be kept 
 
 Fig. 50. — Schaefer's apparatus for heating warm stage. 
 
 at a constant temperature for the study of the circulation. 
 (See Quarterly Mia-oscofi. Journ., Oct. 1870.) 
 
 263. Application of Electricity. — Various compli- 
 cated arrangements have been devised for the transmission 
 of electricity through microscopic objects. The following 
 simple arrangements, however, answer the purpose perfectly. 
 Take a slip of glass 1x5 inches, and of the thickness of an 
 
 Fig. 51. — Gold-leaf electrodes. 
 
 ordinary slide.* Cover one surface with a thin layer of 
 gum-arabic or gold size ; press the moist surface firmly 
 down on gold-leaf ; allow it to dry ; scrape away the gold- 
 leaf, so as to leave two triangles, e e (Fig. 51), with their 
 
 * An ordinary slide might be used for these electrodes, but it is 
 convenient to have the slide so long that its ends project beyond the 
 stage. 
 
HISTOLOGICAL METHODS. 149 
 
 apices pointing towards each other at the centre of the 
 slide, leaving an interval of about a tenth of an inch between 
 them (a) for the reception of the object, which is covered 
 with thin glass in the usual way. 
 
 Electricity may be readily transmitted through the 
 electrodes by clamping the slide at each end with an 
 ordinary brass mounting clip (c c), round which thin wire 
 is twisted. If induced electricity be employed, the wires 
 are attached to the secondary coil of Du Bois Reymond's 
 induction apparatus, with a key interposed in the secondary 
 circuit ; while in the case of voltaic electricity, the wires are 
 attached to the poles of one or more voltaic cells, with a 
 key introduced into the circuit. 
 
 METHODS OF STUDYING LIVING TISSUES. 
 
 264. In studying the normal characters of living tissues 
 when removed from the body of an animal, they are either 
 examined in the fluids that bathe them during life — e.g., 
 blood, salivary, mucus, and pus corpuscles are investigated 
 in their own fluids, — or the tissues are studied in other 
 fluids calculated to produce little or no change in their 
 vital properties. Such fluids are termed " neutral " or 
 " indifferent." 
 
 265. Serous Fluids. — The fluid which bathes most 
 tissues is lymph. Blood serum — seeing that it has a com- 
 position resembling that of lymph — is sometimes employed 
 for moistening living tissues ; but the blood corpuscles 
 that it contains are an inconvenience. Aqueous humour 
 is the most available serous fluid when only a small quantity 
 is desired. It can always be readily obtained from the eye 
 of a sheep or ox. It is somewhat difficult to puncture the 
 cornea without losing the aqueous humour. Beer's knife 
 (Fig. 57) is suitable for the purpose. 
 
 266. Serous fluids are also employed in the examination 
 of non-living tissues when these are prone to undergo 
 change — e.g., nerve fibrils, red marrow cells, spleen pulp, 
 etc. When they are required in considerable quantity, 
 amniotic fluid, pericardial fluid, iodised serum, or dilute 
 
i5o 
 
 PRACTICAL HISTOLOGY. 
 
 albumin, are employed. These may be preserved for a 
 considerable time by placing them, together with a piece of 
 camphor, in glass vessels, which have been thoroughly 
 cleansed by boiling water, to destroy bacterial germs or 
 other causes of putrefaction. 
 
 267. Iodised serum is thus prepared : — Add iCC tincture 
 of iodine and one or two drops of carbolic acid to 100CC 
 
 fresh amniotic fluid, and filter. 
 
 268. Dilute Albumin should only be had recourse to 
 when serous fluids cannot be obtained. It is thus pre- 
 pared : — Dissolve 2 grammes dried sodium chloride in 
 250CC water; add 28 grammes egg albumin, 2*3CC 
 tincture of iodine, two or three drops carbolic acid. Mix 
 thoroughly, and filter. 
 
 269. Salt Solution is a J per cent solution of common 
 salt prepared thus : — Heat sodium chloride to redness, cool 
 it over sulphuric acid, and dissolve 7*5 grammes in 1000CC 
 distilled water. This fluid, though often termed indif- 
 ferent, is not in reality so. It is very convenient, but a 
 
 serous fluid is always to be preferred when indifference is 
 really desired. 
 
 270. Moist Chambers. — Living tissues require to be 
 
 kept moist. For this purpose 
 they are placed in a chamber, 
 the air of which is kept satu- 
 rated with moisture. 
 
 Schultze's modification of Reck- 
 linghausen's moist chamber is a well- 
 known form of this apparatus (Fig. 
 52). It is a glass cylinder — similar 
 to the lower part of an ordinary 
 lamp-chimney — fixed to a plate of 
 glass. The object is laid under a 
 cover-glass ; the air of the chamber 
 is kept moist with strips of wet 
 blotting-paper. The tube of the 
 microscope fits into the cylinder, 
 Fig. 52.— Recklinghausen and Schultze's and evaporation is prevented by a 
 moist chamber. piece of soft lgather interposed be . 
 
 tween the two. In the original chamber devised by Recklinghausen, 
 a piece of thin caoutchouc was tied round a simple glass cylinder 
 
HISTOLOGICAL METHODS. 
 
 151 
 
 below, and the tube of the microscope above. This chamber is con- 
 venient enough for low, but not for high powers ; and in any case the 
 air in the chamber tends to become dry. This difficulty, together with 
 that experienced in using a moist chamber with very high powers, 
 such as the ^V objective, has been entirely overcome by Mr. Dallinger 
 and Dr. Drysdale. 
 
 271. Dallinger and Drysdale *s Moist Chamber. — This 
 apparatus {Monthly Microscop. Journ., vol. xi. p. 97), con- 
 sists of a plate of glass about a tenth of an inch thick, 
 and of a size suitable for the stage of the microscope, room 
 for its movement in all directions being allowed. (A, Fig. 
 
 Fig. 53. — Dallinger and Drysdale's moist chamber. A, Glass stage. B, Glass cell 
 with caoutchouc membrane. C, Vertical section of stage and cell, with lens. % 
 
 53). At one side of the glass plate (a) there is a rectangular 
 arm, to which a brass plate (b) with a ring is fixed. The 
 latter holds an ordinary beaker with water. The glass 
 plate being too thick for the illumination of an object with 
 a powerful achromatic condenser, a piece is cut out of its 
 centre, and thin glass cemented over the aperture (e in A 
 and in C). Moisture is continually supplied by a strip of 
 thin calico (/) spread over the plate and dipping into the 
 water in the beaker. An aperture must be made in its 
 centre about two or three times the breadth of the cover- 
 glass (g) employed. The inventors found an aperture -J-J- 
 inch for a cover-glass of £ inch work well. A thin piece of 
 caoutchouc, with an aperture in its centre, is tied over 
 a glass cylinder (B) about 1 
 
 J inch in diam. and f inch in 
 
i 5 2 PRACTICAL HISTOLOGY. 
 
 height. The cylinder is set upon the cloth, and the lens 
 (immersion) is screwed down through the aperture in the 
 elastic membrane, as represented in vertical section in C, 
 where a is the thick and e the thin glass plate of the moist 
 calico, g the cover-glass upon the object, h the glass cylinder, 
 and m the caoutchouc membrane in contact with the objec- 
 tive immersed in a drop of fluid on the cover-glass. Any 
 evaporation from the chamber is fully compensated by the 
 passage of fluid through the cloth by capillary attraction 
 below the margin of the cylinder. 
 
 272. When it is necessary to preserve the living tissue 
 at a constant temperature above that of the surrounding 
 air, a hot stage (Fig. 49) must be had recourse to, and it 
 seems likely that the moist chamber last described (Fig. 53) 
 might be placed on a hot stage of suitable dimensions, and 
 the effect of heat upon tissue-growth thus observed under 
 high powers for a lengthened period. 
 
 METHODS OF HARDENING THE TISSUES. 
 
 Some tissues are too soft, others too hard, to permit of 
 their being cut or dissected; it is therefore necessary to 
 alter their consistence. 
 
 273. Drying and Boiling were formerly much resorted 
 to, but are now rarely employed. The former may be used 
 for tendon (§ 104), when transverse sections are wanted 
 merely to show the relations of the various systems of 
 fibres ; moreover, it is sometimes advantageous to partially 
 dry a tissue that has been hardened in alcohol before at- 
 tempting to cut it. If, however, protoplasm and other soft 
 matters have not been previously hardened, they are gene- 
 rally spoilt by desiccation. Boiling is useful for hardening 
 the fibres of the crystalline lens (§ 236), and also those 
 of muscle, when coarse dissections are to be made of the 
 latter. 
 
 274. Chromic Acid is much employed as a hardening 
 agent. A dilute solution of it in water may be used alone 
 for hardening the lung, spleen, uterus, muscle, cornea, 
 
HISTOLOGICAL METHODS. 153 
 
 spinal cord, and nerve ; a dilute solution of chromic acid 
 with potassium bichromate is used for the brain, stomach, 
 and intestine. A solution of chromic acid in dilute alcohol 
 is used for the retina, cochlea, and skin (see pp. 4 and 5). 
 It is not well adapted for the liver, kidney, lymphatic gland, 
 crystalline lens, and vitreous humour. 
 
 Chromic acid hardens the tissues in from two days to 
 two months. The tissue should be cut into small pieces 
 and placed in a small quantity of fluid for the first eighteen 
 hours. This should be changed at the end of this period, 
 and a large quantity of fluid substituted. The process of 
 hardening by means of chromic acid appears to be analogous 
 to the process of tanning. The tissue becomes tough, like 
 leather. The chromic acid is removed from the fluid, and 
 possibly forms some compound in the tissue, hence a large 
 quantity of the chromic solution is necessary. The solu- 
 tion employed is always dilute ; the same object cannot be 
 attained by using a strong solution of the acid, for in that 
 case an impervious crust forms around the outer part of the 
 tissue, while the centre remains soft and becomes rotten. 
 When the tissues are sufficiently hard, they are removed 
 from the acid and placed in methylated spirit, if it be 
 necessary to preserve them until sections are made. Very 
 delicate tissues that are apt to spoil in the chromic acid, 
 e.g. the embryo, should be placed in methylated spirit ere 
 they are sufficiently hard. The spirit completes the process 
 of induration. Spirit removes much of the yellow colour 
 given to the tissues by the chromic acid. 
 
 Chromic acid is a very hygroscopic substance, and 
 therefore, as it is very difficult to keep dry, it is advisable 
 to keep it dissolved. A 1 per cent solution is most 
 convenient, and can be diluted at any time. 
 
 275. Picric or Carbazotic Acid is a hardening agent 
 introduced by Ranvier. A saturated solution made with 
 cold water is employed. The pieces of tissue must be 
 small and the quantity of fluid large. 
 
 It does not render the tissues so hard as chromic acid, 
 but at the same time its tendency to produce shrivelling is 
 less. It is recommended in § 205 as a hardening agent 
 
154 PRACTICAL HISTOLOGY. 
 
 for the salivary glands and pancreas. While hardening 
 albuminous tissues it removes calcareous matter ; on this 
 account it is useful in the preparation of bone (§ 1 1 9). It 
 gives the tissues a deep yellow tinge, which may, however, 
 be removed by immersion in water or in alcohol. In the 
 case of chromic acid the colour is much more difficult to 
 remove. 
 
 276. Miiller's Fluid is thus prepared : — 
 
 Potassium bichromate, 2*5 grammes. 
 
 Sodium sulphate, 1 gramme. 
 
 Distilled water, 100CC. 
 This fluid is very useful for hardening the liver, kidney, 
 ovary, etc. (pp. 4 and 5). It is sometimes termed Miiller's 
 eye fluid, on account of its having been originally employed 
 for hardening the retina. For this purpose, however, it is 
 inferior to a mixture of chromic acid and alcohol. After 
 the use of Miiller's fluid for a month or so, the hardening 
 is carried still farther by the employment of rectified spirit. 
 
 277. Potassium Bichromate is the hardening agent 
 in Miiller's fluid. This substance may, however, be employed 
 alone. A 1 or 2 per cent solution in water is useful for 
 hardening glands ; from two weeks to a month are required. 
 Hardening is carried still farther by immersion in rectified 
 spirit, which also removes much of the colour. 
 
 278. Alcohol is useful when rapid hardening is re- 
 quired. It acts by removing water and coagulating 
 albumin. It is one of the oldest of indurating agents. It 
 is specially useful for yellow fibro-cartilage (§ 114) and 
 gland tissues. The ordinary rectified spirit (sp. gr. 0*838), 
 and absolute alcohol are the best forms. Methylated spirit, 
 often recommended, is to be avoided. The tissues should 
 be placed first in equal parts of rectified spirit and water 
 for a day ; then in rectified spirit for forty-eight hours, and 
 finally in absolute alcohol for some hours, to complete the 
 hardening. The tissues spoil if they are allowed to remain 
 too long in the alcohol, unless they have been previously 
 hardened in chromic, picric, or osmic acids, or potassium 
 bichromate and such like. Alcohol is now rarely employed 
 alone, but it is very useful for completing the hardening of 
 
HISTOLOGICAL METHODS. 155 
 
 such organs as the liver, kidney, intestine, etc., when 
 these have been previously partially hardened by the agents 
 just mentioned. 
 
 279. Chromic Acid and Alcohol. — A solution of 
 chromic acid in dilute alcohol is prepared thus : — 
 
 Chromic acid, 1 gramme. 
 
 Water, 20CC. 
 
 Dissolve, and slowly add rectified spirit, 180CC. 
 This fluid, devised by my former assistant Dr. Pritchard, 
 is excellent for hardening the retina, membranes of the coch- 
 lea, skin, and possibly some other organs, (pp. 4, 5, and 6). 
 
 280. Sections of organs hardened in the above sub- 
 stances generally require to be rendered transparent. Gly- 
 cerine, or its diluted form — Farrants' solution, clove oil, or 
 turpentine, are commonly employed, and the tissues are 
 preserved in glycerine, Farrants' solution, or dammar. 
 
 281. Osmie Acid (Os O4) is a very valuable hardening 
 agent introduced by Max Schultze. It occurs in commerce 
 in small vitreous-like masses kept in sealed tubes. It is 
 most conveniently kept as a 1 per cent solution in distilled 
 water, preserved in well-stoppered bottles protected from the 
 light. It is excessively poisonous, and its disagreeable, 
 heavy, pungent vapour is dangerously irritating to the 
 conjunctiva and nostrils. Employed in solutions of -^ to 1 
 per cent it is of great service for hardening embryonic 
 tissues, nerve fibres, retina, connective tissue corpuscles, 
 epithelium, and epidermis, testis, etc. It fixes the tissue 
 elements without producing a granular precipitate or causing 
 shrivelling. It blackens fat and the medullary matter of 
 white nerve fibres, and it also darkens, though to a much 
 less extent, albuminous tissues. From ten minutes to two 
 days are required. The pieces of tissue should always be 
 small. After hardening they are washed in distilled water, 
 and are then placed in rectified spirit, or sections are made 
 and mounted in a saturated solution of potassium acetate, 
 or in Farrants' solution. The former is the best (Schultze), 
 for glycerine renders them too transparent. 
 
 28 1 a. Silver Nitrate, Gold Chloride, Palladium 
 Chloride, are also used for hardening tissues, but they will 
 
156 PRACTICAL HISTOLOGY. 
 
 be referred to under the head of staining agents (§§ 327, 
 
 33°>33i-) 
 
 282. Freezing is a method of very great value. With 
 
 its aid, lung, kidney, spleen, lymphatic gland, vitreous 
 
 humour, brain, etc., may all be removed from the living 
 
 animal, frozen, and sliced, within fifteen minutes. 
 
 Freezing is suitable for hardening the brain and spinal 
 cord, only when slices of the fresh tissue are desired for the 
 isolation of nerve cells, etc. These organs are best hardened 
 in chromic acid, potassium bichromate, etc., not because 
 they can be more readily cut when hardened by these 
 substances, but for the reason that, when the tissue has 
 been previously hardened by these agents, the slices do not 
 go to pieces when they are manipulated, as they are apt to 
 do when the unhardened frozen tissue thaws after freezing. 
 For the same reason, it is advantageous, in the case of the 
 kidney, lung, liver, spleen, and such like, to have the pro- 
 cess of hardening carried to some extent by Muller's fluid, 
 potassium bichromate, chromic or picric acid, before freezing 
 them for the purpose of section. The special cases where 
 freezing is of service have been indicated in the preceding 
 demonstrations. 
 
 Indeed, speaking generally, freezing is of most service 
 — 1. In enabling us to quickly harden and cut fresh tissues 
 when the permanent preservation of these is not a primary 
 consideration. 2. In completing, with a view to section, the 
 hardening of tissues already partially indurated by other 
 means ; so that — a. The process of hardening is shortened ; 
 b. The tendency to shrivelling of the tissue by the harden- 
 ing fluid is lessened, because of its shorter exposure to its 
 influence ; c. The sections, when they thaw, are still suffi- 
 ciently indurated by their treatment previous to freezing, to 
 permit of their successful manipulation and preservation. 
 Freezing may be employed after any of the hardening 
 agents described in the preceding paragraphs, but in the 
 case of alcohol, it of course must be completely removed 
 by soaking the tissue in water ere the freezing can be 
 accomplished. 
 
 Freezing has hitherto been had but little recourse to, 
 
HISTOLOGICAL METHODS. 157 
 
 owing to the clumsy and inconvenient method hitherto 
 adopted. This has simply consisted in placing the tissue 
 on tinfoil, or in a platinum capsule, and setting it in a 
 freezing mixture. It is very difficult and tedious to freeze 
 any large piece of tissue in this way, and when it is removed 
 from the freezing mixture, for the purpose of section, it 
 begins to thaw, and thereby gives rise to so much incon- 
 venience, and to results so unsatisfactory, that the method 
 of freezing has been almost neglected. The method is now, 
 however, rendered simple and satisfactory with the aid of 
 the freezing microtome (Fig. 58). It is convenient to study 
 its mode of employment in § 302. 
 
 METHODS OF SOFTENING THE TISSUES. 
 
 283. Removal of Calcareous Matter. — Chro??iic and 
 nitric acid both harden albuminous tissues, but soften those 
 which owe their hardness to the presence of calcareous 
 salts. Bone and tooth may be softened in the following 
 fluid :— 
 
 Chromic acid, 1 gramme. 
 
 Water 200CC. Dissolve and add 
 
 Commercial nitric acid, 2CC. 
 This chromic and nitric fluid gives far better results than 
 dilute nitric or hydrochloric acid used alone, for while the 
 nitric acid more especially removes the calcareous matter, 
 the chromic acid especially hardens the bone protoplasts. 
 This fluid produces excellent results. For details, see § 15. 
 Small pieces of bone may also be softened in a 
 \ per cent solution of chromic acid, or in a solution of 
 picric acid, kept saturated by the presence of superabundant 
 crystals. The latter is preferable. This method is useful 
 for fcetal bone (§11 9). 
 
 284. Softening of Connective Substances. — Dilute 
 alcohol (rectified spirit * 1 part, water 2 parts) is of much 
 
 * Ranvier recommends alcohol at 36 of Cartier ; this is very 
 nearly the strength of ordinary rectified spirit, the sp. gr. of which is 
 0-838. 
 
158 PRACTICAL HISTOLOGY. 
 
 service for softening white fibrous tissue (Ranvier), when, 
 e.g., it is desired to separate muscular fibres. It also 
 softens the cementing matter between epithelial cells, and 
 thus permits of their ready isolation. 
 
 285. Boiling — by converting white fibrous tissue into 
 gelatine — is useful for permitting of the isolation of mus- 
 cular fibre in the making of coarse dissections. 
 
 286. Dilute sulphuric acid. — White fibrous tissue is 
 rapidly dissolved by maceration, at a temperature of 46°C, 
 in dilute sulphuric acid (commercial acid 1 part, water 
 1000 parts). This method is very useful in making coarse 
 dissections of muscle. The cement connecting the fibres 
 of the lens may be dissolved by boiling for five or ten 
 minutes in dilute sulphuric acid (1 per cent). 
 
 287. Acetic acid and glycerine (glycerine 1 ounce, 
 glacial acetic acid 5 drops) is recommended by Beale for 
 softened white fibrous tissue, in tracing the course of nerve 
 fibres with high powers. The white fibrous tissue is ren- 
 dered very transparent and gelatinous, while mejdullated 
 nerve fibres are rendered granular. In this way the nerve 
 elements are readily recognised. This mode of tracing 
 fine nerves has now, however, been largely superseded by 
 the method of staining them with chloride of gold. 
 
 288. Iodised serum (§267) is recommended by Schultze 
 for the maceration of white nerve fibres. The cement be- 
 tween the fibrils is softened and dissolved. 
 
 289. Chromic acid, potassium bichromate. — Although 
 these substances have already been described as hardening 
 agents, they are also used for softening connective substances. 
 \ per cent solution of chromic acid softens the material at 
 the sides of the prisms of striped muscle, and so facilitates 
 cleavage into fibrillae (§ 141). 
 
 A very dilute solution of chromic acid (one or two parts 
 to 10,000 of water) is also an excellent macerating agent 
 for the separation of epithelial cells. So also is a very dilute 
 solution of potassium bichromate (one or two parts to 
 1000 of water). These very dilute fluids should have a 
 few drops of carbolic acid added to prevent putrefaction of 
 the tissue. 
 
HISTOLOGICAL METHODS. 159 
 
 28 9 a. Method of Digestion. — Artificial digestion in 
 a fluid containing pepsine and hydrochloric acid has been 
 used for causing striped muscle to cleave into discs. It may 
 also be employed for softening and clarifying white fibrous 
 tissue. The manner in which it may be applied to the skin 
 in studying the arrangements of its elastic fibres is detailed 
 in § 223 b. 
 
 MECHANICAL DISSOCIATION OF TISSUE ELEMENTS. 
 
 290. Tissue elements may be separated by various 
 mechanical methods. 
 
 a. By dissection with needles. — Those most suitable are 
 thick sewing needles fixed in strong handles (Figs. 54 and 34). 
 
 Fig. 54. — Dissecting needle. 
 
 Delicate textures and organs should be dissected under 
 fluid. 
 
 b. By shaking. — This is a most useful method of 
 dissociation, e.g., the lymph corpuscles may be readily 
 freed from the adenoid tissue in a section of lymphatic 
 gland in this way (§11 o). The shaking is performed in a 
 test tube containing some desirable fluid. 
 
 c. By pressure under the cover-glass, one is often able 
 to separate tissue elements so as to display them effectively. 
 The mode of demonstrating nerve cells in the fresh brain, 
 detailed in § 247, c, is a good example of this method, and 
 there are many others with which every one who works with 
 the microscope for some time, soon becomes familiar. 
 
 It will be readily understood that the various fluids for 
 softening connective substances facilitate the mechanical dis- 
 sociation of tissue elements. 
 
 METHODS OF MAKING SECTIONS. 
 
 291. Saw. — Sections of unsoftened bone are made with 
 a fine saw. The scratches thereby produced, are got rid of, 
 and the sections at the same time ground sufficiently thin 
 upon a stone, after the manner already described (§ 118). 
 
i6o 
 
 PRACTICAL HISTOLOGY. 
 
 292. Scissors are in almost constant requisition for 
 removing small portions of soft tissues for microscopical 
 preparation. The most convenient form is that shown in 
 
 Fig- 55- 
 
 Fig. 55. — Fine scissors for microscopical purposes. 
 
 293. Valentin's Knife was formerly much employed 
 for making thin slices of organs, such as the kidney, lung, 
 stomach, etc., in the fresh condition. It consists (Fig. 56) 
 of two parallel blades. In using the knife, these are first 
 clamped in close apposition by the wedge a. They are 
 then opened to any desirable distance by the screw b, and 
 are, of course, fixed by the forces of the wedge* and the 
 screw acting in opposite directions. The thickness of the 
 
 Fig. 56. — Valentin's knife. 
 
 slice depends on the distance between the blades. The 
 sections obtained by this knife are far inferior to those made 
 with a freezing microtome, but nevertheless it is useful when 
 the microtome cannot be employed. 
 
 294. Single-bladed knives. — A razor and a scalpel 
 are the knives most commonly used for cutting any tissue 
 which by softening or hardening has been brought to a suit- 
 able consistence. The " army razors," made by Heifor of 
 Sheffield, are the most convenient for all ordinary purposes. 
 They are made of good steel — a most essential point — and 
 their blades are thin and broad enough to give good sections. 
 Similar razors, made for section-cutting by Weiss of London, 
 
HISTOLOGICAL METHODS. 161 
 
 are still better. They are, however, only suitable for making 
 sections of tissue held in the hand, their blades being too 
 thin for use with a microtome. For ordinary purposes it 
 is not necessary to have the razor ground flat on one side 
 — (the right when the face is down). It does well enough 
 to have the blade concave on both sides, provided it be 
 broad enough to receive the whole section ere it come 
 against the thick back of the blade. If one attempt, how- 
 ever, to make large sections without the aid of a machine, 
 then a long broad-bladed knife, flat on both sides is neces- 
 sary, but it is only a waste of time to attempt anything of 
 the sort without a microtome for the purpose of keeping the 
 tissue fixed and the blade gliding steadily and evenly during 
 the process of section (see § 302). 
 
 Beer's knife is a useful instrument for puncturing the 
 cornea when a drop of aqueous humour is wanted. An 
 ordinary scalpel is not suitable for this purpose. Beer's 
 knife has a thin triangular blade tapering to a fine point. It 
 may also be used for making sections in place of a scalpel. 
 
 Fig. 57. — Beer's knife. 
 
 295. Wetting the Knife. — The knife must always be 
 wet, in order that the slice of tissue may float over its surface, 
 a Water, b salt solution, c methylated or rectified spirit, d 
 absolute alcohol, are used for this purpose. Of these, c and 
 d are the best, but they are only to be used in cases where 
 spirit may be added to the tissue without altering it — e.g., 
 when the tissue has been hardened in spirit, Mutter's fluid, 
 or chromic acid. 
 
 296. Hand-sections. — If the tissue and the knife be 
 both simply supported by the hands, the sections are, for 
 convenience, often termed hand-sections. The tissue is 
 held between the thumb and fore and middle fingers of the 
 left hand, and the forefinger is used as a table for support- 
 ing the back of the knife. The knife should never be held 
 like a pen, and it should never be pushed hut always drawn 
 from heel to point obliquely through the tissue. The sec- 
 
 M 
 
1 62 PRACTICAL HISTOLOGY. 
 
 tions should always be manipulated with camel-hair pencils. 
 Much practice is required before dexterity is attained, but 
 it is very important to thoroughly practise this mode of 
 section ; for, when only small slices are required, they can 
 be made very perfectly in this simple way. 
 
 297. Imbedding. — The piece of tissue is often so 
 small that it is impossible to hold it in the hand, and it is 
 sometimes so brittle that it is apt to go to pieces when cut. 
 To overcome these difficulties, it is necessary to imbed the 
 tissue, so as to support and hold it firm during the section. 
 
 298. Imbedding in Carrot. — A carrot is an excellent 
 imbedding agent. For holding in the hand a piece of 
 softened bone or hardened spinal cord, etc., it is extremely 
 convenient. A hole rather smaller than the organ should 
 be scooped in the carrot, near its centre. An incision 
 should then be carried from the hole to the periphery, so 
 that the hole may be enlarged for the admission of the 
 organ, round which the carrot closes like a hollow spring. 
 The carrot does not cling either to the knife or to the texture; 
 and the knife is wetted at every slice by passing through the 
 can'ot. It is not suitable, however, for delicate tissues. 
 
 299. Imbedding in elder pith is recommended by 
 Ranvier. A hole somewhat larger than the tissue is made 
 in the pith, and the piece of tissue — e.g., a hardened nerve 
 — is placed in it. The whole is then immersed in water, 
 to cause the pith to swell up and clasp the tissue firmly. 
 
 300. Imbedding in Paraffin. — Paraffin is the imbedding 
 agent most commonly employed. Ordinary solid paraffin 
 is too hard. Five parts of it should therefore be mixed 
 with one part of hog's lard with the aid of a gentle heat. 
 The addition of a little clove oil renders the mixture less 
 liable to adhere to the knife. A mixture of equal parts of 
 bees'-wax and sweet oil is often recommended, and no 
 doubt it answers very well for hand sections ; but it is not 
 so suitable as paraffin for imbedding in a microtome, be- 
 cause the wax and oil, having a higher melting point than 
 the paraffin mixture, contracts so much when it cools, 
 that it separates from the metal around the well of the 
 microtome. 
 
HISTOLOGICAL METHODS. 163 
 
 Melt the paraffin mixture in a water bath, at as low a 
 temperature as possible, pour it into a paper cone or paper 
 saucer, or into the well of a microtome. With forceps, dip 
 the tissue into the paraffin, at once remove it, and, when 
 the layer of paraffin has cooled, replace it finally in the 
 paraffin. By this means the overheating of the tissue will 
 be avoided. Tissues to be imbedded in paraffin should be 
 as dry as possible, otherwise the supporting substance will 
 not cling to the tissue. The water may be removed from 
 the surface of the tissue by immersing it for a short time in 
 spirit, and then allowing it to dry. 
 
 The great fault of paraffin is, that its slices get in the 
 way, and often spoil the slice of the tissue while it is being 
 made ; moreover, it is difficult to get rid of. The sections 
 require to be picked out from its debris, or this must be 
 washed away from them. This implies considerable loss of 
 time. 
 
 301. Imbedding in Gum. — a. This method, which we 
 owe to Briicke, is a very valuable one. The solution of 
 gum should be as thick as possible. It is made by pouring 
 cold water on " picked " {clean) gum-arabic, and stirring it 
 occasionally. It is extremely useful for imbedding the lung, 
 and other tissues of irregular configuration, where an- inter- 
 stitial and not merely an external support is necessary. 
 Any alcohol i7i the tissue must always be carefully removed 
 by immersion in water for twenty-four hours or so previous 
 to placing them in the gum. 
 
 b. For imbedding the lung, a small piece that has been 
 previously hardened by chromic acid in the usual way, and 
 from which all spirit has been removed, is placed for thirty- 
 six or forty-eight hours in a thick solution of gum. It is 
 then transferred to methylated spirit, in which it soon be- 
 comes sufficiently hard to permit of easy section. 
 
 c. For the retina, cochlea, small intestine, and other 
 parts with delicate tissues needing careful support, the 
 following method is used : — Place the tissue in a cone of 
 bibulous paper containing thick solution of gum for six to 
 twenty-four hours. Then set the cone with its contents in 
 methylated spirit for forty-eight hours or so, to remove the 
 
164 
 
 PRACTICAL HISTOLOGY. 
 
 water and so stiffen the gum. Lastly, remove the paper, 
 imbed in paraffin, and make sections. 
 
 d. The gum may be readily removed from the sections 
 by immersion for some time in water. 
 
 302. Microtome. — In making hand-sections with such 
 a knife as a razor, success depends on the steadiness of the 
 operator. It is, however, always difficult to hold the tissue 
 and the knife perfectly steady, and when a large section is 
 wanted — say of kidney, spinal cord, or brain — a great many 
 
 Fig. 58. — The freezing microtome. 
 
 sections have to be thrown away as imperfect. This diffi- 
 culty is overcome by employing a microtome. Various 
 microtomes have long been in use. These, however, it is 
 unnecessary to describe, for the freezing microtome invented 
 by the author presents advantages which were not possessed 
 by any previous instrument. 
 
 The freezing microtome (Fig. 58) consists of a plate of 
 gun-metal (b), with a circular opening in its centre. The 
 opening leads into a well (a), closed inferiorly by a brass 
 
HISTOLOGICAL METHODS. 
 
 165 
 
 plug (k, Fig. 59), capable of being moved up or down by 
 means of a screw (d). The tissue to be frozen is placed in 
 the well, and sections are made by gliding a knife through 
 the tissue that projects above the level of the brass table. 
 
 Fig. 59. — Vertical section of freezing microtome. R, Transverse section of the 
 
 knife employed. 
 
 The thickness of the sections is regulated by an indicator 
 (e). A freezing mixture is placed in the box (c), the water 
 from which flows away by the tube (h). The microtome 
 is clamped to a table by the screw (f). The tissue is seen 
 in a, Fig. 59, and the freezing mixture in g of the same 
 figure. In the instruments now made, the brass box is 
 covered with gutta-percha. 
 
 The freezing microtome is essentially the well-known 
 microtome devised by Mr. Stirling, Sub-Curator of the Ana- 
 tomical Museum of the University of Edinburgh, modified 
 for freezing. It serves a double purpose. 1. It serves the 
 same purpose as the microtome of which it is a modifica- 
 tion ; that is to say, it may be used for cutting unfrozen tissues 
 imbedded in paraffin in the usual way. 2. For cutting 
 tissues hardened by freezing. The second method of using 
 
1 66 PRACTICAL HISTOLOGY. 
 
 the machine will be more readily comprehended after a 
 description of the first 
 
 303. When the microtome is used without freezing, the 
 tissue may be imbedded in paraffin or in carrot. 
 
 a. When paraffin is employed the temperature at which 
 the paraffin is melted should be just sufficient for the pur- 
 pose and no more, in order that the tissue may be heated 
 as slightly as possible, and also in order that the paraffin 
 may, on cooling, shrink to the smallest possible extent. 
 With a pair of forceps dip the tissue — previously exposed 
 to the air for a short time in order to dry its surface — into 
 the melted paraffin, at once withdraw it, and, when the 
 paraffin crust has cooled, replace it permanently in the well 
 of paraffin, close to the margin from which the face of the 
 knife is to moved in the process of section. The side 
 nearest the operator is most convenient. A mixture of 
 equal parts of bees'-wax and sweet oil is much recommended 
 for the same purpose as the paraffin in the above case. 
 Certainly the wax and oil mixture is excellent for imbedding 
 tissues, and it can be cut with ease. But it melts at a 
 higher temperature than paraffin, and, owing to the great 
 thermal expansion, it retracts from the side of the tube of 
 the machine, and so the wax cylinder becomes loose. The 
 only way in which this can be prevented is by heating the 
 machine to a like temperature before introducing the wax. 
 This is tedious ; and, inasmuch as it is unnecessary in the 
 case of paraffin, this is to be preferred. Even with this, 
 however, the paraffin cylinder is apt to become a little loose, 
 and to turn round in the machine : hence it is important 
 that there be an eccentric hole in the brass plug (k, Fig. 59) 
 with a projecting wooden pin fixed in it. The rotation of 
 the paraffin cylinder is thus prevented. 
 
 b. When carrot is used a cylinder of it to fit the well of 
 the microtome must cut with a cork-cutter, and the tissue 
 imbedded as already described in § 298. 
 
 304. The knife must have a back with straight edges, so 
 that it may not tilt in the process of section. The surface 
 that rests on the plate should always be hollow, and it is 
 convenient to have the upper surface the same (r, Fig. 59), 
 
HISTOLOGICAL METHODS. 167 
 
 unless the sections be very large, when it should be flat or 
 slightly convex. The concavity is convenient for carrying 
 a pool of spirit, over which the section floats ; the. floating is 
 essential. It is convenient to keep the back of the knife 
 towards the operator, and either to draw it steadily through 
 the tissue from heel to point, or to push it in the opposite 
 direction. The blade of the knife should not be too thin, 
 otherwise it tends to bend into the well. A considerable 
 amount of dexterity is required, but this is soon gained by 
 practice. 
 
 305. When the microtome is used for freezing , the freez- 
 ing box requires to be covered with a non-conductor, such 
 as a thick jacket of felt, flannel, or gutta-percha. The 
 latter is much to be preferred, and an instrument having 
 this covering should be obtained. The plug (k, Fig. 59) 
 is unscrewed and oiled to prevent its becoming fixed during 
 the freezing. The tissue and an imbedding fluid are placed 
 in the well. If a watery fluid be employed, it becomes 
 crystalline when frozen, and is therefore badly adapted to 
 serve as the imbedding agent. 
 
 306. The difficulty is, however, entirely overcome by 
 the method of imbedding in gum (§ 301), as suggested to 
 me by my late assistant, Dr. Pritchard. A thick solution of 
 gum when frozen does not become crystalline, and can be 
 cut like cheese. If there be any alcohol in the tissue it 
 must be thoroughly removed by immersion in water for six 
 to twenty-four hours, according to the size of the tissue, 
 before it is placed in the gum. 7/ is always advantageous 
 to allow the tissue to soak in the gum for twelve to twenty- 
 four hours before freezing, in order that the gum may permeate 
 every part of the tissue and p?-event the formation of a crystal- 
 line co?idition within the frozen tissue. Thick gum solution 
 is poured into the well, and, when a film of ice has formed 
 at the periphery, the tissue should be introduced and held 
 against the advancing ice until it is fixed. In this way the 
 tissue may be secured in any position for the process of 
 section. A plate of cork with a weight on it is placed on 
 the microtome plate to prevent the entrance of heat, and 
 also of salt from the freezing mixture. 
 
168 PRACTICAL HISTOLOGY. 
 
 307. Small quantities of finely powdered ice and salt 
 are placed alternately with the aid of a small spoon in the 
 freezing box, and are stirred round the well, the tube H 
 being carefully kept open to allow the water to escape from 
 the melting ice. When snow can be obtained, it is, of 
 course, used instead of ice. The powdering of the ice is 
 rather tedious, but it may be readily done in that form of 
 sausage machine where two cylinders with spiral grooves are 
 rolled against one another. Before putting the ice into 
 this apparatus it should be wrapped in a thick cloth and 
 broken with a wooden mallet. The freezing process is 
 really very simple, and can be fully carried out in from ten 
 to twenty minutes. Of course the period is shorter the 
 colder the surrounding air. 
 
 308. It is not necessary to wet the knife, for this is 
 thoroughly done by the thawing gum. The sections often 
 contain a number of air bubbles ; these, however, together 
 with the gum, are soon removed by placing them in dilute 
 alcohol (rectified or methylated spirit 1, water 2 parts)* 
 If the sections are to be preserved for a time until they are 
 examined or mounted, they are transferred from the dilute 
 alcohol to rectified spirit. When sections are apt to be 
 spoilt by manipulation, they should be placed on the slide before 
 they thaw. They can then be readily washed by making a 
 pool of dilute alcohol around them, which may be changed 
 two or three times. 
 
 309. The more one employs the freezing microtome, 
 the more clearly does one perceive the great assistance 
 which it offers to the histologist. A number of tissues may 
 be frozen and cut at the same time. Delicate organs, such 
 as the retina, the embryo, villi of the intestine, lung, trachea 
 with its ciliated epithelium, may all be readily cut without 
 fear of their being destroyed by the imbedding agent, as is 
 apt to be the case when paraffin, pith, and carrot are em- 
 ployed. The cases to which freezing is applicable have 
 already been detailed in § 282. It must be carefully borne 
 in mind, however, that in the case of a tissue saturated with 
 water spicules of ice are formed, and after section various 
 crevices may sometimes be seen which have resulted from 
 
HISTOLOGICAL METHODS. 169 
 
 these spicules, or from the laceration of the tissue due to 
 the knife having come in contact with them. This difficulty 
 is overcome by a thorough saturation of the tissue with gum 
 previous to freezing, as described in § 306. 
 
 310. Alterations of the freezing microtome. — The freezing 
 microtome was invented and first described by me in the 
 Journal of Anatomy and Physiology, May 1871. The 
 instrument there figured was simply a Stirling's section- 
 cutter, to which I adapted a freezing box. I soon found, 
 however, that the box was too small, the well too large, and 
 the screw imperfect. I therefore altered the apparatus, and 
 described the new form (Fig. 58) in the Lancet (July 1873). 
 The only further alteration that I have made is to have the 
 freezing box entirely covered with gutta-percha, the escape 
 tube (H, Fig. 58) made larger, and the cutting-table made 
 longer between the well and the end nearest the operator. 
 These are real improvements, which make the instrument 
 work in a most satisfactory manner.* At the same time, 
 it may be stated that the identical instrument described in 
 the Lancet has been in daily use in my laboratory for three 
 years. We use no other, because it really has always 
 answered the purpose remarkably well. It has often been 
 suggested that the freezing box should project at the right 
 as well as at the left side of the machine. This arrangement 
 I have carefully avoided from the first, because it interferes 
 with the hand in using the knife, and certainly it is not 
 necessary. 
 
 METHODS OF INCREASING THE TRANSPARENCY OF 
 
 THE TISSUES. 
 
 311. The transparency of the tissues may be increased 
 — 1. By impregnating them with fluids which strongly 
 refract light, e.g. glycerine, turpentine, clove oil, Canada bal- 
 
 * The instrument thus modified is made by Mr. Gardner, instru- 
 ment maker, South Bridge, Edinburgh, and I can no longer recommend 
 the instruments of other makers, for they have shown themselves much 
 more disposed to supply the instruments for which they happen to have 
 had " castings " made than to adopt my suggestions". 
 
170 PRACTICAL HISTOLOGY. 
 
 sam, dammar. 2. By partially or completely dissolving 
 certain elements of the tissues, so as to permit of others 
 being seen. Acetic acid, caustic potash or soda, are used for 
 this purpose. These partially or completely dissolve the 
 soft albuminous parts of most tissues. 
 
 312. Glycerine and acetic acid may be used together as 
 recommended by Beale. (Glycerine 1 oz., glacial acetic 
 acid 5 drops.) 
 
 313. Acetic acid. — The acid most commonly employed 
 is the ordinary pyroligneous acid. It contains 28 per cent 
 of anhydrous acid (or 33*3 per cent of glacial acid). It is 
 important for ordinary purposes not to employ the acid in 
 too concentrated a form. Pyroligneous acid 1 part, water 
 2 parts, is the dilution most useful for ordinary purposes, 
 but so great a dilution as 4 or 5 drops to an ounce of 
 water is sometimes very "serviceable, e.g. for showing the 
 nerve terminations in striped muscle (§ 160, Kolliker) and 
 for slowly softening white fibrous tissue. 
 
 314. Caustic potash is sometimes used for rendering 
 albuminous tissues transparent, and for softening connecting 
 substances. A 30 per cent solution of the caustic alkali 
 in distilled water is the most generally useful solution 
 (Moleschott). Ewart and Thin {Journ. of Anatomy and Physi- 
 ology, vol. x. p. 223) have recently recommended a solution 
 of caustic potash in an equal weight of distilled water as a 
 means of preparing the fibres of the crystalline lens. 
 
 315. Glyceri?ie, acetic acid, and potash, being all miscible 
 with water, may be employed to clarify tissues from which 
 water has not been removed. Oil of cloves, or other essen- 
 tial oil, turpentine, creosote, carbolic acid, render most tissues 
 transparent. As these do not mix with water, it must be 
 previously removed by drying the tissue, or by immersion 
 in alcohol, and then allowing the alcohol to escape. 
 
 316. Tissues hardened in chromic or picric acid, and 
 in potass, bichrom., are rendered somewhat opaque, and 
 must in general be clarified. This is usually done by 
 adding a, clove oil ; b, turpentine ; c, glycerine. These 
 fluids have different powers — a is stronger than b, and b is 
 stronger than c. Glycerine is employed for tissues that 
 
HISTOLOGICAL METHODS. 171 
 
 must not be made too transparent, e.g. unstained spleen, 
 alimentary canal, lung, liver. Generally speaking, clove oil 
 and turpentine are too powerful as clarifying agents for 
 tissues not stained with such dyes as carmine and logwood. 
 When glycerine is used, the tissue is preserved in it. 
 When clove oil or turpentine is employed, the tissue is 
 preserved in dammar or Canada balsam. 
 
 METHODS OF STAINING THE TISSUES. 
 
 317. In staining the tissues, there are two points to be 
 especially borne in mind. 1. The staining fluid may affect 
 some parts of the tissue more than others, or it may stain 
 some parts, and others not at all ; e.g. the nucleus is more 
 deeply tinged with carmine or magenta than the surrounding 
 protoplasm, epithelial cement is deeply stained by silver 
 nitrate, nerve fibrils by gold chloride. 2. The staining 
 fluid may affect the tissue with tolerable uniformity, and 
 prove serviceable by merely rendering very transparent 
 colourless parts more evident. 
 
 318. Carmine. — The use of this valuable dye for 
 histological purposes was first recommended by Gerlach. 
 It tinges protoplasts, the axial cylinders of white nerve 
 fibres, but not the modified protoplasm of which cilia and 
 muscular fibre consist. It is useful to have two fluids. 
 
 a. Weak carmine fluid, an exceedingly good carmine 
 fluid for general purposes, is a modification of that proposed 
 by Beale. Beale's fluid is thus prepared : — 
 
 Carmine . . .10 grains. 
 
 Strong ammonia . . 30 minims. 
 
 Glycerine ... 2 ounces. 
 
 Rectified spirit . . J ounce. 
 Place the carmine in a test tube, add the ammonia, 
 boil for a few seconds, let the solution stand for an hour 
 with the test tube open, add the water, filter, add the spirit 
 and the glycerine, and allow the mixture to stand exposed 
 to the air until the odour of ammonia is scarcely percep- 
 tible, then keep it in a stoppered bottle. This fluid is, 
 owing to its density, slow in its action, and, although it 
 
172 PRACTICAL HISTOLOGY. 
 
 may yield excellent results, it is very uncertain in its opera- 
 tion. Tardiness and uncertainty of action may be avoided 
 by retaining all the ammonia in the fluid, and by diluting 
 the fluid when it is to be used. Therefore, prepare the 
 fluid thus : — Place the carmine in a test tube, add the 
 ammonia, bring to the boiling point, add the water and 
 filter, then add the spirit and glycerine, and preserve in a 
 stoppered bottle. When about to be used it should be 
 diluted from two to seven times with water. The last is 
 the most generally useful dilution. The fluid should always 
 be filtered after dilution, and the tissues placed in a large 
 quantity of it from 2 to 24 hours. This fluid is therefore 
 not at all suitable for rapid staining, but carmine staining 
 is generally most beautiful when it takes place slowly. 
 
 b. Strong carmine fluid. — It is useful to have a strong 
 carmine fluid for rapidly staining nerve tissues. It is thus 
 prepared : — 
 
 Carmine 1 gramme. 
 
 Strong ammonia . . iCC. 
 Dissolve in a test tube, with the aid of a gentle heat, and add 
 
 Distilled water . ' . 23CC. 
 Filter and preserve in a stoppered bottle. For ordinary 
 purposes it tinges too rapidly, and for tissues unhardened 
 in chromic acid or Miiller's fluid it contains too much 
 ammonia. Therefore, should this fluid be used for staining 
 in the ordinary way, expose it in a beaker to the air for 
 two days, to let the excess of ammonia escape, then dilute 
 it with five times its bulk of distilled water, filter, and pre- 
 serve in a stoppered bottle. 
 
 319. Carmine staining with palladium, — When tissues, 
 and especially nerve tissue, have been overhardened in 
 chromic acid, carmine may entirely fail to stain them. In 
 such a case the following method (Merkel) is of much service. 
 Place a large drop of \ per cent watery solution of palladium 
 chloride on a slide, and on another slide a large drop of a 
 strong ammoniacal solution of carmine (§ 318, b). Allow 
 the section to remain in the palladium for about two 
 minutes. Wash it in water and place it in the carmine 
 fluid for about three minutes. Then wash in water. 
 
HISTOLOGICAL METHODS. 
 
 173 
 
 320. Carmine is, however, a somewhat uncertain dye, 
 and it is often difficult to get good results, especially with 
 tissues hardened in chromic acid. This substance, together 
 with picric acid, potassium bichromate, and Muller's fluid, 
 must always be removed by washing in water previous to 
 staining. 
 
 321. After staining, the superfluous pigment is removed 
 by washing in water acidulated with 1 per cent hydro- 
 chloric or glacial acetic acid, or in rectified spirit 60 parts, 
 water 39 parts, hydrochloric acid 1 part {Pritchard). The 
 acid brightens the colour. 
 
 Tissues stained with carmine may be mounted in Far- 
 rants' solution, glycerine, or dammar. 
 
 322. Picro- carmine. — The picro-carminate of am- 
 monia introduced by Schwarz and Ranvier, is an excellent 
 staining agent, and is prepared thus : — Take 100CC of a 
 saturated solution of picric acid. Prepare an ammoniacal 
 solution of carmine by dissolving 1 gramme in a few CC 
 water, with the aid of an excess of ammonia and heat. 
 Boil the picric acid solution on a sand-bath, and when boil- 
 ing add the carmine solution. Evaporate the mixture to 
 dryness. Dissolve the residue in 100CC water, and filter. 
 A clear solution ought to be obtained ; if not, add some 
 more ammonia, evaporate, and then dissolve as before. 
 
 A double staining is produced. Nuclei are stained by 
 the carmine, while the picric acid gives a yellowish tinge to 
 muscle, epithelium, and epidermis, etc. Sections of skin 
 may be beautifully stained by this fluid : the epidermis, 
 hair, and muscles of the hair follicles are rendered yellow, 
 while the cutis vera is reddened. If carmine staining 
 alone be desired, the stained tissues are placed in dis- 
 tilled water, by which the yellow colour from the picric 
 acid is speedily removed. 
 
 323. Logwood is, like carmine, suitable for staining 
 tissues for permanent preparations. Generally speaking, it 
 stains the same tissue elements as carmine. The tint is 
 violet, and on this account it is not nearly so fatiguing to the 
 eye as a red dye. The tissues may either be fresh, or they 
 may have been hardened in chromic acid or alcohol. It 
 
1 7 4 . PRACTICAL HISTOLOGY. 
 
 stains the nuclei of epithelium, and the protoplasts of 
 connective tissue remarkably well ; nerve cells and axial 
 cylinders, the lung, skin, cornea, tongue, etc., can be 
 beautifully tinged by this agent. It is, however, a somewhat 
 perplexing substance, on account of the difficulty expe- 
 rienced in getting a staining fluid capable of giving a pure 
 violet tinge.* It was introduced by Boehmer. Kleinen- 
 berg's receipt for the solution is the best (Foster and Bal- 
 four's Elements of Embryology, p. 248). 
 
 • A. Make a saturated solution of crystallised calcium 
 
 chloride in alcohol (70 per cent), and add alum to 
 saturation. 
 
 B. Make a saturated solution of alum in alcohol (70 
 
 per cent). 
 
 C. Add A to B in the proportion of 1 :8. 
 
 D. Make a barely alkaline saturated solution of hema- 
 
 toxylin in water. 
 
 For staining place eight or ten drops of D in a watch- 
 glass half filled with C ; allow the tissue to remain in it 
 until stained of sufficient depth ; then wash in water. 
 Diffuse staining may be removed by immersion in rectified 
 or in methylated spirit, or in a J- per cent solution of alum. 
 
 Tissues stained with logwood are mounted in Farrants' 
 solution, glycerine, or in dammar. 
 
 324. Magenta (rosaniline nitrate) is. an extremely use- 
 ful dye, because of its rapidity of action and the sharp 
 definition which it gives to objects. Like carmine, it stains 
 protoplasts, the axial cylinders of white nerve fibres, etc. 
 Unlike carmine, it fades, and is therefore not suitable for 
 permanent preparations. Nevertheless, the colour can be 
 retained for a long time by mounting the stained tissue in 
 one-third per cent watery solution of corrosive sublimate. 
 Two fluids are necessary. 
 
 a. Ordinary magenta fluid prepared thus — 
 
 Magenta crystals . . 1 Decigramme. 
 
 Rectified spirit . . 9CC. 
 
 Distilled water . . 213CC. 
 
 * A good logwood staining fluid may be obtained from Martindale, 
 chemist, New Cavendish Street, London, W. 
 
HISTOLOGICAL METHODS. 175 
 
 Dissolve the magenta in the water, and then add the spirit. 
 Preserve in a stoppered or well-corked bottle. This fluid 
 is used for the tissues generally. 
 
 b. Strong magenta fluid prepared thus — 
 
 Magenta crystals . . 1 Decigramme. 
 
 Distilled water . . 15CC. Dissolve and add 
 
 Rectified spirit . . 5CC. 
 
 Glycerine . . . 20CC. 
 This fluid, devised by my former assistant, Dr. Ferrier, 
 is used for staining blood corpuscles. Being of a specific 
 gravity, similar to that of the liquor sanguinis, the coloured 
 corpuscles of non-mammalian vertebrates alter but little in 
 shape while they become stained. 
 
 325. Aniline Blue-Black* has been recently recom- 
 mended by Mr. Sankey (Quarterly Microscop. Journ., vol. 
 xvi. p. 95) for staining the nerve cells of the brain and 
 spinal cord. The tint which it gives to them is a blue-gray. 
 He gives the following formula : — Dissolve 5 centigrammes 
 of the dye in 2CC water, pour it into 99CC methylated 
 spirit, and filter. The sections of brain stain in a few 
 minutes. The sections are clarified with clove oil and 
 mounted in dammar in the usual way. Mr. Lewis' 
 (Quarterly Microscop. Journ., p. 73) recommends deep 
 staining with an aqueous solution of the dye (^ to 1 per 
 cent), and the subsequent removal of superfluous pigment, 
 by immersion for twenty minutes or so in an aqueous solu- 
 tion of chloral hydrate (1 to 10 per cent). The chloral is then 
 removed by washing in water, and the sections mounted in 
 dammar (see § 247, c). 
 
 326. Madder. — Under the title "purpurine" the fol- 
 lowing fluid is recommended by Ranvier for staining car- 
 tilage cells : — Add 1 gramme of alum to 200CC distilled 
 water ; bring them to the boiling point in a porcelain basin, 
 and then add " some " extract of madder previously pow- 
 dered, and rubbed up with a little water. The ebullition is 
 continued, and a portion is dissolved. It is filtered while 
 
 * This is a cheap substance, and may be obtained from Messrs. 
 Read, Holliday, and Sons, Huddersfield, or at 15 Fenchurch Street, 
 London. 
 
i;6 , PRACTICAL HISTOLOGY. 
 
 hot, and the filtrate is allowed to trickle into 60CC 
 rectified spirit. Slices of cartilage are allowed to remain in 
 the fluid from 24 to 48 hours. They are then washed in 
 water, and mounted in glycerine. The nuclei and nucleoli 
 of the cells are brightly coloured, the cartilage matrix 
 faintly so. He also recommends its use in studying intra- 
 cartilaginous ossification. It . stains tissues hardened in 
 chromic acid. 
 
 327. Silver Nitrate. — His, but more particularly 
 Recklinghausen, introduced this substance into histology. 
 It is of great value for staining the epithelium of serous 
 membranes, lymphatics, and blood-vessels. It also stains 
 the matrix of connective tissue and of cartilage, and often 
 the nuclei of cells. In addition to staining, it fixes albu- 
 minous matter, and is thus valuable by facilitating the 
 preservation of delicate tissues, e.g., adenoid tissue of 
 lymphatic gland, epithelium of intestine. A solution of the 
 salt is commonly used, but the solid form is also sometimes 
 employed for the cornea. 
 
 328. The 01'dinary silver process. — A \ per cent solution 
 in distilled water is usually employed, but somewhat weaker 
 solutions may also be used. In silvering a tissue — e.g., the 
 mesentery of the frog, or omentum of guinea-pig or cat — dip 
 it two or three times into distilled water to remove sodium 
 chloride. Place it in the silver solution from one to three 
 minutes. Remove the superfluous silver by washing in 
 distilled water. Place the washed tissue in glycerine or in 
 distilled water, but glycerine is better, and expose it to 
 diffuse daylight until it becomes slightly brown. Mount in 
 glycerine, or in glycerine jelly. 
 
 By exposure to light the silver salt is reduced, and the 
 tissues are thereby blackened. If the immersion of the 
 tissue in the silver solution be of very brief duration, only 
 the outlines of the epithelial cells are blackened. The 
 cells appear to be united by a colourless " cement," which 
 readily reduces the silver salt when acted on by light. If 
 the silver act for some time longer, the nucleus and general 
 substance of the epithelial cells is blackened, also the 
 nuclei of fat cells, connective tissue corpuscles, etc. 
 
HISTOLOGICAL METHODS. 177 
 
 Tissues that have been slightly silvered may be stained 
 with logwood or carmine ; the ammonia of the latter, how- 
 ever should be neutralised with acetic acid, and the tissue 
 washed in acidulated water (§ 321). 
 
 329. Silver process for blood-vessels. If a frog be taken, 
 kill it by stunning it on the head : expose the heart, snip 
 off its apex, and allow it to bleed thoroughly. Push a glass 
 or brass cannula from the ventricle into one of the aortae, 
 and inject a stream of distilled water to wash out the blood 
 with its chlorides : inject J per cent silver nitrate solution, 
 allow it to remain for 8 or 10 minutes, and then wash it 
 out with distilled water. The most convenient parts to 
 take are the mesentery and bladder. These are therefore 
 removed and exposed to the light, as stated in § 328. If, 
 however, it is desirable to silver also the epithelium covering 
 the mesentery, this is done after the injection, as described 
 in § 328. In the case of a warm-blooded animal, such as 
 a guinea-pig, the water and the silver solution are both 
 heated to 39 C. The fluids are injected into the aorta. 
 
 A solution of silver nitrate may also be injected into 
 lymphatic gland, areolar tissue, testis, lung, etc., for the 
 fixing and staining of tissue elements. The necessary 
 apparatus for injection is described in § 344. 
 
 330. Chloride of Gold (auric chloride) is of much 
 service for staining nerve fibrils, connective tissue corpuscles, 
 cartilage cells, etc. It is not merely a staining, but, like 
 silver nitrate, is also a hardening agent. Unfortunately its 
 action as a dye is somewhat uncertain. 
 
 a. Ordi?iary Method. — Place the tissue, not later than 
 fifteen minutes after its removal from the body, in \ per 
 cent solution of auric chloride in distilled water, until it 
 becomes of a pale yellow colour. From fifteen minutes to 
 two hours will be required, according to the thickness of 
 the tissue. Transfer to very dilute acetic acid (1 or 2 per 
 cent) for five or ten minutes. Lastly, place the tissue in 
 distilled water, and expose to diffuse daylight, until a steels 
 gray or violet tint appear. This results from the reduction 
 of the gold salt. The reduction is hastened by the acetic 
 acid. The tissue may also be exposed to the light in dilute 
 
 N 
 
178 PRACTICAL HISTOLOGY. 
 
 acetic acid or in glycerine. The time of exposure varies 
 with the intensity of the. light; from one to two days 
 usually suffice. Mount the stained tissue in glycerine, or 
 in glycerine jelly. The preparations deteriorate however. 
 
 b. Method for the Co7'nea. — The gold method was intro- 
 duced by Cohnheim for staining the nerves of the cornea. 
 The following method, devised by Klein {Monthly Micro- 
 scopical Joum., vol. vii. p. 157), gives better results than 
 Cohnheim's in this case. Remove the cornea from a 
 rabbit or guinea-pig within fifteen minutes after death, and 
 place it in J per cent gold solution, from one and a half to 
 two hours in the case of the rabbit, and rather more than 
 an hour in the guinea-pig. Wash in distilled water, and 
 expose to the light in the water for twenty-four or thirty- 
 six hours, the water being changed twice or thrice. Then 
 transfer the cornea to dilute .glycerine (pure glycerine 1 
 part, distilled water 2 parts) for two or three days. Lastly, 
 place the cornea in water, and gently brush away any pre- 
 cipitate that may have formed; make sections obliquely 
 horizontal or vertical, and mount in glycerine. A cornea 
 so treated is ash-gray or slightly violet. 
 
 331. Chloride of Palladium was introduced by F. F. 
 Schultze for staining non-striped muscle. It renders the 
 fibres brownish yellow. An aqueous solution of 1 per 
 1000 is employed, to every 20CC of which a drop of 
 hydrochloric acid is added, in order to preserve it. For 
 staining, three or four days are required. Schultze recom- 
 mends this as a hardening agent for the lens and the 
 retina. Its use as an adjuvant in the carmine staining of 
 nerve tissue hardened in chromic acid has been alluded to 
 in § 319. 
 
 332. Osmic Acid. — The value of this substance for 
 blackening, more especially medullary substance of nerve 
 and all fatty matter, has been already alluded to in § 281, 
 where it has been described under the head of hardening 
 agents, because of its especial value as such. 
 
HISTOLOGICAL METHODS. ' 179 
 
 METHODS OF INJECTION. 
 
 I. — INJECTION MASSES FLUID AT THE ORDINARY 
 TEMPERATURE. 
 
 333. Aqueous Solution of soluble Prussian Blue. 
 
 — Soluble Prussian blue can now be procured in the 
 market. The following, however, is the method in which 
 it may be prepared (Briicke). 
 
 A. Dissolve 217 grammes potassium ferrocyanide in 1 
 litre distilled water. 
 
 B. Dissolve 100 grammes ferric chloride in 1 litre 
 distilled water. 
 
 C. Make a cold saturated solution of sodium sulphate. 
 Mix 1 volume of A with, two volumes of C, and mix 1 
 
 volume of B with 2 volumes of C. Pour B C very gradu- 
 ally into A C, stirring all the while, and let stand for a 
 day. Pour off supernatant fluid, and filter off the remain- 
 ing fluid from the precipitate. Wash the precipitate upon 
 the filter, with repeated small quantities of distilled water, 
 until it flows through distinctly blue. Press and dry the 
 deposit. 
 
 For the purpose of injection, a 2 per cent solution of 
 the blue in distilled water is employed. The fluid may be 
 used cold, or it may be heated to the temperature of the 
 body. After injection, prevent the blue from diffusing 
 through the capillary walls by immersion in spirit (90 per 
 cent). If the colour fade, immersion in turpentine, especi- 
 ally in that which has been exposed to the air for some 
 time, or in acid, restores it. Mount in dammar ; glycerine 
 must be avoided. 
 
 334. Beale's Prussian Blue injection. 
 
 A. Mix 1 oz. glycerine with 4 oz. water. 
 
 B. Add 60 minims tincture of ferric chloride (British 
 pharmacopoeia) to 1 oz. of solution A. 
 
 C. Dissolve 1 2 grains potassium ferrocyanide in 1 oz. 
 of solution A. 
 
 Pour B into C very gradually, with constant stirring and 
 
i8o PRACTICAL HISTOLOGY. 
 
 shaking of the mixture, then add the remainder of solution 
 A, and also i oz. rectified spirit. The injected tissues are 
 preserved in acid glycerine (glycerine i oz., hydrochloric 
 acid two drops). 
 
 Prussian blue injections must not come in contact with 
 alkali. The above injection masses are excellent when it 
 is of no moment to have the vessels fully distended, but 
 merely to have the indication of pigment within them, to 
 aid in their recognition amidst other tissue elements. 
 
 334A. Asphalt and Chloroform. — Ludwig has recently 
 employed a mass, consisting of asphalt dissolved in chloro- 
 form and filtered, for the injection of the bile ducts. The 
 merit of this fluid is, that chloroform being an extremely 
 mobile fluid, flows readily along the vessels, and that it 
 rapidly evaporates and leaves them filled by a solid black 
 mass. 
 
 335. Alcannin and Turpentine.— A solution of al- 
 cannin in turpentine or in chloroform is used by Ludwig 
 for injecting lymphatics. The solution is of a bright red 
 colour. Both the turpentine and the chloroform solutions 
 flow readily. When the latter is employed, the chloroform 
 evaporates and leaves the alcannin in the vessels. 
 
 336. Solution of Silver Nitrate. — The employment 
 of this has already been referred to in § 329. 
 
 2. — INJECTION MASSES SOLID AT THE ORDINARY 
 TEMPERATURE. 
 
 337. Carter's Carmine and Gelatine Mass. — This 
 — the best of all the carmine masses — is prepared thus : — 
 
 Carmine, 60 grains. 
 
 Strong ammonia, 120 minims. 
 
 Glacial acetic acid, 86 minims. 
 
 Solution of gelatine (gelatine 1, water 6 parts), 2 oz. 
 
 Water, ij oz. 
 
 Dissolve the carmine in the ammonia and water with the 
 aid of gentle heat, and filter; add to this i-| oz. of hot 
 gelatine solution, and mix thoroughly. Add the acid to 
 
HISTOLOGICAL METHODS. 
 
 181 
 
 the remaining J oz. of gelatine solution, and drop this into 
 the heated carmine mixture with constant stirring. The 
 acid is added in order to precipitate the carmine and 
 prevent its diffusion through the walls of the capillaries. 
 The presence of the gelatine prevents the precipitated 
 particles from assuming a coarse granular form. For sec- 
 tion, the tissues are hardened — first, in equal parts of 
 rectified spirit and water, with the addition of i per cent 
 hydrochloric acid ; then in rectified spirit ; and, lastly, in 
 absolute alcohol. Sections are then made, and mounted in 
 dammar. 
 
 33S. Prussian Blue and Gelatine Mass. — 
 
 f Gelatine, 33 grammes. Distilled water, 200CC. Dis- 
 A \ solve with the aid of a gentle heat (using a water-bath). 
 
 j Soluble Prussian blue, 4 grammes. Distilled water, 
 
 ( 300CC. Dissolve. 
 Add B to A gradually. The tissues are hardened for 
 section and preserved, as stated in § 337. 
 
 339. Injection of Blood-vessels. — Syringe. — A brass 
 syringe S (Fig. 60), capable of holding from four to six 
 
 M 
 
 Fig. 60.— Injection syringe (S)>ith mercurial manometer (M). 
 
 ounces of fluid, is very often used^for injection. It is 
 usually provided with brass nozzles (n) of various sizes, for 
 vessels of different calibre, and a stopcock (s) which may 
 be separated from the nozzle and from the syringe. The 
 nozzle is tied in an artery ; the stopcock-piece is pushed 
 into it ; both are filled with injection-mass by means of a 
 pipette; and the stopcock turned. The syringe is then 
 filled— care being taken to expel all air by holding the 
 
1 82 
 
 PRACTICAL HISTOLOGY. 
 
 nozzle uppermost while a drop or two of injection is forced 
 out. A few drops are allowed to fall into the stopcock- 
 piece to completely fill it. The two are united, the stop- 
 cock turned, and the injection slowly performed. The 
 piston of the syringe must never be pushed, but always 
 screwed down, to ensure steadiness of pressure. This 
 apparently simple process is very apt to fail because of the 
 difficulty of judging of the amount of pressure applied. 
 If it be too great, capillaries are over-distended or ruptured, 
 and the injection spoilt. This is easily avoided, however, 
 by attaching — as is sometimes done — a pressure-gauge to 
 the syringe. This is an ordinary mercurial manometer (M) 
 connected to the stopcock-piece of the syringe by an elastic 
 tube containing air, and a stopcock /. When both stop- 
 cocks are open, and the fluid expelled from the syringe, a 
 part of it passes into the elastic tube, compresses the air, 
 and elevates the mercury in the one limb of the manometer 
 above that in the other, and thus indicates the pressure 
 under which the fluid is being driven into the vessels. The 
 syringe, though convenient for rapid injection, is, neverthe- 
 less, tedious ; and therefore Ludwig was led to devise a 
 pressure-bottle apparatus. 
 
 340. Pressure-bottles. — A simple but efficient apparatus, 
 
 Fig. 61.— Pressure-bottle injection apparatus. 
 
 essentially similar to that used by Ludwig for prolonged 
 injections, is shown in Fig. 61. c represents a zinc or 
 copper water-bath with a perforated tray, on which the 
 
HISTOLOGICAL METHODS. 183 
 
 animal or organ is laid, and covered with glass (g). The 
 water is warmed by an ordinary Bunsen's burner, and its 
 temperature is indicated by a thermometer (/). Two Wolff's 
 bottles (B) — one containing, say a carmine mass for inject- 
 ing arteries, and the other a Prussian blue mass for veins — 
 stand in the water, so that when a hot injection has to be 
 made, the mass may be readily warmed. The masses are 
 expelled from them by air driven from a large Wolff's bottle 
 A, by water flowing into it from a tap (w). The pressure 
 exerted upon the air in A is indicated by a mercurial 
 manometer M, and its amount, of course, depends upon 
 the rapidity with which the water is allowed to flow. Glass 
 nozzles, each with a small shoulder, as in n, Fig. 60, for 
 securing them in the vessels, are tied in the indiarubber 
 tubes from the injection-bottles (B); and when one or other 
 injection is not required, it is cut off by the stopcock of its 
 exit tube (c). The corks of the bottles are of vulcanised 
 indiarubber. The lengths of the glass tubes penetrating 
 them are indicated in the Fig. The elastic tubing requires 
 to be very carefully tied over the glass to prevent leakage. 
 Such an apparatus is of great service, for its action is 
 automatic, and the pressure can be very finely graduated. 
 
 341. Injection. — The blood-vessels should be injected 
 immediately after death from chloroform. When the 
 breathing is just about to cease the right auricle should be 
 opened to allow the vessels to empty. Previous to injection 
 the vessels may be washed out with a stream of f per cent 
 salt solution, heated to 40 C. if the animal be warm-blooded. 
 The use of the salt solution, however, is not essential. 
 
 If a gelatine injection be used, it must of necessity be 
 heated to about 40 C, and the animal must also be kept 
 warm ; but even when an injection that is fluid at ordinary 
 temperatures is employed, it should be heated 40 ° C. or so, 
 because of the vascular spasm which a cold fluid is apt to 
 occasion. 
 
 It is very important always to begin with a low pressure, 
 say J- an inch of mercury, and to increase it very gradually 
 to 3 or even to 4 inches. When the injection is complete 
 the artery and vein are ligatured, and the organ placed 
 
1 84 PRACTICAL HISTOLOGY. 
 
 in cold methylated spirit, to cause the gelatine to consolidate 
 rapidly, and so prevent its escape. After several hours, or 
 a day, the preparation is removed from the alcohol, and 
 treated as may be desirable. 
 
 342. For the injection of the kidney the organ should 
 not be removed from the animal, because of the anastomoses 
 between the renal and lumbar vessels. The nozzle should 
 be placed in the renal artery close to the aorta. 
 
 343. For the injection of the liver from the portal vein, 
 and the lung from the pulmonary artery, salt solution should 
 first be injected, the right and left auricles of the heart be- 
 ing respectively opened to permit the blood to escape. 
 
 344. Injection of Lymphatics. — The puncture method 
 for the injection of lymphatics was introduced by Ludwig, 
 and simply consists in thrusting the sharp needle-like nozzle 
 of a Wood's syringe (the syringe of Pravaz of continental 
 writers) into various textures, and driving the fluid wherever 
 it will go. e.g. In the case of the testis, if the nozzle be thrust 
 through the tunica albuginea after death, and the injection 
 driven into the gland substance, it very readily finds its way 
 into the lymphatics, and runs along them even as far as the 
 thoracic duct, which may be thus injected. The chain of 
 lymphatics in the leg of the dog can be easily demonstrated 
 by simply thrusting the nozzle of the syringe into the pad 
 of the foot after death, and pushing the fluid onwards by 
 compressing the limb from below upwards. The lymphatics 
 of mucous membranes and aponeuroses may be injected in 
 the same manner. A watery solution of Briicke's blue 
 (§ 333)) or tne turpentine or chloroform solution of alcan- 
 nm (§ 335)> or asphalt (§ 334A) may be employed. For 
 ordinary demonstrations the blue is the best. 
 
 345. As the chloroform and turpentine used in these 
 fluids is apt to destroy the cement of the fastenings of the 
 syringe, they may be injected by an apparatus made of a 
 glass tube drawn to a fine point attached to a piece of 
 caoutchouc tubing, which is filled with the injection mass, 
 and closed by a ligature at the other end. On pressing the 
 elastic tube with the finger the fluid is expelled. 
 
 346. Injection of Bile Ducts. — Inject the portal vein 
 
HISTOLOGICAL METHODS. 185 
 
 with carmine and gelatine. Previous to this, open the com- 
 mon bile duct, so that the distension of the blood-vessels 
 may press as much of the bile out of the ducts as possible. 
 Then inject the bile capillaries from the common bile duct 
 with an aqueous solution of soluble Prussian blue. It is 
 not necessary, however, to previously inject the veins. The 
 liver of the rabbit is well adapted for injecting the bile ducts. 
 A pressure of from o'8 to 1 inch mercury is usually sufficient 
 for the ducts, but it may require to be gradually increased 
 1 "6 inch. The fluid being driven against the bile contained 
 in the liver, an injection of the whole liver must not be 
 attempted, but the injection should be discontinued as soon 
 as a few lobules on the surface of the liver have become 
 distinctly blue. After injection, place the liver in cold 
 methylated spirit. Mount the sections in dammar. 
 
 METHODS OF PRESERVING THE TISSUES. 
 
 347. The Dry Method. — Hair, and sections of dried 
 bone and tooth, are sometimes mounted in air. The differ- 
 ence between the refractive powers of the tissues and the 
 air is, however, so great that the outlines are extremely 
 dark. They show better when mounted in some more re- 
 fractive medium than air, e.g. glycerine, glycerine jelly, or 
 dammar. When a tissue is mounted in air, a ring of dam- 
 mar or white zinc cement rather smaller than the cover-glass 
 is made on the slide, and allowed to nearly dry. The tissue 
 is then placed within the dammar cell (a, Fig. 62) and the 
 cover-glass placed over all and pressed upon the dammar, 
 to which it adheres. If the portion of tissue be thick, a 
 glass cell is used (fr, Fig. 62). 
 
 3 47 a. Dammar and Canada Balsam. — a. All water 
 must be removed from the tissue by drying or by immer- 
 sion, first in rectified spirit and then in absolute alcohol. 
 The alcohol is driven away by floating the tissue upon oil 
 of cloves, or turpentine, in a watch glass or on a slide, and 
 then the balsam of dammar is added, and the cover-glass 
 put on (see § 152). 
 
 b. If Canada balsam be employed, prepare it as follows : — 
 
1 86 PRACTICAL HISTOLOGY. 
 
 Place some pure Canada balsam in a saucer or other shallow 
 vessel ; cover the vessel with bibulous paper to exclude 
 dust ; dry it in an oven at a temperature not above 65 ° C, 
 until, when it cools, it becomes as hard as ice ; dissolve this 
 crystalline balsam in benzole. The old-fashioned method 
 of mounting things in balsam was so troublesome, that one 
 avoided it as much as possible. Undried balsam was taken 
 and rendered quite liquid by heat. The slide, the cover- 
 glass, and the tissue to be mounted, all required to be heated. 
 It was difficult to get rid of air bubbles and to avoid over- 
 heating the tissue. But by using a limpid solution of per- 
 fectly dried balsam, all difficulty disappears. 
 
 c. Dammar is now much employed as a substitute for 
 Canada balsam. It renders the tissues more transparent 
 than balsam. The following is an excellent method for 
 preparing the dammar fluid : — Dissolve \ oz. dammar resin 
 and \ oz. gum mastic in 3 oz. benzole, and filter. If rapid 
 filtration be desired, use twice as much benzole, and then 
 evaporate the fluid to half its bulk. Chloroform and tur- 
 pentine are by some recommended as solvents of these resins, 
 but the dammar fluid so made is turbid. If, however, it be 
 prepared as above recommended, it is perfectly clear. Bal- 
 sam or dammar is suitable for mounting unsoftened bone 
 and tooth, hair, brain, and spinal cord, and indeed most 
 tissues that have been hardened in alcohol or chromic acid, 
 which require to have their transparency much increased. 
 It renders softened bone and tooth too transparent. Speak- 
 ing generally, it is more suitable for mounting stained than 
 unstained tissues. The latter are often rendered too trans- 
 parent. The tissues very perfectly preserved in either of 
 these substances, because all water is removed. 
 
 348. Glycerine is not adapted for white fibrous tissue 
 and blood-vessels, unless they have been hardened in 
 chromic acid. Otherwise it causes the white fibres to swell 
 up and lose their normal features, but it is well suited for 
 tissues that are to be examined by very high powers. Liver, 
 lung, alimentary canal, and skin, after having been hardened 
 in chromic acid or alcohol, show better in glycerine than in 
 balsam or dammar, unless they have been stained with car- 
 
HISTOLOGICAL METHODS. 187 
 
 mine or logwood. It is suitable for mounting tissues that 
 have beeen stained with silver nitrate or gold chloride. For 
 tissues containing vessels injected with Prussian blue, use 
 two drops H CI in 1 oz. glycerine, to preserve the colour. 
 It is somewhat difficult to mount things in glycerine. Air 
 bubbles are difficult to eliminate, and if the glycerine pass 
 outside the cover-glass, it is difficult to get rid of it. It can 
 be removed most readily by sucking it up with a fine glass 
 pipette, Fig. 45). It is often necessary, however, to transfer 
 the preparation to a new slide, on which a smaller drop of 
 glycerine has been placed. The faults of glycerine are — 1, 
 a tendency to render the tissues too transparent ; 2, a diffi- 
 culty in getting rid of superfluous fluid; 3, a difficulty in 
 getting rid of air bubbles. 
 
 349. Farrants' Solution is an excellent substitute for 
 glycerine in many instances indicated in the preceding 
 demonstrations, because of its feebler tendency to render 
 the tissues transparent. It consists of " equal parts of gum- 
 arabic, glycerine, and a saturated solution of arsenious acid " 
 (Frey). Owing to its containing gum it becomes hard, so 
 that the cover-glass becomes fixed at its margin. In 
 mounting preparations with this fluid, the covered object is 
 allowed to lie for a day before the varnish is applied, so 
 that the cover may be fixed and thereby prevented from 
 shifting during varnishing, as so often happens with gly- 
 cerine. 
 
 350. Glycerine Jelly is a good preservative agent for 
 many tissues, such as lung, blood-vessels, tooth, bone, 
 cochlea, etc. The tissues must be steeped in weak spirit 
 (§ 35 2 ) previous to mounting. They cannot be transferred 
 from glycerine to glycerine jelly directly. Fungi are very 
 apt to grow in some kinds of glycerine jelly that are sold. 
 Rimmington's glycerine jelly is the best. Its composition 
 is unknown, but it appears to contain glycerine, gelatine or 
 isinglass, and carbolic acid. 
 
 351. Potassium Acetate. — A saturated solution of this 
 substance has long been used by botanists. It is the best 
 medium for osmic acid preparations (Schultze). Glycerine 
 renders these too transparent. The fluid may be prepared 
 
1 88 PRACTICAL HISTOLOGY.' 1 
 
 thus : — Dissolve 56 grammes potassium acetate in 28CC 
 hot water. When cold, add 2CC camphorated spirit, and 
 filter. 
 
 352. Weak Spirit — consisting of rectified spirit 1 part, 
 distilled water 3 parts — was formerly much employed. 
 Muscle, connective tissue, and blood-vessels, can be well 
 preserved in it for a time, but sooner or later they become 
 granular and useless. 
 
 352A. Rectified Spirit may be used for mounting soft- 
 ened bone and tooth. Glycerine renders these organs too 
 transparent if they are unstained. 
 
 353. Naphtha and Creosote fluid is useful for pre- 
 serving urinary casts (§ 222). 
 
 354. Cells for Microscopic Objects. — Cells are made 
 of tissue paper, tinfoil, cardboard, dammar varnish, or white 
 zinc cement, or of glass. A paper cell is made by cutting 
 with a punch a paper circle the size of the cover-glass. A 
 hole is then cut in the centre with a punch of smaller size. 
 A cell of dammar or white zinc varnish (a, Fig. 62) is made 
 
 Fig. 62.— Cells. 
 
 by tracing a ring of the substance on the slide with the aid 
 of Shadbolt's turn-table (Fig. 6$), and then allowing it to 
 dry. A glass cell (b, Fig. 62) usually consists of a section 
 of a glass tube cemented to a slide with marine glue. Some- 
 times a sunk glass cell (c) is employed. It is merely a 
 depression ground in the glass. Delicate tissues that are 
 apt to be spoilt by the weight of the cover-glass should be 
 placed in a cell of tissue paper or gold-size. 
 
HISTOLOGICAL METHODS. 
 
 189 
 
 Fig. 63.— Turn-table. 
 
 355. Turn-table. — The dammar or white zinc cells 
 above mentioned are made with the aid of Shadbolt's turn- 
 table (Fig. 63), consisting of a 
 rotary brass disc with a series of 
 concentric lines around its centre. 
 There are clips for fixing a slide, 
 and a wooden board for support- 
 ing the whole. The slide is 
 centred and fixed by the clips. 
 A brush dipped in the varnish is 
 held over one of the rings, and 
 the table turned rapidly. The 
 apparatus is also used for putting a ring of varnish round 
 the margin of the cover-glass in sealing up a preparation. 
 
 356. The spreading out of Sections. — In dealing 
 with large sections or with membranes there is often consi- 
 derable difficulty in spreading them out. This difficulty 
 may, however, be readily overcome by floating them on to 
 the slide by the method described in § 87. 
 
 357. Mounting-clip. — In the case of sections that can 
 bear some pressure, and which are difficult to keep perfectly 
 
 flat, a mounting-clip may be ap- 
 plied until the cover-glass is 
 fixed by glycerine jelly, dam- 
 mar, or some cement (Fig. 64). 
 It consists of a brass wire 
 bent so as to form a spring. 
 The end that is applied to the 
 fig. 64-Mounting-ciip. cover-glass is covered with 
 
 leather. In the case of bone, tooth, or cartilage, it may be 
 used, but the section is apt to be spoilt unless the spring 
 be feeble, and, indeed, in most cases no such thing need be 
 used. 
 
 358. Cements. — When the object is mounted in 
 Canada balsam or dammar, no cement is required ; but if 
 any other preservative substance be employed, even glycerine 
 jelly — for it is hygroscopic — the margin of the cover-glass 
 must be varnished. Dry the slide with bibulous paper, 
 then paint on a layer of gold-size, allow it to dry, and lastly 
 
190 PRACTICAL HISTOLOGY. 
 
 cover this with white zinc cement. The latter gives an 
 elegant finish to the preparation, and is at the same time an 
 excellent varnish. Shadbolt's turn-table (Fig. 63) is often 
 employed in applying the varnish. Glycerine is apt to ooze 
 through the varnish after a time ; when this happens, run a 
 stream of cold water over the preparation, then dry and 
 paint on a fresh layer of zinc cement. 
 
 359. Mode of keeping Preparations. — The slides 
 should lie upon the back, not upon the edge. All prepara- 
 tions should be carefully labelled. The things shown should 
 be mentioned, the date, and the name of the mounting fluid 
 ought also to be stated. Never keep a bad preparation, and 
 don't attempt to preserve everything. 
 
INDEX. 
 
 Acetic Acid, 170 
 
 „ and glycerine, 158, 17b 
 
 Achromatic condenser, 38 
 Adenoid tissue, 78 
 Adipose ,, 77 
 Adjustment, coarse and fine, 32 
 
 ,, of objective, 24 
 
 Albumin, dilute, 150 
 Alcannin and turpentine injection, 180 
 Alcohol, absolute, 154 
 
 „ dilute, 284 
 
 ,, methylated, 154 
 
 „ rectified, 154 
 
 ,, weak, 188 
 Angle of aperture, 23 
 Aniline, 175 
 Areolar tissue, 74 
 Arteries, 100 
 Asphalt and chloroform injection, 180 
 
 Bacteria, 54 
 
 Beale's carmine fluid, 171 
 
 ,, Prussian blue injection, 179 
 Beer's knife, 161 
 Blood, circulation of, 102 
 Blood corpuscles of Bird, 58 
 „ Fish, 58 
 
 Newt, 55 
 „ Man, 58 
 
 ,, effect of acetic acid, 56, 59 
 
 alcohol, 57 
 electricity, 61 
 gases, 61 
 magenta, 57, 59 
 syrup, 57 
 ' tannic acid, 57, 60 
 enumeration of, 62 
 
 Blood corpuscles, feeding of, 61 
 
 ,, structure of, 60 
 
 Blood-crystals, 65 
 Blood-vessels, 100 
 Bone, 82 
 
 Brownian movement, 51 
 Briicke's dissecting microscope, 36 
 
 Calcic Phosphate, 124 
 Camera lucida, 44 
 Canada balsam, 185 
 Capillaries, no 
 Carmine staining fluid, 171 
 
 ,, injection, 180 
 Carrot, for imbedding, 162 
 Cartilage, 79 
 Casts, 124 
 
 Cells, for mounting, 188 
 Cements, 189 
 Cerebellum, 139 
 Cerebrum, 139 
 Chalice cells, 72 
 Chromic acid, 152, 158 
 
 „ and spirit, 155 
 
 Ciliary motion, 69 
 Circulation of the blood, 102 
 Cochlea, 134 
 Condenser, achromatic, 38 
 
 ,, bull's-eye, 28 
 
 Connective tissue, 73 
 Cornea, 128 
 Cotton fibres, 53 
 Cover-glass, 1 
 
 „ cleaning of, 41 
 
 ,, effect of, 24 
 
 Creosote and naphtha fluid, 188* 
 Crystalline lens, 130 
 Cystine, 124 
 
192 
 
 INDEX. 
 
 Dammar, 185 
 
 Darwin's dissecting microscope, 36 
 
 Description of objects, 41 
 
 Development, 143 
 
 Diaphragm of microscope, 29 
 „ lymphatics of, 106 
 
 Digestion, method of, 159 
 
 Dilute alcohol, 284 
 
 Disc-bearing tissue, 54 
 
 Dissecting microscope, 36 
 
 Dissociation by needles, 159 
 „ pressure, 159 
 
 „ shaking, 159 
 
 Drawing, 43 
 
 Dry lens, 25 
 
 Dry method of mounting, 185 
 
 Ear, 134 
 
 Elastic tissue, 73 
 
 Electricity, application of, 148 
 
 End organs, 99 
 
 Epidermis, 66 
 
 Epithelium squamous stratified, 65, 66 
 
 „ „ simple, 68 
 
 „ ciliated, 69 
 
 ,, columnar, 71 
 
 „ transitional, 73 
 
 Eye, 128 
 Eye-piece, 26 
 
 Farrants' Solution, 187 
 Fibrocartilage, 79 
 Fibrous tissue, yellow, 73 
 white, 74 
 Focus of a lens, 12, 15 
 Freezing, 156 
 
 ,, Microtome, 164 
 
 Gas-Chamber, 144 
 Gases, application of, 144 
 Glycerine, 169, 170, 186 
 
 „ jelly, 187 
 Gold chloride, 177 
 
 ,, size, 189 
 Granules, 51 
 Gum for imbedding, 163 
 
 H^emin, 65 
 Haemoglobin, 65 
 Hair, 126 
 Hand sections, 161 
 Hardening, methods of, 152 
 Hartnack's Microscope, 33 
 Heat, application of, 145 
 Hot stages, 146 
 
 Illumination by Achromatic light, 83 
 ,, artificial light, 36 
 
 „ daylight, 30 
 
 ,, polarised light, 39 
 
 Imbedding in carrot, 162 
 „ gum, 163 
 
 „ ice, T64 
 
 ,, paraffin, 162 
 
 ,, pith, 162 
 
 „ wax and oil, 162 
 
 Injection masses, 179 
 ,, methods, 181 
 Intestine, 115 
 Iodised serum, 150 
 Irrigation, 144 
 
 Kidney, 120 
 
 Lamp, 36 
 Lenses, 11, 21 
 
 ,, faults of, 19 
 Linen fibres, 54 
 Liver, 117 
 
 Living tissues, study of, 149 
 Logwood staining fluid, 173 
 Lung, no 
 Lymph gland, 107 
 Lymphatics, 105 
 
 Madder staining fluid, 175 
 Magenta ,, 174 
 
 Magnifying power, estimation of, 48 
 Malassez and Potain's pipette, 62 
 Marrow, 87 
 
 Medulla oblongata, 137 
 Micrometer eye-piece, 49 
 
 „ stage, 47 
 
 Micromillimeter, 50 
 Microscope binocular, 35 
 
 „ compound, 17, 33 
 
 „ dissecting, 36 
 
 „ monocular, 33 
 
 „ simple, 16 
 
 ,, estimation of magnifying 
 
 power, 46 
 
 ,, preparation of, 40 
 
 „ testing of, 39 
 
 Microtome, 164 
 Mirror, 28 
 
 Moist chambers, 150 
 Mounting clip, 189 
 
 „ of specimens, 185 
 Mucin, 66, 73 
 Mucous tissue, 79 
 
INDEX. 
 
 193 
 
 Mucus, 65 
 Miiller's fluid, 154 
 Muscle, 90 
 
 ,, nerves of, 99 
 
 Naphtha and Creosote fluid, 188 
 Needles for dissection, 159 
 Nerve cells, 97 
 
 ,, fibres, gray, 96 
 
 „ ,, white, 94 
 
 ,, fibrils, 97, 98 
 Nose, 136 
 Nose-piece, 31 
 
 Object, preparation of, 41 
 ,, size, estimation of, 48 
 Objective, 22. 
 
 ,, dry and immersion, 25 
 Ocular, 26 
 Osmic acid, 155, 178 
 Osteoblasts, 85 
 Ovary, 142 
 Oxalate of lime, 124 
 
 Pacini's Corpuscles, 99 
 Palladium chloride, 178 
 Pancreas, 119 
 
 Paraffin, for imbedding, 162 
 Penicillium, 53 
 Periosteum, 85 
 Picric acid, 153 
 Picro-carmine, 173 
 Pipette, ordinary, 144 
 
 ,, Malassez and Potain's, 62 
 Pith for imbedding, 162 
 Polariscope, 39 
 Potassium acetate, 187 
 
 ,, bichromate, 154 
 
 ,, hydrate, 170 
 Preparation of tissues, preliminary, 3 
 Preservation of tissues, 185 • 
 Pressure-bottle injection apparatus, 182 
 Prickle cells, 67 
 Prism for drawing, 45 
 Prostate, 142 
 
 Prussian blue injections, 179 
 Purpurine staining fluid, 175 
 
 Razor, 160 
 
 Reagents, application of, 144 
 
 ,, bottles for, 1 
 
 „ list of those required, 2 
 
 Rectified spirit, 154, 188 
 Reflector, parallel-plate, 45 
 Retina, 130 
 
 Salivary Corpuscles, 66 
 
 ,, glands, 119 
 Salt solution, 150 
 Sanderson's warm-stage, 147 
 Schaefer's heating apparatus, 148 
 Schneiderian membrane, 136 
 Scissors, 160 
 Sections, making of, 159 
 
 ., mounting of, 189 
 Serous epithelium, 105 
 
 „ fluids, 149 
 
 ,, membranes, 149 
 Shadboldt's turn-table, 189 
 Silver nitrate, 176 
 Size of object, estimation of, 48 
 Skin, 125, 66 
 Slide, 1 
 
 Sodium hydrate, 170 
 Softening, methods of, 157 
 Spinal cord, 137 
 Spleen, 107 
 Squared glass, 46 
 Stage of the microscope, 30 
 
 ,, warm „ 145 
 
 Staining, methods of, 171 
 Starch, 53 
 Stomach, 113 
 Stomata, 105 
 
 Strieker's warm-stage, 146 
 Sulphuric acid dilute, 158, 
 Suprarenal body, no 
 Syringe, 181, 184 
 
 Taste Bulbs, 113 
 
 Tendon, 75 
 
 Test objects, 39 
 
 Testis, 141 
 
 Thyroid gland, 109 
 
 Tissues, preparation of, 3 
 
 Tongue, 112 
 
 Tooth, 88 
 
 Torula, 52 
 
 Tradescantia, 51 
 
 Transparency, methods for increasing, 169 
 
 Triple phosphate, 124 
 
 Turn-table, 189 
 
 Turpentine, 170 
 
 Urates, 124 
 Uric acid, 124 
 Urinary deposits, 123 
 Uterus, 142 
 
 Valentin's Knife, 160 
 Vapours, application of, 44 
 
 O 
 
194 
 
 Varnishes, 189 
 Veins, 100 
 Vitreous humour, 79 
 
 Warm-stage, Simple, 145, 
 „ Strieker's, 146 
 
 INDEX. 
 
 Warm-stage, Strieker and Sanderson's, 
 
 147 
 Weak Spirit, 188 
 
 Yeast, 52 
 
 Zinc Cement, 190 
 
 THE END. 
 
 Printed by R. & R. Clark, Edinburgh. 
 
" s