OUTLINES 
 
 OF 
 
 PRACTICAL HISTOLOGY, 
 
 BEING THE NOTES OF THE HISTOLOGICAL SECTION 
 OF THE CLASS OF PRACTICAL PHYSIOLOGY 
 
 HELD IN THE UNIVERSITY OF EDINBURGH. 
 
 WILLIAM RUTHERFORD, M.D., F.R.S.E., 
 
 PROFESSOR OF THE INSTITUTES OF MEDICINE IN THE UNIVERSITY OF EDINBURGH, 
 EXAMINER IN PHYSIOLOGY IN THE UNIVERSITY OF LONDON. 
 
 LONDON: 
 
 J. &. A CHURCHILL, NEW BURLINGTON STREET. 
 PHILADELPHIA : LINDSAY AND BLAKISTON. 
 
 1875. 
 IAU rights reserved.] 
 
QMS 5 
 
PREFACE. 
 
 THE first edition of these Notes was published in the 
 Quarterly Microscopical Journal for January 1872. Various 
 circumstances having delayed the appearance of my " Text- 
 Book of Practical Physiology," I have been induced to issue 
 a new edition of the Notes, for the purpose of assisting 
 those who are attending a course of Practical Histology for 
 the first time. The present edition is considerably altered 
 from the first, various new methods having been intro- 
 duced. All figures of tissues, and most of those of appa- 
 ratus, are reserved for my larger work. Pathological 
 histology is almost entirely omitted, as this subject is 
 taught practically by my colleague the Professor of 
 Pathology. As the time at the disposal of most stu- 
 dents of medicine is very limited, a course has been 
 adopted, whereby students in classes, numbering from 
 twenty to thirty, can be conducted through the leading 
 points of practical histology in about thirty lessons, each 
 lesson extending from an hour to an hour and a half. As 
 
IV PKEFACE. 
 
 it is impossible in this short time to prepare all the tissues, 
 special demonstrations previously made by the teacher are 
 resorted to, in order that the introduction to the subject 
 maybe as complete as possible. The special 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. These Notes especially concern the course 
 which is introductory to an advanced study of the subject, 
 and the reader is desired to bear in mind that they are 
 primarily intended for the students of the University of 
 Edinburgh. 
 
 It is not professed that these Notes are more than an 
 outline of the subject of Practical Histology. They merely 
 give the points to which every student of medicine ought 
 to attend. Care has been taken to render all instructions 
 as definite as possible. Descriptions of structure are 
 generally left to the ordinary text-books of descriptive 
 histology. The structure of the microscope is also omitted. 
 When the student desires a full account of the various 
 methods proposed for the examination of the various 
 tissues and organs, let him consult Frey on " The Micros- 
 cope," and Beale "How to work with the Microsope." 
 Such works should, however, only be used for reference ; 
 for although admirable encyclopaedias, they can scarcely 
 be regarded as guides for the beginner. Carpenter's book 
 on " The Microscope " will be found an excellent treatise 
 
PREFACE. V 
 
 on the structure of the microscope and its accessories, as 
 well as on botanical and zoological histology. 
 
 When the student finds in the following Notes no state- 
 ment about the preservation of a specimen, it is to be 
 inferred that it is needless to preserve it. 
 
 In the course of the demonstrations the student is 
 desired to read the general considerations which follow 
 them ( 75). 
 
ABBREVIATIONS. 
 
 H. Means high power. A combination of lenses, such as Hartnack's 
 ocular No. 3, and objective No. 7, with the tube of the microscope 
 drawn out, magnifying about 300 diam., is the high power most 
 useful for the student. 
 
 L. stands for low power. Hartnack's ocular No. 3, objective No. 
 3, tube drawn out, magnifying about 50 diam. 
 
 D. stands for demonstration, and indicates that the student is to 
 prepare a microscopical demonstration. 
 
 V. S., L. S., and T. S., respectively mean vertical, longitudinal, 
 .and transverse section. 
 
 ERRATA. 
 
 P. 10, line 1 1 from top, for p read p f . 
 P. 49, last line of large type, dekte (a.) 
 
Hartnack's No. IIlA Microscope. A, Eyepiece. D, Coarse adjustment. C, Con- 
 denser. H, Fine adjustment. J, Objective. L, Stage. M, Diaphragm. N, Mirror. 
 
OUTLINES 
 
 OF 
 
 PRACTICAL HISTOLOGY. 
 
 INTRODUCTION. 
 
 1. In the histological section of the class of Practical Physiology 
 in the University of Edinburgh, the student is supplied with a 
 Hartnack's No. IIlA microscope, having a No. 3 and a No. 7 
 objective, and a No. 3 eyepiece the No. 4 eyepiece commonly 
 supplied by Hartnack being intentionally omitted as of little 
 value. The highest magnifying power attainable with these 
 lenses is 300 diameters ; a power sufficiently high for most 
 purposes for the beginner. He is also supplied with the following 
 fluids for the preparation and preservation of the various tissues : 
 1. Distilled water. 2. Salt Solution. 3. Magenta Solution. 
 4. Carmine Solution. 5. Logwood Solution. 6. Absolute 
 alcohol. 7. Clove oil. 8. Acetic acid. 9. Glycerine. 10. 
 Weak spirit. 11. Potassium acetate solution. 12. Canada 
 balsam, or Dammar mounting fluid. 13. Gold size varnish. 
 14. Dammar varnish. Other re-agents are only occasionally 
 employed, and are provided when required. 
 
 2. The student is desired to bring to the class a razor ground 
 flat on the side next the right hand when the face of the razor 
 is held downwards (if the student is left handed, the left and not 
 
 A 
 
the right side of the razor should be flattened) ; a pair of strong 
 needles in handles ; scalpels, forceps, scissors (such as are found 
 in the ordinary dissecting case) ; three or four mounting clips ; 
 three or four camel-hair brushes of small size; two or three 
 dozen ground class slides, 3 by 1 inch ; J oz. extra thin circular 
 cover-glasses, f -inch broad ; and a box to hold say fifty prepara- 
 tions. The student is thus enabled to preserve for himself the 
 principal specimens made during the course. 
 
 3. At a future time, when the student undertakes histological 
 work in private, he would do well to procure a Hartnack's 
 microscope. This microscope is recommended because its defin- 
 ing power is excellent, its stage is not too high, and the whole 
 instrument is small and easily used. Before purchasing a 
 microscope it should invariably be tested by a competent 
 observer. A drop of fresh human blood is perhaps the best 
 test object. Unless the margins of the coloured corpuscles 
 appear perfectly sharp the definition is imperfect. The only 
 colour exhibited by a single coloured corpuscle is pale yellow : 
 if therefore an isolated corpuscle when exactly in focus shows a 
 red or green halo the lenses are not achromatic. 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 fiat. 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. 1 
 
 The advanced student should, in addition to the above lenses, 
 procure Hartnack's No. 9 immersion objective, and, for a still 
 higher power, Gundlach's No 8 immersion objective. Both are 
 good lenses, and the latter is remarkably cheap. Gundlach's 
 lenses, however, vary so much in quality that great care must be 
 exercised in making a selection. 
 
 1 Hartnack's microscopes may be obtained from Bryson, Optician, 60 
 Princes Street, Edinburgh; Tisley and Spiller, 1 72 Brompton Road, London, 
 S.W. ; Baker, 244 High Holborn, London, W.C. 
 
3 
 
 Admirable lenses are made in this country by Boss, Powell 
 and Lealand, Browning, and others, but their great expense 
 places them within the reach of only a few persons. 
 
 The advanced student should also obtain a stage and a 
 Hartnack's eyepiece micrometer, a freezing microtome, Valentin's 
 knife, Chevalier's camera lucida, Strieker's hot stage, and, if need 
 be, a colza oil or paraffin lamp, with a chimney of pale blue 
 glass for night-work. He may also procure Hartnack's polarising 
 apparatus, turn-table, and a small syringe for injections. 
 
 An achromatic condenser is not required, unless for very 
 exceptional work. Other accessories are, speaking generally, of 
 very little use. 
 
 PREPARATION OF TISSUES PREVIOUS TO THEIR EXAMINATION. 1 
 
 4. The following fluids require to be prepared : I. f per cent. 
 Salt Solution. Dissolve 7.5 grammes of dried ordinary NaCI 
 in 1000CC distilled water. II. 1 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 methylated spirit. IY. 
 Chromic and Bichromate Solution. Dissolve 1 gramme chromic 
 acid, 2 grammes potass, bichrom. in 1200CC water. V. Chromic 
 and Nitric Fluid. Chromic acid 1 gramme, water 200CC, then 
 add 2CC nitric acid. VI. Mailer's Fluid. Dissolve 25 grammes 
 potass, bichrom. and 10 grammes sodium sulphate in 1000CC 
 water. 
 
 5. Most of the requisite tissues and organs may be obtained 
 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 
 
 1 This section is designed to assist the student when he undertakes 
 histological work in private. 
 
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 J per cent, chromic acid fluid ; tie it to pre- 
 vent the escape of the fluid, and place the distended lungs in 
 the same fluid, and cover them with cotton wool. Change the 
 fluid at the end of 18 hours. Allow them to remain in this 
 fluid for a month, then transfer to meth. sp. until required. 
 
 7. Open by a linear incision the oesophagus, stomach, small 
 and large intestine, and wash them with salt solution. Place 
 a portion of the small intestine in chromic and bichromate fluid 
 for a fortnight (change the fluid at the end of 18 hours), and 
 then in meth. sp. until required. Place a portion of oesophagus, 
 large intestine, and stomach in \ per cent, chromic acid for three 
 weeks or a month (change the fluid at the end of 18 hours), and 
 then in meth. sp. until required. Place a portion of the stomach 
 in Miiller's fluid until required for the preparation of non-striped 
 muscle, and preparations of the gastric follicles. Open the 
 bladder, wash it with salt solution, and prepare it in the same 
 manner as the small intestine. 
 
 8. Eemove the kidneys; divide one transversely and the 
 other longitudinally, and place them in Miiller's fluid. Change 
 the fluid at the end of 18 hours. At the end of four weeks 
 transfer them to meth. sp. until required. They will be ready 
 after having been a fortnight in the spirit. It is well to prepare 
 the kidney of the dog in the same way. 
 
 9. Cut half of the liver into small pieces, and prepare it in the 
 same way as the kidneys. Place the tongue, divided trans- 
 versely into five or six pieces, spleen, uterus, some thin muscles 
 from the limbs or abdomen, in \ per cent, chromic acid. Change 
 the fluid at the end of 18 hours. At the end of a month place 
 in meth. sp. 
 
 10. Place testis of dog freely incised, and ovaries of cat or 
 dog, in Miiller's fluid. Change at the end of 18 hours, and at 
 the end of three weeks transfer to meth. sp. 
 
11. .Remove both eyes. Divide them transversely behind the 
 crystalline lens. Kemove the vitreous. Place the posterior 
 halves in the chromic and spirit solution III. Change at the 
 end of 18 hours. Transfer to meth. sp. at the end of ten days. 
 (Pritchard). Place the crystalline lens in Muller's fluid for five 
 weeks, and then in meth sp. Place the cornea in J per cent, 
 chromic acid for a month, and then in meth. sp. 
 
 12. Cautiously open the cranial and spinal cavities. Eemove 
 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 meth. sp. for 18 hours. Transfer the spinal cord to J 
 per cent, chromic acid for six or seven weeks. Change at the 
 end of 18 hours. Prepare the sciatic nerve in the same manner. 
 Place the brain in chromic and bichromate fluid. Change at 
 the end of 18 hours, and then once a week, until the brain is 
 hard and tough. If it be not tough and leathery at the end of 
 six weeks, place it in J per cent, chromic acid for a fortnight, and 
 then in meth. sp. 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. Pro- 
 cure a portion of human cerebrum (vertex), as fresh as possible, 
 and treat it in the same manner as the cat's brain. At the end 
 of six or seven weeks transfer all the tissues to meth. sp. 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. 
 
 13. 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 transversely in 
 two or three places, and put them into the chromic and nitric 
 fluid. Change the fluid repeatedly until the bone is just suffi- 
 ciently softened, and then transfer it to meth. sp. If the soften- 
 ing is not completed in a month, double the quantity of nitric 
 acid in the fluid. The bone must not, however, remain 
 

 
 iii the softening fluid after all calcareous matter has been 
 removed. 
 
 14. Place a piece of human scalp, skin from palmar surface of 
 finger, and skin of dog (for muscles of hair follicles), in chromic 
 and spirit fluid. Change at the end of 1 8 hours. At the end 
 of a month transfer to meth. sp. 
 
 15. Eemove the petrous portion of the temporal bone, open 
 the tympanum, pull the stapes from the oval fenestra, and place 
 the cochlea in chromic and spirit fluid. Change it at the end of 
 18 hours, and again on the seventh day. But if a copious 
 brown precipitate 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 it in meth. 
 sp. In the guinea-pig the cochlea forms a very evident pro- 
 jection 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 guiriea-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. 
 
 16. Chromic acid requires from one to six weeks to harden 
 tissues. It is important not to leave them too long exposed to 
 its influence, otherwise they are very difficult to stain with 
 carmine, and they may become friable and useless for the purpose 
 of section. Therefore they are transferred to meth. sp. when the 
 hardening is quite or nearly complete. 
 
 17. 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. 
 
 THE MODE OP WORKING WITH THE MICROSCOPE. 
 
 18. Prepare the Microscope. Before proceeding to prepare 
 
the object for examination : a. See that the proper lenses are 
 upon the microscope, b. Clean the tube, examine the lower 
 surface of the objective, and the upper surface of the ocular, and 
 see that both are clean, c. If the objective foe Hartnack's No. 
 7 (one-sixth of an inch focus), place it half an inch above the 
 level of the stage ; if it be Hartnack's No. 3 (one-inch focus), 
 place it two inches above the stage. As objective No. 7 will 
 presently be required, fix that on the microscope, d. 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 micro- 
 scope, but by a dimness of the object which may be under 
 examination. e. Clean one or more slides and cover-glasses. 
 If it be found dificult to clean new cover-glasses, place them 
 in a beaker ; cover them with ordinary commercial sulphuric 
 acid ; heat to the boiling point in a water bath ; allow the acid 
 to cool, and then wash the glasses with water. The latter should 
 never be laid fiat on the table, but should rest on their edges until 
 required. 
 
 19. Prepare a Simple Object. Place a small drop of milk 
 upon a slide, and let a cover-glass fall obliquely upon the drop. 
 The cover-glass may be applied most carefully by holding it near 
 the margin with a pair of forceps. The small brass forceps 
 supplied with Hartnack's microscope is well adapted for this 
 purpose. If too large a drop be taken, remove the superfluous 
 fluid from the edge of the cover-glass by means of blotting 
 paper. 
 
 20. Find the Focus. The No. 7 objective is on the micros- 
 cope, and it has been placed half an inch above the stage that 
 is, far outside its focal distance (one-sixth of an inch). Place 
 
8 
 
 the object on the stage, and bring it into focus ; in so doing, 
 keep the slide moving gently by means of the left thumb and 
 forefinger, seize the coarse adjustment with the right hand, and 
 while the eye looks through the microscope, lower the body of 
 the microscope very carefully in a spiral manner until the object 
 is visible. Take care not to go within the focal distance, other- 
 wise the object or the objective may be damaged. WJien the 
 object can be seen, however dimly, use the fine adjustment and 
 bring it accurately into focus. But never use the fine adjust- 
 ment until the object is visible. 
 
 21. Describe the Object. It is very important that the 
 student should be educated in the method of describing micros- 
 copic objects. To facilitate description, he should attend to the 
 following : 1. Shape. 2. Size. 3. Border. 4. Upper and 
 under surface. .5. Colour. 6. Transparency. 7. Contents. 
 8. Effects of re-agents. 
 
 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 corpuscles 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. Size. Attend in this case to the relative, 
 but omit the absolute size. Border. Is it smooth and regular, or 
 notched? 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 peritoneum is under observation, it is important. 
 Colour. When exactly in focus, are the corpuscles coloured or 
 colourless ? Transparency. 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 are not 
 distinctly visible. Contents. Does the corpuscle appear to be 
 
homogeneous, or can included particles be seen on carefully 
 focussing through the whole depth of the corpuscle 1 
 
 22. Drap the Object. It is very important that the student 
 should embrace every opportunity of making drawings of micros- 
 copical objects, for drawing necessitates a thorough inspection 
 of an object, and it impresses its features deeply upon the 
 memory. Excellent paper for drawing is the smooth but not 
 too highly glazed :i antique note paper " made by Pirie and Son, 
 Aberdeen. The pencils used are H.B. and H.H.H.H. The 
 shading should not be produced by lines, as in ordinary drawing 
 and wood engraving, but it should be a continuous shade pro- 
 duced by a rapid to and fro movement of the pencil. The 
 student should draw the object just as he sees it without altering 
 its apparent size. In making drawings of complicated subjects, 
 for the purpose of publication, it is often necessary to reduce the 
 size, and to fill in the details of one drawing from many speci- 
 mens, but with this the beginner has nothing to do. The only 
 
 Fig. 1. Chevalier's camera lucida, with Hartnack's microscope. 
 
10 
 
 practice advisible in his case is to draw tilings just as lie 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 difficult to 
 get the exact size and the general form without a camera. 
 Chevalier's camera lucida (Fig. 1.) is the best. 
 
 The eyepiece of the microscope is removed, and the camera 
 inserted in its place, and fixed in any position with a screw (6'). 
 The rays penetrate a prism (p), undergo total internal reflec- 
 tion, emerge, are collected by a special eyepiece, and pene- 
 trate a second prism (p), 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 Fig. With Chevalier's camera the stage of the micros- 
 cope is kept horizontal, so that it is perfectly serviceable in the 
 case of fluids. 
 
 Beale's reflector is also a good arrangement for drawing (Fig. 
 2). It consists of a slip of neutral tint-glass (?*), placed at an 
 angle of 45 in a metallic support made to fit over the ordinary 
 eyepiece. This is the cheapest camera, but it does not give so 
 sharp an image as that above mentioned, and the microscope 
 must be inclined. 
 
 Fig 2. Drawing with Beale's Reflector. 
 
11 
 
 In using either apparatus 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 not be too brightly 
 illuminated, othenvise the point of the pencil will not be clearly 
 seen. It is generally advantageous to close one eye. To all 
 drawings a statement of the magnifying power should be added, 
 and also a statement of the lenses used, e.g. Milk corpuscles 
 x 300. Hartnack obj. 7, oc. 3. Tube drawn out. 
 
 Add acetic acid, observe its effects on the milk 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. 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 inferre d 
 that each milk globule has an envelope that is soluble in acetic 
 acid. From other chemical considerations this envelope is 
 believed to consist of casein, a substance soluble in an excess of 
 acetic acid. 
 
 23. Estimate the magnifying power of the microscope. Let 
 the ]S"o. 7 objective and the JSTo. 3 ocular be on Hartnack's 
 microscope, with the tube fully elongated ; what is the magnifying 
 power 1 Place a stage micrometer on the stage, and focalise a 
 space that has in the process of manufacture been determined 
 to be one-thousandth of an inch (O'OOl in.) Hold the points of 
 a pair of compasses on a level with the stage, 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 one space of the micrometer ; then ascertain 
 with the aid of an inch scale, to what extent the 0*001 inch has 
 been enlarged. Suppose it to be magnified to 0'3 inch; then 
 0'3 ^0*001 =a magnifying power of 300 diameters linear. The 
 tube may be shortened and the magnifying power again estimated, 
 
12 
 
 and it will be found that the shorter the tube the lower the 
 power. 
 
 24. Estimate the size of an object. This may be most 
 accurately done with the aid of an eyepiece micrometer. This 
 consists of a glass slip, with a fine scale engraved upon it. 
 Hartnack's is the best. Measure the breadth of the human 
 coloured blood corpuscle ; use the No. 7 objective, elongate the 
 tube, and substitute the micrometer ocular for the ordinary one. 
 Bring a single coloured corpuscle into focus. It is seen through 
 the micrometer lines. Suppose its broad diameter covers three 
 of the smallest divisions. Push in the tube ; the corpuscle does 
 not now cover so much of the micrometer ; hence the value of the 
 divisions of the ocular micrometer varies with the magnifying 
 power, and it is necessary to determine the value of the ocular 
 micrometer divisions for all the powers employed. Therefore 
 substitute a stage micrometer for the blood, elongate the tube, 
 and focalise a 1/1 000th inch division. Suppose it to cover ten 
 divisions of the ocular micrometer, then evidently each division 
 must, with this magnifying power, be equivalent to 1/1 0,000th 
 inch. This ascertained, substitute the blood for the stage 
 micrometer. Suppose that the broad diameter of a coloured 
 corpuscle covers three divisions of the ocular micrometer, 
 then 3/10,OOOths=l/3333 inch. The corpuscles vary slightly 
 in size ; the average size being l/3200th inch. This figure must 
 be remembered, for the breadth of the human coloured blood 
 corpuscle is commonly taken as the histological unit of size. 
 
 HlSTOLOGICAL DEMONSTRATIONS. 
 
 25. Granules aad Brownian movement. (H.) Eub 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 which has no light centre. The 
 term coarse granule is applied to a particle of irregular shape 
 having a light centre surrounded by a dark border. The dif- 
 
13 
 
 ference between the optical characters of the former and those of 
 the latter is entirely owing to size. Brownian motion is exhib- 
 ited by all minute particles floating in a fluid. The motion is 
 probably owing to thermal currents in the fluid ; the solid 
 particles merely indicating the directions of these currents. 
 
 26. Hairs from stamens of Tradescantia virginica. (H.) a. 
 Examine in water ; observe the cells, wall, nucleus, protoplasm, 
 vacuoles, and movements of the protoplasm. Notice that the 
 protoplasm is a colourless finely granular jelly. 
 
 b. Heat, and observe that a gentle heat accelerates the proto- 
 plasmic movement. A hot stage suitable for roughly heating 
 a microscopic object may be made of a plate of tin, 9 x 2 \ inches, 
 with a small hole in the centre. Strieker's hot stage is 
 employed when it is desirable to carefully graduate the tempera- 
 ture and keep it constant. 
 
 27. Germinating yeast. 1 (H.) 
 
 a. Examine different forms of the torulae. The vacuole must 
 not be mistaken for a nucleus. 
 
 b. Add magenta, and observe that as the pigment flows under 
 the cover-glass the buds stain most rapidly. The protoplasm in 
 many of the torulse becomes stained, while the envelope 
 through which the staining agent must necessarily pass to 
 reach the protoplast remains 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 torulse never become stained. 
 These are probably the oldest ones. Observe that the envelope 
 is not equally thick in all the torulae. In the youngest 
 
 1 If beer yeast (tomla cerevisise) cannot be obtained, procure German 
 yeast, and sow it in sugar and water, or in the following modification of 
 Pasteur's fluid : 
 
 Water . . 8000 
 Cane sugar . 1300 
 Ammonium tartrate 100 
 
 Potassic phosphate . 20 
 Calcic phosphate . 2 
 Magnesium sulphate . 2 
 
 Keep the yeasty fluid gently warmed before the fire for a few hours. 
 
15 
 
 31. Linen fibres. (H.) Examine in water. These are not 
 twisted like cotton fibres. 
 
 32. Disc-bearing tissue. (H.) Examine a very thin vertical 
 section of an ordinary cedar pencil, or of. a piece of common fir 
 in water, and observe the disc-like depressions on the fibres. 
 
 33. Bacteria. (H.) Erom a putrefying infusion of some 
 albuminoid matter, e.g. muscle. Mince some muscle, 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 36 hours or so. Examine a particle of the 
 pellicle that forms upon the fluid with the microscope. Observe 
 the definite direction of the movements of the elongated bacteria 
 as long as they are alive. Add magenta. The fluid stains and 
 kills them. When dead, they exhibit only Brownian move- 
 ment. The direction of this movement is indefinite. When 
 living, the elongated bacteria wriggle like snakes, and thereby 
 move in definite directions. The latter motion is vital, and 
 results from contractility. 
 
 30, 31, 32, and 33 are examined because of the frequency of 
 their occurrence in the urine, and other fluids examined by the 
 medical microscopist. 
 
 34. Blood of newt. 
 
 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. 
 
 D 1 (H.) a. Examine the coloured corpuscles. With the 
 more obvious characters notice this, that the corpuscle 
 at first appears to be homogeneous, but after a time a 
 light oval spot appears in the centre of many ; this is 
 the position of the nucleus. 
 
 b. Examine the colourless corpuscles. There are several 
 varieties of these. Some are very finely granular nuc- 
 leated masses of protoplasm, as large, or even twice as 
 
16 
 
 large, as the coloured corpuscles. These are the common 
 white corpuscles. Others are coarsely granular. These 
 are not nearly so numerous as the first. A third set 
 consists of nucleated protoplasts, much smaller and finely 
 granular than the others. Study the amoebiform move- 
 ments of some of the large common finely granular 
 corpuscles. If they move sluggishly, apply heat with 
 the ordinary hot stage. Examine Strieker's hot stage 
 and the mode of using it. 
 
 c. Add acetic acid at edge of cover-glass, and gently 
 raise the cover to break the coagulum, and so allow the 
 acid to penetrate. Notice that the coloured corpuscles 
 suddenly become enlarged ; they lose colour, and the 
 nucleus becomes evident. The general mass of each white 
 corpuscle becomes very transparent, while the larger 
 ones usually exhibit three nuclei. The amoeboid motion 
 of the white corpuscle ceases. 
 
 D 2 (IT.) The same blood. Cover, and add water at margin, 
 of cover-glass. The coloured corpuscles lose their colour, 
 and often become more or less globular owing to 
 endosmose. 
 
 D 3 (H.) The same blood. Add strong syrup. Mix the- 
 blood and syrup with a needle before covering. The* 
 coloured corpuscles shrivel, owing to exosmose. 
 
 D 4 (H.) The same blood. Before covering add the special, 
 magenta fluid used for staining corpuscles (see p. 63). 
 It may be necessary to add afterwards a little of the 
 ordinary magenta fluid. The nuclei of coloured and 
 colourless corpuscles are stained. 
 
 D 5 (H.) The same blood. Add at margin of cover-glass 
 fresh solution of tannic acid (2 grains in 1 oz. warm 
 water. Allow it to cool). The coloured corpuscles 
 often become globular, and after a time the haemoglobin 
 becomes slowly or suddenly extravasated from the cor- 
 puscle in the form of a bud adherent to the wall, or in 
 
17 
 
 the form of fine granules scattered irregularly around 
 the wall. With l-25th inch objective it may be seen, 
 after the action of tannic acid, that the coloured cor- 
 puscle has a definite envelope, and this envelope may 
 sometimes be found ruptured where the bud has formed. 
 
 For D 2, 3, and 4, frog's blood does perfectly. 
 
 D 5 can also be done, though not so well, with the frog's 
 corpuscles. 
 
 35. Blood of bird. (H.) Examine, and then add acetic acid. 
 
 36. Blood offish. (H.) This must be quite fresh. 
 
 37. Human blood. (H.) 
 
 D 1. Examine a. Coloured corpuscles, b. "White corpuscles. 
 c. Fibrin, d. Add acetic acid at side of cover-glass, and 
 observe that the coloured corpuscles become globular 
 and lose their colour, while the white corpuscles become 
 very transparent, and show in the larger ones usually 
 three nuclei. 
 
 D 2. The same blood. Allow the drop to partially dry 
 before putting on the cover-glass, in order to get crenation 
 of the coloured corpuscles. 
 
 D 3. The same blood. Add to the blood, before applying 
 the cover-glass, the special magenta fluid for staining 
 blood corpuscles. No nucleus is revealed in the coloured 
 corpuscle, but in the white corpuscle three nuclei usually 
 appear. 
 
 D 4. The same blood. Add tannic acid. Mix the drop 
 of blood with a drop of the tannin solution (same as for 
 newt's blood) before applying the cover-glass, and watch 
 the change in the coloured corpuscles. The appearances 
 are similar to those noticed in the newt's coloured 
 corpuscles. Frequently, however, a delicate envelope 
 may be seen outside the bud of extravasated haemoglobin. 
 
 Examine human blood, through which induced electricity 
 has been passed, by the following arrangements. Take 
 B 
 
18 
 
 an ordinary slide, wet one surface with gum, press it 
 firmly down on gold leaf, allow it to dry, scrape away 
 the gold leaf so as to leave two triangles with their apices 
 pointing towards each other at the centre of the slide, 
 and reaching to an eighth of an inch from each other. 
 Attach wires to two mounting clips. Place the slide on 
 the stage and clamp each gold leaf triangle with a clip. 
 Attach the wires to the secondary coil of a faradic 
 machine. Place a drop of blood between the points of 
 the gold leaf electrodes and apply a cover-glass. Trans- 
 mit faradic shocks and watch effect. The coloured 
 corpuscles become globular and lose their colour. The 
 cause of this is probably electrolysis. 
 
 38. Pus. (H.) Examine and then add acetic acid. After 
 the action of the acid the larger corpuscles usually show three 
 nuclei, as in the case of the larger white blood corpuscles oi 
 human blood. 
 
 39. Saliva. (H.) Examine squamous epithelium and salivary 
 corpuscles. Notice the Brownian motion of the granules in the 
 latter. The salivary corpuscle appears to be a mucus corpuscle 
 modified by the imbibition of fluid. Add magenta, and observe 
 its effect on the cells. Observe the delicate fibrils of coagulated 
 mucin. 
 
 40. Mucus. (H.) Examine mucus corpuscles obtained from 
 urine, and compare with salivary and pus corpuscles. A mucus 
 corpuscle has usually a single globular nucleus, sometimes, 
 however, there is more than one. 
 
 41. Squamous epithelium. (H.) Omentum of guinea-pig or 
 cat previously stained with silver nitrate. Mount in glycerine. 
 (This will be wanted for areolar tissue, fat-cells, and perhaps 
 vessels, on a future day.) Study the method of staining ( 111). 
 
 42. Ciliated epithelium. 
 
 D 1 (L. & H.) Cilia from gill of salt-water mussel. Exa- 
 
19 
 
 mine in sea water. Apply heat by means of the ordinary 
 hot stage, and observe the accelerating effect of a moderate 
 temperature on the ciliary motion. 
 
 D 2 (H.) Study the action of chloroform on the ciliary 
 motion (Lister's experiment). For this purpose take a 
 slide with a glass cell, or make a cell from a ring of putty, 
 and place it upon a slide. Place a small piece of the 
 mussel's gill in a drop of sea water on the under surface 
 of the cover-glass. Place this over the putty cell. 
 Examine the cilia, then place a drop of chloroform on 
 the floor of the cell, and watch the effects of the vapour. 
 The motion gradually comes to a standstill. Study the 
 mode of applying vapours to microscopic objects with 
 Strieker's stage. 
 
 D 3 (H.) Scrape mucous membrane of frog's pharynx, and 
 examine ciliated cells in J per cent, salt solution. (Or, 
 kill a cat by a blow on the head : chloroform must not 
 be given, and scrape the mucous membrane of the 
 trachea.) Notice the clear band at the broad end of the 
 cell where the cilia are seen. If ciliated epithelium be 
 stained with carmine or magenta, the nucleus and the 
 finely granular protoplasm around it become stained, 
 but the cilia and the clear band remain unstained. In 
 this the latter resemble the contractile matter of muscle. 
 An excellent preparation of ciliated epithelium for preservation 
 will be obtained in the transverse section of trachea (see 51). 
 
 43. Columnar epithelium, (H.) Scrape the mucous mem- 
 brane of the small intestine of cat, prepared as directed in 7. 
 Diffuse the scraping in water with the point of a knife or needle ; 
 cover and examine. Notice the finely striated band at the 
 broad extremity of the columnar cell. This is more clearly seen 
 with a l-25th lens. This preparation need not be preserved, for 
 a section of the intestine ( 59) gives all that it shows. 
 
 44. Transitional epithelium. (H.) Examine a preparation 
 
20 
 
 made by scraping the mucous membrane of cat's bladder, pre- 
 pared as directed in 7, and preserved in glycerine. In this 
 variety of epithelium the cells may be squamous, columnar, or 
 quite indefinite in shape. 
 
 The various forms of glandular epithelium will be examined 
 in the different glands. 
 
 45. Elastic tissue. (H.) Lig. nuchse of calf. Snip off a small 
 piece with scissors, place it on a slide, add a drop of water, and 
 tease it thoroughly with needles, cover and examine. Remove 
 the cover-glass, and add acetic acid. It does not affect elastic 
 fibres. This tissue may be preserved in weak spirit or glycerine, 
 but it may also be preserved by the acetic acid already added ; 
 therefore, take care that the space under the cover-glass is com- 
 pletely filled with the acid. All air bubbles must be removed. 
 With bibulous paper thoroughly dry the slide outside the 
 cover-glass, and then, paint a ring of gold size around and over 
 the edge of the cover. When this has dried, put on a second 
 coat, with or without the aid of a turntable. When this has 
 dried, put on a final coat of dammar varnish. (See 127.) 
 
 46. Areolar tissue. 
 
 D 1 (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 the substance being strongly 
 refractile. 
 
 b. Take off 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 position 
 
21 
 
 excepting a clear jelly. The nuclei of the connective 
 tissue protoplasts 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 how- 
 ever be preserved, as specimens will be obtained in sections 
 of the alimentary canal. 
 
 The splitting of the white fibres into fibrils is easily effected 
 after maceration for a fortnight or so in a 10 per cent, 
 solution of salt, often changed, or in lime or baryta 
 water. (Eollett.) These agents appear to soften or 
 dissolve a cement which unites the fibrils. 
 
 D 2 (H.) Examine in a drop of aqueous humour (see 77) 
 a preparation of intermuscular fascia from the leg, or of 
 subperitoneal tissue from the lumbar region of a frog 
 recently killed. Remove a small portion of the tissue 
 with scissors and gently spread it out on a slide in the 
 aqueous humour. 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 connective tissue 
 protoplasts proper may perhaps also be seen. 
 
 D 3 (H.) Examine the preparation of omentum already 
 made, and observe the connective tissue corpuscles 
 amidst the fibres, and the epithelium here and there 
 clasping the bundles of fibres. Wandering cells (no 
 longer moving of course) may sometimes be seen singly 
 or in groups here and there on the surface of the bundles. 
 
 47. Tendon. (H.) T. S. dried tendon of sheep. The tendon 
 should be dried until it is just dense enough to be readily sliced. 
 Make an excessively thin section with a scalpel wetted with 
 
22 
 
 methylated spirit. Water does not adhere to the knife. 
 Examine the section in water. Observe the compressed branch- 
 ing spaces between the transverse sections of the longitudinal 
 fibres of the tendon. Some of the spaces contain nucleated 
 protoplasts. Bundles of fibres are seen running transversely 
 around and between the longitudinal bundles. (This need not 
 be preserved.) 
 
 Examine T. S. tendon hardened in chromic acid, and pre- 
 served in glycerine. In this, the spaces between the 
 bundles are not so compressed as in the dried tendon. 
 Examine L. S. tendon hardened in chromic acid. 
 
 48. Adipose tissue. (H.) Examine the preparation of omen- 
 turn of guinea-pig or cat already made. The nucleus of the fat 
 cell may have been stained by the silver, and if so, it will be 
 readily seen. 
 
 Examine a preparation of adipose tissue previously carmin- 
 ised or stained with logwood and preserved in glycerine. 
 Magenta does not stain the nuclei of the fat cells readily. 
 Such a preparation may be made by freezing a portion of 
 fresh skin, slicing it, and then staining it with carmine. 
 
 49. Adenoid tissue. (II.) a. Examine an extremely thin sec- 
 tion made with the freezing microtome of a fresh mesenteric 
 gland of cat or dog. Brush away lymph corpuscles with a small 
 camel-hair pencil, or, what is better, shake the section gently in 
 a test-tube containing j per cent, salt solution, in order to see 
 the branched connective tissue corpuscles of which adenoid 
 tissue consists the branches of the corpuscles, however, having 
 been transformed into fibres. Sections of lymph glands may be 
 advantageously stained with logwood (see 110). Preserve in 
 glycerine. (This preparation will be wanted on a future day.) 
 
 Study the mode of using the freezing microtome (see 85). 
 
 50. Mucous tissue. (H.) Examine a preparation of vitreous 
 humour. (Such a preparation may be made by slicing a frozen 
 eye and preserving the section of the vitreous in potass, acet.) 
 
23 
 
 In the peripheral part of the vitreous the tissue consists of a 
 jelly-like matrix, containing branched connective tissue cor- 
 puscles. Towards the centre of the vitreous no protoplasts can 
 be found in the jelly. 
 
 51. Cartilage. (H.) 
 
 D 1. With scissors make a section of white fibrocartilage 
 from inter vertebral disc of sheep, tease it slightly in salt 
 solution, and examine it in this fluid. The matrix con- 
 tains numerous fibrils like those of white fibrous tissue. 
 The protoplasts are rounded and enclosed in a capsule 
 (cartilage capsule). The cells may be found in various 
 stages of fission. (The capsule around the protoplast is 
 most evident with a 1-2 5th lens.) Preserve in glycerine. 
 
 D 2. Section of yellow fibre-cartilage from epiglottis of 
 sheep, hardened for a day in methylated spirit (nothing 
 else hardens this so well). Examine and preserve in 
 glycerine. When examined with a very high power it 
 is evident that this tissue consists of cells, imbedded in a 
 matrix formed of a spongy material. Here and there its 
 processes resemble the fibres of elastic tissue, but it is 
 erroneous to speak of the matrix as consisting of elastic 
 fibres imbedded in a granular matter. The apparent 
 granules are the cut ends of spicules of the spongy 
 matter. 
 
 D 3. T. S. tracheal rings of cat, hardened in chromic acid 
 (see 6). Mount in glycerine. Examine hyaline car- 
 tilage and perichondrium. This is a valuable specimen if 
 the tissue be cut with the aid of the freezing microtome, 
 and its surfaces therefore not destroyed by imbedding in 
 paraffin. It will show ciliated epithelium, glands in 
 mucous membrane, hyaline cartilage and fibrous tissue. 
 
 D 4. T. S. costal cartilage of adult. Kept in methylated 
 spirit for a day or so. Make the section through a point 
 of calcification. Preserve in glycerine. Some of the 
 
24 
 
 cells may have undergone fatty degeneration. Close to 
 the point of calcification, the cells will be found in rows 
 owing to proliferation. The matrix between the rows 
 will probably be found to be fibrillated owing to its 
 having been transformed into a calcified fibrous tissue. 
 D 5. V. S.. articular cartilage and bone from head of softened 
 bone of cat. (L.) Examine cartilage and cancelli. (H.) 
 Examine bone corpuscles in lacuna, canaliculi, osteo- 
 blasts in the cancelli, formation of bone corpuscles from 
 cartilage corpuscles. Examine and mount in glycerine. 
 Glycerine is apt to render softened bones too transparent ; 
 when such is the case, mount in meth. sp., and use thick 
 dammar as the cement. This preparation ought to be 
 one of much value. I have seen no preparations of 
 softened bones so beautiful as those prepared in the 
 manner indicated in 13. Examine a preparation of 
 osteoblasts under a 1-2 5th inch lens. 
 
 52. Bone. (H.) T. S. softened long bone of cat. (Treat as 
 D 5, 51.) Examine Haversian canals and the concentric 
 arrangement of the lacunae around these ; the difference between 
 the dense and the cancellated bone; the lamellae and their 
 arrangements ; the periosteum. 
 
 Examine T. S. and V. S. of unsoftened long bone. Such 
 sections are made with a fine saw, and are then ground 
 sufficiently thin upon a hone. They are best mounted 
 dry (in air). 
 
 53. Tooth. V. S. softened tooth and jaw of cat. Treat as 
 D 5, 51. (L.) General arrangement of tooth in alveolus, pulp 
 cavity, dentine. (H.) Dentine and crusta petrosa. The enamel 
 disappears under the influence of the acid. The odontoblasts 
 may possibly be found on the inner aspect of the dentine, while 
 their external processes the fibres of Tones may be seen pass- 
 ing into the dentine. 
 
25 
 
 Examine sections of unsoftened tooth made in the same 
 way as the above. (L.) In V. S. notice arrangement of 
 enamel fibres ; coloured lines in enamel, and contour lines 
 in dentine, and the curvatures of the dentinal tubules. 
 (H.) In V. S. notice the transverse markings in the fibres 
 of the enamel. Bundles of the fibres will probably be found 
 to have been cut transversely. These will probably show 
 that each fibre is of a more or less hexagonal shape. 
 Observe the fine secondary branchings of the dentinal 
 tubules, giving rise to a feathery appearance. This is 
 usually most evident near the outer part of the dentine. 
 In this situation look for interglobular spaces. T. S. of 
 dentinal tubes will probably be found at some part of the 
 dentine, if not, examine a V. S. of tooth where the 
 section has been made immediately external to the pulp 
 cavity. 
 
 54. Muscle. (H.) 
 
 D 1. Strip off a small piece of non-striped muscle from 
 intestine or stomach macerated for two months or so in 
 Miiller's fluid. Separate the fibres by teasing. Preserve 
 in glycerine. It is difficult to see the nucleus unless the 
 fibres be stained. Examine frog's bladder carminised. 
 Notice the spindle-shaped and the tri-radiate fibres. 
 
 D 2. Eresh striped muscle of frog. Dissect it with needles 
 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, ot stretched be- 
 tween the ends of fibres broken across, or prolonged as a 
 funnel-shaped process from the end of a ruptured fibre. 
 Add acetic acid. It renders the sarcous substance very 
 transparent and reveals the nuclei of the sarcolemma. 
 The sarcolemma itself, being of the nature of elastic tissue, 
 is unaffected. 
 
 D 3. V. S. (with a razor) striped muscle of cat or rabbit, 
 
26 
 
 hardened in chromic acid. Place the section in a drop 
 of glycerine, tease with needles and examine the trans- 
 verse stripes of the fibres and the fibrils. 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 to 
 cleave transversely. The transverse cleavage takes place 
 through a faint dim stripe running across the clear stripe. 
 (This faint stripe can rarely be seen in this muscle with- 
 out the use of a very high power.) In this preparation 
 sarcous elements may be seen. These are oblong particles 
 resulting from a transverse cleavage of the fibrils. Pre- 
 serve in glycerine. The fibrils of striped muscle may be 
 readily obtained from the muscle of the lobster by harden- 
 ing it in meth. sp. for 24 hours and teasing. 
 
 An excellent preparation of T. S. and V. S. striped muscle 
 will be obtained from hardened tongue (see 59). 
 
 Examine T. S. striped muscle, carminised striped muscle, 
 and injected muscle. 
 
 D 4. Muscle from the leg or thorax of water beetle (Dy- 
 tiscus Marginalis). Add a drop of salt solution, gently 
 separate the fibres, and examine immediately. Notice 
 the light and dim stripes, Schaefer's "muscle rods" 
 with their heads in the light stripe. A power of 
 1000 diameters is required to see the "rods" clearly. 
 Study contractions. Even a f per cent, salt solution 
 rapidly alters the fibres, and, therefore, for prolonged 
 study they should be surrounded by their own juice only 
 (Schaefer). 
 
 Examine striped muscle of Dytiscus under 1-2 5th lens to 
 see "muscle -rods." 
 
 55. Nerve-tissue. 
 
 D 1 (H.) Eresh nerve of frog. Eray out one end of the 
 nerve with a needle in salt solution or aqueous humour, 
 
27 
 
 and examine axial cylinder and white sheath ("white 
 substance of Schwann"). The grey sheath (" sheath of 
 Schwann") may sometimes be seen prolonged as an 
 infundibuliform process from the end of a ruptured fibre. 
 
 D 2 (H.) Nerve of frog hardened in osmic acid (one-fifth 
 per cent.) for two days. Wash in distilled water for a 
 day, tease, and preserve in potass, acet. The acid darkens 
 the white sheath. Ranvier's nodes may be readily seen 
 in the course of the fibres. 
 
 D 3 (H.) L. S. with scissors white matter fresh spinal cord 
 of cat or sheep. (Add no fluid.) An extremely small 
 piece must be taken. 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 grey 
 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 inexperienced observer. 
 
 Examine fine white nerve fibres in frog's bladder, car- 
 minised, and blood-vessels injected with Beale's 
 glycerine and Prussian blue, and preserved in glycerine 
 and acid. (Glycerine 1 oz., glacial acetic acid 5 drops.) 
 The acid renders the white sheath granular, and by this 
 enables one at once to distinguish delicate medulated 
 nerve fibres from white fibrous tissue. 
 
 D 4 (H.) T. S. sciatic nerve of cat hardened in chromic 
 acid. Stain with carmine in the manner recommended 
 in 72. After staining and washing remove the water 
 by immersing first in methylated spirit in a test or 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 
 
28 
 
 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. Watch with L. the clarifying 
 effect of the oil. 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 down. 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. 
 
 Study (H.) the section of nerve prepared as above. Notice 
 the divided nerve fibres and the connective tissue (peri- 
 neurium, neurilemma.) 
 
 Examine (H.) nerve fibrils in cornea stained with gold 
 (see 112). (H.) Grey fibres from sympathetic nerve 
 prepared as follows. Tease and preserve in glycerine a 
 portion of the cervical sympathetic of a rabbit recently 
 killed. 
 
 D 5 (H.) Section of fresh Gasserian ganglion of sheep 
 with scissors. Examine in salt solution. The nucleus, 
 nucleolus, and general protoplasm of the cell can be 
 readily seen. The processes are usually broken off. 
 
 The capsule that surrounds each cell may be demonstrated 
 thus : make sections of a frozen Gasserian ganglion 
 (fresh), and stain with carmine or magenta. 
 
 Examine cells of spinal cord carminised, to see processes 
 of nerve-cells. 
 
 The fibrillar structure of the processes of nerve-cells may 
 be shown as follows. Cut the fresh spinal cord of a 
 calf into pieces about a quarter of an inch in length. 
 Place these for a month in a one per cent, potass, 
 bichrom. solution. Eemove a thin slice of the grey 
 matter of the anterior horn with scissors, tease with 
 needles, stain with carmine, and mount in glycerine. 
 
29 
 
 Or freeze the fresh cord of the calf; make transverse 
 sections. Place them in l-5th 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. 
 
 Examine a preparation of the pear-shaped cells of sympa- 
 thetic ganglia. These may be prepared 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. 
 
 56. Blood-vessels. (H.) 
 
 D 1. Pia mater of sheep. With scissors remove a slice J 
 inch or so in depth from the surface of a cerebral con- 
 volution. Lay it upon a slide, with the pia mater touch- 
 ing 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 and 
 examine. Observe the nuclei in the capillaries. The 
 elastic membrane, the muscular fibres, and the tunica 
 adventitia in the larger vessels. Add acetic acid. The 
 tunica adventitia swells up, becomes transparent (hyaline), 
 and the nuclei of its connective tissue corpuscles appear. 
 The muscular fibres become very transparent and show 
 their nuclei. The elastic membrane is then more evident. 
 
 Examine elastic membrane of blood-vessels isolated. This 
 may be readily prepared from the basilar or other larger 
 artery at the base of the sheep's brain. Take a short 
 piece of the artery, lay it open, add a drop of acetic acid, 
 tease with needles, and mount in glycerine. 
 
 Examine blood-vessels with silvered epithelium (see p. 64), 
 and T. S. blood-vessels. A preparation of T. S. blood- 
 vessels may be readily obtained by making a T. S. of 
 
30 
 
 hardened spinal cord with the pia mater intact (see 72), 
 or a Y. S. of tongue (see 59). 
 
 57. Circulation in frog's web and mesentery, a. Inject one, 
 two, or three minims according to size of frog of a watery 
 solution of curara, each minim of which contains 0'032 milli- 
 gramme of the poison. 
 
 b. Tie a soft cotton thread round the longest toe, close to its 
 tip, and another round the toe next to, and on one or the other 
 side of this. Lay the frog on its back and stretch the web gently 
 over a triangular window in a piece of cardboard (A. Fig. 3) by 
 drawing the threads attached to the toes gently through the 
 slits a and a. The web must not be permitted to dry. 
 
 Fig. 3. Frog-plate of cardboard. The slits c and c' are only used when 
 the frog is not curarised and requires to be secured in a bag. 
 
 c. (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. 
 
 d. (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 col- 
 oured corpuscles move principally in the centre of the stream, 
 while the colourless corpuscles may often be found adhering to the 
 
31 
 
 wall of the vessel, or moving tardily along it. The peripheral 
 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. 
 
 e. Apply to the web a particle of strong mustard freshly made 
 into a paste with water. Wash it off at the end of twenty 
 minutes. While the mustard is acting, expose the mesentery 
 by a vertical incision on the left side of abdomen, a vein immedi- 
 ately below the axilla must be avoided, and also the stomach 
 and duodenum. With forceps gently draw out the intestines, 
 and pin it to a cork crescent close to a window in the cardboard 
 (B. Fig. 3). 
 
 Examine with L. and H. arteries, veins, and capillaries. For 
 prolonged observation it is necessary to place a slip of thin glass 
 over the window in the cardboard, and also to cover the 
 mesentery with a small cover-glass. It should be wetted with 
 aqueous humour. 
 
 g. Wash off mustard and look at inflamed web with L. and H. 
 
 The diapedesis of the white corpuscles can be best seen in the 
 tail of a tadpole, rendered unhealthy by keeping the animal for 
 some days in a small quantity of water without changing it. 
 
 Study the mode of injecting vessels by a water pressure 
 apparatus, and also with a syringe (see 114). 
 
 58. Lung. 
 
 D 1. (H.) Examine the section of trachea already prepared. 
 Study ciliated epithelium and the layers of epithelial 
 cells below those that are ciliated; the mucous glands 
 and the cartilage. 
 
 D 2. a. Make sections of chromic acid lung across a bronchus 
 visible to the naked eye. Sections of lung, either fresh 
 or hardened, are lest made by freezing. Examine and 
 
32 
 
 preserve in glycerine. (Sections may be stained with 
 logwood and mounted in glycerine or dammar.) 
 b. (L.) Notice the general characters of T. S. bronchus and 
 large vessels in order to recognize them readily with the 
 high power. (H.) Examine epithelium of air vesicles 
 and the fibrous tissue around these; T. S. bronchus 
 showing epithelium, glands, muscle, cartilage; T. S. 
 pulmonary artery and vein. The wall of the vein is 
 thinner than that of the artery. Adenoid tissue may in 
 some sections be seen around the larger blood vessels and 
 in the bronchial mucous membrane. Examine a prepara- 
 tion of lung with blood vessels injected. 
 
 59. Alimentary Canal. 
 
 D 1. V. S. chromic acid tongue of cat. Examine and pre- 
 serve in glycerine (L.) Papillae, muscle, (H.) Epithelium, 
 T. S. and V. S. striped muscle, T. S. and perhaps V. S. 
 vessels, fibrous tissue. (Sections may be stained with 
 carmine or logwood and mounted in dammar.) 
 
 Examine (H.) V. S. gustatory disc of rabbit's tongue for 
 taste bulbs. This may be made from a tongue hardened 
 in absolute alcohol. Stain with logwood, and mount in 
 dammar. 
 
 Examine (L.) V. S. human tongue showing fungiform and 
 filiform papillae. (L.) Y. S. injected tongue of cat or 
 man. 
 
 D 2. V. S. chromic acid stomach of cat. This is best 
 made by freezing. Preserve in glycerine. (L.) Eelations 
 of mucous, sub-mucous, and muscular coats. (H.) Eolli- 
 cles, muscularis mucosa, sub-mucous coat. Blood vessels, 
 L. S. and T. S. non-striped muscle. 
 
 The various structures may also be beautifully seen in a 
 stomach, macerated for six weeks or so in Miiller's fluid 
 ( 7). This is well suited for the demonstration of the 
 cells in the peptic follicles. In a very thin V. S., columnar 
 
33 
 
 cells may be seen at the patent extremity, while two 
 kinds of cells are seen in the caecal portion of the tube, 
 the so-called " peptic cells " placed externally, and the 
 continuation of the columnar cells internal to these. In 
 the csecal part of the follicle the columnar cells are often 
 much shortened and irregular. 
 
 D 3. T. S. Small intestine of cat hardened in chromic and 
 bichromate fluid ( 7). Preserve in glycerine. (L.) 
 Mucous, sub-mucous, and muscular coats. (H.) Columnar 
 epithelium of villi, and Lieberkiihn's follicles, muscularis 
 mucosa. 
 
 D 4. T. S. Large intestine of cat hardened in chromic acid 
 ( 7). Preserve in glycerine. (L.) Same as above. (H.) 
 Lieberkiihn's follicles. 
 
 Sections of stomach and intestine, if carminised, may be 
 mounted in dammar. 
 
 Examine (L.) V. S. injected stomach. T. S. injected small 
 and large intestine. Inner aspect of small intestine 
 injected. T. S. duodenum carminised showing Brunner's 
 glands. T. S. small intestine carminised showing Peyer's 
 follicles ; and V. S. small intestine showing valvulae 
 conniventes. 
 
 (H.) Contents of Peyer's follicle, seen in a T. S. or V. S. 
 carminised small intestine with blood vessels injected. 
 
 60. Liver . 
 
 D 1 (H.) Cells of fresh cat or rabbit's liver. Examine in 
 salt solution. 
 
 D 2 (H.) Cells of liver of ox. These are usually fatty. 
 
 D 3. Thin section of liver of cat hardened in Miiller's fluid 
 and spirit. Make the section across some of the larger 
 vessels. The sections are best made by freezing. Preserve 
 in glycerine. (L.) Lobules. (H.) Appearances and 
 relations of cells one to another and to capillaries 
 C 
 
34 
 
 capsule of Glisson, T. S. portal vein, one of the larger 
 
 bile ducts, and hepatic artery. 
 D 4 (L.) Section of liver of rabbit or cat injected from portal 
 
 vein, and hardened in meth. sp. Preserve in glycerine 
 
 or dammar. 
 Examine liver of rabbit (H.) with bile capillaries injected 
 
 with soluble Prussian blue, and blood capillaries injected 
 
 with carmine and gelatine, preserved in dammar. (L.) 
 
 Liver with hepatic artery and venous system injected. 
 
 61. Kidney. 
 
 D 1. Y. S. Malpighian pyramid of dog or pig's kidney, 
 previously hardened in Miiller's fluid and spirit. Examine 
 and preserve in glycerine. The section is best made by 
 freezing. It can also be tolerably well made with 
 Valentin's knife. (L.) Arrangements of tubules in 
 cortex and medulla, pyramids of Ferrein, Malpighian 
 bodies. (H.) Epithelium of convoluted and straight 
 tubules, Malpighian bodies and capsule, and larger blood- 
 vessels. 
 
 D 2 (H.) Scrape the cut surface of cortex of fresh kidney 
 of sheep to get isolated glomeruli, epithelium, and 
 basement membranes. Examine in salt solution, and 
 then add acetic acid, and notice effect on glomerulus. 
 
 D 3 (H.) T. S. Malpighian pyramid of Muller's fluid kidney 
 near its apex. Examine and preserve in glycerine. (The 
 section is best made from a frozen kidney, but may also 
 be made with a Valentin's knife.) Study connective 
 tissue matrix, and T. S. tubules. 
 
 D 4 (L. and H.) V. S. Malpighian pyramid of injected 
 kidney. See afferent and efferent vessel of glomerulus 
 and the vasa recta. Mount in glycerine or dammar. 
 
 62. Urinary deposits. (H.) The various forms of these are 
 studied in the chemical division of the class of Practical Physi- 
 ology. It is convenient however to study here the methods of 
 
35 
 
 mounting them, in order that the student may prepare specimens 
 similar to those which he will afterwards be shown. 
 
 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 supernatant fluid by the dis- 
 turbing influence of the pipette. For the ordinary examination 
 of the deposit it is generally convenient 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 prevents the diffusion of the deposit under the weight 
 of the cover-glass. If Hartnack's No. 7 objective and No. 3 
 ocular be used, shorten the tube fully, as the power is a little 
 too high with the tube elongated, that is to say, the lens re- 
 quires, in the latter case, to come so near the cover-glass that 
 the whole depth of the cell is not visible. 
 
 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 shallow dammar or balsam cell, 
 made by painting a ring of fluid dammar, or Canada balsam, on a 
 slide. With the aid of a pipette place a particle of the deposit 
 in such a cell, add a drop of the preservative fluid, and seal up. 
 
 a. Urates. Mount in weak spirit or glycerine solution (see d). 
 
 b. Cystine. Mount in dilute acetic acid (ordinary pyroligneous 
 acid 1 part, water 20 parts). 
 
36 
 
 c. Triple phosphate. Mount in strong ammonia 1 part, water 
 6 parts. 
 
 d. Uric acid and oxalate of lime. Mount in glycerine and 
 camphor ivater, equal parts (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 
 dammar varnish, to prevent the hygroscopic jelly from attracting 
 moisture from the air. As the jelly answers admirably, the use 
 of Canada balsam or dammar is unnecessary ; and this is fortu- 
 nate, for the use of these media in this connection is attended 
 with considerable difficulty. 
 
 e. Casts and epithelium. Pus and mucus may be preserved 
 in naphtha and creosote fluid prepared as follows, (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 
 or three weeks, occasionally shaking or stirring the mixture, then 
 filter, and preserve in corked or stoppered bottles (Beale). 
 
 63. Lymphatics. (H.) Examine (a) a silvered preparation of 
 the septum cysternaB lymphatics? magnse of the frog, preserved 
 in glycerine, showing the stomata, or apertures of communication 
 between the cavity of the peritoneum and that of the lymph sac. 
 (b) A silvered preparation of the centrum tendineum of the 
 rabbit, preserved in glycerine, showing lymphatic vessels with 
 their epithelial lining, (c) A silvered preparation of the mesentery 
 of the rabbit preserved in glycerine, showing lymphatics stained. 
 
37 
 
 Study the method of staining ( 111), and the puncture method 
 of injecting lymphatics ( 116). 
 
 64. Lymphatic gland. (H.) Examine the section previously 
 prepared. Study the relations and appearances of the trabeculae, 
 lymph paths, and glandular tissue. 
 
 Excellent sections of lymphatic gland may be obtained by 
 freezing a gland of the dog or cat, after hardening for a few days 
 iii l-6th per cent, chromic acid. 
 
 65. Spleen. 
 
 Examine, without the microscope, the cut surface of fresh 
 spleen of ox, to see pulp and splenic corpuscles ; then 
 scrape the pulp away, in order to see the trabeculse. 
 
 Section of chromic acid spleen of cat ; mount in glycerine. 
 (L.) Trabeculae, capsule, splenic corpuscles. (H.) Struc- 
 ture of trabeculse, splenic corpuscles and pulp. 
 
 Sections of spleen tinged with carmine may be mounted in 
 dammar. 
 
 66. Thyroid gland. (L. and H.) Examine sections of human 
 thyroid gland injected and uninjected. The uninjected gland is 
 best hardened in Miiller's fluid for a month, and then in meth. 
 sp. for a fortnight. Preserve 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. 
 
 67. Suprarenal Capsule (L. and H.) 
 
 Examine V. S. suprarenal capsule prepared as follows : 
 Harden in Miiller's fluid for a month, and then in me- 
 thylated spirit for a fortnight. Mount the sections in 
 glycerine or dammar. 
 
 68. Skin and Epidermic Structures. 
 
 I) 1. V. S. skin of palmar surface of finger hardened in 
 chromic acid. Preserve in glycerine, or stain with car- 
 
38 
 
 mine or logwood, and mount in dammar. (L.) Horny 
 layer and rete mucosum of epidermis, cutis vera, papilla, 
 possibly sweat glands. (H.) Epidermis, papillae, touch. 
 (Wagner's) corpuscles, fibrous tissue, vessels, fat cells, 
 possibly nerves and Pacinian corpuscles. 
 Excellent preparations of the skin may be made by the fol- 
 lowing method devised by my assistant, Dr. William Stirling : 
 Mix ICC pure hydrochloric acid with 500CC water at 38 C. 
 (100 F.), and add 1 gramme pepsine. 1 After keeping the mix- 
 ture 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 38 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. 
 
 Examine (L.) V. S. skin of human heel for sudoriparous 
 glands. (L.) Y. S. skin from back of sheep's neck for 
 sebaceous glands. (L.) V. S. skin of dog, showing the 
 sudoriparous glands opening into the hair follicles (Wm. 
 Stirling). (L. and H.) V. S. skin of dog for erector 
 muscle of hair follicle. (H.) V. S. skin of finger for tactile 
 (Wagner's) corpuscles. (L. and H.) Pacini's corpuscles. 
 
 1 The pepsina porci, prepared by Bullock and Reynolds, Hanover Street, 
 Hanover Square, London, should be employed, if the student does not desire 
 to prepare it from the stomach himself. 
 
39 
 
 Pacini's corpuscles 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, how- 
 ever, be found with far greater readiness in the mesentery of the 
 cat, where they are evident to the unaided eye as small, clear, 
 oval, bead-like bodies. They are best studied in the fresh con- 
 dition, being difficult to preserve. Glycerine is a tolerably good 
 preservative medium. 
 
 D 3 (H.) Human hair. 
 
 D 4 (EL) Hair of sheep. 
 
 D 5 (H.) Hair of rabbit. 
 
 D 6 (H.) Feathers of moth or butterfly. 
 
 Mount preparations 3, 4, and 5 in dammar. 
 
 Examine (H.) V. S. and T. S. human scalp, stained with 
 carmine or logwood, for the contents of hair follicles. 
 
 69. Eye. 
 
 D 1. V. S. chromic acid cornea of cat. Preserve in gly- 
 cerine. (H.) Anterior epithelium, and the dense fibrous 
 band corresponding to Bowman's membrane in the human 
 eye. Posterior epithelium and the elastic lamina (mem- 
 brane of Descemet). Fibrous tissue and corneal cor- 
 puscles. 
 
 Examine cornea of guinea-pig, showing nerves and corneal 
 corpuscles stained with gold. (See 112.) 
 
 D 2 (H.) Fibres of crystalline lens of ox, cat, or dog, boiled 
 for ten minutes in dilute sulphuric acid (1 per cent.). 
 Tease and preserve in glycerine or potassium acetate. 
 
 Examine (H.) T. S. fibres of lens, hardened in Muller's 
 fluid and spirit, and preserved as in D 2. 
 
 D 3 (H.) Hexagonal pigment cells from eye of cat or dog, 
 hardened in Muller's fluid or chromic acid. Preserve as 
 in D 2. 
 
 D 4 (H). Retina hardened in chromic acid and spirit. Freeze, 
 and make V. S. of retina alone, or of all the tunics of the 
 
40 
 
 eye. Preserve in glycerine, or carminise and mount in 
 dammar. Study 1. Inner limiting membrane and fibres 
 of Mutter. 2. Fibrous layer (fibres of optic nerve). 3. 
 Cellular layer (nerve cells). 4. Inner granular layer. 
 5. Inner nuclear layer. 6. Outer granular layer. 7. 
 Outer nuclear layer. 8. Outer limiting membrane (it is 
 difficult to see this). 9. Jacob's membrane : the rods 
 are readily seen, the cones are seen with difficulty. 
 
 D 5 (H.) Eetina of frog, hardened in \ per cent, osmic 
 acid for 36 hours, washed in distilled water for 24 hours. 
 Cut with scissors or by freezing. (This method of pre- 
 paration is well adapted for showing outer and inner 
 segments of rods, outer limiting membrane, etc.) Pre- 
 serve in potass, acet. 
 
 Examine preparations showing (L.) Y. S. optic nerve enter- 
 ing eyeball ; V. S. ciliary muscle, iris, and sclerotic : 
 inner aspect of injected ciliary processes. (H.) Branched 
 pigment cells of choroid. (The vitreous has already 
 been examined, 50.) 
 
 70. Coclilea. 
 
 Examine V. S. cochlea. (L.) Observe the modiolus, lamina 
 spiralis ossea, limbus laminae spiralis, basilar membrane, 
 ligamentum spirale, rods of Corti, membrane of Eeissner, 
 scala vestibuli, scala media, and scala tympani. (H.) 
 Observe inner and outer rods of Corti, cells of Corti, 
 nerves, membrana reticularis, membrana tectoria, together 
 with the parts before mentioned. Examine also (H.) a 
 superficial view of the heads of the rods of Corti and the 
 membrana reticularis. 
 
 The following will be found an excellent method of preparing 
 the cochlea, devised by my former assistant, Dr. Urban Pritchard, 
 to whom I gave this subject for a graduation thesis (Edinburgh 
 Prize Thesis, 1871) : 
 
 The structure of Corti's organ may be investigated in the fresh 
 
41 
 
 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 methylated spirit into the tympanic cavity with an ordi- 
 nary subcutaneous syringe, thrust through the membrana 
 tympani. 
 
 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, devised by Dr. Pritchard ( 4, Solu- 
 tion III.). This fluid is used in the manner indicated in 15. 
 
 In the case of foetal cochlea?, 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 hydro- 
 chloric acid diluted with water ten times, but far better results 
 are obtained by using the bone-softening fluid already men- 
 tioned. (See 4, Solution V.). 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 ordinary meat-jack. 
 With the aid of this device, even a large cochlea may be softened 
 in a few days. 
 
 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 Strieker's 
 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 
 ( 85), for four or five hours, then immerse the cone in meth. 
 
42 
 
 sp. 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 ( 102) in a microtome, 
 with the modiolus of the cochlea placed at right angles to the 
 axis of the tuhe 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 other mode of 
 section, the slices should be immediately placed in meth. sp. 
 
 The sections may be stained with magenta, logwood, gold, 
 silver, and carmine. The last two yield the best results. Car- 
 mine may be successfully used in the following way. Mix equal 
 parts of water, methylated spirit, and Beale's carmine fluid, with 
 all the ammonia retained (see 107). (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 meth. sp. 
 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 
 weak spirit (meth. sp. 1 part, distilled water 3 parts). Next 
 cover them with an acid washing fluid, consisting of proof spirit 
 (rectified spirit 5 parts, water 3 parts), containing 1 part hydro- 
 chloric acid in 100 parts proof spirit. After washing in this 
 fluid for six or eight hours, until all diffuse staining is removed, 
 cautiously pour off the fluid and preserve the sections in ordinary 
 meth. sp. 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 ( 111). 
 
 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 weak spirit, but 
 if glycerine jelly be used for mounting, this precaution is un- 
 necessary. The best preservative media are glycerine, glycerine 
 jelly, potassium acetate, and dammar or Canada balsam. The 
 
43 
 
 ast two are only resorted to when the sections have been stained. 
 For unstained, and even for stained sections, glycerine jelly is a 
 good preservative medium. In mounting vertical sections of 
 the cochlea, the modiolus should always be placed across the 
 glass slip to facilitate subsequent microscopical study. 
 
 71. Sckneiderian Membrane. 
 
 Examine (H.) olfactory cells from nose of newt prepared as 
 follows : Harden the nose for a week in one per cent, 
 potass, bichrom. Tease a small portion of the mucous 
 membrane and preserve in glycerine. Y. S. Schneiderian 
 membrane of cat or dog, prepared in the same way as the 
 cochlea, and preserved in glycerine or glycerine jelly. 
 Observe epithelium and glands. 
 
 72. Spinal cord and brain. 
 
 D 1. T. S. spinal cord of cat hardened with chromic acid 
 as directed in 12. Wash the section in water, and car- 
 minise by the following method, here adopted because of 
 its rapidity. Place a large drop of J per cent, watery 
 solution of palladium chloride on a glass slide, and on 
 another slide a large drop of an ammoniacal solution of 
 carmine (ammonia ICC, carmine 1 gramme. Dissolve 
 with the aid of a gentle heat, add 23CC distilled water, 
 and filter). Allow the section to remain in the palladium 
 for two minutes. Wash it in water, and place it in the 
 carmine fluid for three minutes. Wash in water, and 
 mount in dammar, as described in D 4, 56. Observe 
 (L.) grey and white matter, nerve roots if present, fissures. 
 (H.) nerve cells, T. S. nerve fibres, neuroglia, blood- 
 vessels in the pia mater, and in the substance of the cord, 
 the central canal arid its epithelial lining. 
 With regard to the staining of nerve tissue, hardened with 
 chromic acid, it is to be remembered, that the longer the exposure 
 of the tissue to the influence of the acid, the less readily is it 
 stained with carmine ; indeed, after a very prolonged action of 
 
44 
 
 chromic acid, carmine has scarcely any effect unless it be preceded 
 
 by the action of palladium chloride (Merkel). In any case, the 
 
 method above recommended yields the most rapid results. The 
 
 staining is most perfect, however, when the tissue has not leen 
 
 over-hardened, and when it is sloicly tinged by a dilute solution 
 
 of carmine alone. A dilution of Beale's carmine fluid, with all 
 
 the ammonia retained, answers the purpose admirably (see 107). 
 
 Examine (L. and H.) T. S. spinal cord unstained. V. S. 
 
 spinal cord carminised, showing a nerve root crossing a 
 
 white column. (L.) T. S. injected spinal cord. (L. and 
 
 H.) T. S. medulla oblongata, 
 
 D 2. V. S. cerebellum hardened in chromic acid, carminise 
 and mount in dammar. Observe (L.) the relations of 
 the various layers, and the general appearance of the 
 convolutions, (H.) the flask-shaped cells at the inner part 
 of the outer grey layer, the external processes of these 
 cells, their branchings and the neuroglia with its nuclei 
 amidst which the branches disappear ; cells of the inner 
 grey layer, nerve fibres and nuclei in the white matter. 
 D 3. V. S. convolutions from vertex of human cerebrum, 
 hardened in chromic acid and carminised. Mount in 
 dammar. (H.) Observe the pyramidal cells. The cells 
 are seen to have a pyramidal shape only when the section 
 is vertical as regards the cells. 
 Examine (L.) V. S. injected cerebral convolutions. 
 
 73. Generative organs. 
 
 D 1. (H.) With a scalpel or scissors remove a few tubuli 
 from the testis of cat or dog, hardened in Miiller's fluid. 
 Tease and mount in glycerine. A complete section of 
 the organ may be made by freezing. 
 
 Examine (H.) spermatozoids. (L. and H.) Section of 
 ovary of cat showing Graafian follicles and their contents, 
 prepared as recommended in 10. The ovary should 
 remain in meth. sp. for forty-eight hours after removal 
 
45 
 
 from Miiller's fluid. The hardening should then be com- 
 pleted by immersion in absolute alcohol for forty-eight 
 hours or so. Imbed in paraffin, and make sections. 
 The sections may be mounted unstained in glycerine, or 
 they may be stained with carmine or logwood, and mounted 
 in dammar. 
 
 D 2. (L. and H.) T. S. uterus of cat hardened in chromic 
 acid. Preserve in glycerine. Study the uterine glands. 
 
 74. Development. 
 
 Examine a series of preparations of the embryo chick, 
 hardened with chromic acid, stained with carmine, and 
 mounted in dammar. A description of the mode of pre- 
 paring the chick is quite beyond the limits of these 
 notes. The student will find a full account of this 
 subject in the Elements of Embryology, by Foster and 
 Balfour. (London: Macmillan. 1874.) 
 
 GENERAL CONSIDERATIONS REGARDING 
 HISTOLOGICAL METHODS. 
 
 RELATIONS OF THE TISSUES TO SURROUNDING MEDIA. 
 
 75. When it is not desirable to examine the textures in their 
 living condition, they should be placed as fresh as possible in 
 preservative media. At death, the optical characters of many 
 tissues undergo rapid change. Generally speaking, transparency 
 diminishes. 
 
 Some tissues, such as hair, tooth, and bone, may be examined 
 in air. This, however, is generally objectionable; for, owing to 
 the great disparity between the refractive powers of the air and 
 the tissues, the outlines are too broad and too dark. 
 
 Some fluids alter the normal characters of the textures, e.g., 
 the blood corpuscles swell in water, while in a concentrated 
 solution of sugar or salt they shrivel. Acetic acid causes the 
 
46 
 
 fibrils of white fibrous tissue to assume the appearance of a 
 transparent jelly ; chloroform dissolves the medullary substance 
 of a white nerve fibre, while water coagulates it. In selecting a 
 medium in which to place a tissue, the physico-chemical nature 
 of the tissue and the medium must therefore be taken into 
 account. The points of greatest importance in this connection 
 are : a. The effect of the medium upon the vitality of the 
 tissue. b. The hardness or softness of the tissue, c. The 
 solubility of the tissue in the medium, d. The chemical rela- 
 tions between the tissue and the medium, e. The transparency 
 of the tissue. /. The refractive index of the medium. 
 
 Nonpreservative media employed in the examination of the tissues. 
 
 76. To study the normal characters of living tissues, examine 
 them in the fluids which bathe them during life, or in fluids 
 which do not alter their vital properties. Blood, salivary, mucus, 
 and pus corpuscles are examined in their own fluids. 
 
 77. Serous fluids. The fluid which bathes most tissues is 
 lymph. Blood serum seeing that it has a composition re- 
 sembling that of lymph, is sometimes employed for moistening 
 living tissues ; but the blood corpuscles which 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. 
 
 78. Serous fluids are also used for the examination of various 
 delicate tissues, even when these are not living, when they are 
 required in considerable quantity, amniotic fluid, pericardial 
 fluid, iodised serum, or dilute 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. Iodised serum is some- 
 times employed for macerating nerve cells and white nerve 
 fibres, in order to show the " primitive nerve fibrils " (Schultze). 
 
47 
 
 It is thus prepared : add 1 part tincture of iodine to 100 parts 
 fresh amniotic fluid ; filter, and place a small piece of camphor 
 in the fluid. 
 
 79. Dilute albumin may also be employed for the maceration 
 of delicate tissues. It should, however, only be had recourse to 
 when serous fluids cannot be obtained. It is thus prepared : 
 dissolve 2 grammes dried sodium chloride in 250CC water ; 
 add 28 grammes egg albumin, 2 - 3CC tincture of iodine, and 
 filter. Place a small piece of camphor in the fluid (Frey). 
 
 80. Salt solution. This is a f 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. 
 
 The serous fluids, dilute albumin, and salt solution, are some- 
 times termed "neutral" or "indifferent" fluids, on the supposi- 
 tion that they do not alter the normal appearances of the 
 tissues. This, hoivever, is only true of the serous fluids. 
 
 81. Water is only employed for dense tissues, such as elastic 
 tissue, hair, and epidermis. It destroys the normal characters 
 of soft tissues. 
 
 The above-mentioned fluids are not adapted for the permanent 
 preservation of the tissues. 
 
 The preservative fluids, those which clarify, and those 
 which stain the tissues, are also used as media in which to 
 examine them. 
 
 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. 
 
 82. Drying and boiling were formerly much resorted to, but 
 are now rarely employed. The former may be used for tendon, 
 when transverse sections are wanted merely to show the relations 
 
48 
 
 of the various systems of fibres ; moreover, it is sometimes 
 advantageous to partially dry a tissue that has been hardened 
 in alcohol before attempting to cut it. Excellent sections of the 
 skin may be obtained in this way. If, however, protoplasm and 
 other soft matters have not been previously hardened they are 
 hopelessly spoilt by desiccation. Boiling is useful for hardening 
 the fibres of the crystalline lens, and also those of muscle, when 
 coarse dissections are to be made of the latter. 
 
 83. Freezing is a method of much value. With its aid, 
 sections of any desirable thinness may be readily made of all the 
 soft parts of the body in a perfectly fresh condition. Lung, 
 kidney, spleen, lymphatic gland, vitreous humour, brain, &c., 
 may all be removed from the living animal, frozen and sliced in 
 a few minutes. 
 
 Freezing is suitable fpr hardening the brain and spinal cord, 
 only when slices of the fresh tissue are desired for the isolation 
 of nerve cells, &c. These organs are best hardened in chromic 
 acid, potassium bichromate, &c., not because they can be more 
 readily cut when hardened by these agents, but for the reason 
 that, when the tissue has been previously hardened in 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 
 process of hardening carried to some extent by Muller's fluid, 
 potassium bichromate, or chromic acid, before freezing them for 
 the purpose of section. The special cases where freezing is of 
 service have been indicated in the preceding demonstrations. 
 
 Freezing has been had but little recourse to, 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 
 
49 
 
 section, it begins to thaw, and thereby gives rise to so much in- 
 convenience, and to results so unsatisfactory, that the method of 
 freezing has been almost neglected. The method is rendered 
 simple and satisfactory with the aid of the freezing microtome. 
 When this apparatus becomes generally known, freezing will be 
 resorted to as the best method for obtaining sections of fresh or 
 imperfectly hardened tissue. 
 
 Fig. 4. The Freezing Microtome. 1 
 
 As the freezing microtome is a novel instrument, it is here 
 figured for the assistance of those who may not yet have seen it. 
 It (Fig. 4) consists of a plate of gun metal (B), with a circular 
 opening in its centre (A). The opening leads into a well (E), 
 
 1 The Microtome here figured is a modification of that originally de- 
 scribed by me in the Journal of Anatomy and Physiology, May 1871. One 
 or two alterations have been proposed by others, but I cannot regard these 
 as anything but the reverse of improvements. The apparatus as manu- 
 factured by Young, surgical instrument maker, North Bridge, Edinburgh, 
 answers admirably. 
 
 D 
 
50 
 
 closed inferiorly by a brass plug (K, Fig. 5), 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 over the top of the well. 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 
 
 B A 
 
 Fig. 5. Yertical Section of Freezing Microtome. 
 
 tissue is seen in A, and the freezing mixture in c. B is a 
 section of the knife employed. 
 
 The freezing microtome is essentially the well-known micro- 
 tqme devised by Mr. Stirling, Sub-Curator of the Anatomical 
 Museum of the University of Edinburgh, modified for freezing. 
 It serves a double purpose. 1. For cutting tissues hardened in 
 the ordinary way by chromic acid or spirit, &c. 2. For cutting 
 tissues hardened by freezing. The second method of using the 
 machine will be more readily comprehended after a description 
 of the first. 
 
 84. WJien the microtome is used in cutting an unfrozen tissue, 
 
51 
 
 pour into the well a mixture of solid paraffin (five parts), and 
 hog's lard (one part), melted by a gentle heat. The temperature 
 employed should be just sufficient for the purpose 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 be 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 heat- 
 ing 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, how- 
 ever, 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. 5) with a projecting 
 wooden pin fixed in it. The rotation of the paraffin cylinder is 
 thus prevented. 
 
 85. When the microtome is used for freezing, surround the 
 freezing box with a thick jacket of felt or flannel to prevent the 
 entrance of heat. Unscrew the plug (K, fig. 5), and oil both its 
 exterior and interior, so that it may not become fixed by the 
 freezing. The tissue to be frozen, together with an imbedding 
 fluid, are placed in the hole. If a watery fluid be employed, it 
 becomes crystalline when frozen, and is therefore badly adapted 
 
52 
 
 to serve as the imbedding agent. This difficulty, however, is 
 entirely overcome by using a solution of gum arabic. Frozen 
 mucilage can be sliced as readily as a piece of cheese. The use 
 of gum water for this purpose was suggested to me by my late 
 assistant Dr. Urban Pritchard, and it is a suggestion of much 
 value. The solution of gum is thus made : Add to 10 oz. of 
 water 120 minims of camphorated spirit, and 5 oz. of picked 
 (clean) gum arabic; when the gum has dissolved, strain the 
 fluid through calico or tow, and preserve it for use in a corked 
 bottle. The gum solution should be placed in the microtome 
 well first, 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. Lay a piece of thin 
 vulcanised indiarubber upon the microtome plate, so as to cover 
 the well, and prevent the entrance of heat and of salt from the 
 freezing mixture. Secure the indiarubber by a weight. Place 
 in the freezing box small quantities of finely powdered ice, and 
 salt alternately; constantly stir them with a glass rod around 
 the well, and keep the tube (H) open to permit of the egress of 
 water from the melting ice. This 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. It 
 is possible, especially in winter, to have the tissue frozen too 
 hard to permit of its being readily cut. It splinters when it is 
 too hard. This is prevented by discontinuing the further 
 addition of the freezing mixture, or by dropping water or a 
 | per cent, salt solution at the ordinary temperature on the 
 surface of the frozen tissue, or by heating the razor slightly. 
 
 A number of tissues may be frozen and cut at the same time. 
 If they have been previously immersed in spirit, they must be 
 steeped for a night in a large quantity of water ere they can be 
 frozen. In all cases, where it is possible, it is well to steep the 
 tissues for an hour or two in the solution of gum before freezing 
 them, in order that the gum may thoroughly penetrate the tissue. 
 
53 
 
 A great advantage of the freezing method for delicate organs, 
 such as the retina, trachea, embryo, etc., is that the imbedding 
 agent can be readily removed from the tissue by immersion in 
 water. 
 
 86. A razor answers for all ordinary purposes. The blade 
 should always be hollow on both surfaces (E, Fig. 5). It is a 
 mistake to employ a flat knife, for it is scarcely possible to keep 
 the surface of the brass table of the machine smooth enough to 
 permit of the knife lying quite flat. The operator should cut 
 outwards from himself by drawing the knife obliquely through 
 the tissue, which should be cut at one sweep. This is not 
 possible with a frozen tissue if the ice be too hard. For an un- 
 frozen tissue, imbedded in paraffin, the knife should be wetted 
 with methylated spirit. This should be dropped upon the knife 
 from a funnel with an elastic tube depending from it, clamped 
 by a Mohr's clip. The funnel should be suspended at a con- 
 venient height above the machine. The spirit keeps the blade 
 perfectly clean, and it forms a pool upon it, which enables the 
 section to float over the blade. In the case of freezing, it is not 
 necessary to wet the knife, for the melting ice readily does so. 
 
 87. Chromic acid is a much used hardening agent. It is 
 however not well adapted for kidney, liver, lymphatic glajid, 
 vitreous humour or crystalline lens, and it is apt to spoil the 
 epithelium of the intestine. One-fifth per cent, watery solution 
 is employed for the embryo, J per cent, solution for the spinal 
 cord, stomach, cornea, skin, lung, etc. A mixture of chromic 
 acid and potassium bichromate (see IV., 4) is used for the brain. 
 Chromic acid solution in weak spirit is used for the retina, coch- 
 lea, etc. (see III., 4). 
 
 Chromic acid hardens the tissues in from two days to two 
 months. The tissue should be 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 
 
54 
 
 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 same. object 
 cannot be attained by using a strong solution of the acid, for in 
 such a 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 suffi- 
 ciently hard. The spirit completes the process of induration. 
 Spirit removes much of the yellow colour given to the tissues 
 by the chromic acid. 
 
 88. Picric or Carbazotic acid, I per cent, watery solution, 
 hardens more rapidly than chromic acid, but the chromic acid is 
 to be preferred, because it does not colour the tissues so much. 
 
 89. Alcohol has been recommended for gland tissues. They 
 should be first placed in equal parts of methylated spirit and 
 water for a day, then in methylated spirit for a similar period, 
 and finally in absolute alcohol. It is most useful, however, for 
 completing the hardening of tissues, e.g. the kidney, intestine, 
 etc., that have been partially hardened in Muller's fluid or 
 chromic acid. 
 
 Sections of tissues hardened in alcohol, chromic, or picric 
 acids, generally require to be rendered transparent ; for this pur- 
 pose clove oil, turpentine, or glycerine, are commonly employed, 
 and when clarified, the tissues are mounted in glycerine, dammar, 
 or Canada balsam. 
 
 90. Muller's Fluid (potassium bichromate 2J parts, sodium 
 sulphate 1 part, distilled water 100 parts) is used for harden- 
 ing the liver, kidney, crystalline lens, stomach, ovary, testis, etc. 
 It requires from three to six weeks, and the tissues require to be 
 
55 
 
 afterwards frozen or placed in methylated spirit, or it may be, in 
 absolute alcohol, to render them sufficiently hard. Mount the 
 unstained tissues in glycerine, or stain them and mount in 
 dammar. 
 
 91. Osmic Add ( T Vth to 1 per cent, in distilled water) for 
 hardening the retina, epithelium, and white nerve fibres. It 
 hardens tissues in from twelve to forty-eight hours. When hard, 
 place them in distilled water for a day or two. Mount the 
 tissues in a saturated solution of potassium acetate (Schultze). 
 
 Methods of Softening the Tissues. 
 
 92. Removal of calcareous matter. Chromic 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 water containing a-half per cent, 
 chromic, and one per cent, nitric acid. (See V., 4, and 13.) 
 This fluid hardens the protoplasts of the tissue while it removes 
 calcareous matter. The results of its use are extremely satis- 
 factory. 
 
 93. Softening of connective substances. It is sometimes 
 desirable to soften or dissolve various connective substances, 
 such as white fibrous tissue, the cement which connects the 
 fibres of the lens and the fibrils of striped muscle. White 
 fibrous tissue may be softened : a. By boiling in water, b. By 
 maceration, at a temperature of 116 Pah., in dilute sulphuric 
 acid ( T ^th per cent.); these methods are useful for coarse dis- 
 sections of muscle, c. By maceration in glycerine (1 oz.), and 
 glacial acetic acid (5 drops) (Beale). This is valuable for 
 enabling us to trace fine nerves in many tissues. The white 
 fibrous tissue is rendered very transparent and gelatinous, while 
 medullated nerve fibres are rendered granular. In this way the 
 nerve elements are readily recognised. This mode of tracing 
 fine nerves has now been largely superseded by the method of 
 staining them with perchloride of gold. d. By prolonged 
 
56 
 
 maceration in a 1 per cent, solution of potassium bichromate 
 or Muller's fluid. This permits of the isolation of the gland 
 elements of kidney, intestine, stomach, etc., when specimens are 
 to be prepared by teasing. The cement between the fibrils of 
 the nerve axial cylinder may be dissolved : e. By maceration in 
 the above acid glycerine. /. By maceration in iodised serum. 
 The cement joining the fibrils in striated muscle may be dis- 
 solved by prolonged maceration in J per cent, chromic acid. 
 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.). 
 
 94. Methods of separating tissue elements, a. By dissection 
 with needles. Delicate textures and organs should be dissected 
 in some fluid, such as water, or f per cent, salt solution, b. By 
 gently pressing a small piece of tissue under a cover-glass, after 
 placing the tissue on a slide in the usual way. 
 
 Methods of Cutting the Tissues. 
 
 95. Some tissues are too soft, while others are too hard, to 
 permit of satisfactory section. "When such is the case, harden 
 or soften them by the methods above described. 
 
 96. Saw. Sections of unsoftened bone and tooth are made 
 with a fine saw. The sections are then ground sufficiently thin 
 upon a hone. 
 
 97. Scissors are often useful for removing a thin portion of 
 unhardened tissue. 
 
 98. Valentin's Knife has two parallel blades, the distance 
 between which can be adjusted by means of a screw. The thick- 
 ness of the slice depends on the distance between the blades. This 
 knife is useful for slicing soft organs, such as the liver, 
 kidney, and lung, in their fresh condition. Sections of fresh 
 organs made with this knife are, however, far inferior to those 
 made with a single-bladed knife used with the freezing micro- 
 tome. 
 
57 
 
 99. Single-bladed Knives. A scalpel and a razor are the 
 knives most commonly used for cutting any tissue which "by 
 softening or hardening has been brought to a suitable consistence. 
 The razor should be ground flat on one side if it is not to be 
 used with a microtome. Eor a right-handed person the flat side 
 should be that facing the right when the cutting edge of the 
 razor is held downwards. In giving this direction, it is pre- 
 sumed that the concave side of the knife will be held uppermost 
 in the process of section, so that it may hold a pool of fluid over 
 which the slice may readily float. When a microtome is not 
 employed, the razor should be pulled, not pushed, obliquely 
 through the texture, and the cutler should be instructed to bear 
 this in mind in setting the blade. 
 
 100. Gutting Instruments must always be wetted, a, Water, 
 b, salt solution, c, methylated 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, Miiller's fluid, or chromic acid. The sections must float 
 over the surface of the knife, and they must always be manipu- 
 lated with sable or camel-hair brushes. 
 
 101. Imbedding in Carrot. The piece of tissue is often so 
 small that it is impossible to hold it in the hand, and it is some- 
 times 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. Carrot may 
 be used for this purpose. For holding in the hand a piece of 
 softened bone or hardened spinal cord, etc., it is extremely con- 
 venient. A hole rather smaller than the organ should be scooped 
 in the carrot, near its centre. Ari 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. 
 
58 
 
 102. Imbedding in Paraffin. When a very firm support is 
 required, paraffin is better than carrot. Ordinary solid paraffin 
 is too hard, but if five parts solid paraffin be mixed with one 
 part hog's lard, its consistence becomes suitable for the purpose 
 of section, and when a section-machine is employed, it is much to 
 be preferred to a mixture of equal parts of beeswax and sweet 
 oil for the latter having a higher melting point, contracts so 
 much when it cools, that it separates from the wall of the box of 
 the section-machine. 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. 
 
 103. Imbedding in Gum. Very delicate tissues may be im- 
 bedded in a strong solution of gum (Strieker). Remove all al- 
 cohol from the tissue to be imbedded, by immersing it in water 
 for twenty-four hours. Place the tissue for four or five hours in 
 a cone of blotting paper containing mucilage (see 85). Then 
 set the cone with its contents in methylated spirit, for forty-eight 
 hours or so, to remove the water and thereby stiffen the 
 gum. This may be more rapidly done by absolute alcohol. 
 Lastly, imbed in paraffin and make sections. 
 
 104. Microtome. In making sections the student ought to 
 practise as much as possible the ordinary method of holding the 
 tissue in the one hand and the knife in the other. If the piece 
 of tissue be small it is generally advantageous to imbed it in 
 carrot or in the paraffin mixture. 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 
 
59 
 
 many sections have to be thrown away as imperfect. This 
 difficulty is overcome by using a microtome. Various micro- 
 tomes have long been in use. The freezing microtome, 1 already 
 described, serves the purpose admirably. (See 83.) 
 
 The knife used with the microtome may be an ordinary razor, 
 concave on both sides, but with the face and back perfectly 
 straight from point to heel. For sections more than a third of 
 an inch in diameter the blade of a razor is too short. For large 
 sections, therefore, the blade of the knife should be a foot in 
 length, and two and a half inches in breadth. The side of the 
 blade (the left) that rests on the table of the microtome should 
 be concave, while that which receives the slice should be flat, or 
 so slightly concave, that the movement of the slice of tissue over 
 the blade will not be retarded by its edge coming in contact with 
 the blade when it approaches the back. A pool of fluid over 
 which the slice may float is essential. (See 86.) The knife 
 should be steadily drawn from heel to point through the tissue, 
 but with the face turned from the operator, so that he may cut 
 away from himself. 
 
 Methods of increasing the Transparency of the Tissues. 
 
 105. 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 balsam, 
 dammar. 2. By partially or completely dissolving certain 
 elements of the tissues, so as to permit of others being seen. 
 Acetic acid, caustic soda or potash, are used for this purpose. 
 These partially or completely dissolve the soft albuminous parts 
 of most tissues. Glycerine and glacial acetic acid may be used 
 together. (G. 1 oz., acid 5 drops.) (Beale.) The acetic acid 
 
 1 The microtome invented by His has been praised by some as the best 
 of all. It is only adapted for cutting unfrozen tissues. The author is un- 
 acquainted with it, but Professor Kolliker, who may be regarded as a 
 sufficient authority on such matters, has laid it aside as inferior to that 
 recommended by the author. 
 
60 
 
 commonly used is ordinary pyroligneous acid. The solution of 
 soda is, caustic soda 1 part, water 20 to 30 parts. These four 
 fluids all mix with water, hence they are employed to clarify 
 tissues from which water has not been removed. 
 
 Turpentine, clove, or other essential oil, creasote, and 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. 
 
 Tissues hardened in alcohol or chromic acid are rendered 
 opaque, and must in general be clarified. This is generally 
 done by adding, a, clove oil ; b, turpentine j c, glycerine. These 
 fluids have different powers a is stronger than b, and b is 
 stronger than c. Glycerine is employed for tissues that 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. 
 
 106. 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 proto- 
 plasm, epithelial cement is deeply stained by silver nitrate, nerve 
 iibrils 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. 
 
 107. Carmine. The use of this valuable re-agent 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 
 
Gl 
 
 two fluids. a. Weak carmine fluid. An exceedingly good 
 carmine fluid, for general purposes, is the following modification 
 of that proposed by Beale. Beale's fluid is thus prepared. 
 Carmine 10 grains, strong ammonia 30 minims, glycerine 2 oz., 
 distilled water 2 oz., rectified spirit J oz. 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 
 perceptible, then keep it in a stoppered bottle. This fluid is, 
 owing to its density, slow in its action, and, although it may 
 yield excellent results, it is uncertain in its operation. Tardi- 
 ness and uncertainty of action may be avoided by retaining all 
 the ammonia in the fluid. 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 the fluid 
 is to be used, dilute it seven times with water (Atkinson), filter, 
 and place the tissues in a large quantity of the fluid 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. Tissues which have been steeped in 
 chromic acid, potassium bichromate, or Miiller's fluid, must 
 always be thoroughly washed in water ere they are placed in 
 carmine. Remove superfluous pigment by washing the stained 
 tissues in water, or proof spirit (rectified spirit 5 parts, water 3 
 parts), to which 1 per cent, of hydrochloric acid has been added 
 (Pritchard), and mount in pure glycerine or dammar (see 119). 
 Beale's method is to stain the tissues with his carmine fluid 
 undiluted, then to wash them for a day or two in dilute 
 glycerine (glycerine 2 parts, water 1 part), and lastly, to mount 
 them in acid glycerine (glycerine 1 oz., glacial acetic acid 5 drops, 
 or 2 drops hydrochloric acid in place of the acetic acid). 
 The acid brightens the colour, b. Strong carmine fluid. It is 
 useful to have a strong carmine fluid for rapidly staining nerve 
 
G2 
 
 tissues by the method recommended in 72. This is prepared 
 as follows. Dissolve 1 gramme carmine in ICC strong ammonia 
 in a test tube with the aid of a gentle heat, add 23 CC distilled 
 water, filter, and preserve in a stoppered bottle. This fluid is of 
 the proper strength for staining by the method above referred 
 to ( 72). Eor 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 preserve in a 
 stoppered bottle. 
 
 108. Picro-carmine. A double staining, with picric acid and 
 carmine, may be obtained by a fluid prepared as follows 
 (E-anvier). Drop a strong ammoniacal solution of carmine into 
 a saturated solution of picric acid until the fluid is neutralised. 
 Then filter, and preserve in a stoppered bottle. The picric acid 
 gives a yellow tinge to epithelium and muscle, while the 
 carmine stains nuclei, 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. 
 
 109. Magenta (rosaniline nitrate) is useful for staining tissues 
 rapidly in the ordinary process of examination. 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 re- 
 tained for a long time by mounting the stained tissue in one- 
 third per cent, watery solution of corrosive sublimate. Two 
 fluids are necessary a. Magenta crystals 1 decigramme, rectified 
 (not methylated) spirit 9CC, distilled water 213CC. 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. The following fluid, devised by my former 
 
63 
 
 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 nonmammalian vertebrates 
 alter but little in shape while they become stained, b. Dissolve 
 1 decigramme crystallised magenta in 15CC distilled water, and 
 add 5CC rectified spirit, and 20CC glycerine. 
 
 110. Logwood is like carmine, suitable for staining tissues for 
 permanent preparations. Speaking generally, it stains the same 
 elements of the tissues as carmine and magenta. The tint is 
 violet. The tissues may either be fresh, or they may have been 
 hardened in chromic acid or alcohol. It stains the nuclei of 
 epithelium, and the protoplasts of connective tissue remarkably 
 well ; the lung, skin, cornea, tongue, etc., can be beautifully 
 tinged by this agent. Bb'hmer, who introduced logwood stain- 
 ing into histology, used haematoxyline ; but a fluid prepared as 
 follows is cheaper, and gives admirable results. Make an 
 infusion of logwood thus, pour 200CC cold water on 20 
 grammes logwood chips, and allow to stand at the side of the 
 fire for six hours or so. The infusion must not reach the boiling 
 point. Filter, and add to the filtrate a sufficient quantity (about 
 350CC) of a J per cent, watery solution of alum, to give a fine 
 purple colour. Then add 30CC rectified spirit. Before staining, 
 filter the fluid. A fresh tissue is tinged by this in three or four 
 minutes. It is better, however, to tinge a tissue more slowly 
 than this, therefore place ten or a dozen drops of this fluid in a 
 watch-glass, half filled with water. From half an hour to an 
 hour will be required. "Wash the tissue in water, or in J per 
 cent, alum solution, to remove superfluous pigment, and mount 
 in glycerine or dammar. 
 
 111. Silver Nitrate. Eecklinghausen introduced this sub- 
 stance into histology. It is of great value in staining non- 
 stratified squamous epithelium of serous membranes, lymphatics 
 and blood-vessels. Use \ per cent, solution in distilled water. 
 In staining a tissue, e.g. the frog's mesentery, dip it two or three 
 
64 
 
 times into distilled water to remove the sodium chloride. Place 
 it in the silver solution from one to three minutes. Remove 
 the superfluous silver by washing in common water until the 
 water ceases to become milky. Place the washed tissue in 
 glycerine, and then expose to diffuse daylight until it becomes 
 slightly brown. Mount in glycerine, or in glycerine jelly. In 
 order to stain the epithelium of blood-vessels, wash out the 
 blood-vessels of a newly-killed animal with distilled water, then 
 inject J per cent, solution of silver nitrate, allow it to remain 
 in the vessels for five minutes or so, and then wash it out with 
 distilled water. If the animal be warm-blooded, the water and 
 the silver solution must be heated to 100 Fahr. Lastly, inject 
 a warm solution, gelatine (gel. 1 part, water 6 parts), and expose 
 to the light, and mount as above mentioned. This is a modifi- 
 cation of Woodward's process by my former assistant Dr. 
 Pritchard. 
 
 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. 
 The silver process has been of great value in the investigation 
 of the lympathic system and blood capillaries. 
 
 112. Chloride of gold (auric chloride) is of much service for 
 staining nerve fibrils, connective tissue protoplasts, cartilage pro- 
 toplasts, etc. The colour varies from bluish-grey to violet. 
 Place the tissue, not later than fifteen minutes after its removal 
 from the body, in one-half per cent, solution of auric chloride in 
 distilled water, until the tissue 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 
 
65 
 
 (1 or 2 per cent.) for five or ten minutes. Lastly, place the 
 tissue in distilled water, and expose to the light, until a steel-grey 
 or violet tint appear. This results from the reduction of the gold 
 salt. The reduction is hastened by the acetic acid. 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 gold method was introduced by Cohnheim for staining 
 the nerves of the cornea. The following method, devised by 
 Klein (Monthly Microscopical Journal, 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 precipitate that may have formed , make 
 sections obliquely horizontal or vertical, and mount in glycerine. 
 A cornea so treated is ash-grey or slightly violet. 
 
 113. Osmic acid } introduced by Max Schultze, is, as we have 
 seen, a valuable hardening agent. Its property of blackening the 
 white substance of Schwann and all fatty matter, as well as, 
 though to a less extent, albuminoid matter, renders it an 
 important staining agent. For the method of using it see 91. 
 
 Methods of Injection. 
 
 114. Injecting apparatus. The fluid may be injected by 
 means of a syringe. The piston of the syringe must be slowly 
 screwed, not pushed, down, in order to prevent jerking. For 
 most purposes the syringe has given place to a far more efficient 
 apparatus, consisting of Wolff's bottles. The fluid is driven into 
 
 E 
 
66 
 
 the vessels by water-pressure, which can be regulated with the 
 greatest nicety. There is no jerking, no refilling of a syringe, 
 no haphazard application of pressure, as is the case with the 
 syringe. This valuable method we owe to Ludwig. Always 
 begin with a low pressure and increase it gradually. In the 
 case of the blood-vessels of a rabbit, the pressure may be raised 
 to 110 millimetres (4J inches) of a mercurial column. 
 
 115. The injection of blood-vessels. I. Injection masses 
 fluid at ordinary temperatures, a. Watery solution of soluble 
 Prussian blue. Soluble 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. Dis- 
 solve 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 gradually into A C, stirr- 
 ing all the while, and let stand for a day. Pour off supernatant 
 fluid, and filter off the remaining 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, maceration in turpentine or in acid brings it back. 
 Mount in dammar, b. Beale's Prussian blue injection. A. 1 oz. 
 glycerine added to 4 ozs. water. B. 60 minims tincture of ferric 
 chloride added to 1 oz. of solution A. C. 12 grains potassium 
 ferrocyanide dissolved in 1 oz. of solution A. Pour B into C 
 very gradually, with constant stirring and shaking of the mix- 
 ture, then add the remainder of solution A, and also 1 oz. of 
 spirits of wine. Mount in acid glycerine (glycerine 1 oz., H Cl 
 
67 
 
 2 drops). Prussian blue injections must not come in contact 
 with any alkali. These injecting fluids are excellent when a 
 perfect injection is not desired. II. Injection masses solid at ordi- 
 nary temperatures, a. Carter's carmine and gelatine injection. 
 This, the best of all the carmine injections, is prepared thus : 
 60 grains carmine, 120 minims strong ammonia, 86 minims 
 glacial acetic acid, 2 oz. solution of gelatine (1 of gelatine to 6 
 of water), 1J oz. water. Dissolve the carmine in the ammonia 
 and water with the aid of gentle heat, and filter ; add to this 1^ 
 oz. of hot gelatine solution, and mix thoroughly. Add the acid 
 to the remaining half ounce of gelatine solution, and drop this 
 into the heated carmine mixture with constant stirring. For 
 the purpose of section, harden in weak and then in strong 
 alcohol. Mount the injected tissues in dammar, b. Prussian 
 blue and gelatine injection. A. Dissolve, with the aid of heat, 
 33 parts of gelatine in- 200 parts distilled water. B. Dissolve 
 4 parts soluble Prussian blue in 300 parts distilled water. 
 Gradually add B to A. For hardening the injected tissues, to 
 permit of sections being made, use first weak and then strong 
 alcohol, containing 1 per cent. H Cl, or use a J per cent, solution 
 of chromic acid for some weeks, and then alcohol. Preserve the 
 injected tissues in dammar. 
 
 Inject the vessels immediately after death by chloroform. 
 Previous to injection the vessels may be washed out with j 
 per cent, salt solution. This, however, is not essential. The 
 injection and the salt solution, if it is employed, should be 
 heated to the temperature of the blood (100 F.) in the case of 
 a warm blooded animal. Tie the vessels when the injection is 
 completed, and plunge the injected parts into cold meth. sp. 
 
 116. The injection of lymphatics. Use a solution of Alcannine 
 in turpentine (Ludwig), or a watery solution of soluble Prussian 
 blue. For the injection of lymphatics, Lud wig's method of 
 puncture is adopted. Fill a subcutaneous syringe with the fluid, 
 thrust the nozzle into the tissue, and slowly drive the fluid 
 
68 
 
 wherever it will go. The lymphatics of the testis and spermatic 
 cord of the dog can be readily injected by simply thrusting the 
 nozzle of the syringe through the tunica albuginea. 
 
 117. The injection of bile ducts. Inject the portal vein with 
 carmine and gelatine. Previous to this, open the common 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 20 to 
 25MM mercury is usually sufficient for the ducts, but it may 
 require to be gradually increased to 40MM. 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 
 meth. sp. Mount the sections in dammar. 
 
 Methods of Preserving or Mounting the Tissues. 
 
 118. The dry method. Hair, and sections of dried bone and 
 tooth, are sometimes mounted in air. The difference 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 refractive medium than air, e.g. 
 meth. sp. glycerine, glycerine jelly, or dammar. When a tissue 
 is mounted in air, a ring of dammar rather smaller than the 
 cover-glass is placed on the slide, and allowed to nearly dry. 
 The tissue is then placed within the dammar cell, 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. 
 
 119. Dammar and Canada balsam. All water must be 
 
69 
 
 removed from the tissue by drying or by immersion, first in 
 methylated spirit, and then in rectified spirit or absolute alcohol. 
 The alcohol is driven away by floating the tissue upon oil of 
 cloves, or oil of turpentine, and then the balsam or dammar is 
 added, and the cover-glass put on (see 56, D 4). If Canada 
 balsam be employed, prepare it as follows. 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 150 Fahr., 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 require to be heated. It was difficult to get 
 rid of air bubbles and to avoid overheating the tissue. But by 
 using a limpid solution of perfectly dried balsam, all difficulty 
 disappears. 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 J oz. gum 
 mastic in 3 ozs. benzole, and filter. If rapid filtration be desired, 
 use twice as much benzole, and then evaporate the fluid to half 
 its bulk. Chloroform and turpentine 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. Balsam 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. 
 Speaking generally, it is more suitable for mounting stained 
 than unstained tissues. The latter are often rendered too trans- 
 parent. The tissues are very perfectly preserved in either of 
 these substances, because all water is removed. 
 
70 
 
 120. 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 carmine or logwood. It is suitable for 
 mounting tissues that have been stained with silver nitrate or 
 gold chloride. For tissues containing vessels injected with 
 Prussian blue, use 2 drops H Cl 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. It is often necessary, however, to transfer the 
 preparation to a new slide, on which a smaller drop of glycerine 
 has been placed. 
 
 121. 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 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. Eimmington's glycerine jelly is the best. 
 Its composition is unknown, but it appears to contain glycerine, 
 gelatine or isinglass, and carbolic acid. 
 
 122. Potassium acetate. A saturated solution of this sub- 
 stance 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 thus : dissolve 
 56 grammes potassium acetate in 28CC hot water. When 
 cold, add 2CC camphorated spirit, and filter. 
 
 123. Weak spirit (meth. sp. 1 part, distilled water 3 parts) 
 
71 
 
 was formerly much employed. Muscle, connective tissue and 
 blood vessels can be well preserved in it for a time, but very 
 often they become granular and useless. 
 
 124. Methylated spirit may be used for things which have 
 been hardened in spirit, if they do not require to be clarified. 
 Softened bone shows best in spirit. 
 
 1 25. Naptha and creosote fluid is useful for preserving urinary 
 casts (see 62, e.) 
 
 126. Cells for microscopic objects. For dry objects use cells 
 made of tinfoil, paper, cardboard, dammar varnish, or glass. 
 For wet objects use glass cells, or cells made by painting a ring 
 or square of dammar varnish upon a glass slide, and allowing it 
 to dry. Delicate tissues that are apt to be spoilt by the pressure 
 of the cover-glass should be placed in a dammar cell. The glass 
 cells are cemented to the slide with marine glue. It is most 
 convenient to buy them fixed to the slide. 
 
 127. 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, the margin of the 
 cover-glass must be covered by cement. First, paint on a layer 
 of gold size, and allow it to dry, then cover this with a layer of 
 dammar varnish. The dammar mounting fluid, thickened by 
 drying, is the best dammar varnish. If a tissue, e.g. a slice of 
 softened bone, be mounted in meth. sp., use thick dammar varnish 
 as the only cement, otherwise it will be apt to run in and spoil 
 the preparation. The slide must be dried with bibulous paper 
 ere the varnish is applied. 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 dammar. 
 
72 
 
 128. Mode of keeping preparations. The slides should lie 
 upon the back, not upon the edge. All preparations should be 
 carefully labelled. The things shown should be mentioned, the 
 date and name of the mounting fluid ought also to be stated. 
 Never keep a bad preparation, and do not attempt to preserve 
 everything. 
 
Rutherfor 
 
 Outlines 
 histology