P:m COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD HX00025275 Digitized by the Internet Archive in 2010 with funding from Columbia University Libraries http://www.archive.org/details/courseofelementaOOfear A COUESE OF ELEMENTAEY PRACTICAL HISTOLOGY £- A COURSE OF ELEMENTARY PBACTICAL HISTOLOGY BY WILLIAM FEARNLEY Uonfion MACMILLAN AND CO. AND NEW YORK 1887 F3\ Richard Clay and Sons, london and bungay. TO E. KLEIN, M.D., F.R.S. WITH THE MOST SINCERE REGARDS OF HIS PUPIL THE AUTHOR. PREFACE This work is not only intended for medical students working in a well-furnished laboratory under a teacher, but for students of all denominations who can com- mand the means and have the wish to construct for themselves a histological cabinet for study and future reference. With this object in view it differs from all other text- books of histology in several particulars, but notably these : It contains a list of all the apparatus required ; the classification of the structures which have to be collected is novel ; and it contains much detail in the various processes anticipating the absence of a teacher. Every process has been thoroughly tested, so that however imperfect these processes may be, such as they are, they can at least be relied upon. The work should be gone through thus : First collect the necessary apparatus ; next work through the exer- cises ; then collect and prepare the tissues ; and finally Vlll PREFACE. prepare the permanent slides. Few of the demonstra- tions of Part II. can be prepared altogether beforehand : most will require some preparations at the time; but this in the nature of the case is unavoidable. Amongst many others I am more especially indebted to the writings of the following physiologists : Brunton, Foster, Frey, Huxley, Klein, Purser, Ranvier, Ruther- ford, Schafer, and Stirling. I am also indebted to Mr. Martin J. Cole for some of the processes, and to Mr. T. P. Collings for the able manner in which he has executed the drawings and wood-engravings. In conclusion I ask much forbearance on the part of teachers, students, and readers generally, and inform them that the immense amount of laboratory work and the recording of results have all been conducted between the numerous and unavoidable interruptions of a family- medical practice. W. F. London, 1st May, 1887. CONTEXTS. PART I. PAGE A. Miceoscopy 1 — 33 B. Appabatcs, Reagents, and some Minor Opera- tions 34 — 61 C. Teasing 61—63 D. Irrigating 63 E. Gases and Yapours 64 F. Applying Heat to Tissues 65 G. Injecting 65—83 H. Dissecting 8S— 92 I. Happening and Softening 93 — 102 J. Embedding 102 — 117 K. Section-Cutting 117 — 131 L. Staining 132—141 M. Dehydrating and Clearing 112 — 143 N. Mounting 144—150 0. Practical Exercises 150 — 161 P. The Collection and Preparation of Material . 162 — 204 Q. Two Short Methods of Preparing Sections. . 204 — 207 b CONTENTS. PART II. PAGE INTRODUCTION 209 I. The Vegetable Cell 211—215 II. The Animal Cell 216—219 III. Amphibian and Human Blood-Corpuscles . 220—222 IV. Action of Reagents on Blood-Corpuscles . 223 — 227 V. Epithelium 227—232 VI. Areolar and Adipose Tissue . . . 232 — 235 VII. "White Fibrous and Yellow Elastic Tissues 235 — 238 VIII. Cartilage 238—242 IX. Bone 243—247 X. Development of Bone 248 — 251 XI. Muscle 251—255 XII. Nerve-Fibres 256—259 XIII. Blood-Vessels 260—262 XIV. Lymph Spaces and Channels . . . 263 — 265 XV. Lymphatic Glands, Tonsils, and Thymus . 266—268 XVI. Skin, Hair, and Nails 269—277 XVII. Trachea, Bronchi, and Lungs . . . 278—282 XVIII. The Teeth and Tongue .... 283—286 XIX. The Salivary Glands and the Pancreas . 287 — 288 XX. Oesophagus, Stomach, and Duodenum . . 289 — 292 XXI. Small and Large Intestines . . . 293 — 297 XXII. The Liver 298—302 XXIII. The Ductless Glands 303—306 XXIV. The Kidneys 307—312 XXV. The Ureter, Bladder, and Male Genera- tive Organs 313—317 XXVI. The Female Generative Organs. . . 317—324 XXVII. Terminations of Nerve-Fibres . . . 324 — 326 CONTEXTS. xi PAGE XXVIII. The Spinal Cord, Nerve-Cells, and Ganglia 327—332 XXIX. Oegans of Taste and Smell . . . 333—335 XXX. Organs of Vision xxxi. 336 " S46 XXXII. Organs of Hearing 346 — 354 XXXIII. The Brain and Medulla Oblongata XXXIV. | . 354—356 APPENDIX 357—359 PEACTICAL HISTOLOGY. PART I. A.— MICROSCOPY. A 1. — The Laboratory. A room of the house facing the north should, if possible, be chosen. It need not be a large room, but it should be provided with a large cupboard and plenty of shelving and have a cold- water tap in it. It ought to have a long and broad fixed work-table, of sufficient height, a hand-breadth below the worker's elbow, to allow work to be conducted with comfort in the standing position. It is convenient to have a wooden, rectangular, oblong trough let into it to act as a nest for the microscope and the microscope-lamp. This trough should be about one-fourth wider than the base of the microscope, and should have its long axis the whole breadth of the table : its depth may be six cm. The microscope need never be removed from this recess, but should be covered by a large old silk handkerchief when not in use. B i> PRACTICAL HISTOLOGY. A 2. — An Index Needful. There are so many little things required in a histological laboratory, few of these being required for use at any one time, that drawers and shelves are especially needful. An index, such as a " Where is it ? " — a book sold by stationers for register- ing addresses — is almost an essential. The drawers and shelves must be labelled with distinctive marks, such as letters of the alphabet or numerals ; then everything taken into the laboratory whose proper place is not on the work-table should have a place in a drawer or shelf selected for it, and the thing under its proper index letter ; thus, paraffin melting at 37° C. under letter P, put in its own place. Whenever this particular paraffin is wanted the index shows its whereabouts, and it should be put back in its place after use by reference to the index if necessary. This simple precaution will save time and keep the laboratory free from accumula- tions of odds and ends which invariably get in one's way whilst working. A 3. — A Diary is Essential. When hardening or treating tissues in any way, so many hours or so many days are prescribed. We perhaps place fifty tissues in five or six different media on the same day : the times of changing and the media into which they are to be changed must be arranged at the time. When these very various times come it is impossible to re- member. We therefore turn to the various days and mark down what is to be done. This necessitates a daily reference to the diary to see what requires doing. After clearing the work-table for the day's work, a most necessary proceeding for those not MICROSCOPY. 3 gifted with the habit of putting away everything at the time of using, the very first operation should be a reference to the diary, and a carrying out of the things to be done as therein indicated for that day. All resolutions, e.g., to let a tissue have a day's or two days' further immersion in its present medium, should be noted at the time, and placed under the proper day, a day to come of course. Both diary and index should be kept either upon the work-table or within easy reach. A 4. — The Compound Microscope-Stand. The following advice is intended for those who know little or nothing of the instrument, and is therefore dogmatic rather than argumentative. Every microscopist, excluding those w r ho merely seek amusement from the instrument, uses a small simple stand for the greater portion of his w r ork ; but when he is obliged to use powers higher than an eighth and bring out their full capabilities he requires a more complicated and therefore more expensive stand. Now, this requires more money than the average student of medicine can afford ; therefore it is customary to recom- mend a small stand which will do, as above stated, the greater portion of the work, and to show the student those rarer microscopical features requiring very high powers with a more complicated instrument, the pro- perty of the laboratory or of his teacher. This work is intended as much for junior practitioners working in a private laboratory of their own as for medical students so called, therefore, wdiilst placing the small working stand, as it is entitled to be placed, in the B 2 4 PRACTICAL HISTOLOGY. front rank, the more complicated instrument will also be recommended. First, then, a small working stand is a prime neces- sity. There are scores of these to be bought, of differ- ent patterns, but the one which comes nearest to my idea of a good stand is the recently-introduced "Star" instrument of R. and J. Beck. Tig. 1. — Beck's " Star " microscope. This instrument, shown by Fig. 1, is made with a simple draw-tube coarse adjustment as well as with a rack-and-pinion one as shown. The latter is to be preferred on account of its allowing the use of the double nose-piece carrying the two working powers constantly in use. MICROSCOPY. O A 5. — Substage Condenser. "With small working stands not fitted for a substage condenser of the best kind, Wenham's half sphere is excellent, and costs only five shillings. It is used by simply touching its flat surface with the tip of the tongue, and making this surface adhere to the under surface of the slip, imme- diately beneath the object to be illuminated. Some small Fig. 2. — Baker's student's microscope. stands have an adapter to receive one of the objectives as a condenser : a one-fourth, one-fifth, or one-sixth inch objective makes a very good condenser. Such a stand is shown by Fig. 2, made by Baker, of High Holborn, with which I have worked for some years. A G. — The Stand Condenser. This is by no means a necessity, but for high-power work it is of G PRACTICAL HISTOLOGY. great use. By its use the greater portion of the rays of light from the lamp- wick can be collected and con- centrated on the mirror of the microscope. It should, Fig. 3. — Baker's microscope-lamp. however, when not in use always be put away, and not allowed to stand on the work-table, or it gets in the way. MICROSCOPY. 7 A 7. — The Microscope-Lamp. No better lamp for night-work or dark days can be chosen than the one represented by Fig. 3, and it is not expensive either. It is so constructed that the light may be lowered so near the level of the table that direct illumination, the mirror being pushed aside, can be instantly resorted to when occasion requires. A 8. — Eye-pieces or Oculars. Two eye-pieces are required. They should be the A and C eye-pieces if of English make, or the 2 and 4 if of foreign make. The student will now see that whilst English opticians name their objectives by their focal distances, one-inch, one- sixth inch, &c, and the oculars by letters of the alphabet, the Continental opticians just do the reverse. Another peculiarity is this : an English optician speaks of a deep and shallow eye-piece ; by the former he means a powerful and by the latter a weak eye-piece. This nomenclature is puzzling to a beginner, who finds the shallow eye-piece very long and the deep one very short. A 9. — The Two Working Powers. These powers or lenses are in constant use, and should be cleaned and screwed upon a double nose-piece, as we are constantly requiring to use first one, then the other. Thus it is usual to take a general survey of the whole of a tissue, which can only be done with a two-inch, one-inch, or at most a two-thirds or a half-inch ; then to inspect a small part of it at closer quarters, which can only be done with a more powerful lens, such as a quarter, fifth, or sixth-inch. For the low power a two-inch is too low, and we 8 PRACTICAL HISTOLOGY. choose either a one-inch, a two-thirds, or a half-inch. For a higher power, called by authors of histological works high-power, a quarter-inch, a fifth, or a sixth will do. Strange as it may seem, this wide range of choice in the two working lenses is almost a matter of indifference. The usual powers are either the one-inch and the quarter-inch, as recommended by English opticians, or the half-inch and sixth-inch, as recom- mended by foreign opticians, or those who work with French or German objectives. The custom of the day is to prefer Zeiss's powers, and laboratories therefore are mostly supplied with his A and D lenses, the former being a two-thirds and the latter a one-sixth inch. If the student chooses English powers, let them be a one-inch and a quarter-inch ; if he prefers Zeiss's powers, let them be the A and D lenses, or his AA and DD lenses — these are the same in power, but better lenses, and only cost a few shillings more in each case. A 10. — The Magnifying Power and Angles of Lenses. If a vulvar fraction having 1 for its numerator be made of the focal distance of the lens, we only require to put a after the denominator to ascertain the magnifying power of the lens. Thus a one-fourth magnifies forty, a one-tenth magnifies 100 times, and so on, and one inch or y amplifies ten times. When, therefore, we remember this, that the power, say a one-fourth power, gives an image of forty diameters only, and we are viewing the object with a one-fourth lens and a C ocular, and perhaps getting an amplifi- cation of 200 linear, it is the ocular which is amplifying MICROSCOPY. 9 the image formed by the lens five times. We must never lose si^ht of this fact in the choice of our oculars, because the more the image is amplified the less sharp the definition. In other words, when good definition is required we use a weak ocular, such as an A or a B, and we of course have less amplification. On the other hand, when we require high amplification we use a powerful ocular, such as a C or a Continental 4, and we recede, so to speak, from good definition the higher in amplification we go. On this account, lenses or powers are tested by deep or powerful eye-pieces, because it is only a well-corrected lens which will give an image sufficiently perfect to bear high amplification, because of course all the imperfections of an image become more and more prominent the more it is amplified. Lastly, the angle of aperture has nothing to do with amplification. Thus a cheap one-fourth lens with a low angle, say of 50°, and costing a sovereign, amplifies as much as a one-fourth lens with an angle of 100°, and perhaps costing five sovereigns. Whilst mentioning the subject of angles we may further remark that low-angled lenses have greater penetrating power than higher ones ; that is, the focal plane is deeper, or, in other words, the depth of the piece of the object seen at the same time without altering the distance of the lens is greater. For instance, suppose we were viewing with a one-fourth power the letters a, b, c, d, placed one under the other, the nearest being a. We might see the a, b, c, d all at once if the lens had an angle of 50° ; we might see a, b, c all at once if the angle were 60° ; a, b, if the angle were 90°; and only a if the angle were 100°. 10 PRACTICAL HISTOLOGY. For histological purposes we must not have too high- angled lenses, but we must avoid the low-angled lenses. The one-inch cannot well have too high an angle, the two-third inch should have an angle of 36°, the half- inch 40°, the one-fourth inch 75° to 90°, the one-sixth inch 110°, the one-eighth inch 110° to 120°. The angles of the oil-immersions we shall refer to afterwards, when discussing special lenses. A 11. — The High-Power Lens. An exceedingly small but important proportion of histological work can only be done by the use of a high power, such as a twelfth or fifteenth-inch. All powers higher than an eighth should have water or oil between their front lens and the object viewed, because water and oil refract light more strongly than air. When nothing is placed be- tween the lens and the object viewed, the lens is called a dry lens, because air only is interposed ; w T hen oil or water is interposed, the lens is called either a water- immersion or an oil-immersion lens, according to the medium used. A 12. — Some Good Dry Lenses. Swift and Son's new high-angled one-inch lens is a most remarkable one. This and Zeiss's D or DD form an excellent set of working lenses. Zeiss's A and D, or AA and DD, are much used in English laboratories. Beck's half-inch at 40° and their one-sixth inch are excellent as every-day powers. Any one of the above sets screwed on a double nose- piece, other things being equal, will satisfy all the needs of the histologist, If the student hesitates as to choice, MICROSCOPY. 1 1 he had better adopt the street urchin's mode of settling the matter by tossing up one of the coins of the realm. A 13. — Immersion Lenses. All powers higher than a sixth, or at most an eighth, should be oil-immer- sion lenses. Dry or air lenses are quite superseded by immersion lenses, and of the two forms of immersion lenses, water and oil, water-immersions are almost things of the past, and quite as much superseded by oil-immersions as dry or air lenses are superseded by immersions. Up to a few years ago it was thought that immersion lenses owed their superiority to the greater amount of light they transmitted by working in a medium, water or oil, with higher refractive properties than air. The history of the discovery that this was not the cause of their superiority is exceedingly interesting. This history was published at length in the Journal of the Royal Microscopical Society, by the able editor, Mr. Frank Crisp. I have to thank Mr. Crisp for the loan of the woodcuts in illustration of the subject; If between the reflectin^-mirror and the stao;e of the microscope we interpose a very small opening in the diaphragm, and on the stage lay a grating of ruled lines, on removing the eye-piece and looking down the tube we observe a series of images of the aperture, like Fig. 4, all circular in homogeneous light, but the outer ones consisting of spectra in white light. The small pencil admitted through the dia- phragm is " diffracted." We next lay upon the stage a slide, such as Fig. 5, consisting of, let us say, a 12 PRACTICAL HISTOLOGY. circle containing both wide and narrow lines ruled on glass. Removing the eye-piece as before, we have of course, on looking down the tube, the appearance presented in Fig. 6, the coarse lines giving diffraction spectra twice as close and numerous as those caused by Fig. 4. the fine lines. The reason for this we have already seen ; the present point is, what influence these dif- fracted rays have upon the image, and it is here that the experiments just referred to are so important and ■'3000O0C00: ,30000 Fig. Fig. 6. interesting First of all, by a diaphragm at the back of the objective, such as that in Fig. 7, let us cover up all the diffraction spectra, allowing only the direct or central white pencil to reach the conjugate focus, or MICROSCOPY. 13 image-point. On replacing the eye-piece, all the fine ruling has disappeared, leaving only the general outline of the object, as in Fig. 8. By suppressing the diffracted rays, therefore, the fine detail or structure of an object is obliterated. Fig. Fig. S. Secondly, let us adjust behind the objective a dia- phragm like Fig. 9, which allows all the lower spectra in Fig. 6 to pass to the image-point, but suppresses every alternate spectrum of the upper set, diffracted Fig. 0. Fig. 10. by the coarse lines. The image now appears as in Fig. 10, the upper set of lines to all appearance being identical with the lower set. Precisely in the same way, if we substitute a diaphragm like Fig. 11, stopping 14 PRACTICAL HISTOLOGY. off yet another half of the alternate spectra, the lines are again apparently doubled, and we " see " Fig. 12, though the actual object remains the same. In these experi- ments therefore, while retaining the central pencil of light Fig. 11. Fig. 12. throughout, we have created apparent detail or structure in the object by suppressing certain of the spectra. Still further, however, let us take a slide which, when magnified, resembles Fig. 13, or a "crossed grating.'' We get with this from the small aperture rectangular Fig. 13. Fig. 14. spectra somewhat like Fig. 14, but in addition there are other diagonal spectra, caused by the regularly- recurring intervals diagonally across the squares. Con- structing a diaphragm like Fig. 15, which allows only the MICROSCOPY. 15 central pencil and two of these diagonal spectra to pass, the vertical and horizontal lines of the object have vanished, to he replaced by Fig. 16. This experiment is troublesome, the diaphragm having to be prepared Fig. 15. Fig. 16. with extreme care ; but the results, first deduced from theory, have been rigorously verified. Now the microscopic student knows that many ob- jects, by their minute and regularly-recurring structure, cannot fail to give strong dirTractive effects. The well- fig. r, Fig. IS. known Phurosigma angulatum will serve as an example of the practical effect of the foregoing considerations. It gives three sets of dirTractive spectra arranged as in Fig. 17. As each set is produced by something 16 PRACTICAL HISTOLOGY. resembling lines at right angles to it, the three sets of lines in the object must be arranged mainly as in Fig. 18 ; but it will be obvious, from what has gone before, that by selecting different sets of spectra with or without the central beam the apparent images will differ widely. It is also manifest that all these images cannot represent the true structure. If, however, we are pretty sure that we have all the characteristic spectra, and their position and relative intensity can be calculated, then the resultant image can also be calcu- lated ; and so far as all the spectra are included it will Fig. 10. represent the real object. Mr. Stephenson 1 records an extraordinary instance of such calculation in the case of this identical P. angulation. A mathematical student who bad never seen a diatom, taking the spectra alone, worked out from calculation the drawing given in Fig. 19 as the result. Now, the small markings be- tween the hexagons had never been seen in P. angulatum by anybody. But on Mr. Stephenson making special examination of a valve, stopping out the central pencil 1 Journal of the Royal Microscopical Society, vol. i. 1878, p. 186. MICROSCOPY. 17 so that its superior illuminating effects might not over- power the others, these small markings were found exactly to exist, though they were so faint as to have eluded all observation until mathematical calculation from their spectra had shown that they must be there. The general conclusion is, therefore, that we can have no true image of an object whose structure is sufficiently fine to give diffraction sj)ectra, unless all the diffracted rays, or rather perhaps all the truly characteristic sets of spectra, are collected ; and the image will more or less resemble the object, in proportion as the spectra are all collected, or at least sufficient of these charac- teristic spectra. As we have found before that the finer the grating the more widely deflected are the diffracted spectra, we can now readily understand how, as regards minute structure especially, collection of the widest possible angular field of rays from the object is a point of the utmost importance for correct delineation, quite irrespective of greater illumination ; and it is in this respect that immersion objectives have such an enormous advantage. Of course these considerations only apply to struc- ture of a certain degree of fineness. With more coarse- ness, all the diffracted spectra which are visible may be collected by a moderate angle. But when we reach a certain fineness it will be seen that the imagfe in a microscope of small angular aperture can be no true representation of the object at all, but is due to peculiar selective conditions. This may be well show r n by an experiment with c 18 PRACTICAL HISTOLOGY. Amphipleura pellucida. "With a homogeneous-immersion objective of large aperture, focus the object under an illumination so oblique as to show up all the lines clearly. Then remove the eye-piece as in previous experiments ; and, placing the eye at the conjugate focus or image-point of the objective, the direct beam will of course emerge obliquely as a bright spot ; whilst on the side of the field, and close to its margin, will be seen a faint bluish light, the inner portion of the first di fraction spectrum. Only a portion of one spectrum, observe ; and that so near the margin that it must be lost with any objective of much less angle. If now a small bit of paper be adjusted on the back lens of the objective so as to stop this blue light and no more, the illumination is demolished by an almost infinitesimal jDortion, and the diatom is still visible, apparently as brightly illuminated as before. But the characteristic striation, which caused, and was therefore imaged by, the diffracted light, is gone, just in the same manner as was demonstrated in Fis^s. 7 and 8. A 14. — Some Good Oil-immersion Lenses. Swift and Son make a good one-twelfth inch with great working distance which only costs £6. Lately, Reichert, of Vienna, has brought out a one-fifteenth costing £9, and one of the same focal distance costing only £5. These are all good lenses. Leitz led the way by bringing out a one-twelfth inch for £5 5s. This is a good lens but for its working distance, which is so short that it renders the lens useless almost. The first three lenses are excellent in every respect ; all have good working distances. MICROSCOPY. 19 A 15. — Choice Outfits. For those who can afford it, any one of the three following outfits will leave the purchaser nothing to desire, however affluent in circumstances : — 1. Beck's Pathological Stand. This stand is small and answers the double purpose of daily and occasional work. Fig. 20. — Beck's pathologi cal stand. Their half-inch at 40° and their one-sixth inch should also be taken as ordinary working lenses. Also their highest-angled substage condenser. The glass stage is c 2 20 PRACTICAL HISTOLOGY. a beautiful piece of mechanism, but a plain brass stage is supplied in its place if desired. Fig. 21. — Nelson's student's stand. 2. Nelsons Student's Stand. This also answers every purpose. Swifts' one-inch and their one-fourth inch MICROSCOPY. 21 should be taken as the working lenses, and their highest- angled substage condenser. It will be seen that the stage is cut away. It is also provided with a sliding bar which acts as a finder when working with high powers, or searchiug a slide. 3. Bakers New Stand. I regret I have no wood- cut of this. It also is an excellent stand. Zeiss's A A. and DD lenses should be chosen as the working powers, and Zeiss's condenser. A double nose-piece to carry the two working lenses is to be added, whichever be chosen. For a high power, one of the first three oil-immersions I have named should be the high power taken with any of the three outfits. Such outfits cost almost £30, and are not generally bought by students; but those in practice who do not care to be always changing and who want an outfit for life cannot for money be better equipped. The very large elaborate stands are quite unfit for ordinary work, whilst those I have mentioned are very especially suited for ordinary work, and answer every purpose in special work. A 16. — The Magnifying Powers of the Micro- scope. After obtaining his microscope, oculars, and objectives, one of the first things the student must do is to construct a table of amplifications with all the different combinations at his disposal. Thus, suppose he has Beck's Star microscope, their half-inch and one-sixth inch objectives, and two eye-pieces, he will construct a table like the one we here give. 22 PRACTICAL HISTOLOGY. Objective. Eye-piece. Shallow, i Deep. 1 ( Half-inch Tube in j ( One-sixth inch x i Half-inch Tube out ) ( One-sixth inch Fig. 22. Suppose lie has this table completed and suspended within sight of his work-table, and he is using his one- sixth inch objective and a deep eye-piece, with the tube in, then on looking at the table he will see the magni- fication he is using by lookiug at the figures where the cross is placed. To fill up the spaces or figure values left open in the table, take a stage-micrometer divided in O'l and 0"01 mm., also a pair of compasses with fine points, then, with the half-inch objective on and the shallow eye-piece in and the tube short and vertical, look down the tube with one eye, keeping the other eye open, and open the compasses until the two points coincide with any two of the lines of the stage-micrometer, taking care to hold the compass-points on a level with the stage- micrometer. Now apply the compass-points to a milli- 1 A shallow eye-piece is one of low power, and longer in length than a deep (stronger) one. MICROSCOPY. 23 metre measure, and the apparent size is obtained. This apparent size divided by the real size (number of milli- metre spaces of the stage-micrometer which the points of the compass appeared to stretch over) will give the magnifying power. Proceed in the same way for each of the other combinations, and when the card is filled up hang it within sight of the work-table. Of course, should he have more eye-pieces and more objectives, he will simply have to extend the table in a manner at once obvious. Continental eye-pieces are known as numerals, and objectives as letters of the alphabet — a state of things equally easy to tabulate. A 17. — To Measure Objects under the Micro- scope. Professor Schafer x advises the following plan : — Put a stage-micrometer (which is a glass slide ruled in the centre with the lines 01 and O01 mm. apart) under the microscope in such a manner that the lines run left and right (the rnicroscoj)e must not be inclined). Focus them exactly. Put a piece of white card on the table at the right of the microscope. Look through the instrument with the left eye, keeping the right eye ojDen. The lines of the micrometer will appear projected upon the paper. Mark their apparent distance with pencil upon the card, and afterwards make a scale of lines in ink the same interval apart. A magnified representation is thus obtained of the micrometer scale. Mark upon it the number of the eye-piece and of the objective, and the length of the microscope tube. This scale-card will serve for the measurement of any object without the further use of the micrometer. To measure 1 Essentials of Histology, p. 6. '24< PRACTICAL HISTOLOGY. an object, place the scale-card upon the table to the right of the microscope, and view the object with the left eye, keeping the right eye open. The object appears projected upon the scale, and its size in 1 or O01 mm. can be read otf. It is important that the same objective and eye-piece should be employed as were used in making the scale, and that the microscope tube should be of the same length. A 18. — Drawing Microscopic Objects. Without a single exception, every teacher is agreed that nothing impresses the appearance of the structures as seen under the microscope and leads to exact observation so well as sketching these appearances. Every student, whether he can sketch or not, is bound in common justice to himself to sketch in his own way what he sees under the microscope, however clumsily : such sketches impress him as he can be impressed in no other way. If he only draws what he sees, he cannot get wrong, however poor his production. With a very little practice, however, he will soon acquire the habit of sketching ; and will then need some, at all events, of the apparatus here recommended. 1. Camera Lucida. The histologist as a rule never works with the tube of his microscope inclined : he nearly always works with the tube vertical, not that the latter position is an easier one, but because the stage of the microscope must be horizontal for dissecting, teasing, examining fluids, and so forth. As many of the structures to be depicted would be disarranged by tilting the microscope tube and with it of course the stage, a prime necessity of his camera lucida must be adapta- MICROSCOPY. 2a tion for use with a vertical tube. We recommend Zeiss's, as in Fig. 23, and in order that the drawing may be kept within reasonable limits, the drawing-paper has to be elevated. 2. Wooden Desk. This is made so as to hold the drawing-paper at an angle of 15° above the level of the stage in front of the microscope, so that the drawing once made may not have to be reduced. I ig. 23. — Zeiss'.s camera lucida. 3. Zinc or Copper Stencil Plate. This had better be a square with sides of 11 cm., having a circle cut in it 9 cm. in diameter. We use this to make a black border for the drawing before commencing to draw, which will prove of the greatest assistance whilst drawing, as will be presently pointed out. 4. Paper. The drawing-paper had better be chosen to take water-colours, then we can if we like use a 26 PRACTICAL HISTOLOGY. little colouring. It should be hard, thin, smooth, and unglazed. 5. Lead-Pencils. A HB, also a HHHH pencil, will be required. 6. Lit! to graphic Pens. About half a dozen of these should be got, and each well mounted in a separate holder. 7. Sable Brushes. We should have at least six of these, with hairs short and coming to a tme point. Four are required to be set apart for use with Indian ink, carmine, yellow, and blue respectively. The brushes used for these must not be used for any other colours, and each must be marked with the name of the pigment it is kept for. Besides the four sables set apart a.s specialists, we require two or three for general use, sables to apply any other of our pigments except the four named above. These must always be rinsed in water, never placed in the mouth. 8. Pigments. These should be few and of the best quality, and in cakes, not moist colours. The cakes must be kept well separated to prevent dirtying, or chipping each other. Again, only transparent colours must be chosen. These had better be Payne's grey, Antwerp blue, carmine, scarlet lake, yellow ochre, Hooker's greens No. 1 and No. 2, and raw sienna. The colours to be avoided are those which load and become opaque when dry ; such as vermilion, cadmium, the umbers, emerald green, and Vandyke brown. He should test the cakes by taking a perfectly clean porcelain palette, and rubbing a little of each side by side upon it, and allowing all to dry ; when dry, reject any if dull and dusty, and have them MICROSCOPY. 27 exchanged. Lastly, he should buy a piece of the very best Indian ink he can possibly find. The above are all he will really require, except a palette, india-rubber, an erasing knife, and so forth. Of course, if he intends to sketch Avithout colours, he will at once see that a camera lucida, paper, a stencil plate, and Indian ink, with a sable or two, a support to hold the paper, and the lead-pencils, will only be required. To those who have never sketched, the following hints will be useful. First, take a rectangular piece of paper twice the size of the stencil plate ; then, having placed the stencil plate upon its left-hand half, run a lead-pencil around its outer square and its inner circle. Flood the space so marked with clean water and a clean large sable ; then, when the paper has so far dried as to have a sodden appearance, place it on an inclined plane, such as the wooden desk, and wash over it a solution of Indian ink ? made thick and black by a good deal of rubbing down in water. The brush in putting on this or any other wash must be w T ell filled, a commencement made at the top of the surface, and care taken never to retouch the surface already washed over with the sable. If not black enough, a second lot of ink may be washed over after the first has quite dried. When this black outline is dry, place the paper on the drawing-board, and raise it or low T er it as required ; the rim of the circle and the rim or margin of the field as seen through the camera must exactly coincide. The student will find the black circle a great help to him in several wavs. Just to convince himself, however, let 28 PRACTICAL HISTOLOGY. him try a drawing without its aid. Besides being an aid in drawing, it is optically useful by contrast, and improves his sketch in appearance. The stand must bear the paper at an angle of about 15°. He then adjusts the lights, taking care to have the paper most illuminated. Then, having a very fine point indeed upon his hardest or HHHH pencil, he commences to draw an outline of what he sees, without removing the point of his pencil from the paper. The moment he has done this — namely, lightly indicated the outlines, or a sufficiency of them, of his object — he removes the camera : it is done with. He now fills in typical parts, after getting from his text-book all information about these, with his pencil, by keeping his eyes alter- nately on the field and on his paper. If he determines upon using pigments, he should glance over a few simple directions in the most elementary works on the subject, and he will find no difficulty whatever in doing at least as much as is required for educational purposes. Lastly, he should mark the typical parts with the small letters of the alphabet, and, on the right half of the paper, index or briefly describe each typical part. I, in the name of all the teachers who ever taught, again assure him that such productions, however clumsy and crude, will impress him more than hours of reading. Before commencing to place letters upon his drawing, he ought to tabulate upon a piece of clean waste paper the typical parts to be indicated, taking the information from a text-book, and arranging them in the order of their importance, as typifying what is to be particularly observed. In other words, the things peculiar to the MICROSCOPY. 29 tissues should be lettered and described first : for instance, in the case of lung, the bronchi and alveoli, &c, should come before arteries, veins, nerves, or areolar tissue ; in kidney, again, the divisions, corticular and medullary, should come before the Malpighian cor- puscles and renal tubes, and these again before the epithelia. He ought in the same circle to sketch bits of important parts under high amplification, and in all drawings indicate the amplification either by the sign of multiplication followed by a numeral, or, what is perhaps better, give the name of the ocular and objec- tive ; thus, if he be using Zeiss's glasses, he would put, we will say, Zeiss's A A 2, or simply A A 2, which means of course the AA (two-thirds inch) power and number 2 ocular. If he be using English glasses, he will put, we will say, oc. A, obj. J. Of course he ought to state whether the tube of the microscope is in or out at the time. By keeping a table of amplifications of his microscope pinned up near his work-table he will see at a glance the value of the indices above described. A 19. — Photo-micrography. The use of dry- plates has simplified this art and brought it within the reach of those whose time is very fully occupied. For truthfulness in detail it is far before sketching ■ but unlike sketching, it does not educate the observing faculties, or it does so to a much less extent. An excellent essay on the process, and the appa- ratus required, is given in The Methods of Microsco'pical Research (Bailliere and Co.). It is from the pen of Mr. Frederick Greening, whose beautiful photo-micrographs 30 PRACTICAL HISTOLOGY. illustrate the pathological series of Studies in Micro- scopical Science published by the same firm. This essay is remarkable in several ways : notably in a description of a method to prevent flare in the body tube : in the full explanation of the process, and explanation of points in which beginners get wrong. o o o Another work, a small three-shilling hand-book on Photo-micrography, by Jennings and Maddox, is pub- lished by Messrs. Carter and Piper, 5 Furnival Street, Holborn, E.C., and is more recent than the former. The camera recommended by Greening is much the same as the one by Swift and Son here shown (Fig. 24). The latter work has a full notice of the remarkably good microscopic object-glasses made by this firm. These objectives are corrected for photo-micrography, which does not make them any the worse for ordinary work with the microscope. Presuming that the student who wishes to photo- graph will procure these two works, I shall make no further remarks except to draw attention to a most efficient source of light for photo -micrography. It is an incandescent lamp and constant battery, the latter being the invention of Mr. F. W. Branson, F.C.S. Usually a plano-convex lens is used to converge the light on the object, or substage condenser; but for low powers, instead of an ordinary lamp, an opal or frosted incandescent lamp is used without the intervention of the lens. A switch for making and breaking contact is supplied with it. The quantity of the two solutions required to charge the battery suffices for about fifty hours' light at a cost of about one penny per hour. o.: PRACTICAL HISTOLOGY. The battery, solutions, lamps, and switch are sup- plied by Mr. C. Baker, 244 High Holborn, W.C. In using the battery care is to be taken that a sufficient quantity of current is used ; the zincs must be lowered so as to } 7 ield a light rich in actinic rays ; in fact the nominal light of 5 candle-power may be doubled with advantage. The solutions supplied with Fig. 25. — Improved constant battery. the battery obviate the necessity of re-amalgamating the zincs ; hence the battery, which is free from fumes, only requires the solutions replacing when these are exhausted. The current is switched on to the lamp for the purpose of focusing, then broken whilst the sensitive plate is substituted for the focusing screen. The circuit MICROSCOPY. 33 is once more made during the time of exposure, after which the lio-ht is switched off for the closure and removal of the dark slide. A small shutter fitted below the stage and moved by pneumatic or electric means may be substitute'! for the switch if desired. ]) B. -APPARATUS, REAGENTS, AND SOME MINOR OPERATIONS. B 1. — These are arranged in groups either on account of the sources from which they may be procured, or for other reasons which will appear in each case. The present list of requisites can all be procured of those opticians who sell microscopes and their belongings. a. A Compound Microscope, with rack-and-pinion coarse adjustment and double nose-piece: two oculars: two working powers : one high power, not less than one- twelfth inch, which should be an oil-immersion, will be required. A substage condenser, or a substage piece into which the higher of the two working powers can be screwed : a stand condenser : a microscope-lamp : a stage-micrometer, and a camera lucida will also be required. b. A Dissecting -Microscope. For those who can afford it the one here shown is an excellent instrument, and is supplied with two achromatic lenses marked 10 and 20, indicating the linear amplification in either case. The lower power is by far the most useful, though occasionally the higher power is required. It may be as well to say that unless the student can afford a really good dissecting microscope, such as the REAGENTS AND MINOR OPERATIONS. '.)o one here shown, or the one used in the biological class recently conducted by Professor Huxley, and sold b} r Baker, he had far better learn to dissect with the compound body without any other aid whatever. The reversing of movements and positions is exceedingly confusing at first ; but after a very few trials — each trial should not extend over a quarter of an hour — he will Fig. 26. — Reichert's dissecting-mieroscope. get into the way of using the compound body, and in a few weeks become a far better dissector than any one only using an ordinary dissecting-microscope can possibly be. Thus his very poverty will be the means, as poverty frequently is, of making a better man of him in the loner run . D 2 3G PRACTICAL HISTOLOGY. c. Glass Slips. In England these are three inches long by one inch broad, and are called three-by-one slips, or simply slips. When they have mounts upon them, they are then called slides. The ones recommended are made of the best flatted crown glass. About 2 gross will be needed, or H gross at least. d. Cover-Glasses. Only No. 1 covers should be used, and of these all measuring over '006 inch are to be placed in a pill-box and not used for permanent slides. These thicker glasses are useful for the rough-and-ready examination of objects ; also for use with moist cham- bers, irrigation, and similar processes. Round covers are to be chosen on account of the extra facility they afford when ringing is required. The student should get half an ounce each of half- inch and three-fourths inch. The reason for rejecting covers thicker than "006 inch is that objectives with no correction collar are corrected to work through this thickness of glass. e. Cover-Glass Forceps. Special forceps are sold for grasping and laying on cover-glasses. The forceps have a very broad grasping surface, and can hold the cover firmly without breaking it. Baker sells them. /. Cover-Glass Measurer. By specially ordering covers of a prescribed thickness, i.e. covers not thicker than •006 inch, the student can do without a cover-glass measurer. Should he however choose to measure his own covers, the instrument sold by Ross and Co. is the best. It costs two guineas. g. Watch-Glasses. Half a dozen ordinary-sized and two large-sized watch-glasses will be required. REAGENTS AND MINOR OPERATIONS. 37 h. Writing Diamond. It is better to write or scratch the permanent slide with a single mark and to keep a full record of the slide in a book than to label the slide as is usually done. These labels are liable to drop off and can never be made, in many cases, to contain information enough concerning the slide. Labels are indispensable in the case of bought slides of course. i. Dissecting Case. This must not be such a one as is used by medical students, but must be the one used in biological work. Baker sells the one used in the South Kensington classes. It costs twenty-five shillings, and contains : — A strong pair of scissors ; a fine pair ; a strong pair of forceps, and a fine pair ; two razors ; four scalpels; a pair of bone forceps ; needles fixed in handles, kc. ; all of these being absolutely requisite in our case. j. Microtome. k. Hypodermic Syringe. This is required for in- jecting fluids into the areolar tissue, and other pur- poses. Messrs. R. and J. Beck sell a good one for five shillings, which they call Koch's syringe, because it can be taken to pieces and sterilised by heat. /. Beers Knife. This is useful for cutting fringes off sections whilst they lie on the slip, also for taking away the cornea. It is not indispensable. in. Bottle of Asphalt. n. Bottle of Gold, Sue. o. Warm Stage. The student should procure a warm stage. That of Reichert is excellent but costly. It is heated by warm water : it has a thermometer, and is altogether a superior instrument. Stanley makes one 38 PRACTICAL HISTOLOGY. for less than half the amount of Reichert's, heated by a flame. This also has a Centigrade thermometer. In case the student should not care to purchase either, he can construct one for himself out of a piece of stout copper plate the thickness of that used by engravers for visiting-cards. The plate is to be cut into a rectangular piece, three inches by two, with a half-inch hole in its centre, and a stout long limb left in the middle of one of the lon£ sides standing out at rii»ht anodes to the long axis of the piece just described. Cacao butter is used in A' 1 Fig. 27. — Reichert's warm stage. place of a thermometer. A little piece is placed on the plate near the slip, and the flame of a spirit-lamp is removed when signs of melting appear in the butter. B 2. — The following articles are grouped because of most, if not all, being sold by dealers in chemical apparatus. a. Spirit- Lamp. b. Air-Gas Burner. A small size answers well. c Very small Glass Mortar and Pestle. d. Wash-Bottle. One is here depicted. REAGENTS AND MINOR OPERATIONS. 39 c Gas or Vapour Bottle. This may be like the wash-bottle, with the two tubes reversed. The external end of each tube should have a piece of india-rubber tubing placed upon it, one for a clamp, the other for receiving a further length of glass tubing. / Siphon Bottles. Winchester quarts — with sound corks each perforated with two holes, one for air to get through into the bottle, the other to admit a long glass tube reaching to the bottom of the bottle — make good Fro. 28.— Wash-buttle. bottles for siphon purposes. The glass tube should be bent downwards immediately above the cork, and have upon it a long piece of india-rubber tubiug. This requires the use of a clamp, or a stop-cock. These bottles should be placed aloft, and are useful for holding salt-solution, and for injecting proposes. (j. Drop Reagent Bottles. A very great saving of time and of reagents is effected by the use of a German re- agent bottle marked "Patent L— H." These bottles 40 T'R.UTICAL BISTOLOGY. are made to contain 15, 20, and 30 grammes, the last size being the most convenient. A single drop, or a number of drops, of their contents can be instantly obtained by giving the stopper half a turn and invert- ing the bottle. Two deep grooves are cut on opposite sides of the lower half of the stopper; then, by giving the stopper half a turn, air enters a hole, a, in the middle of the back of the bottle-neck and travels down one groove of the stopper. By inverting the bottle the fluid Fig. 2:».— Drop reagent and stain bottle. enters the other groove of the stopper, and gets into a third groove cut in the inside of the neck of the bottle, extending from the lip. It is impossible to overrate the value of these little bottles in histological work. We must in using them always remember to give the stopper half a turn before replacing them after use : we should also prevent the stoppers getting fixed. It. Beakers. Two or three beakers, each holding half REAGENTS AND MINOR OPERATIONS. 41 a litre, should be obtained. If the student intends bathing tissues in paraffin, three or four very small beakers are useful. These may be less than a fourth the size of the former. i. Test-talcs. A few of these are desirable. Six are sufficient. j. Short Test-tides. These are extremely useful: one is here shown. They are about an inch long, i 'f I ig. 30.— Short test-tube. or little more, and just wide enough to admit a small cork, half to three-quarters of an inch. A dozen will not be too many. /.■. Stirring-Rods. One or two of these are required ; made of glass, of course. /. Two Glass Funnels. m. Kest of Cork-Cutters. n. Glass Syringe. One holding an ounce is the best size. o. Glass Tubing. The tubing such as is used for babies' feeding-bottles is best for our purposes. Two pounds should be obtained. 42 PRACTICAL HISTOLOGY. p. A small Three-cornered File. This is required to cut the 2d ass tubing. q. India-rubber Tubing. The small black tubing such as is used for babies' feeding-bottles is best. About three yards will be needed. A piece of india-rubber tubing six inches long for fixing to the middle neck of the three-necked Wolff's bottle is also needed. This piece should just admit the nozzle of the Higginson syringe. The air-gas burner will also require a length of india-rubber tubing. r. Three Brass Clamps. They must be spring clamps. s. Two White Breakfast Saucers. t. Pieces of Cork. A few pieces of sheet cork, and a block of cork such as that depicted holding the rat, are needed. Both kinds are sold by leather-sellers. u. A Set of Gramme . Weights and Scales. v. Measure Glasses. A graduated 500 c.c. measure and a graduated 10 c.c. measure are needed. A graduated 1 c.c. pipette had also better be obtained. w. A Sand-Bath. This the student can make for himself out of a tin lid which he fills with Calais sand. x. A Water-Bath. A biscuit-tin, soldered so as to hold water, does admirably. y. An Iron Tripod. One that will hold the baths will be needed. B 3. — The next article, distilled water, is about the most important in the histological laboratory. Some wholesale chemists sell a very pure distilled water ; but the student had better rig up a small still for himself, unless he is exceptionally placed. When distilled water is not essential, I have used the word tap- water, REAGENTS AND MINOR OPERATIONS. 43 or simply water. The distilled water, especially for nitrate of silver staining, must be very pure. B 4. — The following list of things forms a group because of their being mostly chemicals. a. Acetic, hydrochloric, nitric, and sulphuric acids are all needed. The acetic must be the glacial kind. A pound of each may be obtained. Each should be in a well-stoppered gloss bottle. h. Ammonium tartrate, half an ounce ; powdered white arsenic, half an ounce ; borax, half a pound ; calcium phosphate, half an ounce, are required. c. Canada balsam, half a pound ; picked gum arabic, one pound, will both be required. d. Strong solution of ammonia is also needed. About half a pound will be sufficient. It should be in a well- stoppered glass bottle. e. Sulphate of magnesia, half a pound ; potash alum, one pound, will be needed. Both should be powdered. /. Potassium phosphate, half a pound ; shellac, four ounces ; sodium carbonate, half a pound ; sodium chloride, half a pound ; sodium sulphate, half a pound ; sulphate of copper, two ounces ; finely-powdered zinc white, as it is called, one ounce, will also be needed. Many of the above articles are not needed in such large quantities, but they are mostly so cheap that druggists object to sell less quantities. B 5. — The following articles are also procured from the chemist : they are all essentials. Absolute alcohol, one pound ; rectified spirit, two pounds ; methylated spirit, one gallon ; ether, four 44 PRACTICAL HISTOLOGY. pounds; chloroform, half a pound; glycerine, which should be Price's, two pounds ; xylol, quarter of a pound ; benzol, one pound. The chloroform may be the methylated of Duncan and Flockhart ; the ether may also be Howard's methylated '730, which costs very little, and answers admirably for the freezing microtome. If the ordinary chemist has not the xylol, it will be procured by him. B 6. — This group contains one or two special articles which the chemist can easily get if he has none in stock. The articles are all essentials. Picric acid, one pound ; chromic acid, quarter of a pound ; chromate of ammonium, quarter of a pound ; bichromate of ammonium, one pound ; bichromate of potash, one pound. The picric acid in crystal is highly explosive and must be kept away from lights ; the chromic acid is highly deliquescent. B 7. — The following are also essentials. Rectified oil of turpentine, two pounds ; creasote, half a pound ; oil of cloves, two ounces ; cedar-wood oil, four ounces. Care is to be taken to have the clove oil and creasote as colourless as possible. B 8. — The following group is composed of articles used for staining. a. Carmine, two ounces ; hoematoxylin, one gramme; logwood chips, finely ground, one pound. Messrs. Richardson, of Friar Lane, Leicester, sell the best carmine I ever use at half-a-crown per ounce. REAGENTS AND MINOR OPERATIONS. 45 b. Osmic acid, one gramme ; gold chloride, one gramme; silver nitrate, two drachms. The two former are sold in sealed tubes. I would advise the student to purchase a bottle of osmic acid solution, 1 per cent., from the opticians, in preference to dissolving the acid himself. e. Eosin, iodine green, safranin. rosein, Spiller's purple > anilin blue-black, methyl anilin green ; a few grammes of each will be required. These should all be from the laboratory of Dr. Georg Griibler, Leipsic. Baker is his agent. Each dye is sold in a small bottle which costs a shilling. B 9. — A diary ; a " Where is it ? " ; a few sheets of thick, white, bibulous paper ; a sheet or two of paper dead black on one side and gummed on the other; six camel-hair brushes ; one large camel-hair brush. Stationers keep these articles. The six camel-hair brushes have hairs half an inch long surrounded by tin and mounted on black wooden handles. In other words, they are small or fine paint-brushes. The large camel-hair brush is for use when silvering the diaphragm. B 10. — The following are all requisite : — Cotton-wool, bleached, one sheet ; lillikin pins, a large paper of about 200 ; half a yard of sateen ; half a yard of fine flannel ; one or two knots of white filoselle. B 11.— Hot Filter. This will be needed only if the student intends making his own glycerine jelly. It is made by Messrs. James Allen and Son. B 12. — Two or three sets of embedding L's. These are sold by the Cambridge Scientific Instrument 46 PRACTICAL HISTOLOGY. Company, St. Tibbs' Row, Cambridge, and cost haif-a- crown a pair. B 13.— Paraffin wax (136° F.), two pounds, (110° F.), two pounds ; Allen and' Hanburys' chrisma, one pound. The two paraffins, or paraffin waxes as the makers call them, are sold by Messrs. Johnson and Son, 175 Bishopsgate Street Without, London, E.C. The higher melting-point wax costs eightpence, and the lower sevenpence per pound. The chrisma is sold in four- pound tins. B 14. — White wax, one pound, and olive oil, one pound, will also be required. B 15. — Gelatine will be required in two forms if the student makes his own glycerine jelly; thus he will require four ounces of French gelatine, " colle de Paris," and two or three two-ounce packets of Swinborne's patent calves'-feet gelatine for making carmine-gelatine injection mass. Celloiuin, two or three tablets, will be required if the student wishes to embed in this elegant and efficient way. E. Schering's celloidin is the best, and may be obtained from Corbyn and Co., Oxford Street. B 16. — Some pieces of plate glass, each the size of a crown piece, a rasp and a water-of-Ayr stone the student will need if he wishes to make his own preparations of bone and teeth by rubbing down. I advise him not to waste his time doing this, but rather to send a shilling and a penny to Messrs. Cole and Son, 171 Ladbroke Grove Road, London, W., for any specimen of this description he may need. REAGENTS AND MINOR OPERATIONS. 47 There is no special educational value in making these preparations. B 17. — The student will either have to purchase an injecting syringe and cannula?, or he may get my injecting apparatus : they both cost about the same ; but, whilst injecting with the syringe is most uncertain and difficult, injecting by constant pressure is easily acquired and very certain in its results. Suppose he adopts the constant-pressure method he will require the following apparatus : — Injecting bath; gas regulator; thermometer; a three-necked and a two-necked Wolff's bottle ; a mano- meter ; stop-cock and set of cannulse; a Higginson's syringe ; some copper wire and pliers ; also a foot or more of lead pipe the size of the tube used for babies feeding-bottles ; and a gas-jet. The bath is made of copper, tinned inside, by Messrs. James Allen and Son, 21 Marylebone Lane, Oxford Street, London. The remaining articles are described in their proper places further on in the book. The thermometer should be a Centigrade one registering up to 300° C. It and the gas regulator, which costs half-a-crown, are sold by Cetti, Castle Street, Holborn, London. It will be seen that the thermometer, which costs six shillings, is indispensable for using with the paraffin bath, and as he can with glass feeding-bottle tubing and a little mercury make the manometer himself, the remaining essentials of the injecting apparatus only cost as much, perhaps thirty shillings, as a good anatomical injecting syringe. 48 PRACTICAL HISTOLOGY. B 18. — The hot- water oven is also made by Messrs. Allen and Son. It or some equivalent will be needed if the student baths his tissues in paraffin. B 19. — Drawing-materials the student will need- He should look over the article on this subject, and determine what he will require, for himself. Photographing-material he will also have to arrange for himself. Of course it is only under exceptional circumstances that the student can indulge in photo- graphing his microscopic objectives. B 20. — The four following articles form a group. He had better make the Farrant's medium himself. Any of the others he can get from Mr. Baker, 244 High Holborn. The xylol balsam is made by Dr. Georg Griibler, the white zinc cement by Mr. Cole, Jun., whose names are to be mentioned, the latter especially, or he may get a white zinc cement that will crack with age. a. Farrant's Medium. This is almost sure to be unreliable if bought ready made ; the student should therefore make it himself. Mr. Cole, Jun., makes an excellent quality thus : — Boil arsenious acid for a few minutes in distilled water in a beaker, then take the liquor so boiled, and when cold add an equal quantity of the best glycerine and mix thoroughly. Next dissolve picked gum arabic in distilled water until the water will dissolve no more. Lastly, mix an equal quantity of the first mixture and the gum solution, and filter through cotton- wool lying in the bottom of a clean glass funnel. b. Glycerine Jelly. The student should either use REAGENTS AND MINOR OPERATIONS. 49 Remington's glycerine jelly, or make it for himself; I advise him to make it himself. The following will be found a good formula and way of making it : — Take of Glycerine (Price's) 50 c.c. Water (thoroughly distilled) 42 c.c. Carbolic Acid Crystals 2 grms. Gelatine, gold label or " Colle dc Paris " . C> grms. 1 new-laid egg. 100 Thoroughly soak the gelatine in the water for twelve hours, then melt it in a water-bath, and add the glycerine and carbolic acid to it. Remove the heat at this stage. Now beat well up the shell and white of the egg, and incorporate it well with the mixture, which must only be about 35 C. whilst so doing ; the object being to diffuse the fluid albumen thoroughly throughout the mass. Again place the mixture in the water-bath, and apply heat until the albumen becomes flaky ; then filter through fine filter-paper in the hot oven, or in the hot filter. The filtrate should be received into a clean, dry, wide-mouthed, ground-stoppered bottle. A small wide-mouthed bottle with an ordinary cork, holding only a few cubic centimetres, should be in regular use to avoid having to frequently heat so large a quantity. The stock bottle should be kept in a cold place always. Care must be taken to thoroughly stir the egg albumen E 50 PRACTICAL HISTOLOGY. into the mixture whilst not too warm, otherwise the albumen will coagulate prematurely, and its mission (to entangle and eliminate all floating solid particles, how- ever minute) will not be carried out. c. Xylol Balsam. To prepare this we take good commercial Canada balsam and expel all moisture from it — render it quite vitreous in fact — and dissolve it in an equal bulk of xylol. The important step of this process is the drying of the balsam without burning it. In order to do so we apply heat for one hour and a half to a thin layer of the balsam. In operating w r e must rigidly preserve the two factors (time, one hour and a half; and the thin layer) : then success is a certainty, the amount of flame and its distance from the balsam being the only uncertain con- ditions. The author takes a round tin lid two inches and a half in diameter and half an inch deep, and fills this to the brim with good commercial balsam. He then sets this in a thin layer of fine sand (a sand-bath) over a Bunsen's gas-burner, and lights the rose-jet, but turns the gas so low that only a faint blue flame exists. The distance from the upper surface of the rose gas-burner to the bottom of the tin holding the sand is two inches and a half. Success the first time trying under such circumstances is almost a certainty. The balsam should be rejected if, after turning off the gas and allowing the mass to get cold, it is either not perfectly brittle, or tinged with brown. In the first case, when the mass is not quite brittle, the amount of flame has been too small ; in the last, too great. When success is obtained — the author once baked six lots in succession REAGENTS AND MINOR OPERATIONS. 51 as an experiment, the third lot only being a failure— the mass is vitreous, has not changed colour, and comes away from the tin by simply pinching the sides of the tin, which fractures the entire mass so that it falls out when the lid is inverted. As we have said, it must be mixed with an equal quantity of xylol. This is easily done by taking a perfectly clean, dry, cylindrical bottle and filling it one- Fig. 31.— Balsam bottle. third full of xylol, then adding the dried balsam bit by bit with constant shaking until the bottle is two-thirds full. AVe keep xylol balsam for use in a glass bottle with a ground-glass stopper and a well-fitting glass cap (see Fig. 31). Another peculiarity of this bottle consists in its having its stopper prolonged as a round glass rod to the e 2 52 PRACTICAL HISTOLOGY. Dottom of the bottle. In filling this using bottle from the stock bottle, we must, of course, filter the xylol balsam through filter-paper, the filter-paper being previously wetted with xylol. The glass cap prevents evaporation so that the glass stopper never gets stuck, neither do crusts of dried balsam form around the cork. These bottles are sold by Griffin, Garrick Street, Covent Garden, W.C., who also sells all the chemical apparatus we may require. d. White Zinc Cement. Amongst the numerous white zinc cements in the market all are worthless except two, so far as I can learn — Cole's and Ziegler's. Mr. Cole, jun., tells me he makes his cement as follows : — He takes a large pickle-jar and fills it with lumps of good com- mercial dammar and then fills it with benzol. He })1 ices the jar in front of a hot fire until the dammar is dissolved. He next takes very finely powdered white zinc and rubs it down in a drying oil, and adds this to the dammar and benzol : lastly, after filtering through dry, clean, thin muslin, he adds a quantity of gold size. This cement never cracks, works beautifully, and what is best of all, it resists the action of oil used with oil- immersion lenses. Baker sells it. Ziegler keeps his formula to himself, but his produc- tion is not nearly so good as Cole's. The only difficulty in making Cole's cement is in getting the zinc of good quality and sufficiently finely ground. B. 21. — The following articles form a group on account of the fact that the student can easily make each for himself. Before commencing to make the articles the student REAGENTS AND MINOR OPERATIONS. 53 should practise working in glass. A few simple directions are here given. Glass-working. A knowledge of about half a dozen simple operations on glass tubing is necessary in the histological laboratory. To cut a piece of glass tube, grasp the tube at the part to be cut ; then, as the part lies on the edge of the table, the tube being held obliquely, draw one way the edge of a three-cornered file guided by the thumb several times across it : turn the tube round, and do the same on the exactly opposite side, then break it over the edge of the table. To bend glass tubing revolve the tube whilst holding it in an air- gas fJame, then quickly withdraw it when the name is yellow and the tube soft, and gently bend it to the required extent. If at one operation the bend is not sufficient, place it again in the flame. To draw out glass tubing as in pipette-making, hold the tube in both hands as before, and apply it to the air-gas flame between the hands, revolving it of course. When the flame is yellow and the tubing soft, quickly withdraw it and as quickly pull it out straight by grasping firmly and pulling the hands straight apart in the exact line of the tube. The softness of the tube and the rapidity and strength of the pull determine the fineness of the tube resulting : thus, if a capillary tube is needed the tubing must be made very soft by heat, and the pull must be vigorous and com- mence immediately the tube has left the flame. To render smooth the ends of a tube, hold the end in an air-gas flame gently revolving it the whole time. This will round off the sharp edge, and will tend to cause the end of the tube to collapse, and therefore lessen its 54 PRACTICAL HISTOLOGY. lumen. If the tube be finely drawn like a pipette to begin with, the end of the tube will be sealed by being placed in the flame, unless air be blown through the tube whilst it is held in the flame. To make a Y~ s haped piece of tubing take a length of tube and apply the flame to the side of the tube, keeping the further end of the tube closed with the point of the finger, whilst the mouth is applied to the near end. When the tube is soft, a strong puff of breath blows a glass bubble at the softened part, which bursts and leaves a small hole in the tube. The end of another piece of tube placed over this hole and both held in the flame will seal and amalgamate the pieces of tubing. When cold, a three- cornered file is used for cutting the pieces into the required lengths. I can imagine the above descriptions causing amuse- ment if read by a glass- worker ; but I give them as sufficiently practical and simple, and as answering all the purposes required. The laboratory must always contaiu a pound or two of the tubing in lengths of about two, or two and a half feet. a. Drying Shelves. In working we have constantly to place slips with exposed tissues on them, or slips freshly ringed and therefore wet, out of the way of dust. A convenient set of shelves is made by taking four pieces of thick plate-glass seven inches long by two inches wide, and interposing six pieces of the same plate glass two inches long and half an inch broad, at their ends. Hollis's glue may be used as a cement. The shelves thus made will be more convenient if REAGENTS AND MINOR OPERATIONS. DO raised upon two other pieces of plate glass glued under the ends, of the same size (two inches by half an inch) as the interposing pieces. b. Mounting Block. It is always quite easy and very desirable to place each section, or piece of tissue, in the exact centre of the slip. I have devised the follow- ing contrivance, and find it to answer better than any other. Take two pieces of thick plate -glass each three inches by one, i.e. the size of our ordinary slips, and stick them together with Hollis's glue. Then rule with Fig. 32. — Mounting block. a writing diamond two diagonal lines on one face of this block. Of course where these lines intersect is the centre of the glass and therefore of the slip. Finally rub lamp-black into the diagonal lines. The block may be placed on a white or a black ground in mounting according to requirements. As a rule, however, no special ground is required. c. Gas or Vapour Cell. Take a slip and ring it with gold size one coat upon another, allowing the last to dry before putting on a fresh one. When it is moder- ately deep cut a small gap with a knife at opposite 56 PRACTICAL HISTOLOGY. sides of the cell. Now draw out a piece of glass tubing to almost capillary fineness : cut with a three-cornered file through the tube, leaving 1 cm. only at the end of the capillary part. Place a piece of india-rubber tube upon this, then lay the capillary part on the slip, and paint sealing-wax varnish (sealing-wax dissolved in methylated spirit) along the capillary tube and the slip it lies upon to a good substantial depth. Of course the capillary tube is to reach the inner rim of the cell, and should not extend beyond the end of the slip. It is better never to remove the india-rubber tubing. d. Moist Cell. Bower and Vines, in Practical Botany (Macmillan), describe a very useful moist cell as follows : — Cut a piece of thick rough cardboard to the size of the glass slip, and punch a round hole in the centre rather less than a round cover-glass of ordinary size. When used for a lengthened observation this is to be placed in boiling water to prevent the growth of fungi ; otherwise it is only to be dipped in warm or even cold water before use. Water is to be dropped on it to supply the place of that lost by evaporation when using the apparatus. e. Cell for Cover-Glasses. Before the commencement of each day's work a stock of ready-cleansed covers ought to be prepared for use. These clean covers are con- veniently kept between two large clean watch-glasses, placed edge to edge. Such a contrivance is greatly im- proved by having a hinge of adhesive plaster. This cell is dust- tight, and capable of being kept very clean. Glasses of "006 inch in thickness and under should be REAGENTS AND MINOR OPERATIONS. 57 kept in it. All covers thicker than '006 inch should be placed in a pill-box : they are excellent for the rough-and-ready examination of tissues in teasing, irrigating, &c. /. Moist Chamber. A good moist chamber is made by taking a small earthenware basin and placing wet blotting-paper on the bottom, inside, then inverting over a dish, or plate, or even over the top of the work- table. Articles such as slips with sections on them covered with a large drop of stain, or watch-glasses full of stains, keep well without loss by evaporation for twenty-four hours or longer. Of course the mouth of the basin must touch all round the flat surface on which it rests. g. Frog Holder. This is described under the article on the Frog further on. h. To Clean Glass Slips. Place the spoiled slides and dirty slips carefully in an old cigar-box which should be placed near the work-table. When this box is full, its contents are carefully placed in a solution of Hudson's extract of soap in a tureen, and in a day or two cleaned first with a rough cloth, then with a dry cloth, and lastly rubbed bright with chamois leather. i. To Clean Cover-Glasses. Place them when quite dry in strong sulphuric acid for an hour; pour off the acid ; let several lots of tap-water be run through them so as to remove most of the acid ; after this lay them on a piece of gauze in a funnel and allow tap-water to run through them till every trace of acid, tested by litmus- paper, is removed. Lastly, remove them to methylated spirit, which should be changed in twenty-four hours in 58 PRACTICAL HISTOLOGY. case of a trace of acid remaining. Keep the glasses in spirit in a wide-mouthed bottle well corked. They should be kept in the spirit till wanted. Cover-glasses when required should be taken out of the spirit, rubbed bright, and then measured before use. For rubbing the spirit off, and polishing, sateen should be used, held between the tip of the index-finger and the thumb. On no account must we bring a second finger into use, otherwise the cover-glass held and rubbed by the three will be broken. The sateen must be scrupulously clean, and used only for this purpose. Fig. 33.— Turn-table. Ringing Slides. A good turn-table is among the most indispensable of the histologist's tools for cell- making, securing mounts against leakage and against the action of the oil used with immersion lenses. In the choice of a turn-table we mast bear this in mind: that the spin we give to the table in using it must always be a gentle spin, not a violent twirl, or our cement will be scattered in a radiating manner all over the slide. If a gentle spin be an absolute essential it follows that the table must run smoothly and revolve for a minute or more without having to be touched REAGENTS AND MINOR OPERATIONS. 59 again. Many turn-tables are worthless on this account : they will not revolve beyond half a minute, even with a violent twirl imparted to them. In ringing avoid putting too much cement in the brush : be careful that the slip does not catch the fingers, or it will be sent spinning far away, and prob- ably spoil the mount upon it : let one coat of cement dry before putting another coat of a different material upon it : be careful to have the cement of proper con- sistence by adding a little of its own fluid solvent to it if it has become too thick : in centering the slip on the turn-table be careful to see that it is the cover-glass that is centred, i.e. the cover-glass ought to coincide with the rings upon the table of the instrument, — a rentle preliminary spin or two is required to determine this. If not centred the cover-glass " wobbles." The brush used must be small. One such brush will last a life-time if we are careful to rinse it in the same fluid the cement is dissolved in immediately after use each time. We cleanse the brush in benzol after using white zinc cement, or asphalt ; and in oil of tuqisentine after gold size, and so forth. The cements used are conveniently kept in wide- mouthed, glass-capped glass bottles, in each of which we keep a short length of solid glass rod to stir the cement before use if required. Cell-making. The simplest form of cell is that made with a fluid put on the slip by means of a turn-table which afterwards solidifies. These are easily made by centering a slip on a turn-table, and ringing it with gold size, asphalt, dissolved caoutchouc, &c. Cells of glass, 60 PRACTICAL HISTOLOGY. brass, tin, or vulcanite, are also easily fixed on the slip by first centering the slip on the turn-table, then making a ring the size of the cell with xylol balsam ; placing the slip out of the way of dust for the xylol to evaporate ; then warming the slip and sticking on the glass, metal, or vulcanite cell. The base, sides, and top of the cell may finally be coated with any reagent which will be unaffected by the fluids we are about to place in the cell : shellac dissolved in alcohol, for instance. To Anaesthetise. Chloroform is the best agent to use, for two reasons : it is rapid and pleasant, and it dilates the blood-vessels. If the student has no proper drop chloroform bottle, an ordinary small bottle can be used with a notch cut in the cork to allow of the chloroform escaping in a rapid succession of drops almost amounting to a stream. Dogs and cats should be placed in a thin-sided bag, such as a pillow-slip, and the bag grasped and con- stricted close above them. The chloroform dropped upon the roof, so to speak, of the bag, that is, immediately beneath the hand, will diffuse itself within the space containing the animal, and anaesthetise it in whatever position it may assume. Rats, guinea-pigs, and animals about their size are conveniently operated upon in a cigar-box which should have gimlet-holes in its ends. The lid of the box must be detached for greater convenience in catching the animal. Rats running in a box or cage are conveniently caught by laying a stout open brown-paper bag on its side in the box. They run into the bag and are easily inclosed and transferred to the cigar-box. TEASIXG. 61 Frogs, newts, and little creatures like them are con- veniently placed under an inverted tumbler into which a few drops of chloroform have been poured. The trans- parent glass enables us to see when life is extinct, or when profound insensibilit) 7 has occurred. If from no higher motive the operator will always cause death or profound insensibility before proceeding to do anything to the animal ; otherwise reflex action or returning consciousness may cause difficulties to arise and false steps to be taken. This almost universal desideratum of the physiologist is carefully concealed by profes- sional anti-vivisectionists who obtain their livelihood by harrowing the feelings of the public. Q— TEASING. C 1. — Normal Fluids. When it is not possible or not convenient to submit tissues to hardening or soften- ing processes, which takes time counted by days or weeks — for example, when one is making a post-mortem examination — we can examine in a rough and imperfect manner the so-called fresh tissues by cutting or snipping off a minute piece and teasing it out with needles. The process is called teasing. In doing so moisture is frequently required by adding fluids that will not alter the structures we are examin- ing, which up to a few minutes or hours ago have been bathed in natural or normal fluids within the body ; 62 PRACTICAL HISTOLOGY. hence we imitate this state of things as nearly as possible by using what are called normal fluids. These are — 1. Blood serum. 2. Aqueous humour from a fresh eye. 3. Three-quarter per cent. (75 p.c.) salt-solution. Now it is evident that uoder ordinary circumstances the two former cannot be obtained ; therefore we mostly use the last. C 2. — Dissociating Fluids. Sometimes the ele- ments of which a tissue is composed cannot be separated without dissolving the parts which bind them. When this is the case the tissue is cut into little pieces such as are used for teasing, and placed in some disso- ciating fluid. There are two such fluids of general application, namely — 1. Ranvier's Alcohol. 2. Osmic Acid Solution (*1 to 1 per cent.). Tissues may be placed in either of these for twenty- four to thirty-six hours, then teased. Besides the two general dissociating fluids there are several special ones mentioned by some authors. The student will do well to use Ranvier's alcohol for all. C 3. — How to Tease a Tissue. Place the tis- sue under a lens, and upon a suitable background. Take two needles fixed in handles and gently separate the elements needed, keeping the eye steadily fixed on the work. Practise teasing with the compound body with an inch objective and a weak eye-piece. Ten minutes' practice at a time, for a very few times, suffices to overcome the difficulties of reversed images. When once mastered this method is the most satisfactory of all. IRRIGATING. 63 Use the same fluid in teasing as the tissues have last been in (normal, or dissociating). By those who cannot or will not master the process of teasing with the compound body some dissecting microscope must be obtained. D._ IRRIGATING. D 1. — The Irrigation of Tissues. When we wish to observe the behaviour of tissues when attacked by reagents we run the reagent under the cover-glass and watch the result. Having placed a cover-glass over the tissue to be irrigated and in sufficient proximity with the slip to command the presence of capillarity, we either place a drop of the reagent we wish to irrigate with at one side or edge of the cover-glass and apply a small piece of bibulous paper to the opposite edge, or we partially fill a pipette with the reagent and allow it to descend upon the edge of the cover-glass, using the bibulous paper as before. A large pill-box full of small triangular pieces of bibulous paper should be kept on the work-table whilst irrigating, and with forceps first one then another corner of the triangle should be presented to the opposite edge of the cover to that at which the reagent enters. 64 PRACTICAL HISTOLOGY. E.— GASES AND VAPOURS. E 1. — To apply Gases and Vapours. When we wish to observe the behaviour of tissues when gases are presented to them, or affect them by vapours, we use a deep cell with a cover-glass for a roof. Such a cell should be deep, so that the tissue or fluid we are ob- serving may be well surrounded by the gas or vapour, and at the same time these may be supplied in the form of a stream ; that is to say, the gas or vapour in the cell or chamber must not be stagnant, but must surround the tissue freely and thoroughly and be able to escape as freely as it entered. One example will illustrate the process. Suppose we take a drop of blood and we wish to observe the beha- viour of the white corpuscles when a gas surrounds any of them, we take not a drop but a droplet in this way : we lightly touch a drop of blood with the middle of the face of a clean cover and a droplet adheres. We now place the cover, droplet downwards, upon the rim of a deep cell, and the droplet is suspended from the roof of the cell, and it can be observed, even its lowest strata, by a moderately high power whilst gas or vapour is admitted to the cell. Solid tissues are treated in the same way ; cilise, for instance. APPLYING HEAT AND INJECTING. G5 F.— APPLYING HEAT TO TISSUES. F 1. — To use the Warm Stage. If the student's means will permit, the warm stage of Reichert, or at least that of Stanley, should be used. Both have good Centigrade thermometers. With the primitive home- made one I have described the amount of heat applied as indicated by the melting of cacao-butter cannot be ascertained with any degree of certainty below or above the melting-point of the butter, which melts at about 35° C. The same precautions as to taking a droplet and the same methods are to be observed as in the application of gases and vapours, E 1. The droplet or tissue should adhere to the cover and the cover should form the roof of a moderately deep cell. G.— INJECTING. G 1. — It is convenient and often necessary to fill the arteries and arterioles of a tissue, or organ, to ascertain their relations to the structure, or the other structures of a part. This is most conveniently done by a mass which will congeal and stiffen in an ordinary tempera- QQ PRACTICAL HISTOLOGY. ture. Such congealing masses are rendered fluid by warmth and coloured by a pigment that will uniformly colour the mass but not escape from the mass'and stain the tissues. Such a composition we have in Dr. Carter's carmine gelatine mass which has now become the almost universal injection-mass. G 2. — Carmine Gelatine Mass. All carmine masses are made after the model of Dr. Carter's mass. Carmine is dissolved in strong ammonia and every trace of this ammonia is got rid of in completing the mass, or the injected specimen will be diffusely stained with the carmine. On the other hand if in getting rid of the ammonia too much acid be used the carmine is pre- cipitated in such coarse granules that the mass cannot be driven into the finer vessels and capillaries. I make this mass in the following way and claim for the method complete sinrplicity and certainty : — Take of Carmine 3 grammes. Liq. Amnion. Fort 6 c.c. Glacial Acetic Acid .... 6 c.c. French Gelatine (gold label) . 7 grammes. Tap-water 80 c.c. Place the gelatine, cut into small pieces, in 50 c.c. of the water until it has dissolved, or swollen to its utmost, which will take at least six hours. At the same time put the carmine into a very small clean mortar and crush all the lumps with the pestle and add the ammonia. Let this stand for a couj)le of hours INJECTING. 67 or so, and give it a good stir from time to time, after which we may pour it and the remaining water (30 c.c.) into a small clean bottle, using the water for rinsing out the mortar in the process. In about six hours Ave place the gelatine and its water in a water-bath, and whilst it is being melted we perform the critical operation on the carmine solution thus : — Hold the carmine bottle in the left hand, using; the end of the fore-finger in place of a cork. Take the measured quantity of glacial acetic acid in a small bottle in the right hand, then, in good light, observe the beautiful dark-purple carmine solution. Xow add the acid, a very few droj:>s at a time, giving the carmine solution a good shake after each addition, and the very instant it changes colour to a carmine red stop adding the acid. The difficulties and waste of time and materials frequently experienced in making this mass arise from attempting to filter the carmine and ammonia : from putting the carmine and ammonia into the gelatine solution before neutralising the carmine and in thus having to rely on the sense of smell in distino-uishinp- the acetic from the ammoniacal odour about the point of neutralisation, which is no easy matter. Immediately before the mixture changes colour the ammonia is neutralised ; therefore, when the change has taken place, the solution is very slightly acid, which is what is required. After the gelatine is thoroughly melted, add the pre- pared carmine solution to it little by little with constant stirring. I usually make many times the above quantity, and f2 ' 68 PRACTICAL HISTOLOGY. pour it into a clean ointment jar; and when it has become cold and stiff, pour methylated spirit on its sur- face. By placing it thus in a cold place well covered over, it will keep almost any length of time. When it has been made and preserved thus, the spirit is to be poured off and the surface dried with bibulous paper. When required for use, place the jar in warm water for a few minutes, then the mass can be shot out as a solid plug into the water-bath. Filtering the mass is not required in making it : indeed no filtering at all is required except at the time of using, when it is better to pass it through a layer of fine flannel — wrung out of hot water — in transferring it to the Wolff's bottle. The best carmine must be used. Glacial, not concentrated, acetic acid is required. The gelatine may either be Coignet's, or Swinborne's patent calves'- feet gelatine. Lastly, tap-water answers the purpose as well as distilled water. I have kept jars of it ready in the way above described for three years : very likely it will keep good even much longer still, but the place it is kept in must be a cold place, otherwise the gelatine will melt and let the spirit into the mass. G 3. — Blue Injection-Mass. This mass is also solid at ordinary temperatures. Unfortunately, specimens injected with it are liable to fade in colour : when, how- ever, we are in a position to dip these in oil of turpen- tine, we can restore the colour wholly, or in part. All specimens injected with the mass should be mounted in the balsam thinned with turpentine. INJECTING. 69 Take of Soluble Prussian Blue ... 2 grammes. Coignet's Gelatine .... 7 „ Distilled Water 91 Rub down the powder in half the water (in successive portions) in a mortar, and pour the solution into a tall narrow vessel with perpendicular sides. Treat the gelatine with the remaining water as in the case of the carmine mass, and when melted add the blue solution little by little with constant stirring to the dissolved gelatine ; then filter through fine flannel. No alkali must come in contact with the Prussian blue. All vessels must be clean, and rinsed out with acidulated water ("2 -per cent. HC1 solution) immediately before coming in contact with the mass or any portion of it. Distilled water is to be used in its preparation : tap- water will not do. Most authors direct the watery solution of the blue to be filtered. As this is most troublesome, and takes some hours, we let the solution remain perfectly still in a narrow tall jar ; and pipette off, or siphon off the fluid, all but the lowest stratum containing the dreo;s. The vessel containing the blue should stand quite half an hour unmoved, and care must be taken not to move it during the process of taking out the dissolved portion. Instead of making the blue, it is better to buy the powder ready made. Many gelatines precipitate the Prussian blue ; therefore, the French gelatine, sold in clear thin sheets, must be used. G 4. — The Injecting Bath. Some few years ago 70 PRACTICAL HISTOLOGY. I devised what has proved to be a most convenient bath for keeping the gelatine mass and the animal, or part to be injected, warm during the process of injection of the blood-vessels by constant pressure. At first I used a much larger bath, but for the past two years I have found ^ ..iiiilllilliiillilliiimw Fig. 34. — Injecting bath, author's pattern. the present form most convenient in every way. It holds comfortably any animal one wishes to inject, from a full- sized cat or rabbit, downwards. The volume of water used is as small as possible, and easily kept at the INJECTING. 71 required temperature by a spirit-lamp with a small flame. It has two departments, namely a deep part for submerging the Wolff's bottle containing the mass, which we will call the well ; and a shallow part on which the animal is laid, which Ave will call the shelf. The well and the shelf will be convenient terms in describing its use and structure. The dimensions of the bath are as follows : — The shelf is 30 cm. long, 15'5 cm. broad, and 9 cm. deep, all inside measurements. The well is 245 cm. deep on three of its sides, and of course 24*5 cm. less 9 cm. on that side which communicates with the shelf. The lenoth o of the well corresponding to the length measurement of the shelf is 12" 5 cm. ; and the breadth of the well corresponding to the same way of measuring the shelf is 10 cm. It stands on three iron legs, the one under the well being 8 cm. in length. This form of bath, and the constant-pressure method described, render injection with congealing masses a very much simpler and more certain method than that by the syringe ; indeed the most accomplished injector with the syringe can never be certain before- hand of his results, after many years' practice ; whereas the practice we have hereafter prescribed will render any ordinary man an accomplished injector in a week, or less, by the constant-pressure method. Apparatus used with the Bath. This consists of a water-bottle (see Figs. 34 and 35), to hold about a litre of water or more ; a Higginson's syringe ; a three-necked Wolff's bottle, which should hold a litre or a little more ; a two- necked Wolff's bottle, holding half a litre ; a mano- 72 PRACTICAL HISTOLOGY. meter ; a spirit-lamp ; and glass or brass cannulse and rubber tubing. The water-bottle we need say nothing about; the Higginson's syringe also is the ordinary one sold for enemata purposes. It fell to my lot to discover a few years ago this extremely delicate method of applying pressure to the pressure-bottle. The pressure can be applied with such accuracy and delicacy that the Fig. 35. — Diagram showing injecting apparatus. mercury in the manometer can be raised almost imperceptibly, or as rapidly as possible, according to requirement. The three -necked Wolff's bottle should be fitted with india-rubber corks, all securely fixed permanently, as they never require to be removed. They should be held down by wire, like soda-water bottle corks. All three corks are perforated and transmit 0'5 cm. glass tubes ; the two outer corks have their tubes INJECTING. 73 short, but the glass tube of the middle cork must extend quite to the bottom of the bottle. It will be seen by the diagram (Fig. 35) that the mass bottle is just the depth of the well of the bath, or rather the well of the bath must be just the depth of the mass bottle, so that the delivery tube from it lies well in the warm water, by being on a level with the bottom of the shelf of the bath. This is important. The mass bottle is a two- necked Wolff's bottle also fitted with india-rubber corks. Half-centimetre glass tubes pass through both corks : one a very short tube, the other continuous with the delivery tube must extend quite to the bottom of the bottle. The cork through which the long tube passes may be permanently wired down, as it never requires to be removed. The other cork, whose short tube is continuous with the pressure-bottle tube, must be removable to admit the filtered gelatine mass. In using the apparatus the gelatine mass remaining after an injection operation must be poured out, and the bottle well rinsed with two or three lots of very warm water, each lot being allowed to pass through the long glass tube and rubber delivery tube when poured out. This secures a clean bottle ready for the next operation. The top of the long glass tube on which the delivery tube fits is best bent at right angles, which prevents a round elbow of the rubber tubing which might stand above the surface of the warm water. In the diagram the glass tube not being so bent the delivery tube is seen far away above the bottom of the shelf of the bath. For a wedge, to keep this bottle from floating up as the mass leaves it, any piece of 74 PRACTICAL HISTOLOGY. wood or cork can be made to do. The spirit-lamp is an ordinary one. It should be so placed that its flame streams up the inner side of the well of the bath. Two ordinary clamps are required, one for the pressure tube, the second for the delivery tube. The mercurial manometer, made with half-centimetre glass tube, should not be too long a one ; then the temptation to apply too much pressure is avoided. One about twenty centimetres is quite long enough. The limb up which the mercury rises should have an index, marked by four distinct straight lines, on the wood which supports the tube, each one inch apart ; the lowest line being on a level with the two surfaces of the resting mercury. Therefore, this allows for six inches of pressure. That is to say, when we apply pressure, the ascending mer- curial column can rise three measured inches, which equals of course six inches pressure. At the very most Ave must never give more than five inches pressure, that is, two and a half measured inches of the ascending limb. Therefore we have a measured half inch of manometer to spare. I prefer the marks in inches rather than centimetres, as there are fewer marks, and each mark can be made more distinct, and therefore more easily read at a distance whilst injecting. It is quite easy to determine the relative height when the mercury stands between any two marks without any extra lines. The cannulas used (see Fig. 36), may be like the one shown at a and sold with anatomical injecting syringes. If so, the stop-cock, h, must also go along with them. This has a beveled end for fitting the cannula, and an INJECTING. 75 end into which fits the beveled end of the syringe. In our case this proximal end is securely fastened into the distal end of the rubber delivery tube with filoselle. When glass cannulas are used, as we have always to keep a pound or two of half-centimetre glass tubing in the laboratory, it is quite easy to make them in the same way we make pipettes. We heat as short a length of the tube as possible by revolving it on its long axis in the flame of a spirit-lamp, or a Bun sen's burner which is better ; then when the flame is quite yellow the heated tube will be quite soft. Now quickly f Fig. 36. — Cannula and stop-cock. remove it from the flame, and with a steady strong pull draw it out in as straight a line as possible. The hotter the length of tube and the more quickly it is drawn out the finer will be the resulting capillary tubing. With a pound of half-centimetre glass tubing and a Bunsen flame we can, in half an hour, draw out some scores of capillary tubes of varying sizes. They will vary very much with the beginner, so that it is quite easy to select a dozen suitable sizes, and cut them with a file in the most suitable part. Each should be cut so as to allow a piece of tube one to two centimetres in length, that has not been drawn out or lessened in diameter, on which to fix our rubber tubing. The drawn-out end of the cannula should, for injecting purposes, be quite 76 PRACTICAL HISTOLOGY. long enough, but no longer than can be avoided. From two to three centimetres is long enough, and yet not so long as to render the cannula fragile. The end cut off with the file should be rubbed smooth on a wetted flag-stone, then held momentarily in the flame. For injecting very small animals, such as frogs and mice, I much prefer brass cannulas because the amount of water, or at least non-injecting fluid, which one has to use to fill the glass cannula and its shank and rubber collar, in making connection and excluding air-bubbles, is very great and dilutes the injecting mass too much The advantages that glass cannulas have over brass ones are two : we can see when the mass is flowing, and we can choose a larger cannula. Their disadvantages are that they require so much non-injecting fluid to fill them at connection time, and they are more difficult to close when the injection is completed and we wish to disconnect. On the whole, I think I prefer a full set of brass cannulas and the accompanying stop-cock which usually go with a good anatomical injecting syringe. The india-rubber tubing should be the black kind used for infants' feeding-bottles. It is best to get a couple of metres of this, as it is used with various instruments. The piece of tube, however, which fits on to the middle neck of the pressure-bottle had better be a wider kind, one centimetre wide, outside measurement, and eight or nine centimetres long. This width just allows the nozzle of the Higginson's syringe to fit tightly. This tube will be of white rubber which has a wire spiral inside to keep open its lumen. This wire of INJECTING. 77 course must be removed. The half-centimetre glass tubing we have described should be selected, and each tube measured, outside measurement, as it varies in diameter very much. The feeding-bottle tubing- is slightly less than half a centimetre wide : this is all the better, as it has to be stretched a little, and therefore fits tighter. It is always best to use a soft ligature when tying this delicate tubing; when tying it is required, filoselle does very well. Hard cotton or ordinary string cuts it. It will be seen that there is only one removable rubber cork, that to the mass bottle already described. After use, before putting the bottle away, this cork should be removed, otherwise it may adhere to the neck of the bottle, and tear when next we wish to take it out. These details, and some details given in the method of injecting, may seem trivial and unnecessary, but we here emphatically say : — Think no attention to minute detail unnecesary in injecting congealing masses into the blood-vessels, or you will fail to get a good injection, and the temper of your temper will be often unnecessarily tried. G 5. — To inject the Blood-vessels of an Animal with a Congealing Mass. It will be better to describe this operation in the order of the steps taken. 1. Fill the water-bath with warm water out of a kettle and set a lighted spirit-lamp under the deep well of the bath, as in Fig. 34. 2. Next melt the carmine gelatine mass and filter it into the Wolff's bottle through flannel. Then tightly cork the bottle and sink it in the well of the bath, and wedge it in with a piece of cork or wood. This is done 7s PRACTICAL HISTOLOGY. to prevent the bottle from rising out of the water as the mass leaves it. 3. Arrange the manometer, pressure-bottle, syringe, and water-bottle as in Fig. 34, and thread an aneurism needle with filoselle. 4. Now deeply narcotise the animal — eat, rabbit rat, or guinea-pig. With suitable strong pins pin down on a cork the legs and head as in Fig. 37. That is, a Fig. 37. — Rat ready for injecting. pin is stuck through each of the four feet, and each foot is stretched downwards. The head is stretched upwards and fastened by a pin through the upper lip. With a pair of scissors incise the skin from the ensiform cartilage to the middle of the neck, and reflect the skin well right and left so as to get a good view of the thorax, especially the sternum and costo-sternal cartilages. Carefully pinch up the flesh over the ensiform cartilage and get one blade of the scissors into the thorax and cut through the costo-sternal cartilages, avoiding the ster- num and the ribs, up to the first rib. Then cut through INJECTING. • 79 the costo-sternal cartilages of the remaining side. The sternum, therefore, will now be attached at its anterior extremity only. Now cut away some pericardium, and make a snip into the right ventricle. 5. We next take a ligature and tie it very tightly around the attached end of the sternum. This effec- tually prevents a fertile source of leakage. Then, instead of cutting the ligature close off, tie a loop and pin this loop down as in Fig. 37, and bring the un- attached end of the sternum under it. 6. Now with a gentle stream of water rinse all blood out of the thorax. We do this best by lifting the cork and its load and holding it under a water-tap. 7. The next is a most important step. Snip off the apex of the heart : this reveals the round left ventricular hole and the rio-ht ventricular slit. Choose the largest nozzle the aorta will admit, and push it gently into the aorta via the left ventricle. Then take the aneurism needle, and with a pair of forceps get the filoselle under the aorta without injuring it, and tie it tightly around the aorta which already contains the nozzle. 8. Lift the animal on its cork and jDlace it on the shelf of the bath. The water, quite warm by this time, should cover — just cover — the entire animal. 9. With a pipette that will go to the very end of the nozzle abstract the blood from the nozzle, and also, with the pipette, carefully fill the nozzle with water taken out of the bath. In doing so take plenty of water in the pipette and push it into the aorta and gradually allow the water to flow out as the pipette is withdrawn. The 80 PRACTICAL HISTOLOGY. object is to fill the nozzle with warm water without allowing any pent-up air to remain. 10. Place a clamp on the tube through which the mass will How, and with the Higginson syringe send sufficient water into the pressure-bottle to raise the mercury in the manometer an inch. 11. The last critical operation has now to be per- formed; namely, making connection between the ani- mal's blood-vessels and the mass. The nozzle is full of warm water : now grasp the delivery tube with the right thumb and forefinger close to the nozzle, and with the left hand remove the clamp. Allow, by relaxing the grasp slightly, the air to get expelled from the delivery tube, and the moment the mass flows make the con- nection. The mercury may now be further raised, little by little, using the right hand for this purpose whilst the left grasps the head and keeps the lips apart that the colour of the lips and tongue may be seen. The deathly pale mouth almost instantly begins to blush, much to our relief, for then we know our mass is flowing. The first redness always appears in the gum between the lower incisors. The mercury may, after the first blush is seen, be raised as quickly as we like, to its full extent, four inches, i.e. two inches of the one limb of the manometer. Indeed he may raise the mercury to five inches at the very end of the process for a few seconds just before clamping the delivery nozzle. 12. The delivery nozzle, its tube having been clamped, may now be withdrawn, the tied -in nozzle secured, and the cork and animal placed in the coldest stagnant water. Water with lumps of ice in it answers INJECTING. 83 only be kept up a few seconds, and this just at the very finish. We know that the blood-vessels are distended, by the swollen dark-red appearance of the conjunctivae. It is only when this stage has been reached that we give the final high pressure. We distend the vessels thus because to fill them only would not do : indeed after the animal has been quite cooled and is ready for dissecting, however distended the vessels may have been in the bath it will be found that now they are only well filled. Should the mass commence running out of the snip in the right ventricle before the vessels are well distended we must ligature the ventricle. Injecting an entire animal is an easy matter or a very difficult one according to the way we set" about it. If the beginner attempts to inject an animal with a con- gealing mass before he has used plain water, or masses fluid in the cold, he may probably be so disheartened that he gives it up believing that he cannot do it at all. Rats are excellent things to practice upon, and we will venture to say that with a couple of days' practice on rats killed with chloroform, using plain water in place of the gelatine mass — provided the steps are rehearsed with the same exactness necessary to inject as we have described, even to the filling of the bath out of the kettle and the placing of the spirit-lamp under the bath, in fact observing every detail — success is a certainty. We repeat that this rehearsal is laborious : that the bath must be emptied and filled for each operation : the bottle, syrino-e, tubing fixed in situ as if taken from the cupboard for each rat : in short for each rat the whole process and every detail observed until each act is taken automatic- G 84 PEACTICAL HISTOLOGY. ally. Suppose success is obtained after two or three rehearsals, we would still advise the student to repeat the process until automatism is obtained. R4sum6. Set the mass to melt in a water-bath : fill the injecting bath with water out of a kettle and set the spirit-lamp going ; filter the mass into Wolff's bottle and apply cork ; wedge the bottle into the bath ; narcotise animal ; if a small animal pin out on cork ; reflect skin and open thorax ; cut away some pericardium and snip right ventricle ; ligature sternum ; remove blood from thorax ; cut off heart apex and tie on nozzle ; remove to bath ; pipette out blood and fill nozzle with warm water ; clamp delivery tube ; raise pressure in bottle ; make connection, and so forth. The water in the bath may be any temperature from 37° C. to 45° C. The operator may choose either glass or metal cannulas An india-rubber collar should be used, whichever he chooses, and this collar as well as the can- nula is to be filled with the warm water when connection is made. When the operator is sure of his ground he will find it an advantage to slit the abdominal wall in the middle line and allow the bowels to float in the warm water. Extravasation of mass from too higdi pressure most frequently occurs in the bowel vessels. Moreover, it is always well to watch how the bowels are receiving the mass. G 6. — To inject the Blood-vessels of Mamma- lian Lungs. For this purpose we proceed exactly as if we were about to inject the entire animal with car- mine gelatine up to the point of inserting the cannula, instead of inserting the cannula in the round hole at INJECTING. 81 best. It may remain an hour or more in this. Then the nozzle having been cut away, the parts may be cut out, placed in 70 per cent, methylated spirit for twenty- four hours, then removed to 90 per cent., there to remain until required for cutting. It will now be well to go over the steps of the process, and point out the errors to be avoided, and the best way of accomplishing each of the numerous operations. We take them in the same order as before. la. The flame of the spirit-lamp must be of ordinary size, not too large, or the water will be too hot by the time the animal is ready to be placed in it. 2a. The india-rubber cork must be wetted, and gently but firmly screwed into the Wolff's bottle. Of course we suppose the fellow-cork to be already in the Wolff's bottle, and tied down with wire permanently, as its removal is never necessary. 4«, 5a. The cork used should be 27 cm. by 8 cm. and 1*5 cm. thick. Such pieces are sold in retail leather- shops. In cutting through the costo- sternal cartilages make first a small snij) immediately below the ensiform cartilage so that the index-finger of the left hand may be gently inserted into the thorax. The finger is pushed along the costo-sternal cartilages, and the blade of the scissors is run along the finger, thus preventing any injury to the parts the scissors might otherwise inflict. Great care must be taken to avoid cutting too close up to the root of the neck, otherwise vessels will be cut and permit copious leakage. The ligature must be placed as close up to the attached end of the sternum as possible. In opening the thorax thus not a single G 82 PRACTICAL HISTOLOGY. vessel is wounded that will allow leakage. The snip in the right ventricle cannot be made too soon; indeed it may be made before putting on the sternal ligature. 6a. If the blood that has flowed into the thorax through the snip in the ventricle be clotted, and can be removed with the fingers, we had better not wash it out. But we must have a clear sight of the aorta, which appears as a white vessel at the base of the heart. 7«. In drawing the filoselle under the aorta in small animals, such as rats, sometimes the filoselle twists the aorta, that is, turns it round and round its long axis. This may be avoided by making the hole beneath the aorta for the filoselle lar^e enough, then o-entlv seizing the aorta and holding it up a little as the thread is being drawn. The elasticity of the aorta pushes the nozzle out of its lumen : this cannot be helped : it is not very difficult to keep the nozzle pushed up witli one's fingers at the same time that the filoselle is bein^ tightened around the aorta. 11a. In making connection be careful to keep the delivery tube in the warm water. The delivery tube when once the gelatine mass is in it, flowing or stagnant must always be kept submerged in the warm water for obvious reasons. Should the inner parts of the mouth remain pale and the mercury of the manometer remain fixed, the mass is not flowing. An air-bubble has got into the connection : we must disconnect and get the air-bubble out with the aid of the pipette and warm water. The pressure at two inches and a half must INJECTING. 87 abdomen close above the diaphragm, or in other words cut the anterior half of the animal away. The reason for the amputation is this: — The liver is extremely lacerable, and in turning up the lobes to get at the bile- duets any cut-off rib or rigid body touching the liver surely lacerates it. By taking the steps we have directed there remains no source of injury to the livei, as all the ribs are removed and the liver lies on the diaphragm exactly as if contained in a soft flaccid bag. AVe now turn up and half over the liver lobes and expose the gall-bladder and the common bile-duct : the latter may be seen close to the portal vein. Place a filoselle ligature tightly around the common bile duct. Now cut off the fundus of the gall-bladder and gently press out as much bile as possible ; then tie in a cannula exactly as if we were doing the same to the aorta. W e now make connection in the manner already described and using a very low pressure, about one inch of mercury, inject our blue mass. Keep the bath going at a proper temperature, and allow the injection to proceed for half an hour or longer at low pressure only. " The blue fluid, driving whatever bile there happens to be left in the ducts before it into the lobules, penetrates first into the interlobular bile-ducts, and from these into the outer parts of the lobules, forcing the bile more and more towards the centre ; here of course there is no escape for it except that a little may pass into the lymphatics and blood-vessels through their walls. Hence it will be understood that the injection can only be made to rill the intercellular biliary passages in the outer part of each lubule." — Schafer. 88 PRACTICAL HISTOLOGY. The entire part of the animal should be placed in cold stagnant water after we have secured the cannula, or its vessel, and made all sources of leakage secure. When quite cold the liver can be cut out and placed in cold methylated spirit for twenty-four hours, then it should be cut into suitable pieces, and select pieces placed in spirit for a week before cutting. It is always more satisfactory to inject the bile-ducts of a liver that has had its blood-vessels previously well injected, because our blue mass has less resident bile to encounter, owing to the distended blood-vessels having forced much of the bile out of the liver entirely. Sections of liver thus injected with Berlin blue must be mounted in turpentine balsam to preserve the colour of the blue as much as possible. Therefore as the turpentine is necessary, the slow mounting or ex- posure process will not do : the cover must be placed upon the liver section, and turpentine balsam used without previous exposure. H.— DISSECTING. H 1. — To dissect and take away Parts of a Small Mammal. When the parts and bits of a small mammal, such as a guinea-pig, rat, rabbit, cat, or small dog, will answer our purpose, we have several advant- ages ; for instance, we can choose our own time : INJECTING. 85 the apex of the heart, however, we insert it in the slit, the former being the left ventricle leading to the aorta, the latter the right ventricle leading to the pulmonary artery. 1. Melt together, and keep warm till required, cacao butter two parts, lard one part. Have also ready a syringe of some kind, with a nozzle that will enter the trachea of the animal we propose to inject. This syringe should also be kept in the warm water. 2. Now proceed as if to inject the entire animal in every particular, but tie the cannula in the pulmonary artery, via the right ventricle. Make connection, and inject with carmine gelatine. 3. After making all sources of leakage secure, such as the left ventricle, carefully remove the " pluck " entire, and, still holding it in the warm water, fill the syringe with the butter and lard and inject the lungs through the trachea. Perhaps all parts of the lungs may not receive the mass, but some are sure to do so and thus provide ample material. The greatest care must be taken to avoid over-distension, which would break down the walls of the alveoli. 4. Now place the doubly- injected lungs in cold methylated spirit, which should be changed in twenty- four hours. 5. Cut sections with a razor moistened with spirit, by simply holding a bit of the tissue in the fingers. Even if we could cut too thin sections in this way we must not cut these sections too thin. 6. Transfer the sections to oil of turpentine, which will dissolve away the butter and lard ; then transfer 8(3 PRACTICAL HISTOLOGY. to methylated spirit and stain in Kleinenberg's haematoxylirj twenty-four hours, and mount in xylol balsam after clearing in clove oil in the usual way. These make most instructive preparations. G 7. — To inject the Blood-vessels of the Liver. The blood-vessels of the liver are nearly always thoroughly injected by the carmine gelatine mass when we have injected the blood-vessels of the entire animal ; therefore, all we have to do after injecting the entire animal, when inspection shows the liver to be well injected, is to proceed to inject the bile-ducts with a gelatine mass coloured by 2 per cent, of Berlin blue instead of carmine. G 8. — To inject the Bile-ducts. We prefer a rabbit for this purpose, but any other small quadruped will do. First inject the entire blood-vessels, and have in a separate water-bath a 2 per cent, solution of Berlin blue gelatine mass. Make the vessels secure, and disconnect after the injection with the carmine gelatine. Empty the Wolff's bottle of the carmine gelatine, and fill it with the blue mass and make everything ready. We suppose of course that the abdominal wall has been slit up in the middle line, and the liver found well injected with the red mass. Most carefully keep the parts well in the warm water and introduce the finger into the thorax and cut the walls of its cavity all round close to the diaphragm. Now get two filoselle ligatures, and secure the thoracic aorta and the vena cava which Ave see stretching like a telegraph-wire from the diaphragm to the heart. Both aorta and cava must be ligatured close to the diaphragm. Now sever the thorax from the DISSECTING. 91 through the entire spinal column and spoil everything. If the student elects to get out the cord by weakening the neural arch, after weakening he introduces one blade of the forceps a short way into the neural canal, and seizes the pedicles and bends and breaks them, first right, then left, all the way down. In getting out the brain, he must take away the cranial vault by little bits, introducing one blade of the forceps a short way each time with great care. '■'Skill to do comes by doing," and we advise the student to practise upon rats first before attempting to get out a brain and cord for subsequent use. After placing the brain and cord in the bowl of fluid* a piece of large nerve such as the sciatic, a muscle (one of the thigh muscles chosen with due regard to its entire circumference being suitable for transverse sections mounted whole), a piece of a long bone (the head, neck, and a short length of the adjoining shaft of the thigh for instance), half the lower jaw if the animal be a cat or dog, pieces of skin, the upper lips and upper eyelids, and the nasal septum are to be taken. The remainder of the carcass can be thrown away. Bv this time the fluid of the bowl must be changed for clean fluid of the same kind. The parts, well sub- merged, may remain in this fluid any time not less than six hours nor more than twenty-four, to suit the convenience of the operator. We now get a wide, shallow vessel, such as a soup- plate, and having poured some Ranvier's alcohol into it prepare the tissues in it as follows : — All parts having a large extent of area btit little depth, such as membranes 02 PRACTICAL HISTOLOGY. skin, lips, eyelids, walls of the hollow viscera (alimentary tube, bladder, &c), are to be pinned out flat on pieces of cork with lillikin pins, or hedgehog spines, taking care to keep mucous surfaces outwards. The solid viscera, such as tongue, liver, spleen, kidneys, brain, spinal cord, &c, must have deep and free incisions made into them in such directions and in such a manner that no part of them is further separated from the hardening agent than a quarter of an inch. In other words, suppose we make incisions across the tongue half an inch apart, no part between the cut surfaces will be further away from the hardening fluid than the distance above prescribed. The direction of all such incisions must be in accord- ance with the subsequent requirements of section- cutting : this is easily remembered by regarding our knife-blade as a section when we are making these incisions ; thus we cut across the long axis of the tongue ; across the long axis of a kidney, but across the short axis also ; across the long axis of the spleen, and so forth, because we only require cross-sections of the tongue and spleen, but sections both ways of the kidneys. Of course when organs are large, such as most livers, and the kidneys of large quadrupeds, the directions of our incisions are of less consequence provided they are half an inch apart, and deep, so that no bit of tissue is further separated from the haidening fluid than a quarter of an inch. By bearing in mind the above principles, the student will be quite well able to place projDerly every part or bit of an animal in its most suitable medium without helplessly depending on detail. DISSECTING. S9 we can kill the animal fasting, or at a suitable time after being fed : we can " fix " the tissues instantly after death, and take what parts and bits we require as our other engagements permit. The animal after fasting, or beino- fed, is to be killed by the inhalation of chloroform. We then tie into the aorta, exactly as if about to inject the vascular system with carmine gelatine, a suitable cannula. This must be done instantly after death. The vena cava is to be opened and kept so ; then the blood-vessels quickly flushed with salt-solution, and instantly afterwards with our " fixing " fluid ("5 per cent, chromic acid solution, picric solution, Midler's fluid). By this means the fix- ing fluid gets at once into the tissues and "fixes" them. The siphon bottles form a most convenient means of thus irrigating the tissues with our salt-solution and fixing agent. We are thus free to use those excellent fixing agents (chromic acid and picric acid), which have little penetrating power. We take a wide shallow earthenware bowl, glazed on the inside, such bowls as are in common household use, and half fill it with the fixing fluid we have been using. We now make an incision through the skin from the symphysis of the jaw to that of the pubis, and reflect it bilaterally : cut through these symphyses and all the superficial structures in the middle line between them, and completely eviscerate from tongue to anus, leaving nothing but the animal as an empty shell, so to speak, and place the entire viscera in the bowl of fluid. With a syringe fill the hollow viscera (lungs, parts of 90 PRACTICAL HISTOLOGV. the alimentary tube, bladder, &c.) with the fluid, using whatever ligatures are necessary, and removing urine, faecal matter, &c, with salt-solution. See to it that the fluid in the bowl well covers all the contained parts. As quickly as possible, get out the eyes, brain, and cord, and place them also in the bowl. In getting out the eyes, cut through each conjunctiva circularly, parallel to the cornea ; hook up one by one the ocular muscles and cut through them.; sever the optic nerve, and the eye without being crushed is got away. Before placing the eyes in the fluid, make a few meridional incisions through the sclerotic with a sharp razor. The brain and spinal cord are always difficult to remove without laceration : the secret of success in this operation consists in faithfully carrying out the follow- ing principles ; we either weaken the neural arch by cutting through the junction of the spinous processes and lamina?, or directly break the neural arch by cutting through the pedicles. First of all fix the animal back upwards upon something (a piece of cork or a board) with stout pins ; divide and well bilaterally reflect the skin from the tip of the nose to the root of the tail, and then get away by cutting and scraping every bit of flesh from the cranial vault and from either side of the superior spinous processes. Thoroughness in doing this will be amply repaid ; indeed it is essential to success. Now with suitable shears (bone forceps, or scissors) cut through either the junction of the spinous processes and laminae, or through the pedicles. In small animals, such as rats and mice, unless the flesh be well cleared away and suitable shears are used, it is easy to cut HARDENING AND SOFTENING. 95 I 2. — Laboratory Alcohols. Students of tins course will do well to get their methylated spirit from a first- class firm. Their rules in the choice of alcohol should be as follows : — 1. Use methylated spirit for making percentage alcohols up to and including 75 per cent. '2. Use methylated spirit for dehydrating sections ; it requires fifteen minutes to accomplish the task; whilst absolute alcohol will do the same in five minutes, but do it no better. 3. Never leave a tissue more than twenty-four hours in an alcohol weaker than 75 per cent, unless disinte- gration is required. 4. In hardening with alcohols where no other reagent is used, commence by placing the fresh tissue in a weaker and pass it through stronger alcohols until 95 per cent, is reached. 5. Always prefer a 95 per cent, alcohol to an abso_ lute alcohol for permanently keeping specimens or sections. 6. Never use methylated spirit for stain mixing. 7. For hardening purposes methylated spirit is in all cases quite good enough. I 3. — Ranviers Alcohol. This is made by adding two parts of distilled water to one part of absolute alcohol or full strength methylated spirit ; usually the latter is taken. I 4. — Seventy-five per cent. Alcohol (75 p.c. Alcohol). This is made by adding 75 parts of alcohol, absolute or methylated, to 25 parts of distilled water. 9G PRACTICAL HISTOLOGY. I 5. — Ninty-five per cent. Alcohol (95 p.c. Alcohol). This is made by adding 95 parts of alcohol or methylated spirit to 5 parts of distilled water. I 6. — Picric Acid Solution. Make a saturated watery solution of picric acid with crystals over. I 7. — To harden in Alcohol. Place fresh tissues in 75 per cent, for twenty-four hours, changing the alcohol two or three times. Then transfer to 95 per cent, for a week, or longer if required. The 95 per cent, must be changed daily, but may be made with methylated spirit. I 8. — To harden in Picric Acid Solution. Place minute pieces for a few hours, varying from eight to forty-eight, in changes of the solution. In all cases the acid may be washed away with tap- water, but the tissues must not be longer exposed to the water than five minutes. They are then transferred to 75 per cent, alcohol, which is to be changed daily for a week or longer. Then they are transferred to 95 per cent, till hardened sufficiently. Some authors say the tissues are to be immersed in changes of alcohol until no more colour comes away. This would require oceans of spirit, and is not necessary. CLASS II. I 9. — Muller's Fluid. Dissolve 25 grammes of bichromate of potash in every 100 c.c. of a 1 per cent, solution of sulphate of soda. I 10.— To harden in Miiller's Fluid. This fluid is quite notorious for its penetrating powers, but takes a HARDENING AND SOFTENING. 03 L— HAKDEXIXG AND SOFTENING. Tissues are either too soft or too hard whilst in the fresh state to be cut, prepared, and mounted for per- manent preservation and therefore require hardening, in a few cases softening, before we can dispose of them further. There are two fluids, namely, alcohol and a saturated solution of picric acid, which harden, and which interfere very little or not at all with after staining. There are again, fluids made with chromic acid or its salts which harden and tan the parts, and if not well abstracted by water and alcohol retard or prevent staining. Lastly, there is osmic acid, which hardens and stains at the same time. It will be best to divide these agents into three classes accordingly. CLASS I. ALCOHOL AND PICRIC ACID. I 1. — Alcohol. There are three kinds of alcohol used in the histological laboratory, namely, absolute alcohol methylated spirit, and rectified spirit. It is necessary to bear in mind what these alcohols are. Absolute alcohol is alcohol without water or any other mixture. Methylated spirit is alcohol or spirits of wine to which 94 PRACTICAL HISTOLOGY. a little wood naphtha is added to prevent people drinking it and cheating the Excise. Rectified spirit is absolute alcohol with lu per cent, of water. In other words, absolute alcohol is rectified spirit from which the 16 per cent, of water has been abstracted. All three may be regarded as full strength alcohols where perfect dehydra- tion is needed : in other words, any of the three may be regarded, for our purposes, as 100 per cent, alcohol ; therefore when a percentage alcohol is prescribed, Ranvier's alcohol for example, the one part of alcohol may be absolute, rectified, or methylated as we choose. Authors who ought to know, Rutherford and Stirling for example, distrust methylated spirit for preserving specimens permanently. Messrs. Cole and Son never use anything else, and their pathological material is kept in the most perfect state possible. I think that any specimens kept in any alcohol whatever must be looked at from time to time, and the spirit changed if a muddiness appears. Whence this muddiness ? those who distrust methylated spirit may ask. The reply is that it will occur occasionally with any alcohol and its specimens, and most likely arises from imperfect harden- in or of the tissue either from the wrong agents used, or from the tissue not having been put into the hardening agents sufficiently soon after death, in which case the protoplasm has undergone changes which have rendered it beyond preservation. It is important to remember that in speaking of or prescribing alcohol the full strength is meant, whether that be absolute, methylated, or rectified. HARDENING AND SOFTENING. 07 long time, counted by weeks, to harden. Pieces of great size or whole organs may be placed in it, but it is best to be moderate, and cut the pieces into the size of 1 cm. cubes, or pieces of that size if of another shape. Place the fresh tissue in the fluid, and change the fluid daily if possible, as it hardens quicker. If desired the fluid may be changed at the end of twenty-four hours, then every fourth day, but at least thirty days will usually be required by so doing, whereas if the fluid be changed daily perhaps half the number of da}'s will suffice. "When hardened, place the tissues in running water for an hour, then in 75 per cent, alcohol, changed once or twice, for a few days, then transfer to 95 percent. When fat in cells is to be preserved, blackened or not with osmic acid, the tissues hardened in Muller's fluid are not to be placed in spirit. I 11. — Klein' s Fluid. Take 2 parts of a one-sixth per cent, watery solution of chromic acid and mix 1 part of methylated spirit with it. I 12. — To harden in Klein's Fluid. Mix the two ingredients fresh each time of using, though the one- sixth per cent, maybe kept in quantity already made. This fluid is of as general application as Muller's fluid. It hardens in from three to ten days, and must be changed at the end of the first twenty-four hours, then daily, or at least every second or third day. The tissues are to be carefully felt and tested daily where chromic acid is used, or they may become brittle and thus spoilt. The fluid penetrates moderately well, and has the great advantage of bringing the various tissues of which H 98 PRACTICAL HISTOLOGY. an organ may be composed to a uniform standard of hardness. The tissues, after being removed from the Klein, arc washed in tap-water for an hour or two, and the harden- ing completed in alcohol, commencing with 75 per cent. I 13. — Bichromate of Ammonia Solution. This is used as a 2 per cent solution in distilled water. I 14. — To harden in 2 per cent. Bichromate of Ammonia. Place the fresh tissues in the fluid and change the fluid very frequently, daily if possible. It requires fifteen, twenty, thirty, or more days to harden, the time largely depending upon the frequency of changing the fluid. The hardening, after getting rid of most of the bichromate with tap-water, is to be completed with alcohols, commencing with 75 per cent. I 15. — Chromate of Ammonia. This is used as a 5 per cent, solution in distilled water. I 16. — To harden in 5 per cent. Chromate of Ammonia. Place the tissues from twenty-four to forty- eight hours in changes of the fluid, then wash a few minutes in tap-water, and complete the hardening in alcohols, commencing with 75 per cent. This solution is used on account of its property of preserving the intra-nu clear network of nuclei. I 17. — ErlickPs Fluid. Dissolve 2*5 grammes of bichromate of potash and 0"5 gramme of sulphate of copper in every 100 c.c. of distilled water. I 1 8. — To harden in Erlicki's Fluid. This fluid is used to harden the brain and cord, which it does in about ten days. It will do so in half the time if we care HARDENING AND SOFTENING. 90 to take the trouble to keep it at a temperature of about 40° C. It must be made fresh each time and filtered before use. It must be changed daily or nearly so, and when the tissues are hard enough the fluid is washed away with tap-water and the tissues finished in alcohols, com- mencing with 75 per cent. I 19. — Chromic and Nitric Acid Fluid. This is made by adding 1 part of strong nitric acid to every 100 parts of a 0*5 per cent, solution of chromic acid. I 20. — To use Chromic and Nitric Fluid. This solution is used for the double purpose of dissolving out the salts of bone effected by the nitric acid, and harden- ing the bone matrix effected by the chromic acid. The fresh bone is placed in repeated changes of the fluid, which must be freshly made each time. The bone is tested with a sewing-needle, which should be able to penetrate the bone without meeting with grit, when the processes will have gone far enough. Ten days may be allowed to lapse before commencing the testing process. The solution is got rid of with tap-water, and the bone placed in alcohols to complete the hardening of the matrix. If sections of the bone render water in which they are placed acid, the water should be repeatedly changed, or 5 per cent, solution of carbonate of soda may be used. H 2 100 PRACTICAL HISTOLOGY. CLASS III. I 21. — Osmic Acid Solution. This is used as a 1 per cent, solution, which is better purchased ready made. Care is to be taken to thoroughly surround the bottle in which it is kept with black paper so as to exclude every ray of light. I 22. — To use Osmic Acid Solution. It is used to stain fat, which it blackens. It must alwavs be borne in mind that strong alcohols dissolve the fat out of its cells ; therefore after hardening a tissue in Miiller's or Klein's fluid, the former being best, sections are to be cut without the tissue being further treated, as is usual with alcohols of increasing strength, and placed from one to twenty-four hours in the osmic solution. When fresh tissues are placed in the solution for hardening and staining only the very smallest bits must be taken, as the acid will not penetrate. GENERAL REMARKS ON HARDENING. Choose a cold, dark place for the purpose. Take the tissues immediately after death, especially in the hot weather of summer, and lose no time in getting them placed in the hardening fluids. Cut them with a very sharp razor on a wax slab in the same plane as that from which sections are to be taken. Except in the case of Miiller's fluid, no tissue must have any portion of its interior further away from the HARDENING AND SOFTENING. 101 nearest surface than '5 cm. or at most 1 cm. In other words, no hardening fluid, Midler's excepted, is to be expected to penetrate beyond "5 cm. If a part be fur- ther from the fluid than this it may be rotten or spoiled before the fluid reaches it. Always suspend the tissues in the upper half of the fluid, and never let them lie at the bottom of it. If they have any blood or dirt about them, wash it off gently with normal salt-solution, not with water. Fig. 3S. — Hardening and preserving bottle. Either place the tissue in a less quantity of the fluid and change this daily, or keep a diary and note in it wdien each has to be changed, and what fluid is to be used at the change. I much prefer the former method. The two-ounce, wide-mouthed bottles (see Fig. 38) suit admirably for hardening purposes if too much tissue be not placed in any one of them. Haifa gross fitted with excellent corks costs six shillings at Messrs. Maw, Son, and Thompson, Aldersgate Street. 102 PRACTICAL HISTOLOGY. On no account keep a tissue or a section in a preserva- tive or hardening fluid that has become muddy. Label each bottle in a very clear, but thorough man- ner, or simply label it with a letter or number and keep a full record in a book. Tissues hardened in alcohol or picric acid have their albumen coagulated ; those hardened in the chromates have their tissues tanned ; therefore when the two former have alone been used the tissues must not be exposed to the softening influences of water, or they go back, that is, become soft again. With regard to those tissues thoroughly hardened by chromates, they can be placed in tap-water, stagnant or running, for hours before being placed in a percentage alcohol. J.— EMBEDDING. When sections have to be taken from a piece of tissue it is necessary that the tissue be held firmly, otherwise the knife cannot do its work. There are two methods of embedding, namely simple or peripheral, and inter- stitial. By simple embedding the periphery only of the substance is grasped : as when we hold a piece of tissue between the thumb and fingers ; or press it between two pieces of carrot, elder pith or amyloid liver ; or pour a liquid over it which will, on becoming cold, harden, such as a mixture of wax and oil. By interstitial embed- ding is meant the infiltration of the entire tissue with a liquid which on s'.mply changing its physical condition EMBEDDING. 103 surrounds and supports every cell and every fibre of the tissue. Thus we place a piece of tissue, after due preparation, in melted paraffin, which will penetrate every space of the tissue however minute ; this paraffin on becoming cold resumes the condition of hardness and solidity it had before being melted, and incloses tightly every cell and fibre of the tissue. The same may be said of celloidin dissolved in alcohol and ether, only heat is not applied. The solution penetrates the piece of tissue, and on removing this to the open air the sol- vents (alcohol and ether) of the celloidin evaporate and leave the celloidin tightly inclosing every cell and fibre. Again, the method of cutting sections in frozen gum is really an instance of interstitial embedding : the liquid gum is first allowed to penetrate the tissue, as much so as the melted paraffin, or the dissolved celloidin ; then it is rendered solid by freezing, and the tissue is cut whilst in this condition. The choice of an embedding method is influenced more by the microtome in which the sections are to be made than by any other consideration. Thus, if a student has a well microtome only, he will not think of embedding in gum and syrup to be afterwards frozen ; on the other hand, should he possess only a Cathcart's microtome, made for freezing only, he will not think of embedding in carrot, or in wax and oil. If every student had the same microtome, it would only be necessary to describe embedding methods suitable for it • but as there are so many different ones it is necessary here to describe methods of embedding to suit any microtome. 104 PRACTICAL HISTOLOGY. There are two methods of simple or peripheral embedding, and two methods of interstitial embed- ding, four embedding methods in all, which are thoroughly efficient, each as far as it goes : one or more of these methods will suit any microtome. These are, embedding in carrot, wax and oil, paraffin, and celloiclin. J 1. — Embedding in Carrot. Carrots may be obtained all the year round nearly, and with very little practice substances from retina, lung, and testicle to spinal cord and shafts of bone — that is, substances of all shapes and consistences — can be held with sufficiently equable pressure to insure sections being made of any degree of thinness up to about 30//. with the ordinary Stirling well microtome. A set of cork-cutters is required, one piece of which must have the same internal diameter as the well of the microtome. Take a sound carrot as large as required : cut a sound piece out of it one inch and a quarter long : place this piece on one end, and, with the cork-cutter the size of the well of the microtome, cut it to fit the well of the microtome tightly ; with a flat-bladed knife divide this piece into two equal semicircular pieces. The further cutting depends on the shape and size of the tissue to be embedded. If a thin flat piece of tissue, such as retina, or cornea, is to be cut, the least possible scraping of the upper half of one of the pieces of carrot only is required. If a piece of lung is to be embedded, a round hole or gauging of the upper part of the face of one of the pieces half the size of this very compressible tissue will be required. If a piece of spinal cord, choose a EMBEDDING. 105 cork-cutter slightly less in its internal diameter, and bore or gauge the upper face of one of the pieces of carrot. In short, one only of the two pieces of carrot is to be bored or gauged, and the piece cut out must be very slightly less in the case of firm bodies, and very much less in the case of compressible bodies, so that when the fellow piece of carrot is placed in correct apposition the piece of tissue may be firmly grasped. When the piece of tissue is irregular in shape, the piece of carrot must be cut with a small cork-cutter used as a gauge and the piece placed in the space with or without the fellow piece of carrot from time to time ; care being taken to gauge tentatively, to avoid taking too much away from any part. The plug thus made is pressed down into the well of the microtome in such a way that the slit in it points right and left as the operator sits, and the half con- taining the tissue is nearest to the operator. The method of cutting the tissue will be described in another place. J 2. — Embedding in Wax and Oil. Take equal quantities by weight of white wax and olive oil, and melt them in a porcelain bowl which has a pouring lip and a short handle. If the temperature of the room be above 66° F., a little more wax may be added ; if below 56° F., a little more oil. In other words, equal parts by weight moke a mixture which cuts admirably in any temperature from 56° to 66° F. The extra hardening, or the extra softening, respectively, by the addition of wax or oil, may quite well be left to guess-work, tempered with ordinary care, as the proportions are after all very elastic ; but the equal quantities must be the starting-point. 106 PRACTICAL HISTOLOGY. The two substances are amalgamated by being heated over a spirit-lamp, or an air-gas flame. The pipkin should rest on a layer of sand on a metal saucer ; in other words on a shallow sand-bath, then as soon as three-fourths of it is melted the flame may be extinguished, and the solid portion, moved about with a needle, will be melted by the already melted mass and the accumulated heat in the sand. Instead of wax and oil, two parts of paraffin wax to one part of chrisma or vaseline may be used ; but I prefer the wax and oil. J 3. — Bathing in Paraffin. In those very numerous cases in histology where very thin sections (3//, to d/jl) have to be made it is best to impregnate the tissue with a suitable paraffin before embedding in it. All the tissues required for this purpose can, with a little forethought be collected, prepared, and bathed in a suitable paraffin at one operation. This saves much time and labour : to have every tissue thus required in one bottle is a con- venience which every student working privately and every teacher must appreciate. Any bit of tissue so preserved can be taken from the bottle and a section from it ; or, in the case of a class, fifty or a hundred sections from it, each not thicker, if occasion requires than 5fi or Sfi, can be under examination in less than a quarter of an hour. Paraffins of two melting-points should be procured, also some of Allen and Hanbury's chrisma, a very pure kind of vaseline. The paraffin of low melting-point will be about 110 c F., that of high melting-point 136° to 140° F. Next to the selection and combination of the paraffins, EMBEDDING. 1 U7 or paraffin with vaseline, the temperature in which it has to be cut is the greatest consideration. To avoid having to bath in a paraffin of higher or lower melting- point it is best to provide for cutting at a temperature of 60° F. in the winter months and for a temperature of 70° F. in the summer; because the temperature of the room in either season is easily regulated to these tem- peratures. Thus, in winter a fire can be lighted if the room is below 60° F., and in the cold weather we often have in summer this can be done and thus bring the room up to 70° F. On the other hand, in hot summer weather if the air of the room has a temperature above 70° F. this will only be during the middle of the day from ten o'clock to four, and even during this time an open window will reduce the temperature so that we can get most perfect sections. Two parts of the paraffin (136° F.) to one part of the paraffin (110° F.) make a mass which cuts perfectly in a room at 70° F. The lower melting paraffin (110° F.) either alone or with one-fifth of its weight of the higher melting paraffin added, cuts well at 60° F. Equal parts by weight of the 136° F. paraffin and chrisma make a mass which cuts well in a room at 55° F. A convenient apparatus for bathing tissues in melted paraffin is the hot-water oven such as the one shown in the annexed figure. It is made of copper and covered with a jacket of felt. The flame may be either an air-gas burner sur- rounded by a mica chimney, or an ordinary gas-burner screwed into a coil of lead pipe. I prefer the latter. Page's gas regulator, which is very simple and inexpensive, may be used to regulate the amount of flame required. 108 PRACTICAL HISTOLOGY. To save time the bath has a large kettle of boiling water poured into it, then filled up with cold water. Also to save time the paraffin should be melted in a porcelain Fii,. 39. — Hot-water oven for bathing in paraffin. pipkin like Fig. 41 before being placed in the bath. We must however in the latter case pour the melted paraffin into the beakers, and allow the amount of extra heat EMBEDDING. 109 above the melting-point to escape before placing in the bath. The small beakers, or short flat-bottomed test- tubes, which hold the paraffin should be supported by something well above the bottom of the bath, so that the paraffin is acted upon by the heated air of the bath only. The thermometer, which had better be a Centigrade one, should be steadily registering the melting-point of the paraffin, or at most 2° above this, for a quarter of an hour at least before placing the tissues in the paraffin. If there be no gas regulator to the metre the operator must be aware of the extra pressure in the gas mains applied on the approach of twilight ; thus on two occa- sions I have left my bath going steadily at 50° C. at 3 jD.m. in winter and have returned at 5 o'clock to find it at 64° or so, and of course my tissues all spoiled. In order to ascertain the melting-point of paraffin — no easy matter — first melt the paraffin in a glass beaker over a spirit-lamp, and allow it to cool a little until signs of congealing appear. Instantly remove it to the water oven, which must be steadily going at the temperature we think nearest the melting-point of the paraffin we are trying. The beaker must be held by a wire frame or tripod well above the bottom of the bath. If in half an hour, not less, the paraffin has congealed it has a higher melting-point ; but if it remains fluid it has probably a lower melting-point. After changing the temperature of the bath until the exact melting-point is reached, we ought to remove the beaker and place a few shavings of the paraffin we are trying upon a piece of glass and lay this on the tripod and watch the effects 110 PRACTICAL HISTOLOGY. of the ascertained heat upon it for half an hour or more. The two paraffins, 110° and 136° F., should be tested on being admitted to the laboratory, and all blended paraffins also. Afterwards they should be placed separ- ately in tin or wooden boxes with their melting-points carefully labelled or scratched upon the box. The variation of the thermometric reading caused by opening the bath, also by introducing paraffin contain- ing more heat than that required to melt it, are two difficulties in the process. The latter is avoided by taking the precaution above alluded to. To prepare tissues for the paraffin-bath it is essential that they be thoroughly hardened and thoroughly de- hydrated ; therefore they must be taken direct, either out of undiluted common alcohol, or absolute alcohol. From the alcohol they are placed directly in creosote, and when thoroughly permeated by this they are, after having all their surfaces touched with bibulous paper to get away as much creosote as possible, placed in the melted paraffin. Thus, suppose we have a cubical- shaped piece of liver 1 cm. in size, a piece of cornea, and a piece of cat's spinal cord 1 cm. in length, to bath in paraffin : the liver will have been hardened in bichromate of potash followed by alcohols of 75 per cent., afterwards full strength; the piece of cornea will probably have been hardened in Miiller's fluid, followed by the two alcohols just named ; and the spinal cord will probably have been hardened in Erlicki's fluid, followed by the two alcohols. In other words, all three tissues are in common alcohol of full strength or in EMBEDDING. Ill absolute alcohol, which has given the last touch of the hardening jorocess to them ; therefore, at any moment, they can be taken from the spirit and placed, with as little spirit as possible, in the creosote. The creosote would thoroughly permeate the cornea in half an hour, the spinal cord in two hours; but the liver would require perhaps four to six hours, perhaps longer, therefore we should let all three be in the creosote at least six hours, and longer if our time permitted. Again, the paraffin would permeate the cornea in half an hour, the cord in one hour, and the liver in three hours, perhaps less ; but we should allow all three to remain in the paraffin-bath quite three hours, and, better, four hours, to be certain of thorough impregnation. Suppose, further, we were staining these three pieces en masse before bathing, we should take them from the spirit as before, and place them, jorobably, in borax carmine, or in Kleinenberg's hematoxylin. The cornea would require to remain in the stain at least twelve hours, the cord thirty hours, and the piece of liver one hundred hours ; therefore, we should let all three remain in the stain four days and nights at least, and, if time permitted, a full week. After taking them out of the stain we should put them in several changes of common alcohol of full strength, to abstract all superfluous colour ; this would take twelve hours, and we should let them remain another twelve hours in a fresh lot of alcohol to thoroughly dehydrate, perhaps using absolute alcohol for this last twelve hours. Lastly, we should take them from the alcohol, put them in the creosote, and bath them in the paraffin as already described. 112 PRACTICAL HISTOLOGY J 4. — Embedding in Paraffin, Wax and Oil, &c. Whether we use wax and oil, paraffin and vaseline, or simple paraffin alone, the process of embedding is the same, therefore one description will do for all. The best way is to make a rectangular trough with either brass embedding |_L> such as are shown in the annexed figure, or paper, or capsule metal ; then, having melted the embedding mass in a porcelain pipkin over a spirit- lamp, or air-gas burner, we pour the mass into the trough so as to overfill it. We now transfix the piece of tissue Fig. 40.— Embedding LL- with a lillikin pin in the direction of the cutting plane (see Fig. 40), and when congelation shows itself either at the bottom or sides of the trough we instantly insert the piece of tissue into the middle of the melted mass and as quickly move it towards the end of the trough until the surface from which sections have to be taken almost touches one or other end of the trough. If the LL are used they should be placed in pairs on glass slips. The mass coming in contact with the cold brass and glass EMBEDDING. 113 soon congeals.- Great care must be taken to avoid placing the tissue in the mass whilst it is too hot : at the same time the mass must be fluid enough, otherwise there will be gaps between the tissue and embedding mass when cold. If there be no necessity for transfixing the tissue with a pin to keep it in the proper relative position for section- cutting, a heated needle may be used for placing the tissue in position after it has been placed in the trough, taking care not to bum the tissue with too hot a needle. Fig. 41. — Porcelain melting-dish and mounted block. The embedding mass must be quite cold and set before the mould is removed. To accelerate the coolino the whole may be placed in cold water or under the tap. After the block has been removed it should be placed on a wax slab and pared with either a sharp razor or a scalpel. First commence by paring and exposing the end of the block the tissue is in. When opaque masses, such as wax and oil, are used, the piece of tissue cannot be seen before commencing to cut away the mass ; therefore some advise a mark to be scratched on the end of the congealing block. This is scarcely requisite, 114* PRACTICAL HISTOLOGY. because the taking away of a very few shavings from either end of the block reveals the presence or absence of the tissue. About a quarter of an inch of the block to form a base should be left. The end of the tissue from which sections are to be cut must be exposed, then the sides must be pared and bevelled (see Fig. 41) in the case of paraffin embedding when ribbons of sections are to be cut. Care must be taken to avoid weakening by over-paring in the case of wax with oil, or paraffin with vaseline masses. If the tissue embedded be recognised before sections are taken from it the name can be scratched on the block with a sharp needle before the block is placed in the bottle or box in which it has to be kept. In all cases the name should be scratched upon the block either before or after cutting. The name should consist of two words, the name of the animal and the name of the tissue, thus : aorta Horse, duocl : Pig, skin Frog. J 5. — Embedding in Celloidin. This is a most simple and most effectual method of embedding, but it requires the knife to be moistened with alcohol, and the edge of the knife to move obliquely in cutting ; therefore unless the knife be fixed in a frame as in Thoma's microtome it is as impossible to cut thin sections as em- bedding in carrot ; indeed it presents no advantages over embedding in carrot, where the knife is held only by the hand, except in those cases where there is no tissue binding the parts together, as in transverse sections of intestinal villi, &c. When however the knife has a mechanical movement, as thin sections of small area can be obtained by celloidin as in paraffin EMBEDDING. 115 embedding. With my Thorn a's microtome I can make d/jl, 4 then in five or ten minutes cut out a piece and place it o 2 196 PRACTICAL HISTOLOGY. in a saucer of Ranvier's alcohol. It must be stained with logwood and pieces mounted in balsam. It is as well to get as much of the silver removed as possible. To do this seize the membrane by its edge as it lies in the Ranvier and trail it to and fro : then place in fresh Ranvier. XXI. 4. — Feed a frog with fat bacon by placing a little bit in the oesophagus every other day three times : then kill the day after the last feeding. Take away the stomach and small intestines : lay them open and notice the difference in the hue of the mucous mem- brane of the stomach and that of the small intestine near the stomach, due to the non-absorption of fat by the former. Place in the moist chamber, then take away a minute piece of the mucous membrane of the small intestine and tease and examine in salt-solution. Notice that the epithelium cells are crowded with fat-globules. Divide the remaining intestine and pin each piece out on cork. Place one in 75 per cent, alcohol for an hour, then in absolute until hardened. Place the remaining half in 0*5 per cent, osmic acid ten minutes : then transfer to Miiller's fluid for three weeks, and pass through alcohols. Both pieces, the spirit piece and the osmic acid one, are to be bathed, embedded^ and cut in paraffin into sections vertical to the mucous membrane. Mount by the shell-lac and creosote method, leaving the osmic sections unstained ; but staining the others with safranin. The sections should be cut about 5yu, in thickness. XXVII. 5. — Cut through the skin in the mid-ventral THE PREPARATION OF MATERIAL. 197 line in a small frog : lift up the flap of skin on one side over the middle of the sternum. A thin band of muscle will be seen running from the skin downwards towards the lower part of the sternum. Cut through the skin above and below the muscle, and tear away the connective-tissue around it. Then keep it stretched and pour 1 per cent, osmic acid solution over it. In a minute or so the muscle will be fixed. With fine forceps tear away carefully from the muscle any connective-tissue that can be seen on its surface and cut it out, beinc- careful to cut the upper end as close as possible to the skin. Place it in osmic solution a few minutes : wash with water : pass through alcohols and mount in balsam. I have taken this and XXI. 4 from Foster and Langley's Practical Physiology, pp. 83, 84. (Macmillan). The preparation is to show the ending of muscle in tendon. XXVIII. 3. — Take away the sympathetic trunk of a frog and stain it with gold chloride thus : — Prepare some 0*5 per cent, gold chloride solution and place it in a watch-glass. Now kill a frog : pin it out on its back and open the abdomen in the usual manner. The sympathetic trunk lies close to the vertebral column on each side of but behind the abdominal aorta. It is a very slender cord with minute semi-transparent pig- mented enlargements (ganglia) on it at short intervals. A further guide to it, as pointed out by Foster and Langley, is the row of small nerve-fibres running trans- versely from the spinal nerves (one from each nerve) to the ganglia. It is so easy for a novice to cut it away without seeing it that I give the above authorities' 108 PRACTICAL HISTOLOGY. way of exposing it. They direct the intestine to be picked up and the mesentery cut through above the kidneys : then a kidney to be pulled up : the peritoneum along its edge to be cut through, and the kidney to be turned over to the opposite side of the body. After gold-staining, place it on a slip and examine with a low power, select the ganglion in which the nerve- cells are seen most clearly and tease this out in glycerine ; remove all connective-tissue : then transfer the cells with a needle to Farrant and mount, using a shallow cell. XXIX. 5. — The posterior nares of the frog are almost close behind the upper lip. Pass a fine probe into a nostril : hold it almost at right angles to the antero- posterior axis of the head, and it will be seen to protrude at the posterior nares. Kill a frog : cut off its head : remove its lower jaw : slit up each nostril and place the head in 5 per cent, chromate of ammonium for forty-eight hours; after this in Ranvier's alcohol twenty-four hours : then in picro-carmine solution twelve hours. Scra]3e away some olfactory epithelium with a Beer's knife : break it up with needles in Farrant and mount, using a shallow cell. XXX. 2. — Cut out with a sharp knife and scissors the two corneas of a frog : stain in gold, and mount each whole, but cut radiating slits in each so as to make it lie as flat as possible. Mount in balsam. XXXI. 2. — Place the eyeball of a frog for twenty- four hours in this mixture (nitric acid and glycerine, of each one part, and add three parts of water). Re- THE PREPARATION OF MATERIAL. 199 move and place in water another twenty-four hours, then break up little bits of the lens in Farrant, and mount, using a shallow cell. XXXI. o. — Remove the eyeball of a freshly-killed frog, and with a sharp razor and scissors divide the eye into an anterior and a posterior half. Place the posterioi half in O'l per cent, osmic acid solution twelve hours, then in 75 per cent, alcohol twelve hours. Break up bits of retina and mount in Farrant, using a shallow cell. XXXI. 4. — With a sharp razor, before removing the eyeball, divide it as in the last preparation, and drop into the cup formed by the posterior half 1 per cent, osmic acid solution. In five minutes place the head in Midler's fluid for three weeks ; then get out the pos- terior half of the eye and take the requisite proceedings for bathing and cutting in paraffin. XI. 3. — The mesentery must be pinned out on cork After passing through alcohols it should be, some portions of it at least, stained in safranin. XXV. 7. — Place two or three testes, small round bodies seen on removing the abdominal viscera, in 5 per cent, chromate of ammonium forty-eight hours ; then place in Ranvier's alcohol twenty-four hours. Lay each on a wax slab and cut it open with a Beer's knife, and squeeze its contents on to a slip. Place some 5 per cent, picro-carmine upon each preparation for twenty-four hours, in a moist chamber ; then transfer with a needle to Farrant on fresh slips, or roughly drain away the stain and add Farrant on the original slips. Use shallow cells. Another way is to allow the semen to dry then mount in xylol balsam. 200 PRACTICAL HISTOLOGY. TRADESCANTIA. I. 8. — Hair of a stamen. Note. — Cell-division may be studied in the sala- mander larva, but it is essentially the same and more easily studied in a plant. I copy the following almost verbatim from Bower and Vines' Practical Botany, p. 25. (Macmillan). In order to study the process thoroughly, the hairs on the stamens of Tradcscantia may be taken. A stamen is to be removed from a bud on a warm day and placed at once in a drop of 1 per cent, sugar-solu- tion on a cover-slip. The cover is then placed over a moist chamber and studied with a power of 500 diameters. A terminal cell of one of the hairs with a large and conspicuous nucleus is to be watched. The nucleus first gradually elongates in the direction of the longer axis of the cell ; it becomes more granular, and its protoplasm aggregates at the poles ; then the nucleus becomes striated, the striae arranging themselves parallel with the longer axis of the nucleus and approach each other at the poles ; thus a characteristic nuclear spindle is produced. The strias are then rup- tured in the equatorial plane and gradually collect at each pole, so that two new nuclei are formed. A layer of granular protoplasm is now found in the equatorial THE PREPARATION OF MATERIAL. 201 plane, which extends peripherally until it reaches the cell-wall. This layer constitutes the dividing wall between the two cells. YEAST. I. 1, 2, 3. 4, 5, 6. — Fresh Brewers' Yeast. Notes. — Sow a little fresh bi ewer's yeast on Pas- teur's fluid in two beakers, lest one should be spoilt. Put aside in a warm place until it begins to froth up. It will keep good for several days, though the operations had better be carried out in one or two days-. Notice : — 1. The varying size of the cells. 2. Their arrangement in clusters or groups. 3. Their shape and mode of union. 4. Their sac, that it is transparent and homogeneous and unstained by magenta. 5. Their protoplasm, that it is less transparent and stains with magenta. 6. Their vacuole : its size, position — sometimes ab- sent. 7. The relative pro]:>ortion f sac, protoplasm, and vacuole. To burst the cells so as to see the sacs and their contents, place the slip on a solid level surface, then press smartly on the cover with the end of a scalpel with a fold of blotting-paper intervening. Place a piece of blotting-paper over one of the beakers of yeast, and examine the cells from day to day to watch their growth and roultijilication. 202 PRACTICAL HISTOLOGY. GERMAN YEAST. I, 7. — German Yeast on potato. Note. — Shake some dry German yeast in distilled water and let it stand to settle, then decant. With a camel-hair brush spread some of the creamy deposit in a thin layer upon each of three or four fresh slices of a sound potato, and place these in a moist chamber. Begin in a week to examine the cells daily to see celt-division. A minute bit is scraped from the potato and diffused with a needle upon a slip in 1 per cent, solution of sugar, and examined with a ^ or -f s inch objective, preferably an oil-immersion. Use a shallow cell. AMCEB^E. II. 1, 2, 3. — Amoebae. Notes. — By irrigating, by applying heat on the warm stage, and by applying gases and vapours and by all other means, the student should make a thorough study of amoebae. They may be -found in small stagnant pools, mud, and damp earth. Seek for them in the slimy sediment of hot-house tanks. If they cannot be found use the white blood-corpuscle of the frog, or newt. Irrigation is easily managed. If the effects of drugs THE PREPARATION OF MATERIAL. 203 are tried on them, the drugs are to be mixed with normal salt-solution. A cover-glass, if leucocytes be used, is allowed only to just touch a drop of blood on a slip. By this means a droplet is obtained and the cover-glass is inverted over the warm stage, or the gas and vapour cell ; then if a drug be tried a drop of the salt-solu- tion containing the drug is placed over the blood. (Brunt on.) Moderate warmth accelerates the movements : a little extra warmth and we have arrest of movements, tetanic contraction or heat tetanus, during which the spherical form is assumed. Movements may be resumed on with- drawing the heat if this has not exceeded 35° C. At 40° C. the motionless sjuhere is in a state of heat rigour in which the protoplasm is coagulated. Motion cannot be induced now by withdrawing the heat. Slight electric shocks increase the protoplasmic movements ; stronger ones cause tetanic contraction ; and numerous or power- ful ones cause coagulation. One per cent, salt-solution first causes increased protoplasmic movement, then causes sudden tetanic contraction and the expulsion of any food they may contain at the moment, and some- times even expulsion of their nucleus (Brunton). Amcebse and leucocytes can be fed by gamboge rubbed down in a drop of water. They should be fed before applying the salt-solution. Sometimes they take a long time to feed ; in these cases the slip should be placed in a moist chamber and time allowed. Very dilute acids and alkalies first increase then arrest protoplasmic movement (Brunton). Oxygen appears to be necessary for their life. 204 PRACTICAL HISTOLOGY. The leucocytes of different animals have different resisting powers to the action of drugs; those of the newt being more resistant to the action of drugs than those of the guinea-pig. It is scarcely necessary to point out that a thorough study, as here indicated, will greatly facilitate the study of the first processes of inflammation. I have taken most of the preceding remarks from Pharmacology, Therapeutics, and Materia Medica, by T. Lauder Brunton (Macmillan), the most fascinating book to the practitioner of medicine who is at the same time a lover of physiology. q.__TWO SHORT METHODS OF PREPAR- ING ALL THE SECTIONS USED IN A HISTOLOGICAL COURSE. L— BY THE FREEZING METHOD. 1. Prepare by hardening, or softening, all the parts of organs and tissues from which sections have to be cut in a complete course of histology. 2. When all are quite ready take a suitable piece of each, taking two or more pieces of each of those requir- ing sections in various planes. Place all the pieces, after due jDreparation, when required, in the gum and syrup. TWO SHORT METHODS OF PREPARATION. 205 3. In twenty-four hours, or longer, prepare the freezing microtome. Take also as many short wide- necked bottles, like Fig. 38, as there are pieces of tissue. Cut each piece of tissue into suitable sections, and pour through a funnel, as already described, each lot of sections into a separate bottle. Change the water frequently to get rid of all the gum and syrup then pour on increasing strengths of alcohol up to 95 per cent. 4. Take out a section from each bottle : examine it to see what it is, then label the bottle from which it is taken. Place the bottles in a cool place, changing the 95 per cent, if it at any time becomes muddy. 5. "Prepare three times as many slips and covers as there are bottles ; appropriately stain and mount three well-selected sections from each bottle and temporarily label each by means of a little piece of gummed paper, and a pencil or ink. 6. With the compound microscope, using an inch or half-inch, or higher power when needed, examine every set of slides, selecting one or two from each. Those selected must now be marked with a writing diamond and the temporary label removed. Should the student not be sufficiently conversant with the subject to dis- tinguish between the better and the worse slides, all the slides should be put away and the selection made under more favourable circumstances later on in the course. The rejected slides can be recovered as clean slips to be used again. 206 PRACTICAL HISTOLOGY. II.— BY THE PARAFFIN METHOD. 1. Collect and prepare all the parts from which sections have to be taken as before. 2. Bath in paraffin at one operation the entire lot and place the pieces in a wide-mouthed bottle. 3. When convenient embed every piece in paraffin, using, if possible, the embedding L's, though paper boxes will do, or boxes made of capsule metal. 4. Cut away all superfluous paraffin from each tissue, and trim it, in the way already described, ready for cutting. 5. Cut a ribbon or two of sections from each block and place them in a clean, dry chip box, but take care to use a separate box for each piece of tissue. The piece of tissue or block should be placed in the same chip box with the ribbons of sections, taking care to avoid rolling the block over the ribbons. 6. Take three prepared slips and covers for each set of sections and mount and otherwise proceed exactly as in the former case. Important Notice. — Although I have described the above processes separately as if either by itself would meet all the requirements of the case, in practice it is best to use both processes for the following reasons : — The freezing method enables us to obtain extensive TWO SHORT METHODS OF PREPARATION. 207 sections ; the paraffin method enables us to obtain exceedingly thin sections, perhaps eight times thinner than by the freezing process. Again, where sec- tions would fall to pieces when cat by the freezing method, the paraffin method is a perfect remedy : for instance, transverse sections of intestinal villi can be well made and mounted by the paraffin method ; but by the freezing method the villi would be scattered in all directions. Of course when the student works with the very perfect but expensive microtome of Thoma the case is different : then if he requires extensive sections he can get them by the paraffin method, or he can very readily get them by using celloidin. My remarks therefore apply to students in general working with ordinary microtomes. For a Third Method of embedding see Appendix. PART II INTRODUCTION. Important Notice. — A space is left below each preparation for the student to fill up in describing the stain used : the mounting medium : the date of prepara- tion : what the slide shows, and any other information which is evidently desirable. The slide itself should only have the two sets of figures scratched upon it with a writing diamond ; thus the slide holding the section of the coats of the stomach of a cat will have XX. 2 scratched upon it. Again, a space is left below each term for the student to fill up in defining the term. He should fill up these spaces before commencing to study the preparations of the lesson, using a good text-book such as the second volume of Quains "Anatomy" Kleins "Elements" Schdfers " Essentials" or Pursers excellent little "Manual." He will then have no need to explain the meaning of these terms when filling up the spaces beneath each preparation. The student should first write what he is goin^ to put in his book on a piece of waste paper. I.— THE VEGETABLE CELL. 1 1. Yeast. — Mount a minute drop of yeast without much pressure. Observe, draw, and measure the cells and groups. 2. Yeast. — Mount a droplet and stain with solution of magenta. 3. Yeast. — Burst the stained cells of the last preparation. 1 Read Huxley and Martin's Practical Biology (Maemillan) for this subject. P 2 212 PRACTICAL HISTOLOGY. 4. Yeast. — Mount a fresh droplet and stain with solution of iodine which stains the protoplasm brown, not blue (showing absence of starch). 5. Yeast. — Mount a fresh droplet and stain with solution of potash. 6. Yeast. — Mount a fresh droplet and mount in a moist cell. 7. German Yeast. — Spread some on fresh slices of potato. THE VEGETABLE CELL. 213 8. Tradescantia. — Place a stamen, taken from a bud on a warm day, in 1 per cent, solution of sugar on a cover-glass inverted over a moist cell. DEFINITIONS OF TERMS. A Protein Compound. Ascopores. Cellulose. 214 PRACTICAL HISTOLOGY. Endogenous Division of Cells. Gemmation. Karyokinesis. Protoplasm. THE VEGETABLE CELL. 21 5 Sac, Torulae. Vacuole. 216 PRACTICAL HISTOLOGY. II.— THE ANIMAL CELL. (AMCEBiE AND COLOURLESS BLOOD-CORPUSCLES.) 1. Amoebae. — (a) Mount a drop of water containing amoebae, and observe their size, outline, and structure. (b) Feed with pigment granules and watch the process of ingestion, (c) Make them disgorge with 1 per cent, salt-solution. 2. Amoebae. — Mount another drop. Mechanically crush to show absence of outer sac. 3. Amoebae. — Mount two drops of water as before on same slip, but under separate covers, and stain one with magenta and the other with iodine. THE ANIMAL CELL. 217 4. Human White Blood-Corpuscle. — Mount a drojilet of blood drawn from the finger-tip. Observe size, form, and structure of the white corpuscles. Run dilute acetic acid under the cover-sdass. 5. Human White Blood-Corpuscle. — Mount other droplets of blood on same slip under two separate covers, and stain one with magenta, the other with iodine. 6. Human White Blood-Corpuscle. — Observe the movements of the white corpuscles when heated to 37° C, afterwards to 50° C. on a hot stage. 218 PRACTICAL HISTOLOGY. DEFINITIONS OF TERMS. Amoeba. Amoeboid Movement. Contractile Vesicle or Vacuole. Diastole. THE ANIMAL CELL. 219 Ectosarc. Endosarc. Proteus Animalcule. Pseudopodium. Systole. 220 PRACTICAL HISTOLOGY. III.— AMPHIBIAN AND HUMAN BLOOD- CORPUSCLES. 1. Frog. — Mount without pressure a droplet of blood, and observe, sketch and measure the corpuscles. 2. Frog. — Make a double-stained permanent pre- paration of newt's or Frog's blood. 3. Human Blood. — Mount a droplet of human blood (drawn from the finger-tip) without pressure. Observe the varieties, form, method of arrangement, colour, &c, of the corpuscles. Sketch and measure some of the corpuscles. AMPHIBIAN AND HUMAN BLOOD- CORPUSCLES. 221 4. Human Blood. — Mount a fresh droplet and run 10 per cent, salt-solution under the cover. DEFINITIONS OF TERMS. Amphibian. Coloured Blood-Corpuscles. Colourless Blood-Corpuscles. 222 PRACTICAL HISTOLOGY. Lymph-Corpuscles. Plasma. Rouleaux. ACTION OF REAGENTS ON BLOOD-CORPUSCLES. 223 IV.— ACTION OF REAGENTS ON BLOOD- COEPUSCLES. 1. Frog. — Run in distilled water under a cover, inverted over a droplet of blood. 2. Frog. — Run in 1 per cent, solution of acetic acid under the cover inverted over a fresh drop of blood. 3. Frog.— Place a droplet of blood and a droplet of 2 per cent, boric acid solution close together on a slip, then cover and watch the effects. 224 PRACTICAL HISTOLOGY. 4. Human Blood. — Run in distilled water under a cover inverted over a droplet of blood. 5. Human Blood. — Run in 1 per cent, solution of acetic acid to a fresh droplet. 6. Human Blood. — Run in 0*2 per cent, potash solution to a fresh droplet. 7. Human Blood. — Place a droplet of 2 per cent, tannic acid solution side by side with a droplet of blood that has been mixed with 0'6 per cent, salt-solution. ACTION OF REAGENTS ON BLOOD-CORPUSCLES. 225 DEFINITIONS OF TERMS. Crenated. Cholesterin. Fibrin. Globulin. Q 226 PRACTICAL HISTOLOGY. Hsematin. Haemoglobin. Lecithin. Nucleus. EPITHELIUM. 227 Nuclear Network. Stroma. V.— EPITHELIUM. [Epithelium in situ occurs in almost half the permanent slides or preparations of histology, so that special preparations may be strictly limited.] 1. Mouth of Operator. — Inside of cheek scraped with a blunt blade, scrapings treated with (a) salt-solu- tion, (b) acetic acid, (c) silver nitrate. Q 2 228 PRACTICAL HISTOLOGY. 2. Rabbit. — Intestinal (columnar) epithelium from small intestine. 3. Rabbit. — Transitional epithelium from bladder. 4. Prog. — Ciliated epithelium from roof of mouth. 5. Oyster. — Gill of fresh oyster, piece of. EPITHELIUM. 229 DEFINITIONS OF TEEMS. Cilia. Columnar Epithelium. Columnar Ciliated. Columnar Non-Ciliated. 230 PRACTICAL HISTOLOGY. Compound Epithelium. Cylindrical Epithelium. Glandular Epithelium. Goblet Cells. EPITHELIUM. 231 Prickle Cells. Scaly Epithelium. Simple Epithelium. Squamous Epithelium. 232 PRACTICAL HISTOLOGY. Stratified Epithelium. Transitional Epithelium. VI.— AEEOLAE AND ADIPOSE TISSUE. 1. Rabbit. — Subcutaneous tissue stained with silver. 2. Rabbit. — Subcutaneous tissue stained with picro- carmine. AREOLAR AND ADIPOSE TISSUE. 233 3. Rabbit. — Subcutaneous tissue stained with ma- genta. 4. Rabbit. — Subcutaneous tissue examined in salt- solution. 5. Rabbit. — Subcutaneous tissue irrigated with 1 per cent, acetic acid. 6. Rat. — Subcutaneous tissue of a } 7 oung rat ex- amined in salt-solution. It will contain fat. 234 PRACTICAL HISTOLOGY. 7. Rat. — Subcutaneous tissue prepared with picric acid and stained. DEFINITIONS OF TERMS. Areolae. Ground Substance. Plasma Cells. WHITE FIBROUS AND YELLOW ELASTIC TISSUES. 235 Stellate Cells. Wandering Cells. VIL— WHITE FIBROUS AND YELLOW ELASTIC TISSUES. 1. Calf. — Transverse and longitudinal sections through tendo Achilles. 236 PRACTICAL HISTOLOGY. 2. Mouse. — Tail tendon stained with logwood. 3. Mouse. — Tail tendon stained with silver. 4. Horse. — Fibres of ligamentum nuchse teased in salt-solution. 5. Horse. — Section across fibres of ligamentum nuchse. WHITE FIBROUS AND YELLOW ELASTIC TISSUES. 237 6. Horse. — Section parallel with fibres of ligamen- tum nuchas. DEFINITIONS OF TERMS. There are no special terms used in connection with these two forms of connective-tissue, but their localities are of special importance. Where is white fibrous tissue found, and what are its functions ? 238 PRACTICAL HISTOLOGY. Where is yellow elastic tissue found, and what are its functions ? VIIL— CAETILAGE. 1. Horse. — Vertical section of a tracheal ring. 2. Horse. — Transverse section of a costal cartilage. CARTILAGE. 239 3. Horse. — Section vertical to surface of the epi- glottis. 4. Pig. — Section vertical to surface of ear-lobe. 5. Hoise. — Section of intra-articular cartilage of stifle joint. 6. Rat. — Section of head of femur including a little of the subjacent bone. 240 TRACTICAL HISTOLOGY. 7. Cuttle-Fish. —Section of a piece of the head cartilage. DEFINITIONS OF TERMS. Articular Cartilage. Cartilage Capsule. Cartilage of Incrustation. CARTILAGE. 241 Cell Territories. Costal Cartilage Elastic Cartilage. Hyaline. R 242 PRACTICAL HISTOLOGY. Intercellular Substance. Perichondrium. Reticular. White Fibro-Cartilage. Yellow Fibro-Cartilage. BONE. 243 IX.— BONE. 1. Sheep. — Section vertical to surface of the shin- bone ground down on a hone, &c. 2. Sheep. — Section vertical to surface of the shin- bone decalcified. 3. Sheep. — Longitudinal section of a decalcified shin-bone. u 2 244 PRACTICAL HISTOLOGY. 4. Horse. — Shreds from deepest part of periosteum of a decalcified parietal bone. 5. Guinea-Pig. — Red marrow taken from a broken (not sawn) rib of a young guinea-pig. DEFINITIONS OF TERMS. Canaliculi. Cementing Substance. BONE. 245 Circumferential Lamellae. Compact Bone. Endosteum. Giant Cells. 246 PRACTICAL HISTOLOGY. Haversian Canals. Haversian Lamellae. Haversian Systems. Interstitial Lamellse. BONE. 24' Lamellated. Myeloplaxe. PerforatiDg Fibres of Sharpey. Periosteum. Spongy Bone. 248 PRACTICAL HISTOLOGY. X.— DEVELOPMENT OF BONE. 1. Kitten. — Section through the ramus of the jaw of a newly-born kitten. 2. Kitten. — Longitudinal section through a meta- carpal bone of a newly-born kitten. 3. Foetal Mouse. — Parietal bone. DEVELOPMENT OF BONE. 249 DEFINITIONS OF TERMS. Bone-Corpuscles. Epiphysal Cartilage. Intermediate Cartilage. Matrix. 250 PRACTICAL HISTOLOGY. Ossification. Ossification, Endochondral. Ossification, Intra-cartilaginous. Ossification, Intra-membranous. Osteoblast. MUSCLE. 251 Osteoclast. Osteogenetic Fibres. XL— MUSCLE. [Note. — Sections of the striated muscle are well seen in the tongue and in numerous other preparations. Except in making the following special preparations I would advise a complete study of living muscle during the physical or graphic course of physiology.] 1. Rabbit. — Bits of unstriped muscle-fibre torn from the colon of the rabbit. 252 PRACTICAL HISTOLOGY. 2. Frog. — Bladder of frog (piece of). 3. Newt. — Mesentery of newt. 4. Mouse. — Transverse section through the ventricles of the heart. 5. Rat. — Vertical transverse sections of the tongue injected with carmine-gelatine. MUSCLE. 253 DEFINITIONS OF TERMS. Cohnheim's Fields. Contractile Substance, Disks. Endomysium. 254 PRACTICAL HISTOLOGY. Fibres. Fibrils. Perimysium. Plain Muscle-Fibre. MUSCLE. 255 Primitive Muscle-Bundle. Primitive Muscle-Cylinder. Sarcolemma Sarcous Element. Unstriped Element. 256 PRACTICAL HISTOLOGY. XIL— NERVE-FIBKES. 1. Horse. — Transverse and longitudinal sections of the metacarpal nerve. 2. Horse. — Teased fragments after treatment by osmic acid. 3. Mouse. — Short lengths of one of the lateral cutaneous branches of the intercostal nerve. NERVE-FIBRES. 257 4. Frog. — Sciatic nerve, fresh, examined in salt- solution. 5. Frog. — Sciatic nerve treated with osmic acid. DEFINITIONS OF TERMS. Axis Cylinder. Lanterman's Notches. 258 PRACTICAL HISTOLOGY. Medullatecl. Mfedullary Sheath Myeline. Neurilemma. NERVE-FIBRES. 259 Ranvier's Constrictions. Ranvier's Internodes. Sheath of Mauther Sheath of Schwann. White Substance of Schwann. s 2 260 PRACTICAL HISTOLOGY. XIII.— BLOOD-VESSELS. 1. Horse. — Transverse section metacarpal artery and vein. 2. Horse. — Transverse section artery and vein of mesentery. 3. Rabbit. — Piece of omentum cut out, which con- tains vessels that can be seen. BLOOD-VESSELS. 261 4. Frog. — Capillaries with artery, vein, pigment-cells, &c, seen on watching circulation in the web. 5. Rabbit. — Arteries and capillaries of omentum, silvered. 6. Rabbit. — Endothelium of humeral artery, silvered. DEFINITIONS OF TERMS. External, Middle, and Internal Coat. 262 PRACTICAL HISTOLOGY. External Elastic Lamina. Internal Elastic Lamina. Valves of Veins. Vasa Vasorum. LYMPH SPACES AND CHANNELS. 263 XIV.— LYMPH SPACES AND CHANNELS. 1. Frog. — Septum cisterna lymphatica magna. silvered. 2. Rabbit. — Part of peritoneal surface of diaphragm of rabbit, silvered. 3. Rabbit. — Central tendon of diaphragm of rabbit (pleural side), brushed and silvered. 264 PRACTICAL HISTOLOGY. DEFINITIONS OF TERMS. Endothelium. Germinating Cells. Intercellular Cement Substance. Juice Canals. LYMPH SPACES AND CHANNELS. 265 Lymph. Lymph Capillaries. Lymph Spaces. Stomata. 266 PRACTICAL HISTOLOGY. XV.— LYMPHATIC GLANDS, TONSILS, AND THYMUS. 1. Rabbit. — Section of a lymphatic gland, silvered. 2. Horse. — Section of axillary gland. 3. Dog. — Section of tonsil. 4. Child.— Section of thymus of child under six months. LYMPHATIC GLANDS, TONSILS, AND THYMUS. 267 DEFINITIONS OF TERMS. Adenoid Tissue. Afferent Lymphatic Vessel. Capsule. Cortical Follicle. 568 PRACTICAL HISTOLOGY. Efferent Lymphatic Vessel. Lymph Path. Lymphoid Cells. Trabecule. SKIN, HAIR, AND NAILS. *269 XVI.— SKIN, HAIR, AND NAILS. 1. Human. — Section vertical to surface of skin, and subcutaneous tissue of the palmar surface of a finger-tip. 2. Human. — Section like the last treated with gold chloride. 3. Human. — Section vertical to surface of the skin of the scalp. 270 PRACTICAL HISTOLOGY. 4. Human. — Section horizontal to surface of the skin of the scalp. 5. Sheep. — Section vertical to surface of hoof of sheep. DEFINITIONS OF TERMS. Arrectores Pili. Ceruminous Gland. SKIN, HAIR, AND NAILS 271 Curium. Cuticle. Cutis. Epidermis. 272 PRACTICAL HISTOLOGY. External and Internal Root-Sheath. Hair-Bulb. Hair-Follicle. Henle's Layer. SKIN, HAIR, AND NAILS. 273 Horny Layer. Huxley's Layer. Medulla of Hair. Meissner's Tact-Corpuscles. 274 PE ACTIO AL HISTOLOGY. Mucous Layer. Pacinian Corpuscles. Papillae. Pith of Hair. SKIN, HATR, AND NAILS. 275 Sebaceous Gland. Shaft of Hair. Stratum Corneum. Stratum Granulosum. T 2 276 PRACTICAL HISTOLOGY. Stratum Lucidum. Stratum Malpiglii. Stratum Mucosum. Stratum Spinosum. SKIN, HATR. AND NAILS. 277 Sudoriparous Gland. Sweat Gland. True Skin. 2 , 8 PRACTICAL HISTOLOGY. XVII.— TRACHEA, BRONCHI, AND LUNGS. 1. Cat. — Section vertical to inner surface and through an entire tracheal ring. 2. Cat. — Section vertical to inner surface and through the cartilages of two or more rings. 3. Kitten. — Section vertical to and including pleura of lung with alveolar epithelium, silvered. TRACHEA. BRONCHI., AND LUXGS. '2~0 4. Rat. — Section across a medium-sized bronchial tube of Iudst injected with carmine gelatine via pulmonary artery. 5. Rat. — Section vertical to and including pleura of a lung injected like the last. DEFINITIONS OF TERMS. What is the Trachea ? What are the Bronchial Tubes ? 280 PRACTICAL HISTOLOGY. What are the Lungs ? What is the Pleura ? Alveolar Passages. Alveoli. TRACHEA, BRONCHI, AND LUNGS. 281 Fibrous Layer of Trachea. Infundibula. Lobes of Lung. Lobules of Lum 282 PRACTICAL HISTOLOGY. Lobular Bronchi. Lymphatics (Peribronchial, Perivascular, Sub- pleura 1). Pulmonary Acinus. Trachealis Muscle. THE TEETH AND TONGUE. 2S3 XVIII.— THE TEETH AND TONGUE. 1. Cat. — Vertical section through decalcified jaw and tooth. 2. Human. — Section of a canine or incisor ground on a hone, &c. 3. Kitten. — Vertical section through jaw and tooth of a newly-born kitten. See X. 1, ante. 4. Cat. — Transverse section entire tongue. 284 PRACTICAL HISTOLOGY. DEFINITIONS OF TERMS. Cement substance. Crown, Neck, and Fang. Crusta Petrosa. Dental Sac. THE TEETH AND TONGUE. 285 Dentine. Enamel. Enamel Organ. Odontoblasts. 286 PRACTICAL HISTOLOGY. Papilla. Peridontium. Pulp. Palp Cavity. THE SALIVARY GLANDS AND THE PANCREAS. 287 XIX.— THE SALIVARY GLANDS AND THE PANCREAS. 1. Rabbit. — Transverse section of the submaxillary gland. This is a true salivary gland. 2. Dog. — Transverse section of the submaxillar o-land. This is a true mucous sdand. e> J 3. Guinea-Pig. — Transverse section of the sub- maxillary gland. This is a muco-salivary gland. 288 PRACTICAL HISTOLOGY. 4. Dog. — Section of a piece of the pancreas of a fed dog. 5. Dog. — Section of a piece of the pancreas of a fasting doer. o o DEFINITIONS OF TERMS. There are no very special terms in connection with these glands. (ESOPHAGUS, STOMACH, AND DUODENUM. 289 XX.— (ESOPHAGUS, STOMACH, AND DUODENUM. 1. Dog. — Transverse section of oesophagus. 2. Cat. — Section through coats of stomach. 3. Dog. — Vertical sections through the mucous membrane of the stomach (1. cardiac, 2. middle, 3. pyloric end) of a fed dog. u 290 PRACTICAL HISTOLOCV 4. Dog. — Vertical sections through the mucous membrane of the stomach (like the last) of a fasting dog. 5. Rat. — Section through the coats of the stomach *& of a rat injected with carmine gelatine (blood-vessels). ()• Pig. — Transverse section of duodenum of a pig. (ESOPHAGUS, STOMACH, AND DUODENUM. 291 DEFINITIONS OF TEEMS. Adventitious Coat. Brunner's Glands. Chief or Central Cells. Muscularis Mucosa. U 2 292 PRACTICAL HISTOLOGY. Parietal Cells. Tunica Mucosa. Tunica Muscularis. Tunica Serosa. Tunica Submucosa, SMALL AND LARGE INTESTINES. 293 XXL— SMALL AND LAKGE INTESTINES. 1. Pig. — Transverse section (vertical to mucous mem- brane) of jejunum of pig. 2. Pig. — Horizontal section of mucous membrane of jejunum of pig. 3. Pig. — Transverse section (vertical to mucous membrane) through a Peyer's patch. 294 PRACTICAL HISTOLOGY. 4. Frog. — Vertical section through mucous mem- brane of intestine of frog fed with bacon-fat. 5. Rat. — Transverse sections through duodenum, jejunum, and colon of a rat with blood-vessels injected with carmine gelatine. 6. Rat. — Strips of longitudinal muscular coat with Auerbach's plexus (stained with gold) adherent. SMALL AND LARGE INTESTINES. 295 DEFINITIONS OF TERMS. Absorption by Villi. Adenoid Tissue. Agminatecl Glands. Auerbach's Plexus. 296 PRACTICAL HISTOLOGY Chyle- Vessels. Intestinal Villi. Lieberkuhn's Glands or Follicles Meissner's Plexus. SMALL AND LARGE INTESTINES. 207 Myenteric Plexus. Peyer's Patches. Solitary Glands. 298 PRACTICAL HISTOLOGY. XXIL— THE LIVER. 1. Frog, — Fresh section of liver, fed frog. Tease and isolate cells and observe shape, appearance, &c, then irrigate with 1 per cent, acetic acid. 2. Pig. — Section of liver vertical to the surface including capsule. 3. Pig. — Section parallel to the surface. THE LIVER. 299 4. Dog. — Section parallel to surface (fed dog). 5. Dog. — Section parallel to surface (fasting dog). 6. Rat. — Section of carmine-gelatine injected liver (blood-vessels). 7. Rat. — Section of carmine-gelatine injected liver (bile-ducts). 300 PRACTICAL HISTOLOGY. DEFINITIONS OF TERMS. What is the Liver ? Bile-Capillaries. Bile-Ducts. Capsule of Glisson. THE LIVER. 301 Hepatic Cell. Hepatic Lobule. Hepatic Vein. Interlobular Vein. o02 PKACTICAL HISTOLOGY. Intralobular Vein. Portal Vein. Sublobular Vein. THE DUCTLESS GLANDS. 303 XXIIL— THE DUCTLESS GLANDS. 1. Dog. — Transverse section of spleen of a fed dog. 2. Dog. — Transverse section of spleen of a fasting dog. 3. Horse. — Transverse sections of the supra-renal capsules. 4. Child. — Section vertical to surface of the thyroid body. 304 PRACTICAL HISTOLOGY. DEFINITIONS OF TERMS I. Spleen. Capsule. Malpighian Bodies. Pulp. Trabecular. THE DUCTLESS GLANDS. 305 II. Supra-Renal Bodies. Cortex. Medullary Portion. Zona Fasciculata. 306 PRACTICAL HISTOLOGY. Zona Glomerulosa. Zona Reticularis. III. Thyroid Body. Acini or Alveoli. THE URINARY ORGANS. 307 XXIV.— THE UEINARY ORGANS. {Kidneys?) 1. Mouse. — Horizontal section through entire length of a kidney. 2. Rabbit,— Vertical section through entire breadth of a kidney. 3. Rabbit. — Vertical section through the cortex. x 2 308 PRACTICAL HISTOLOGY. 4. Rabbit. — Vertical section through medullary rays. 5. Rabbit. — Vertical section through a papilla. 6. Rat. — Vertical section through the entire breadth of kidney, injected with carmine gelatine. THE URINARY ORGANS. 309 DEFINITIONS OF TERMS. What is a Kidney ? Capsule of Kidney. Capsule of Bowman. Columns of Bertini. 310 PRACTICAL HISTOLOGY. Convolutions of Tubes and their Names. Ferrein's Pyramids. Glomerulus. Henle's Limbs, Loops. THE URINARY ORGANS. 311 Labyrinth. Halpighian Bodies. Malpighian Pyramids. Medullary Portion 312 PRACTICAL HISTOLOGY. Medullary Rays. Papillae. Pelvis of Kidney. Vas Afferens, Vas Efferens, Vas .Recta, Venous Arches. THE URTXAEY ORGANS. 313 XXV.— THE URINARY ORGANS (continued). (Ureter, Bladder, and Male Generative Organs.) 1. Horse. — Ureter in transverse section. 2. Guinea-Pig. — Transverse section of bladder. 3. Child. — Section of prostate gland. 3J4 PRACTICAL HISTOLOGY. 4. Child. — Transverse section of penis. 5. Rat. — Transverse section of testis and epidi- dymis. 6. Rat. — An incision into globus major for sperma- tozoa. 7. Newt. — Spermatozoa. THE URINARY ORGANS. 315 DEFINITIONS OF TERMS. Coni Vasculosi. Convoluted Tubes. Corpus Highmori. Mediastinum Testis. 31 G PRACTICAL HISTOLOGY. Rete Testis. Tunica Albuginea. Tunica Vaginalis. Yas Aberrans. THE FEMALE GENERATIVE ORGANS. 317 Yas Deferens. Yasa Efferentia. XXVI.— THE FEMALE GENERATIVE ORGANS. (Ovary, Fallopian Tithes, Uterus, Vagina, Mammary Gland.) 1. Sheep. — Ovum of sheep. 2. Kitten. — Section of ovary of newly-born kitten. . 318 PRACTICAL HISTOLOGY. 3. Rabbit. — Section of ovary of a y oar-old rabbit. 4. Horse.— Transverse section of a Fallopian tube. 5. Cat.— Transverse section through body of uterus, 6. Cow.— Horizontal section through one side of cervix uteri. 7. Cat. — Sections oi mammary gland during lacta- tion. THE FEMALE GENERATIVE ORGANS. 319 DEFINITIONS OF TERMS. Clitoris. Corpus Luteum. Cortical Portion. Discus Proligerus. 320 PRACTICAL HISTOLOGY. Follicle. Germinal Epithelium. Germinal Spot. Germinal Vesicle. THE FEMALE GENEKATIVE ORGANS. 321 Glands of Bartholinus. Graafian Follicle. Liquor Folliculi. Medullary Portion. Y 322 PRACTICAL HISTOLOGY. Membrana Granulosa. Ovum. Primordial Ova. Stroma. THE FEMALE GENERATIVE ORGANS. 323 Tunica Albuginea. Vestibule. Yolk. Zona Parenchymatosa. Y 2 324 PRACTICAL HISTOLOGY Zona Pellucida. Zona Yasculosa. XXVIL— TERMINATIONS OF NERVE- FIBRES. 1. Cat. — Pacinian body from meso-rectum of cat. 2. Cat. — Longitudinal section of a Pacinian body from meso-rectum of cat. TERMINATIONS OF NERVE-FIBRES. 325 3. Duck. — Section vertical to the surface of the tongue of a duck. 4. Rat. — Section like the last. 5. Frog. — Entire pectoral muscle of a small frog. 326 PRACTICAL HISTOLOGY. DEFINITIONS OF TERMS. End-Bulb. End-Plate. Pacinian Corpuscle. Tactile Corpuscle. SPINAL CORD, NERVE-CELLS, AND GANGLIA. 327 XXVIII. —THE SPINAL COED, NEKVE- CELLS, AND GANGLIA. 1. Dog. — Transverse sections of the spinal cord in the cervical, dorsal, and lumbar regions. 2. Horse. — Nerve-cells of the anterior horn of the cord. 3. Frog. — Sympathetic ganglia. (Foster, 113.) 328 PRACTICAL HISTOLOGY. 4. Frog. — Spinal ganglia. 5. Dog. — Transverse and longitudinal section of a dorsal spinal ganglion. DEFINITIONS OF TERMS. Anterior and Posterior Median Fissures of the Cord. Anterior or White Commissure. SPINAL CORD, NERVE- CELLS, AND GANGLIA. 329 Apolar Cell. Bipolar Cell. Central Canal of the Spinal Cord. Cornua of the Cord. 330 PRACTICAL HISTOLOGY. Ganglion. Gelatinous Substance of the Cord. Grey Matter of the Cord. Lateral Grey Masses. SPINAL CORD, NERVE-CELLS. AND GANGLIA. 331 Multipolar Cells. Posterior or Grey Commissure. Posterior Median Fissure of the Cord. Processus Reticularis. 332 PEACTICAL HISTOLOGY. Tractus Intermedio Lateralis. Unipolar Cell. Ventriculus Terminalis. ORGANS OF TASTE AND SMELL. 333 XXIX.— ORGANS OF TASTE AND SMELL. 1. Rabbit. — Vertical section of papilla foliata of the tongue passing across the folise. 2. Rabbit. — Teased preparations of a taste-bud of the rabbit's tongue. 3. Dog. — Transverse section of nasal septum. 334 PRACTICAL HISTOLOGY. 4. Dog. — Teased preparation of olfactory epithelium. 5. Frog. — Teased preparation of olfactory epithelium. DEFINITIONS OF TERMS. • I. Taste. Taste-Buds. The two kinds of Cells. ORGANS OF TASTE AND SMELL. II. Smell. Olfactory Region. The three kinds of Cells. Membrana Limitans Olfactoria. Bowman's Glands. Organ of Jacobson. 336 PRACTICAL HISTOLOGY. XXX.— ORGANS OF VISION. 1. Dog. — Section vertical to surface of upper eyelid. 2. Frog. — Entire cornea treated with gold chloride. 3. Rat. — Piece of cornea treated with silver nitrate. 4. Horse. — Section through cornea, iris, and part of sclerotic. ORGANS OF VISION. 337 DEFINITIONS OF TERMS. I. Eyelids. Cilia. Glands of Moll. Meibomian Glands. Tarsal Cartilage. 338 PRACTICAL HISTOLOGY. II. Conjunctiva. Caruncula Lachryrnalis. End-Bulbs of Krause. III. Cornea. Bowman's Membrane. Fibrse Arcuatae. ORGANS OF VISION. 339 Descemet's Membrane. Lamellae Proper Tissue of the Cornea. IV. Sclerotic. Canal of Schlemm. Funiculus Sclerae. z '1 340 PRACTICAL HISTOLOGY. XXXI. — ORGANS OF VISION {continued), 1. Rabbit. — Section through margin of lens. 2. Frog. — Lens teased and broken up in Farrant. 3. Prog. — Retina teased. ORGANS OF VISION. 341 4. Frog. — Section of retina. 5. Pig. — Section of retina, including fovea centralis. DEFINITIONS OF TERMS (continued). V. Choroid and Iris. Circulus Iridis Major and Minor. Chorio Capillaris. 34*2 PRACTICAL HISTOLOGY. Lamina Fusca. Ligamentum Pectinatis Iridis. Membrana Suprachoroidea. Orbiculus Ciliaris. ORGANS OF VISION. 343 Proper Tissue of the Choroid. Spaces of Fontana. Stratum Yasculosum. The two portions of the Iris. 344 PRACTICAL HISTOLOGY. Vasa Vorticosa. Vitreous Layer. VI. Vitreous Body and Retina. Canal of Petit, Fovea Centralis. ORGANS OF VISION. 345 Layers of the Retina (name them in their order). Macula Lutea. Optic Papilla. Ova Serrata. 346 PRACTICAL HISTOLOGY. Physiological Excavation. Zone of Zinn. XXXII.— ORGANS OF HEARING. 1. Skate. — Sections across one of the semicircuk] canals. 2. Skate. — Longitudinal section through an ampulla. ORGANS OF HEARING. 847 3. Guinea-Pig. — Vertical section through the middle, parallel with long axis, of the cochlea. DEFINITIONS OF TERMS. Ampulla. Auditory Hairs. Basilar Membrane. 348 PKACTICAL HISTOLOGY. Canal of Cochlea. Cells of Deiters. Columella. Cristre. ORGANS OF HEARING. 340 Endolymph. Fibre-Cells of Retzius. Hair-Cells. Helicotrema. 350 PRACTICAL HISTOLOGY Inner Hair-Cells. Limbus. Maculae. Membrane of Reissner. ORGANS OF HEARING. 351 Membraua Tectoria. Organ of Corti. Otoliths. Outer Hair-Cells. 352 PRACTICAL HISTOLOGY. Perilymph. Reticular Lamica. Rods of Corti. Saccule. ORGANS OF HEARING. 353 Scala Tympani. Scala Vestibuli. Semicircular Canals. Spiral Lamina. A A 354 PRACTICAL HISTOLOGY. Spiral Ligament. Tunica Propria. Utricle. XXXIII.— THE BEAIN AND MEDULLA OBLONGATA. 1. Dog. — Section vertical to the surface of the medulla oblongata at the decussation of the pyramids. THE BRAIN AND MEDULLA OBLONGATA. 355 2. Dog. — Section vertical to the surface of the medulla oblongata through the middle of the olivary bodies. 3. Dog. — Section vertical to the surface of the medulla oblongata just above the olivary bodies. 4. Dog. — Section vertical to the surface through the entire cerebellum. A A 2 356 PRACTICAL HISTOLOGY. XXXIV.— THE BRAIN AND MEDULLA OBLONGATA {continued). 1. Man. — Section vertical to the surface and through a cerebral convolution. 2. Rat. — Section through entire head of a rat just behind the eyes. 3. Rat. — Section through entire head of a rat at its thickest part. 4. Rat. — Section through entire head of a rat carried through the cerebellum. APPENDIX. Embedding in Gum and Glycerine. — If the best glycerine be added to the mucilage of gum acacia (B.P.), a trans- parent, infiltrating embedding mass is formed much superior, for our purposes, to any other. In late spring, during the summer, and in early autumn weather the pro- portion of glycerine to be added is 10 per cent. To the mixture 1 per cent, of carbolic acid crystals should be added to keep it. The mixture is used by being exposed, whilst the tissues are in it, to the sun and air just inside the closed window of the laboratory. In four days a tough mass results, when the tissues are to be cut out in blocks, and these blocks are then turned over for further exposure. By the seventh day each block will have become tough enough for cutting, and may therefore be fixed upon a cork, or piece of wood, either by ordinary melted glue or by gum mucilage without any glycerine in it. In the latter case the adhesion obtained will permit of sections being cut next day. A Third Short Method of collecting and cutting all the Tissues required in this Course (see p. 204). — If the tissues be collected, placed whilst fresh in picric acid solu- tion for seven hours, then in repeated changes of 75 per 358 APPENDIX. cent, alcohol for a week, they may be placed in gum and glycerine mixture contained in watch-glasses, dug out singly as blocks on the fourth day, gummed on corks on the seventh day, and cut on the eighth or on any subsequent day one finds it most convenient as they never become too hard. "Whilst in the watch-glasses they must be kept well under the gum and far enough apart to permit of a peri- phery of embedding mass an eighth of an inch broad being left to each block. Whilst cutting the knife must be moistened with methyl- ated spirit of full strength but the sections are to be landed with a camel-hair brush into 70 per cent, methylated spirit. The embedding mass can either be retained during staining if a stain containing 70 per cent, alcohol be used, or the gum can first be removed by placing the sections in water. I have not given this as a method in the body of the book, because in winter and in very wet weather the pro- portion of glycerine has to be ascertained, but will vary from 6 to 10 per cent. However, the student may still use this method with comparative ease in any weather thus : At the same time as the tissues are put to harden boil until tough a piece of liver or kidney and place pieces a twelfth of an inch thick in five watch-glasses filled with the mixture containing respectively 6, 7, 8, 9, and 10 per cent, of glycerine, and proceed as above directed, and try which cuts best. The gum-solution, some Price's glycerine, and a 10 c.c. measure-glass will be re- quired and great care exercised in the proportions, washing out with tap-water the measure-glass between each set of measurements. This mass is like paraffin in its infiltrating and cell and fibre supporting properties : it is superior to paraffin in that it may be used without perfect hardening of the tissues and APPENDIX. 359 saves the tissues from cell-shrinking reagents if we prefer that this should be so. Indeed we need go no higher in the scale of strength than 50 per cent, alcohol, and when fat is to be preserved in cells we can avoid all alcohol. The facility of removal of the embedding material is also obvious. With a knife held in a frame and moved obliquely as fine sections can be got and almost as readily as with paraffin infiltration. For those whose means will not afford a Thoma's micro- tome a well-microtome can be used if the cork has white wax and olive oil embedding mass poured around it. The corks indeed can be fixed by some simple means in any microtome by a little exercise of ingenuity. Notice. — If the gum and glycerine blocks be secured by freezing on a microtome by ordinary gum mucilage, sections of any degree of thinness and area may be obtained with the plane iron. INDEX. Adipose tissue, 232 Alcohol, 93, 95 Ammonia, bichromate of, 98 Ammonia, cliromate of, 9S Amoeba;, 202 Amphibian blood, 220 Anaesthetising, 60 Animal cell, 216 Appendix, 357 Applying gases and vapours, 64 Areolar tissue, 232 Asphalt, 37 B Balsam, bottle for, 51 Battery, constant, 32 Bile-ducts, to inject, 86 Bladder, 313 Blood, amphibian, 220 ,, human, 220 ,, corpuscles, action of reagents on, 223 ,, vessels, 260 ,, ,, to inject, 77 Blue injection-mass, 63 Bone, 243 ,, development of, 248 Boras-carmine, 134 Calf, material from, 166 Camera, 31 Camera-lncidn, 24 Carmine brightening fluid, 135 Carmine-gelatine mass, 66 Cartilage, 238 Cell, animal, 216 ,, making, 59 ,, vegetable, 211 Celloidin, 46 Chemicals, list of, 43 Choice out-fits, 19 Chrisma, 46 Chromic and nitric fluid, 99 Clearing, 142 Collecting material, 162 Compound microscope stand, 3 Constant battery, 32 Cover-glasses, applying, 144 ,, ,, cells for, 56 ,, ,, cleaning, 57 ., ,, measuring, 36 Cuttle-fish, 190 D Dehydrating, 142 Developing bone, 248 Diary essential. 2 Dissection of mammal, S8 Dissociating fluids. 62 Drawing materials, 48 ,, microscopic objects, 24 Dry lenses. 10 Ductless glands, 303 Duodenum, 2S9 E Ehrlich's hsematoxylin, 133 Embedding, 102 ,, in carrot, 104 ,, in celloidin, 114 ,, in gum mucilage, 116 ,, in gum and glycerine, 357 ,, in paraffin, 106, 112 ,, in wax and oil, 105, 112 Eosin. 136 Epithelium, 227 Erlicki's fluid, 93 Eye-pieces, 7 362 INDEX. Fallopian tubes, 317 Farrant's medium, 4S Fibrous tissue, 235 Freezing method, 204 Frog-holder, 57, 195 Ganglia, 327 Gas bottle, 39 Gas cell, 55 Gases and vapours, applying, 64 Gtlatine, 46 Generative organs, female, 317 ,, ,, male, 313 Glass slips, 36 ,, „ cleaning, 57 ,, syringe, 41 ,, tubing, 41 ,, working, 53 Glycerine jelly, 48 Gold chloride solution, 135 H Hair, 269 Hardening, 93 Hearing, organs of, 346 High-power lenses, 10 M Magnifying power and angles of lenses, 3 Magnifying power of the microscope, 21 Mali generative organs, 313 Mammary gland, 317 Material, collecting, 162 Measure-glasses, 42 Measuring microscopic objects, 23 Medulla oblongata, 354, 356 Methods of collecting material, 204, 207 Microscope, lamp for, 7 stands, 3, 34 Microtome, the, 37 Microtomes, 125-130 Mitchell's logwood, 133 Moist chamber, 57 Mounting, 144 ,, block, 55 ,, cell, 56 ,, in balsam, 148 ,, in Farrant, 146 „ in glycerine jelly, 147 ., in watery media, 147 Muller's fluid, 96 ,, ,, hardening in, 96 Muscle, 251 N Nails, 269 Nerve-cells, 327 ,, fibres, 256 ,, terminations, 324 Normal fluids, 61 Immersion lenses, 11, 14 Index needful, 2 Injecting, 65 Injecting bath. 69 Irrigating, 63 K Kidneys, 307 Klein s fluid, 97 ,, ,, hardening in, 97 Laboratory, the, 1 Lamp for microscope, 7 Lenses, 10 List of chemicals, 43 Liver, to inject, 86 Lungs, 278 ,, to inject, 84 Lymph glands, 266 Lymph spaces and channels, 263 O Oculars, 7 Oesophagus, 289 Oil-immersion lenses, 18 Organs of hearing, 346 ,, of taste and smell, 33' ,, of vision, 336. 340 Osmic acid solution, 100, 136 Ovaries, 317 Pancreas, 287 Paraffin method, 206 ,, wax, 46 Photographic materials, 48 Photomicrography, 29 Picric acid, 93 Piero-carmine, 135 Towers, 7 Practical exercises, 150 R Ranvier's alcohol, 95 Reagent bottles, 39 Ringing slides, 58 INDEX. 3G3 Salivary glands, 2S7 Section-cutting, 117 ,, ,, in carrot, 120 , , , , in frozen gum, 1 23 ,, ,, in paraffin, 122 ,, ,, in wax and oil, 121 Section transferring, 1'24 Silver-nitrate solution, 135 Skin, 269 Slips, 36 Smell, organs of, 333 Spinal cord, 327 Spleen, 304 Stand-condenser, 5 Staining, 132 ,, in aniline dyes, 139 ,, in borax-carmine, 137 ,, in Ehrlich's hematoxylin, 136 ,, in eosin, 138 ,, in gold chloride, 138 ,, in Mitchell's logwood, 137 ,, in picro-carmine, 137 ,, in silver-nitrate, 139 ., sections on slips, 140 Stains, list of, 44 Stomach, 289 Tongue, 283 Tonsils, 266 Trachea, 278 Tradescantia, 200 Ureters, 313 Uterus, 317 U Vagina, 317 Vapour cell, 55 Vegetable cell, 211 Vision, organs of, 336, 340 W Warm stage, 37, 65 Wash-bottle, 38 Water-bath, 42 Working powers, 7 Xylol balsam, 50 Table of amplifications, 22 Taste, organs of, 333 Teasing, 61 Teeth, 283 Thymus gland, 266 Thyroid body, 306 Yeast, 201 Yellow elastic tissue, 235 Zinc-cement, 53 THE END, RICHARD CLAY AND SONS, LONDON AND BUNGAY. I J Fesimley F31 ° 0Ur89 ° f •'--t.,* practloal