The Methods oj Microscopical Research DR. GRA THE LIBRARY OF THE UNIVERSITY OF CALIFORNIA PRESENTED BY PROF. CHARLES A. KOFOID AND MRS. PRUDENCE W. KOFOID THE METHODS OF MICROSCOPICAL RESEARCH. THE METHODS OF MICROSCOPICAL RESEARCH A PEACTICAL GUIDE TO MICEOSCOPICAL MANIPULATION SECOND EDITION MUCH ENLARGED AND IN GREAT PART RE-WRITTEN BY ARTHUR C. COLE, F.B.M.S, ALL BIGHTS RESERVED Xonfcon BAILLIEEE, TINDALL & COX 20 & 21, KING WILLIAM STREET, STKAND, W.C. PARIS. MADRID. 1895 PEEFACE TO THE SECOND EDITION. " The Methods of Microscopical Research " formed a portion of the " Studies in Microscopical Science," a serial publication in four volumes, issued and edited by the author, and was embodied (as a treatise) with Yolume II. of that work. The recep- tion accorded to it induced the author to publish it as a separate work. The first edition was rapidly taken up, and the book soon became out of print and unobtainable. In response to a very general demand it is now re-issued, this (the second) edition being greatly enlarged and the work, indeed, almost entirely re-written. It embodies the full results of the author's thirty years' experience in microscopical work, all the formulas from his private note book, all his own special processes and methods of working, and all his simple inventions for perfecting the work and ensuring absolute cleanliness and permanence in preparations. Nothing has been concealed, every- thing has been fully described and explained, and so plainly that the author entertains the hope that the treatise will be found really practical, and so will enable the student and the amateur microscopist to make preparations worth studying and retaining as cabinet specimens. PEEFACE TO THE SECOND EDITION. " The Methods of Microscopical Research " formed a portion of the " Studies in Microscopical Science," a serial publication in four volumes, issued and edited by the author, and was embodied (as a treatise) with Volume II. of that work. The recep- tion accorded to it induced the author to publish it as a separate work. The first edition was rapidly taken up, and the book soon became out of print and unobtainable. In response to a very general demand it is now re-issued, this (the second) edition being greatly enlarged and the work, indeed, almost entirely re-written. It embodies the full results of the author's thirty years' experience in microscopical work, all the formulae from his private note book, all his own special processes and methods of working, and all his simple inventions for perfecting the work and ensuring absolute cleanliness and permanence in preparations. Nothing has been concealed, every- thing has been fully described and explained, and so plainly that the author entertains the hope that the treatise will be found really practical, and so will enable the student and the amateur microscopist to make preparations worth studying and retaining as cabinet specimens. CONTENTS. INTRODUCTION. PAGE 1. On Instruments and their Use 1 2. On Reagents, their Constitution and Action 4 3. On the Methods of Preparation 5 4. On Microscopical Art ... ... ... ... ... 7 CHAPTER I. The Microscope ...^ 10 The Human Eye 13 CHAPTER II. Instruments and their Use 27 CHAPTER III. The Preparation of Animal Tissues 38 CHAPTER IV. On Injecting Blood Vessels, &c 69 CHAPTER V, On Staining Fluids and Staining 77 On Staining Bacilli and Bacteria ... 91 viii. Contents. PAGE Clearing Media ... .., ... ... ... ... 95 Mounting Media and Cements 98 Embedding and Freezing ... ... ... ... ... 109 On Section Cutting and Microtomes ... ... ... 119 CHAPTER VI. On Mounting 132 On the Preparation and Mounting of Insects ... ... 138 On the Preparation and Mounting of Vegetable Sections 141 On the Preparation and Mounting of the Diatomacese ... 144 t CHAPTER VII. On Microscopical Drawing and Painting 161 CHAPTER VIII. On Photo-Micrography 180 THE METHODS OF MICROSCOPICAL EESEABCH, INTRODUCTION. I. On Instruments and their Use. THE investigation of minute structure, whether organic or inorganic, can be exhaustively and satis- factorily accomplished only by the assistance of adequate optical instruments. The organ of vision in man is so constructed that the distance of most distinct vision alternates between eight to ten inches, and the intermediates between these dis- tances, from the normal eye of an adult. This is due to the alterable curvature of the crystalline lens which allows the practically parallel pencil of rays from a distant object, or the divergent rays from an object close to the eye, to be accurately focussed on the retina, or sensitive portion of that organ ; but if the object is brought still closer to the eye than the normal distance of most distinct vision, it gradually 1 2 METHODS OF MICROSCOPICAL EESEAECH. loses its power of accommodation in exerting the strain required to render the crystalline lens suffi- ciently convex to focus the image of the object upon the retina; about two inches from the eye this action becomes impossible. Within the limits of accommodation megascopical characters only can be appreciated. If the eye were capable of altering the curvature of its crystalline lens indefinitely, its power of vision would become illimitable, and tele- scopes and microscopes and other such instruments would be found unnecessary. But the bounds of natural limitation can be conquered by artificial means, and the interposition of a sufficiently convex lens between the near object and the eye alters the direction of the rays of light which proceed from the object so as to bring them within the scope of natural vision, and herein lies the theory on which the microscope has been constructed. The nearer an object approaches the eye, the greater does its visual angle (or angle produced by the intersection of rays, or straight lines, from the extreme points of the object) become, and, consequently, a larger image is focussed upon the retina. Optical instruments, therefore, are required on the very threshold of our " Studies " to enable us to enter the domains of histology and microscopical investigation, and they may be employed directly in a multitude of instances when the objects are of minute size. Equally numerous, however, are the substances and organisms which cannot be thus directly examined, but which require to be sub- jected to special processes and manipulation in order to render them suitable for microscopical examination. In the inorganic kingdom some INTBODUCTION. 3 minerals, rocks, and chemical substances, elemen- tary and otherwise, are found in Nature in such minute particles as to render them suitable for immediate examination and study under the microscope, but, in the majority of instances, it is necessary either to pulverise, grind them down into the thinnest possible sections or sheets, or to precipitate them by chemical means ere they can be brought within the range of observation, and thus the petrologist must be provided with chisels, hammers, files and stones for grinding and polish- ing, and a variety of instruments adapted to the collection and subsequent treatment of specimens, whilst the chemist will require blow-pipes, test tubes, re-agents and balances, &c., &c. For investigations in the organic world, the vegetable histologist must provide himself with a vasculum, a trowel, dipping bottles, and all necessary col- lecting apparatus of all kinds, and' with all the various instruments necessary to facilitate the examination of the unicellular and more delicate forms of plant life, whilst the more highly organised and complex examples will require dissection or sectionising and, in many cases, chemical treatment before they can be examined to any useful purpose. The animal histologist again will find his work inextricably connected with that of his brother botanist, and, to a large extent, will work hand in hand with him, but, as his observations and inquiries progress, there will arise necessities for a collection of tools including scalpels, scissors, bone forceps and saws, needles, forceps of different sizes and forms, section knives, as well as the all-impor- tant and indispensable microtome ; whilst both 4 METHODS OF MICROSCOPICAL RESEARCH. students will require all the staining re- agents and mounting media necessary for the proper treatment and permanent preparation (as slides) of both vegetable and animal specimens, tissues and sec- tions. The study of both organic and inorganic histology is so complicated, that verbal descriptions, however exhaustive and perfect, are, without microscopical demonstration, in many cases, wholly inadequate; diagrams and drawings, therefore, are constantly employed to supplement the descriptions of the lecturer. In the preparation of such valuable aids to verbal description, the microscope is a most valuable, if not an indispensable adjunct, and it has become necessary, equally for the student, the investigator, the observer, the demonstrator and the lecturer, to thoroughly understand the construction and the use of the microscope itself, and of the various apparatus necessary for careful observations and investigations. II. On Re=agents, their Constitution and Action. This is one of the most important branches of Microscopical Technology, and as staining is an art, so a thorough knowledge of the behaviour of the various tissues under the action of chemical re-agents is an absolute necessity alike for the observer and the student. It will be continually found that the optical -means at the disposal of the histologist are insufficient or inadequate to the resolution of structure, and although much may be INTRODUCTION. 5 done with carefully directed and modified light and an equally careful arrangement of magnifying power, yet the utility of the judicious application of re-agents requires only to be understood to be appreciated. The knowledge of the chemical con- stitution, physical properties and modes of manu- facture of re-agents, often leads to the discovery of their specific action on minute and ultimate structure, whether these may consist in a revelation of inherent qualities or in a modification of details. It thus becomes incumbent upon the histologist to most carefully scrutinise the action of every description of re-agent, and equally carefully and systematically to record the results of such ex- aminations. Re-agents may be used in a variety of ways for the elucidation of structure and the detection of the constitution of tissues and bodies. Some are employed as chemical tests, others as staining media to differentiate the tissues or to induce chaoges and appearances whereby certain proper- ties are revealed, whilst others, again, are used as preservative media. To know exactly what to apply and how to apply it for the revelation of specific phenomena, and to obtain the best results is of the very essence of microscopical technique and manipulation. III. On the Methods of Preparation. In order to arrive at a correct knowledge of, and as a means to their study, both organic and in- organic specimens and the great majority of or- ganisms require preparation by special methods. 6 METHODS OP MICROSCOPICAL RESEARCH. Thus, the processes of pulverisation, levigation, sectionising, and grinding of bones, teeth, minerals, and rocks are beset by difficulties of detail which prove to be insurmountable obstacles to the tyro until he shall have acquired the necessary know- ledge and skill, under which they to a great extent disappear, or are more or less easily surmounted. So, also, want of success in section-cutting, stain- ing and mounting specimens is referable to the ignorance or neglect of minute precautions, to the want of absolute cleanliness in all processes, and the absence of the delicacy of manipulation without which success is impossible of attainment. In the opening pages of " How to Work with the Micro- scope," 1 Dr. Beale makes the following observa- tions : " Manual dexterity, although subordinate to many higher mental qualifications, is as essential for the successful prosecution of microscopic obser- vation as it is for that of every kind of experi- mental science. It assists us in the discovery of new means of inquiry and in devising methods by which difficulties may be surmounted. Without skilful manipulation we can neither teach by demonstration facts which have been already dis- covered, nor hope to extend the limits of observa- tion and experimental knowledge. It is not, therefore, surprising that many of the most impor- tant facts which have been recently added to microscopical science, have been discovered by men who had previously well-trained themselves in ex- periment particularly in practical chemistry and minute anatomical dissection. Improvements in 1 Fifth edition, London, 1880, p. 1. INTRODUCTION. 7 the practical details of manipulation almost neces- sarily precede an advance in natural knowledge, and invariably promote and expedite true scientific progress." But although manipulative skill is a very necessary adjunct to microscopical research, an attainment of the understanding of the general principles of action at the outset, sometimes proves to be the most arduous portion of the work, and very often is the only impediment to success. Practice and perse- verance, brought to bear upon previously gained knowledge, are the only royal roads to manual dexterity, and it thus becomes the duty of the instructor to point out, not merely what path ought to be taken, but the various pit-falls which everywhere surround the beaten track, and how best to avoid them. IV. On Microscopical Art. There are two ways in which microscopical objects can be drawn so as to become useful records of research. By the first of these, a rough dia- grammatic representation may be made, without reference to accuracy of form or size ; merely to display the observer's views concerning the struc- ture of the object. The second method is to make an accurate drawing with due regard to the size and form of the object under the microscope. Both of these methods are valuable in themselves, but their usefulness becomes immeasurably enhanced when they are combined so as to afford scope to the artistic skill and scientific knowledge of the draughtsman. O METHODS OF MICROSCOPICAL RESEARCH. There is a great deal of truth in the statement that true art is the outcome of genius, but that does not in any way affect microscopical drawing ; it will be found that patience and practice are suffi- cient to enable the student to overcome every obstacle, and to achieve the most satisfactory results in this department of art. But whilst admitting the perfection to which many great artists in microscopical drawing have attained, and the great assistance to microscopical research afforded by the works of those who, in addition to correctness of vision and observation, possess an educated eye and a hand capable of drawing exactly what they see, give us the benefit of exquisite colouring in addition to correctness of outline and detail, who can sufficiently estimate the incalculable advantages bestowed upon microscopical science by micro-photography, which producing pictures absolutely faultless and free from such slight defects as are inseparable from drawings which, being dependent for their production upon human eyes, brain and nerves, must inevitably be liable to human imperfections, whilst a micro-photo- graph from the hands of a skilled observer and photographer is as inevitably free from all errors and defects, and provides for us an actual and per- fect enlargement in all its features and details colour alone excepted of the image focussed upon the plate ? Micro-photography has indeed proved a boon to the microscopist, and the improvements which have been made and are daily being intro- duced in both apparatus and processes entitle us to believe that valuable and exquisite as are the pictures now produced by photography, the future INTRODUCTION. 9 has greater and even more wonderful results in store for us. It is indeed impossible to overrate the benefits conferred alike upon science, the student, the teacher and the lecturer by this invalu- able art. 10 CHAPTER I. On Instruments and their Use: The Microscope. THE microscope, as an instrument of power in histological research, depends essentially in con- struction, upon its conformity with the laws of light and human vision. It has already been stated that an idea of the size of an object is arrived at through the size of the image focussed upon the retina, and that these dimensions vary in proportion as the object is brought near to, or removed farther from, the eye. When the object is brought close to the eye, its visual angle that is, the angle formed by the crossing of the rays from the extreme points of the object, is larger than when it is placed farther off, and, consequently, the image on the retina is larger also. If this principle were capable of un- limited extension, it would obviously follow, that to continue to increase the magnification of an object, all one would have to do would be to bring the object closer and closer to the eye. But there is a limit to this natural power of microscopical vision in the human subject, and the eye fails signally to accomplish its office when the object is brought ON INSTRUMENTS AND THEIR USE. 11 within about two inches from its surface. The reason of this is, that the crystalline lens of the eye, in assuming a more convex shape through the relaxation of the ciliary muscle, becomes overtaxed at this distance. If, now, a sufficiently convex lens be placed between the object and the eye, so as to enable the divergent rays to be accurately focussed upon the retina, the difficulty will be overcome, and, theoretically, microscopical vision would be illimitable. But, is it so ? Most certainly not. The employment of artificial substances, such as crown and flint glass, diamonds, &c., although they considerably extend the power of sight, do not do so ad infinitum. Here the limitation is purely material, as distinguished from the former instance, the human eye, which is defective not only materi- ally, but physiologically. The worker in the field of microscopical research need not, however, be appalled by these statements, for it will be found that the human organ of vision, in conjunction with the excellent appliances of modern invention, will enable him to approach, and sometimes even to solve satisfactorily, many of those philosophical problems which underlie the evolution of things, both animate and inanimate. In exemplification of this, a few instances may here be recorded. The practical geologist who sallies forth into the field with lens in hand, may gather during his walk a variety of rocks, which, from their cosmological structure, point to an igneous origin ; some of the specimens are coarse grained, whilst others defy the utmost scrutiny of the eye. The microscope is brought to bear upon the question, and he finds 12 METHODS OF MICROSCOPICAL RESEARCH. that a power of 500 diameters is generally the utmost degree of amplification he will require to employ ; but that for all practical purposes powers of from 20 to 100 diameters suffice. "With the assistance of the microscope he is enabled to pro- nounce with decision that the rocks are igneous ; and more, from analytical and synthetical experi- ments he can show that certam coarse varieties, which are thoroughly crystalline (the crystals being simply held together by adhesive and cohesive forces, without the necessity of an interstitial bind- ing substance), are of deep-seated origin, and con- solidated under conditions of enormous pressure and length of time. He is, in like manner, able to affirm of the other varieties, what their mineral constituents are, or have been, and how they came to assume their present states. Thus he builds a part of the fabric of geological philosophy, and with what ? with a comparatively low power of the microscope. To take an example from the organic world ; the questions of the function of various parts of the body, are very often arrived at through a minute study of its members. The form and general ap- pearance of the cells of glands such as the salivary glands, point to the functions they perform, whether they secrete or absorb, and how and when they perform their duties. The study of amoeboid, and even of ciliary motion, under the microscope, does not require a power of magnification much beyond 700 diameters, whilst the life history of the minute forms of life known as germs (bacteria, fyc.), may be readily comprehended by the use of from 700- 1,200 diameters. It is only when such things as the THE HUMAN EYE. ON INSTRUMENTS AND THEIR USE. 13 delicate markings on the siliceous valves of Diatoms, or the artificial ruled lines on glass (Nobert's test) require to be made out, that powers beyond 1,200 diameters become useful ; and it must be admitted that the scientific investigator does not lose any- thing, nor are any of his philosophical deductions vitiated by eschewing such powerful instruments, which, in the hands of the skilful, succeed in amplifying and resolving certain pretty structures ; but, to the ordinary worker are, in reality, impedi- ments to research. On the threshold of this inquiry, it thus becomes evident, that an acquaintance with the general structure of the human eye, coupled with the principles of luminous energy, are necessary adjuncts to an understanding of the microscope, its use and power. The Human Eye. DESCRIPTION OF PLATE. &, Anterior chamber of eye filled with the aqueous humour. c, Cornea, i, iris, p, pupil. cm, Ciliary muscle, cp, ciliary processes. Z, lens. s, Sclerotic coat, c, choroid. r, retina. /, Fovea. RR, superior and inferior recti muscles. 0, Optic nerve entering the sclerotic and choroid coats. v, Vitreous humour. In all studies, whether of pure Microscopy as a Science, or whether of one of those departments of natural history in which the microscope is employed 14 METHODS OF MICROSCOPICAL RESEARCH. as an aid to vision, we must, at the outset, recognise the importance of a study of the human eye. It may be the seat of many imperfections result- ing from misuse, old age, or disease, which are apt to modify the conclusions we may draw from our observations, unless we are careful to study well into what lines such imperfections may lead us. Nature has given us in this organ a means whereby all objects may be compared with each other, more especially as to size, colour, and general characters, and it must astonish the student, who thinks deeply, to find that so little is known definitely as to how we are able to appreciate magnitudes, colours, and forms. It is easy to say that the lenses of the eye focus a picture of the object upon the retina, and the irritations are carried by the optic nerve to the brain, but do we practically realise what this means ? Then, again, unless more of our senses than one are brought to bear upon a matter under considera- tion, we can scarcely form a true opinion upon our subject. Take something which greets our vision for the first time. We know not what it is ; we can see it, it is true, but we have to bring in the aid of other senses before we can arrive at a correct judgment; and even then, our judgment being the result of comparison, and also of experimental contact of substances with our senses so to speak opinions which are formed must, to a certain extent, be modified by the amount of other experience to which our nerve centres have been previously subjected. Take two experts; give to each one a sphere ON INSTRUMENTS AND THEIR USE. 15 composed of lead and tin. Upon asking them what substance they were handling they might probably guess, perhaps not ; they would poise it in their hands, look at it, smell it, try to cut it, perhaps, examine its metallic lustre, and it would be very odd indeed if they could agree as to the composition of the alloy, unless settled by an assay upon the balance. Has it ever occurred to the reader that such pro- cesses as these go on in Microscopy, and that it is necessary to carefully study the organ of vision in order to gain a true insight into the object presented to us ? On reference to plate 1, it will be seen that the eye is a nearly spherical ball, capable of many movements in its socket. It possesses an outer translucent covering called the sclerotic coat (or simply sclerotica) which may be seen at S. This is thick, horny, and opaque, except in its anterior portion. This sclerotic coat envelopes about f of the eye- ball, and in common parlance is called the white of the eye. The anterior transparent portion is called the cornea, and has the shape of a very convex watch glass. It is through this membrane that the light passes to the interior of the eye. The cornea and the interior portion of the sclerotica are covered with a mucous membrane. Behind the cornea is a diaphragm of annular form called the iris ; it is coloured and opaque, the circular aperture in its centre, p, being called the pupil. The iris, i 9 serves the purpose of regulating the admission of light ; it varies in colour in different 16 METHODS OF MICROSCOPICAL RESEARCH. individuals, and is the part referred to when we speak of the colour of a person's eye. Behind the pupil is the crystalline lens, I, having a much greater convexity at its posterior surface than at the anterior. The large posterior chamber is lined by the choroid coat, and this choroid has in front of it a delicate membrane called the retina. The choroid coat consists of a highly vascular membrane containing pigment cells, filled with an intense black mucus, called the pigmentum nigrum. The cavity behind the cornea is filled with a liquid called the aqueous humour, having a refractive index approaching that of 1*3366, while the larger cavity is filled with a transparent jelly, called the vitreous humour, possessing a refractive index of 1*3379, enclosed in a very thin, transparent sac, called the hyaloid membrane. I have now described the principal apparatus of the eye, and may take some of the parts in detail. The crystalline lens is built up of layers, increas- ing in density inwards, the effect of which is to diminish spherical aberration. This lens is enclosed in a transparent capsule, held in position by an elastic membrane. It can be changed in shape by means of a delicate muscular arrangement to adapt its focus for near or distant objects. As glass lenses of varying curves have different focal lengths, so by altering the curves of the crystalline lens we are able to see objects distinctly which are situated in several focal planes. The reader may have noticed that there is a near point at which objects can be seen most distinctly ; this point varies in individuals, but averages from ON INSTRUMENTS AND THEIR USE. 17 8 to 10 inches. As we move farther away from the object, although diminished in size, it may be seen more easily, and with less effort. It would appear, then, that all objects are ren- dered apparently larger, as they continue to approach the eye, but a limit is soon found to this, as at a distance of six inches distinct and easy vision is not possible (except in very abnormal cases). The reason of this is well-known the anterior focal point of a convex lens when shortened lengthens the posterior conjugate focus, so that when an object is brought too near the eye the image of it is projected behind the retina, and the crystalline lens cannot accommodate itself to such extremes. But we know that objects can be seen distinctly at great distances apart, and it may be useful to demonstrate how this is brought about. The real mechanism of accommodation has been much disputed, but the results, as observed, are, that the curvatures of the crystalline lens are altered as the observer adapts his eye to near or remote vision ; increase of curvature, of course, shortening the focal length of the crystalline lens, and being better adapted for near vision, while the shallower curve is necessary for the distant view of remote objects. Helmholtz has shown that the radius of curvature of the anterior surface of the crystalline lens may be varied by means of the muscular arrangement, from 6 to 10 millimetres. We may now cast another glance at the iris. This apparatus is really a continuation of the choroid tunic which lies between the sclerotica and the retina : it ends in front, in what are called 2 18 METHODS OF MICROSCOPICAL RESEARCH. ciliary processes. The small muscular ring sur- rounding the pupil is called the sphincter muscle. Now, the principal use of the choroid tunic, or rather the pigmentum nigrum which it contains, is to absorb those rays of light which have passed through the transparent retina, preventing their reflection, which would interfere with the distinct- ness of the image. By reference to the plate it will be seen that the choroid tunic, the retina, and sclerotica form the three outer rings, while the centre is ramified by nerve filaments and blood-vessels. These nerve filaments and blood-vessels lie in the retina, which really forms a continuation and exten- sion of the optic nerve ; it touches the outer cir- cumference of the iris at the front, and lies open as a cup-shaped disc in the interior of the eye ; it receives the rays of light which have passed in turn through the cornea, aqueous humour, crystalline lens, and vitreous humour, and forms a picture at the focus of these. The nerve fibres of the retina are excited probably by a product of the action of the light picture upon the visual purple, and the irritations are transmitted to the brain by the optic nerve, producing the sensation of vision. The picture produced upon the retina has been compared with that produced by a photographic lens upon a screen or ground glass ; but it will be seen that the instances are not strictly parallel. In the eye the rays falling upon the cornea do not again encounter air, the picture is formed in the highly refractive substance, while in the photo- graphic image air intervenes between the screen and the lens, and between the lenses themselves. ON INSTRUMENTS AND THEIE USE. 19 Then, again, the adaptation of the eye to various distances is obtained by a process so dissimilar to that of the lens in the camera, that it is well no comparison should be instituted. The retina has been previously described as a delicate membrane lining the choroid tunic, inside the sclerotica. Now, if we make a section of the retina, and examine it under the microscope we shall find its structure to be as follows. Starting from the junction of the retina with the vitreous humour, we have : (1) The layer of nerve fibres. (2) The layer of nerve cells. (3) The granular layer. (4) The inner granular layer. (5) The intermediate layer. (6) The outer granular layer. (7) A second fine membrane. (8) The layer of rods and cones. (9) Pigmentum nigrum of the choroid. The retina is the terminal organ of vision, all the apparatus in front of it being merely for the purpose of securing that an accurate image shall be focussed upon it. As to how the luminous im- pressions yield to us such a definite idea of things is a question still under consideration ; many have tried to solve it, but it is open to doubt whether we are any nearer the mark than those philosophers who lived 2,000 years ago. There are several curious properties inherent in the retina. By means of the ophthalmoscope may be seen a point, a little out of the centre, where the optic nerve enters the eye. This spot is totally 20 METHODS OF MICROSCOPICAL RESEARCH. blind, it cannot perceive a trace of light, and if the image of an object falls upon this blind spot, that object is totally invisible e It is at this spot also where the blood-vessels enter the eye, and ramify through nearly the whole of the surface layers of the retina. In the above description points only have been touched which directly bear on good or defective vision. On the other hand, enough has been advanced to show that this organ is liable to im- perfections which may be, and are, extremely liable to modify all our observations made over the tube of the microscope. Now, if we take an ordinary lens of glass and attempt to produce a picture with it, we find the centre alone is plainly visible the lens is afflicted with what is termed spherical aberration, that is, the rays from its periphery are brought to a focus in a different plane to those occupying a central position. But a small amount of spherical aberration is not readily detected by the student. It appears as a haze or fog of light over the object. In the human eye this defect is not observable to any great degree, as the peripheral or more strongly refracting rays are cut off by the iris. Then, again, the curvature of the cornea is ellipsoidal rather than circular, so that the rajs farthest from the axis are least deviated, while the two curves of the crystal- line lens correct, so to speak, the one the other ; and lastly, this lens is of such construction that its refractive power diminishes from the centre to the circumference. Another defect in the eye is due to the different meridians having dissimilar degrees of curvature. ON INSTRUMENTS AND THEIE USE. 21 If a set of concentric circles be observed with one eye, they are seldom all distinct at the same time, and there is produced a kind of Maltese-cross effect, not perceivable, perhaps, in many instances with large circles, but noticeable when drawn to such a size that the outer one is about two inches in diameter. This defect is called astigmatism, and known to oculists as a common cause of headaches. Spasm of the focussing apparatus may derange the spheri- city of the eye, and so affect vision. Strained vision is very subject to this. On the other hand, the same apparatus may be paralysed, and ordinary vision deficient, whilst the focussing of the micro- scope might possibly correct it. Astigmatism has injuriously affected painters ; Turner, for instance, whose later pictures are dis- covered to be slightly distorted, in consequence of the power of accommodation or self -correction having been lost through age. In microscopic drawing, as with the camera lucida, the perspective may be misrepresented, in consequence of astigmatism, and thus endless dis- putes may arise even among the most careful observers. We have now to deal with errors of refrangibility, and it will probably have been assumed that the eye apparatus is entirely corrected for colour. This is not the case, however, except when an object is in exact focus, and the reason that the error due to refrangibility remains practically unnoticed is that the distance between the focal point of the red and violet rays is extremely small. The error due to refrangibility may be noticed by means of the con- 22 METHODS OF MICROSCOPICAL RESEARCH. centric circles already referred to ; by bright day- light adjust the eyes to some object twelve inches away, and without moving the eye insert at a dis- tance of four inches a card inscribed with black circles, when a yellow and blue colouring will be plainly discerned. In order that the reader may thoroughly under- stand the error of refrangibility, the picture afforded by the passage of a solar ray through a prism of glass may be thrown upon a screen ; the rays are deflected unequally, the red least and the violet most. It may be advisable here to state that the degree of dispersion of the rays of white light depends upon the medium through which the ray passes, and this amount of dispersion is measured by the distance of the most prominent dark lines in the spectrum from each other. The diamond disperses much less than crown glass, while the deflection of the ray is greater ; but this is a subject beyond the scope of the present essay. Now, beside these errors, there are others to which the microscopist should devote special atten- tion ; they are caused by small opaque particles existing in the transparent media of the eye-ball. These cast their shadow on the retina, and produce images which appear to exist outside the eye. These extra-retinal images often appear as globules, bacteroid- shaped bodies, or strings of minute pearls, and may be studied by directing the eye to a sheet of strongly illuminated opal glass, through a small aperture made with a fine needle in a piece of thin blackened cardboard. When the microscope is used in a vertical position, ON INSTRUMENTS AND THEIR USE. 23 these globules often gravitate to the centre of the cornea, and even after prolonged use of the inclined tube an observer may often be perplexed by the layer of mucus, or a lachrymal discharge covering the surface of the cornea. Colour is a special sensation excited in the retina by rays of a definite wave length, and the reason why certain objects are presented to our view with colour is that when white light falls upon a given surface, some is absorbed, the remainder being reflected. If the green rays are reflected, then the object appears green, and if the red rays are alone reflected, then the object will be red. The generally accepted theory of colour percep- tion is based on the assumption that three kinds of nerve fibres exist in the retina, the excitation of which produces sensations of red, green and violet, and that modifications of these three sensations yield all intermediate tints. This theory will explain some of the phenomena of colour blindness ; if the nerve fibres which give their special sensation are paralysed, or are absent, the sensation only of the complementary tint will be transmitted with all the defects of the eye. It must not be forgotten that many phenomena con- sist more in errors of judgment than in absolute error of form or sensation. Now in regard to errors of judgment, we must admit that all our estimations are made by compari- son. In magnitude we are guided by the size of the retinal image as determined by the visual angle for position we must have some starting point; and as for distance, every one knows how delusive an inexperienced estimate of this is. At sea, a 24 METHODS OF MICROSCOPICAL RESEARCH. landsman could not judge of the distance of a pass- ing vessel to a few miles, nor could we form any accurate idea of the size of any object emitting practically parallel rays unless we had something to compare it with. We now come to a point which has been much disputed in the study of microscopy binocular vision. The two eyes move together as a system, so that we direct the two lines of regard to the same point in space and consequently see but a single image ; but it is possible to see two if one eye be displaced a little with the finger two images are seen, while if the other be displaced to a corresponding degree the one image is restored. The value of binocular vision may be easily ascertained by experiment. When a picture is presented to the retina of each eye, the compound picture is much brighter than when one retina only is employed. To each point of the retina of one eye there is a corresponding point in the retina of the other, and impressions produced on one of these points are in ordinary circumstances indistinguishable from similar impressions produced on the other. When both retinse are similarly impressed, the general effect is that the impressions are more intense than when one eye only is employed ; and we also get a perception of relief, that is of form in its three dimensions. Take two A eyepieces and look through them to the sky, so that two distinct circles are seen ; now bring them together so that one circle overlaps the other, when this overlapping bi-convex portion will ON INSTRUMENTS AND THEIR USE. 25 be found double the brightness of the remaining portions of the circles. We are indebted to stereoscopic vision for the perception of relief or form in three dimensions, which occurs when the images falling upon the corresponding points of the two retinse are not exactly similar. In looking at an object with both eyes the rays do not run parallel from one side of the object to the eye on that side, but the right eye centres itself to the left side of the object and vice- versa. This may readily be seen by holding up a finger between our eyes and the wall, and looking at the latter. Two fingers may be seen projected on the wall, one of these is seen by the right eye and the other by the left ; but our visual impressions do not inform us which picture is formed by either eye in particular. Now, while steadfastly looking at the wall, close the right eye and the left finger will disappear, while on shutting the left eye, the right finger is rendered invisible. When two similar pictures are presented to the eyes, the impression is more vigorous and looked at with greater ease than when one eye only is employed; vision in this case is called pseudoscopic. Binocular vision should be employed wherever practicable ; it will be found much less trying to the eyes than monocular efforts. It has now been shown that the human eye is extremely liable to imperfections, and being so, strict attention to details is demanded from the micro- scopist. Now, although the human eye is such a wonderful instrument, there are many problems it is unable to solve without extraneous assistance. Take, for example, the bunt of wheat, Tilletia faries. With 26 METHODS OF MICROSCOPICAL RESEARCH. the unaided eye, you will be able to discern nothing more than a black dust, the various details having to be made out by other means. Then again, with ob- jects so minute as the diatom, Amphipleura pellucida, the object itself is almost invisible to the unassisted eye, to say nothing of the beautiful carvings with which the valves are embellished, and which exact for their elucidation the most perfect lenses with which we are acquainted, and the most accurate manipulation of the illumination. You may, indeed, see the contour of many forms of diatoms without extra optical assistance than that afforded us by nature, but not much more than this, as if the eye is approached too closely the picture falls behind the retina and is lost. I have already mentioned the fact that starting with the distance of most distinct vision, continued approach to the eye finally renders the object in- visible, the rays being thrown behind the retina, the mechanism of accommodation being insufficient to produce a curve deep enough to bring the picture to a short conjugate focus. This can, however, be done by interposing a lens, or lenses, between the object and the cornea, so that a virtual image of the object is seen. These lenses form either a simple, or a compound microscope. 27 CHAPTER II. IN continuation of the subject of " Instruments and their Use," the author, holding in the highest estimation that invaluable treatise, Dr. Lionel S. Beale's " How to Work with the Microscope," as a thoroughly practical work, takes the liberty of quoting therefrom the following wise observations, which he considers cannot be too widely dis- seminated. " By describing the results of the in- vestigations of others, a teacher may spread knowledge. By prosecuting original enquiries himself, he may contribute his mite to the gradually increasing stock of information ; but by demon- strating to his pupils the successive steps by which conclusions in scientific enquiries have been at length arrived at, and by describing minutely the methods which have been actually employed in investigation, the teacher not only encourages his pupils to become original observers, and to investi- gate for themselves, but he may succeed in placing them in a position to commence their researches at the point where an enquiry has been abandoned by preceding observers. " The opinion that it is only necessary to place an object in the field of the microscope in order to make out its structure, seems far too prevalent. 28 METHODS OF MICROSCOPICAL BESEARCH. Much of the disappointment suffered by many who are provided with microscopes may be traced to this erroneous idea. Too many look upon the microscope as a mere toy, and microscopical obser- vation as an amusement, by the help of which time may be made to pass away pleasantly. Few are aware of the real interest derived from intelligent investigation, and the instruction afforded, and the facts for contemplation and thought easily to be obtained if only the observer will acquire the neces- sary dexterity and elementary knowledge to enable him to study with success. Many who have be- come interested in what was at first but rough and superficial investigation, have persevered and have at length become excellent observers, who have added new facts to our knowledge, or have ren- dered more accurate information which was already possessed." It is not intended in this work to give minute descriptions of, or to compare one with another the microscopes and various apparatus supplied by the accomplished opticians in this and other countries, the details of which are fully given, and the instru- ments and accessories exhaustively described in so many works upon the microscope. The aim of the present treatise is rather to give thoroughly practical directions for the use of the instruments and apparatus, so that "research" and manipula- tion may go hand in hand. For practical work (as distinguished from delicate observations with the higher powers) the simpler and smaller in other words, the "handier" the microscope which is in constant use the better, and the lower the power that may safely be used to ON INSTRUMENTS AND THEIR USE. 29 accomplish the work, also, the better. The use of the higher powers, and the more delicate accessory apparatus of the microscope follows when the pre- parations are made, and examination and observa- tion commence. As a matter of course, when investigations have to be made during the progress of the work, powers sufficiently high must be used, but let it always be remembered that there is an axiom, " all the best microscopical work has been done with low powers " which, in reference to the aims of this treatise, means all the best and most perfect permanent preparations have been made with low powers, and are afterwards to be studied and examined under the higher powers and con- ditions of light, &c., necessary to their elucidation and use as valuable aids to scientific instruc- tion. For many purposes such as the dissection of the larger insects, or the teasing out of the coarser tissues, a good " simple " microscope will be found extremely useful; but, as a rule, it is better to do almost everything under the compound microscope, and to patiently acquire the necessary skill which enables one to use both ordinary and dissecting needles, small brushes, bristles, tubes, &c., in the field of the microscope whilst using it ; above all things it is important that, if possible, a binocular microscope should be used when many hours are spent in microscopical work, for not only is it natural, and therefore better, to use both eyes, but fatigue to the eyes is thus avoided, whilst it is certain, in respect of those who work with one eye only (unless the eyes are used alternately and often changed) that one eye becomes educated at the expense of the other, and to such an extent, that 30 METHODS OF MICROSCOPICAL RESEARCH. when the monocular instrument is exchanged for the binocular it is often found that the eyes have ceased to be a pair, and do not see alike. Two eyepieces should be available with the work- ing microscope, the ordinary, or A, and a thoroughly good C eyepiece, and two objectives. After thirty years of constant work the author advises that these two powers should be a first rate 1-g- and a J inch. These, with the two eyepieces, will give a series of four magnifications and prove amply sufficient for all ordinary work, and the objectives can be con- veniently used with a double nose-piece, except when dissecting or other such work is being done under the microscope and the nose-piece shall be found to interfere with free manipulation. It is well to take care that the objectives are as nearly as possible of the same length, in order to avoid the risk of breaking the slide under the microscope a catastrophe very likely to occur either from momen- tary forgetfulness of which power may be in use, or hurry in changing the objectives. There is no difficulty in obtaining a 1^ and J inch objectives so arranged as to require but little alteration of the rackwork of the microscope to focus them alter- nately. Whilst a 1-g- inch objective will be found the most useful of all glasses for ordinary work and for most purposes, it will often be found neces- sary in the course of the work to make examinations of its parts and progress by means of the J inch. With English microscopes it is advisable, as a rule, to use English objectives, but admirable glasses of great penetrating power and definition by foreign makers can now be obtained at a much less cost than those of English manufacture. The object- ON INSTRUMENTS AND THEIR USE. 31 glasses made by Zeiss, Reichart and others, can scarcely be surpassed. Flatness of field, penetration, definition, and entire absence of colour are the essentials of a thoroughly good objective, and all objectives not possessing these qualities should be rejected. For working powers objectives with the lower angles of aperture are to be preferred, as it is most desirable to secure as much room as possible for manipulation between the preparation and the objective. For subsequent examination and study, however, high power objectives with high angles of aperture are invaluable, and oil-immersion objec- tives especially so. A thoroughly good fine adjust- ment is all-important to a good working microscope. The stage of a microscope intended for work should be a simple table, either circular or square (pre- ferably the former), so that the specimen may be rotated and all its parts easily got at ; there should be no mechanical arrangements whatever upon it (unless these are removable), so that the hands may have free scope ; and the stage should be of ample size, so that if necessary it may form a rest for the hands whilst working. For exhaustive observations and delicate investi- gations and the study of permanent preparations, the elaborate microscopes and beautiful apparatus now obtainable are of the highest possible value and utility, but for ordinary work they are not only quite unnecessary but too complicated to admit of satisfactory work being done under them with any comfort or convenience, whilst their great costliness and delicacy, and their liability to injury from chemicals and anything like rough usage, render them totally unsuitable as " working " microscopes. 32 METHODS OF MICROSCOPICAL RESEARCH. The following list will prove useful to the student who desires to fit up his laboratory, or workroom, with the most useful and necessary apparatus and requirements for microscopical study, investigations, and the making of preparations. A good simple microscope, which, however, may be dispensed with if the student decides to work entirely with the compound microscope as already advised. A thoroughly good and firm microscope pre- ferably a binocular instrument (which can also be used as a monocular) with large stage, as already described, coarse and fine adjustments, plane and convex mirrors, and diaphragm, and having a fitting under the stage to receive an achromatic condenser of a simple and inexpensive kind, a spot Lens, and a Polariscope. A Bullseye Condenser. Two objectives, 1J and J inch with double nose- piece. Two Eyepieces, " A" and " C." A Camera lucida for drawing objects. A Stage micrometer divided into 100th and 1000th of an inch. A good Paraffin Lamp with metal chimney, and blue glasses to fit into the chimney to moderate the light. A retort Stand. A Spirit Lamp. A small Pestle and morfcar of Glass or agate. A best " Cathcart " Microtome arranged for both the embedding and Ether freezing processes. Two Section Knives and a Plane-iron for cutting Sections. ON INSTRUMENTS AND THEIR USE. 33 A set of Scalpels. Two pairs of ordinary surgical scissors (small and large) and one curved pair. Two pairs of forceps (small and large), and a specially curved pair for manipulating cover glasses. Four " Bulldog " forceps for compressing vessels when injecting. A pair of Bone forceps. Two thick curved needles in handles for lifting Sections. Needles, small and large, and handles for them from which they can be readily removed and replaced when it may be necessary to change them. Needles with flattened points and cutting edges, in handles. Two Sewing Machine needles which will be found most useful for dissecting and for laying out Sections. A Bone saw. An injecting Syringe, complete with Cannulse and Stopcocks. A Hypodermic Syringe for delicate injections. A writing diamond. Two pieces of good cork backed with lead. Two small glass dishes for dissecting under water. Three flasks for boiling. A set of Test Tubes and rack. Short Tubes, with corks, for storing small Sections. Half oz., 1 oz. and 2 oz. Bottles, with Corks, for storing Sections. A Washing bottle. Six Dropping bottles. 3 34 METHODS OF MICROSCOPICAL RESEARCH. Six Dipping Tubes. Six Pipettes. Three Glass rods for stirring. Some small glass tubing. Some small india-rubber tubing. Six small saucers of white porcelain and six large saucers. Six Watch glasses. Three small Black vulcanite photographic trays. These will be found most useful for selecting sections, which have been kept in spirit, when floated upon water placed in the trays. The black background to the white sections renders their selection perfectly easy. Two small and one large Porcelain Photographic Trays, the deeper the better these are for soakiog Sections, when stained with Logwood, in Tap water. Two Glass funnels and a measuring glass for 500 and one for 10 c.c. One gross of 3 in. by 1 in. Glass slips. One oz. each of (mixed sizes) thin glass circles No. 1 and No. 2. Three in. by 1 in. slips with ground out cells. Pure Tin cells, -J- in. and f in. in diameter. NOTE. Glass slips, thin covers and all tools and apparatus required for mounting ; all cements, media and staining fluids, &c., can be obtained at Mr. C. Baker's, 244, High Holborn, London, W.C. LIST OF CHEMICALS REQUIRED. Distilled water the purest obtainable. Glacial acetic acid. Ordinary (best) acetic acid. ON INSTRUMENTS AND THEIE USE. 35 Hydrochloric acid. Nitric Sulphuric Best Methylated Spirit. Alcohol, B.P. Absolute Alcohol. Ammonia B.P. Fort. Ammonium Tartrate. Alum (powdered). Ammonium Chr ornate. Ammonium Bichromate. Arsenious acid. Formic Picric Osmic ,, Acetate of Potass. Bichromate of Potass. Benzol. Borax (powdered). Canada Balsam, ordinary and hardened. Absolute Phenol (Carbolic acid). Carbonate of Potass. Ferrocyanide of Potass. Carmine (best). Celloidin (in Flakes or so-called " shavings "). Chloride of Gold (a 15 grain tube). Chloroform. Chromic acid. Clove oil. Collodion. Ether '720 B.P. Gelatine (" Gold label"). Glycerin. Gold size. 36 METHODS OF MICROSCOPICAL RESEARCH. Gum arable. Hsematoxylin (Crystals). Nitrate of Silver. Potassium in Cylinders for making Liq. Potass. Picrate of Ammonia. Sodium Chloride. Sulphate of Magnesia. Simple Syrup. Turpentine. Xylol. The following tables will be found very useful : FRENCH FLUID MEASURES. The cubic centimetre, usually represented by "c.c." is the unit of the French measurement for liquids. It contains nearly seventeen minims of water; in reality, it contains 16-896 minims. The weight of this quantity of water is one gramme. Hence it will be seen that the cubic centimetre and the gramme bear to each other the same relation as our drachm for solids and the drachm for fluids, or as the minim and the grain. The following table will prove to be sufficiently accurate for microscopical purposes : Cubic centimetres. 1 = 2 = 3 = 4 = 5 = 6 = 7 = Q _ 9 - 10 = 20 = 30 = 40 = 50 = 60 70 80 90 100 17 minims (as near as possible). 34 51 or 1 1 68 85 102 119 136 153 170 340 510 680 850 1020 1190 1360 1530 1700 drachm 8 minims. 25 ,, -L ,, 4HJ , 1 42 , 1 59 , 2 drachms 16 , 2 33 , 2 50 , 5 40 , 1 ounce drachm 30 minims. , 1 3 drachms 20 , 1 6 10 , 2 ou ces 1 , 2 3 50 , 2 6 40 , 3 1 30 , 3 4 20 ON INSTRUMENTS AND THEIE USE. 37 THE CONVEESION OF FEENCH INTO ENGLISH WEIGHT. Although a gramme is equal to 15-4346 grains, the decimal is one which can never be used in microscopy ; hence in the following table it is assumed to be 15f grains, which is the nearest approach that can be made to practical accuracy : Grammes. 1 = 15f grains. 2 - 30* 3 = 46i ,, 4 = 6 If ,, or 1 drachm If grain. 5 = 77 ,,,,1 17 grains. 6 = 92f 1 32f 7 = 107J 1 47* 8 = 1231 2 drachms 3 9 138f 2 18f 10 = 154 2 34 11 = 169f 2 49f 12 = 184f 3 4* 13 = 2001 3 20i 14 = 215f 3 35f 15 = 231 3 51 16 = 246f 4 6| 17 = 261 4 21 18 = 277i 4 371 19 = 292f 4 52f 20 = 308 ,,5 8 30 = 462 7 42 40 = 616 ,,10 16 50 - 770 ,,12* 50 60 - 924 ,,15 24 70 = 1078 ,,17 58 80 = 1232 ,,20 32 90 = 1386 ,,23 6 100 = 1540 ,,25 40 38 CHAPTER III. The Preparation of Animal Tissues. THE majority of animal tissues require hardening to a greater or less extent, whilst some, on the contrary, must be softened before sections can be cut from them. The various processes for embed- ding and infiltrating organs and tissues have, how- ever, recently been brought to such perfection that not only can much of the hardening once found necessary be dispensed with, much time saved and possible injury avoided, but and this is a very important consideration the risk of distortions and the obliteration of important features and properties in the tissues, which it is all important to their study should be carefully preserved, are reduced to a minimum or altogether avoided by the less heroic and more delicate processes now in vogue. Hardening agents, in respect of the effects pro- duced by them upon tissues, &c., form two divisions: (1) those (e.g., alcohol and nitric, and picric acids, &c.), which do not interfere with the subsequent processes for staining the tissues and sections; (2) those (e.^.j osmic and chromic acids, &c.), which do more or less affect the delicacy of the staining and the action of staining re-agents. THE PEEPAEATION OF ANIMAL TISSUES. 39 In hardening tissues it is all important to secure that the specimens shall be thoroughly penetrated by the liquid in which they are placed, and to this end it is necessary to immerse them in an abund- ance of the liquid say into 100 times their bulk, or even more and to divide them into sufficiently small pieces to ensure this result, regard, of course, being had to the attainment of sections of sufficient size to show the structure and features of the various organs this latter consideration, in these days of large sections, being obviously a matter of the greatest importance. The tissues must be as fresh as possible. Parts which have to be treated by the gold or silver processes must be immersed within half an hour after death, or they will cease to be susceptible to the action of re-agents. The time of year also must be a matter of consideration, summer and hot weather being, of course, less favourable to the pre- servation of animal matter than the cold of winter. No tissue can be placed too early in the preservative medium ; all useless matter, such as the contents of the stomach or intestines, must be carefully washed away with f per cent, salt solution. As soon as a fluid has become " fouled " by the tissues placed in it, it should be changed ; as a rule, frequent changes are advisable. All tissues hardening in chromic acid solutions must be examined daily, after the first few days, to prevent them from becoming brittle and thus spoiled. Different tissues placed in the same medium should be separately examined and tested. Some may have become sufficiently hardened and require immediate removal, whilst a more prolonged 40 METHODS OF MICROSCOPICAL RESEARCH. immersion may be necessary for others. No tissue should be allowed to remain in chromium fluids until it is quite hard, if, as is commonly the case, it is necessary to complete the hardening in alcohol to which it should be removed when it has become tough, though still retaining its elasticity. When transferring a tissue to alcohol to complete its hardening, all trace of the medium in which it has already been immersed must be removed by pro- longed soaking in cold water, which should be frequently changed during twelve or twenty-four hours, or until the last washings shall be colourless. Tissues thus prepared must not be put at once into full strength spirit, but first placed in methylated spirit and water ("half and half") for twenty-four hours, thence into pure methylated spirit. It is most important that the shrinkage of the tissues caused by the hardening processes should be uniform as well as gradual; it is therefore again obvious that the greatest care must be taken to use fluids which will penetrate the whole tissue or organ, lest the surfaces merely should become hardened whilst the interior portions remain soft and even become decayed. As a rule it is advisable to commence with weaker, and to advance by degrees to stronger, solutions in order to ensure penetration. A book should be kept in which notes can be made of the nature of the fluids in which organs and tissues are hardening, the dates of changing or substituting fluids, &c. Each bottle should bear a letter or number to corres- pond with the letter or number registered against it in the note book. Tissues and organs may be wrapped in pieces of linen, and a label bearing its THE PREPARATION OF ANIMAL TISSUES. 41 number to correspond with the note book attached to each parcel. In this way many specimens can be hardened together in the same fluid, and much space and waste of fluid avoided without any risk of confusion or error. No hard-and-fast line can be drawn in respect of the time during which the various organs and tissues should remain in the hardening fluids; careful experiments and notes, judgment and experience are necessary in this as in all other chemical work. Again, many tissues require hardening by different processes according to the elements in their structure which it is desired to demonstrate. For instance, to display the cor- puscles and nerves of the cornea it must be treated with gold, whilst in order to show the cell spaces the silver process must be resorted to. The special methods of hardening many of the various tissues and organs will therefore be found included under more than one process. Alcohol. When alcohol is employed as the sole hardening agent it should be used strong (i.e., of 95 per cent.), indeed it will be found necessary in some cases to use absolute alcohol. The specimens are to be immersed in an abundance of the spirit, which must be frequently changed during the first few days. It is advisable in many instances to suspend the specimens, either wrapped in linen or by a strong thread, in the alcohol. Some tissues will become sufficiently hardened in a few days, whilst others, and especially large specimens and whole organs, will require weeks. The more frequently the 42 METHODS OP MICROSCOPICAL EESEARCH. alcohol is changed at first, the more perfect, as well as the more rapid, will be the hardening. Nitric Acid should be used in an aqueous solution of from 5 per cent, to 15 per cent., and the specimens may remain in it from fourteen to twenty-one days. When it shall prove desirable to complete the hardening of a specimen in spirit it may be removed from the nitric acid and placed in the spirit after a much less prolonged immersion in the acid. Great care must be taken to remove from the specimen all trace of the acid by prolonged soaking in water, frequently changed, before placing it in alcohol. Chromic Acid. Make a 1 per cent, solution in water and reduce its strength to J, , or such other percentage as may be required. Specimens must be immersed in large quantities of this solution, in which they may remain for days or even weeks, the length of time during which they should be subjected to its action being determined by the size and nature of the specimen. Mucous tissues harden rapidly in this medium. Nervous tissues, spinal cord and brain require weeks. Bone from which the muscles (but not the perios- teum) have been removed should be steeped in a very weak solution, commencing with a ~ per cent, and increasing to -J- per cent, with frequent, changes during a period of ten days. It is then to be de- calcified by immersion in chromic and nitric fluid until a needle can be passed through it. It is then THE PREPARATION OP ANIMAL TISSUES. 43 to be washed free from all trace of the acids, and transferred to strong spirit. Teeth may be treated in the same manner. In all cases a weak solution should be employed at first and strengthened each time it is changed. The specimens should be examined from time to time, and removed when they shall have acquired the proper condition. The greatest care must be exercised lest they become brittle. When removed from the chromic acid solution they should be soaked in water, frequently changed, for at least twenty-four hours, and then transferred, for keeping, to 95 per cent, alcohol. MUSCLE. A piece of muscle from an animal recently killed is steeped in a small quantity of a i per cent, solution for a week. The cleavage of its sarcous substance is shown by teasing a small piece in glycerine. NERVE. Harden a piece of metacarpal nerve of a horse, or sciatic nerve of a smaller animal for ten days in a i per cent, solution. Stain a bit in logwood, then tease it in glycerine thoroughly. This shows its connective tissue. Chromic Acid and Alcohol. Mix one part of a 4 per cent, solution of chromic acid with two parts of methylated spirit. This should be done when required for use. The following tissues may be placed in this, then transferred to spirit. The time required must be judged according to directions already given: The whole of one cornea, to show stratified epithelium ; a piece of small intestine, to show: non- 44 METHODS OF MICEOSCOPICAL RESEARCH. striped muscle ; heart and pericardium of a small animal ; small arteries and capillaries from the brain of a sheep, after scraping away the brain- substance ; middle-sized artery such as metacarpal of the horse; trachea and lungs. These are gently injected and then immersed in the fluid. The lips, tongue, salivary glands, tonsils, ceso- phagus (distended and tied), stomach (after washing away its contents with f per cent, salt solution), small and large intestines, liver in half-inch cubes, ureter and bladder (distended), ovary, fallopian tubes, uterus (distended per vaginam) may be placed in the solution. Besides the above, which may be taken from either a dog, cat, or guinea pig, the following should be obtained: The thymus gland of an infant; skin of scalp, finger and palm of hand, sole of foot from the human subject; also a nail. The eye of an ox divided transversely just behind the cornea for the ciliary muscle, sclerotic, cornea, and iris; also the choroid and retina. Of course both halves have to be used. The prostate gland and penis of a guinea pig. The cervix uteri of a cow. Mammary gland of an animal near the full period of gestation. The placenta of a cat, or guinea pig. The umbilical cord, which must be cut into pieces an inch long, and hardened for two days in Miiller's fluid before being placed in the present medium. All the above tissues must be daily examined after the third day, and each transferred to spirit after it has become tough. Moreover, the fluid should be changed after the first twenty-four hours, and at intervals of a few days afterwards. THE PREPARATION OF ANIMAL TISSUES. 45 When many different tissues are in fche same jar, the quantity of fluid must be such that the upper surface of the tissues extends half way up the entire fluid, that is to say, the stratum of tissues and stratum of clean fluid over them should be of equal depth. Bichromate of Potass. Make a 2 per cent, solution of bichromate of potass, with ordinary water. Meso-rectum of Cat. Pin this out on cork, and float it cork upwards on the solution for seven days. Liver. After injecting the portal vein with blue gelatine mass, and the hepatic artery with carmine gelatine mass, the liver may be hardened in the solution to toughness, and, of course, finished in alcohol. Spinal Cord of Ox. Pieces an inch long may be placed in the solution frequently changed for from three to five weeks. Spinal Cord of Ox, Horse, or Sheep. If pieces about an eighth of an inch long be macerated two or three days in -J per cent, of the solution, the anterior horn of the spinal cord snipped out with scissors, and teased in carmine solution, then pressed with a cover glass, using Farrant's medium, they will show the isolated multipolar nerve cells very beautifully. The sympathetic nerve cells of the frog can be isolated in the same manner. Cornea of cat, rabbit, or guinea-pig should also be hardened in a 2 per cent, solution for ten days. The lens in 1 per cent, solution for one week. 6 METHODS OF MICEOSCOPICAL RESEARCH. Ovaries of the Cow in small pieces should be macerated in very dilute solution to isolate the large, branched, pigmented cells of the Corpora lutea. Ammonium Bichromate. A 2 per cent, solution made with ordinary water may be employed. This solution is preferred by many to the potass salt solution. It is used in the same way. Columnar epithelium may be prepared as permanent speci- mens by placing a piece of fresh intestine of dog, cat, rabbit, &c., in 1 per cent, solution for two days ; then steeping an hour or two in water and scraping off the epithelium and staining. The cells have to be separated with a needle, and may be mounted in Farrant's medium, or in glycerine jelly. Chromate of Ammonium. A 5 per cent, solution is used. If a newt's liver (in small pieces) and pieces of the small intestines be placed in the solution for forty-eight hours, the liver cells and columnar epithelium may be obtained, as in the case above- mentioned. The goblet cells of Klein can be perfectly preserved in glycerine jelly in this way. The mesentery of the newt may be placed in the solution at the same time, and taken out after twenty-four hours. This shows the non-striated muscle fibre beautifully. The isolated gastric glands of a small mammal may be obtained in the same way by placing pieces of the fresh mucous membrane for three days in the solution. The THE PREPAEATION OF ANIMAL TISSUES. 47 testes of the newt should be placed in the solution for twenty-four hours, then cut into, and their contents squeezed out on to a slide. The sperma- tozoa are thus obtained as a permanent preparation. Another important use of the above solution has been pointed out by Heidenhain. If small pieces of kidney be placed in the solution for forty-eight hours (the cortex should be chosen), the preparation shows the cells of the uriniferous tubules and their peculiarities as no other method, probably, will show them. Miiller's Fluid. This is made by dissolving twenty-five grms. of potass, bichrom. and ten grms. sodse sulph. in 1000 c.c. of water. Miiller's fluid has great penetrating power. It hardens slowly, taking, it may be, from five to seven weeks. It is useful as a commencing agent, to be followed by another of greater shrinking power, such as chromic acid and spirit solution, ordinary alcohol, &c. Very even shrinkage may thus be obtained. For example, if the fresh nasal septum is cut out and placed for two days in Miiller's fluid, then for a week in chromic acid and spirit solution, afterwards in weak, then in pure methylated spirit, the olfactory epithelium will be obtained in excel- lent preservation. After the above explanation no difficulty will be found with the following delicate structures, namely: Developing tooth, the adenoid tissue of lymphatic gland, spleen, thyroid gland, supra-renal-capsules (of the horse, by preference), sympathetic ganglion, 48 METHODS OF MICROSCOPICAL RESEARCH. olfactory epithelium, cochlea, testis, epididymis and vas-deferens, ovary, human placenta, &c. Muller's Fluid and Spirit. Take three parts of Muller's fluid and add to it one part of methylated spirit and keep it in a dark place. The mixture should be made only as re- quired. This is especially useful for the central nervous system. The removal of the brain and spinal cord without injury may here be described. Immediately after death the skin is removed, or at least the skin over the back and neck. Then separate the neck and head from the trunk about the middle of the neck. Next clear away the muscles on each side of the vertebral spines and clip away every spine as close as possible with scissors or bone forceps. Next with ordinary forceps grasp the laminaB, which cover over the spinal cord, one lamina at a. time, and break it outwards by inserting one blade of the forceps wiihin the neural canal, the other on the upper surface of the lamina. Now advance down the spine, breaking the laminse outwards right and left till all the cord with its membranes is exposed. The head portion is dealt with in like manner by breaking off, bit by bit, the top of the skull, insert- ing one blade into the interior, of course, after clearing away the skin and muscles. The spinal cord and membranes of a cat, dog, or rabbit should be carefully taken out and suspended in a deep, narrow vessel. The fluid should be changed at the end of twenty-four hours, then after a week. At the end of this time, divide into pieces THE PREPAEATION OF ANIMAL TISSUES. 49 an inch long, and continue the hardening for an- other week or two, as may be required. It may be replaced by a 2 per cent, solution of bichromate of ammonium for two weeks, and the pieces preserved in spirit, or by Hamilton's method (chloral hydrate, twelve grains, water, one ounce). The cere- bellum, cerebrum, and, of course, the medulla oblongata may likewise be hardened. In the case of the two former, divide into suitable pieces to ensure perfect penetration. The tendo achilles of the calf, and the metacarpal nerve of the horse, each in inch-long pieces ; also the posterior half of the eye-ball of a pig (for the retina) may be hardened with advantage in this fluid. Absolute Alcohol. This should have a specific gravity of 0*795. It hardens in twenty-four hours, with much shrinking. It is used for secretory glands, notably the pancreas, which must be placed direct in it, or the gland may spoil by partial self -digestion. The salivary glands, the lachrymal glands, pieces from both ends of the stomach for the gastric glands, the pancreas in pieces, &c., should be placed in it at once. If the lymphatic gland of a horse, ox, or smaller quadruped is injected with 2 per cent. Prussian blue fluid by Klein's method (introduce a glass pipette filled with the fluid into a lacteal near a mesenteric gland and blow the solution into it), and then placed in absolute alcohol, we have the lymph sinuses well injected. The muscle structure of the beetle or crab is shown well by placing a beetle or the ampu- 4 50 METHODS OF MICROSCOPICAL RESEARCH. tated limb of a crab for a week in absolute alcohol. A bit of muscle is scooped out, stained and mounted, after being teased with needles. Non-striped muscle may be demonstrated thus : Kill a small animal and wash out a length of the , small intestine with salt solution, distend it with absolute alcohol, and tie both ends, then suspend it in the alcohol for twelve hours. With a pair of blunt-pointed forceps now tear off strips from the outer surface, which will include the longitudinal muscle structure ; stain them and mount in balsam. Picric Acid. Make a cold saturated aqueous solution. Small pieces of tissue harden in this in from twelve to forty-eight hours. It is excellent for decalcifying foetal bones which may be left in it, and tested from time to time with a needle, which ought to be pushed easily through them. No time can be specified, in some cases perhaps weeks may be re- quired. Prepare the varieties of cartilage with this solution. The aorta (pieces) of a large animal (horse, or ox), also a cornea to show its fibrous tissue, the thymus gland of an infant, also a lym- phatic gland may all be well prepared by remaining in this solution twenty-four, thirty-six, or per- haps forty-eight hours. The intracellular plexus of fibrils may be well shown, thus : Keep a newt in a little water for three, four, or five days without changing the water. Its outer layer of cuticular epithelium is shed as a cast of the entire animal. Place the film in the solution for twenty-four hours, then wash in plain water till no colour is given off, THE PREPAKATION OP ANIMAL TISSUES. 51 and preserve in common alcohol till required. A small piece of this, stained in picro-carmine and mounted in Farrant's medium, shows the above- named structure beautifully. The fibres of white fibrous tissue may be shown to advantage, thus : Tear off fine strips of tendo achilles of an animal and place them for twenty-four hours in the solu- tion. By " teasing " a little piece in water the white fibres held together by cement substance are well displayed. Osmic Acid. This is purchased as a 1 per cent, solution and diluted with distilled water to a ^, a J per cent, solution, &c., as required. It is a hardening agent which also stains fatty matter, and is, therefore, useful in blackening the medullary nerve fibres. It is very poisonous, very expensive, and soon spoiled by exposure to light. It must be kept in a well stoppered bottle, which should be of black glass or covered with black paper, so that not a particle of light shall be admitted. Substances placed in it harden in from four to thirty-six hours, and may be prepared and mounted in either glycerine, glycerine jelly, or Farrant's medium. Very little pieces, not larger than half a grain of wheat, are to be used of any tissue, and the bottle in which they are placed must also be very small ; the short glass tubes in which homoeopaths keep their smallest pilules answer well. With the above cautions and directions we may enumerate the tissues which are treated with advantage by osmic acid. In a 1 per cent, solution 52 METHODS OP MICROSCOPICAL RESEARCH. immerse costal cartilage of a kitten, puppy, or young rabbit, rat or guinea pig, for twelve hours ; a piece of the sciatic nerve of the frog for ten minutes ; the non-medullated nerve running in the wall of the splenic vein of an ox, also a piece of human placenta. A 1 per cent, solution may be injected into a testis with a hypodermic syringe, and the whole organ may then be placed in alcohol. The following are best treated with a -J- per cent, solution, namely : liver cells of a dog or small animal (the liver is cut across and the cut surface scraped) are placed in the solution for an hour. Areolar tissue is demonstrated by injecting a -J- per cent, solution into the groin of a puppy or kitten immediately after death. A bulla is thus formed, which must be snipped out with scissors, spread on a slide stained with logwood and then covered. A per cent, solution may be forcibly injected into the anterior horn of a fresh spinal cord. The part is cut out, macerated for two days in dilute alcohol, and the multipolar cells isolated by " teasing," after staining with picro-carmine. A J per cent, solution (the strength most frequently employed) may be used for showing mucous tissue ; thus : inject the axilla, or groin, of a very young embryo, as described above. Pieces the size of a pea of the following may be hardened in a J per cent, solution : Submaxillary gland, fresh pancreas, pieces from each end of the stomach, small intestines, supra-renal cap- sules of the horse, human skin. Place a piece of the anterior horn of a fresh spinal cord for ten days in a ifeth per cent, solution, then, after washing away the greyish deposit, place it in an equal THE PREPARATION OF ANIMAL TISSUES. 53 quantity of glycerine and water for a fortnight ; stain with weak magenta solution, " tease " in glycerine, and cover (Stirling). Miscellaneous Preparations. Salt Solution. This is made as a f per cent, solution, that is, 75 grm. in 100 c.c. of water. It is used for washing away foreign matter from organs before immersing them in other fluids, and for examining fresh tissues. In so using it, a very small drop is placed on a slide, and in it is immersed a minute piece of the structure about to be examined. "Tease" this out with needles; then put over it a cover glass. The following are to be studied : Fresh columnar epithelium from the small intestines ; ciliated epithe- lium scraped from the roof of the frog's mouth; ciliary motion may be seen in the yellowish coloured gills of the common salt water mussel. Ligamen- tum nuchse of ox ; subcutaneous connective tissue ; adipose tissue ; red marrow from a long bone ; striped muscle (best seen in sartorius of the frog) ; nerve fibre, sciatic of the frog ; fresh pia mater ; fresh spleen (the ox spleen by preference) ; thymus gland ; kidney ; Gasserian ganglion ; placenta ; decidua, &c., may all be so treated. Glycerine. Margarine crystals may be obtained by steeping morsels of fat for twenty-four hours in glycerine. These post-mortem products appear as delicate needles. 54 METHODS OP MICROSCOPICAL RESEARCH. Water. Squamous epithelium may be stained in magenta solution and examined in water. To obtain it, scrape the inside of the cheek with a blunt knife. The surface of the cheek may be also scraped and the scraping examined in water and afterwards irrigated with a 5 per cent, solution of liquor potassae. Dilute Alcohol. Mix two parts of water with one of rectified spirit. This is a useful dissociating solution recom- mended by Ranvier. Olfactory, ciliated and transitional epithelium, may be prepared as permanent preparations thus : Place a small piece of fresh trachea of a small quad- ruped for ciliated a piece of fresh bladder for transitional the head of a frog, with the nostrils slit up, for olfactory epithelium, in the solution for two days. The parts are then scraped and the scrapings stained and mounted in glycerine, or glycerine jelly. Stirling " fixes " the ciliated epithelium by placing the scraping in a 1 per cent, solution of osmic acid. Non-striped muscle may be obtained by taking out the bladder of a fresh- killed frog and distending it with the solution, then placing it for twenty-four hours in the solution. After brushing away the mucous membrane with a camel-hair brush, stain the bladder in picro- carmine, and mount a piece in Farrant's medium. Isolated heart muscle fibres of the frog's and mam- malian heart can be obtained by the same process. THE PREPARATION OF ANIMAL TISSUES. 55 Purkinje's fibres can be obtained by snipping out bits of them (seen as fine transparent lines on the inner parts of the walls of the ventricles of the heart of oxen and sheep), and placing them in the solution for two days. The villi of human placenta can be isolated by soaking a piece of placenta two days in the fluid. Chromic Acid and Nitric Acid Solution. This is made by adding 1 c.c. of strong nitric acid to every 100 c.c. of the J per cent, solution of chromic acid. It is used chiefly as a decalcifying agent. Tooth, intervertebral disc with subjacent bone, articular cartilage with subjacent bone^ costal cartilage of an old person, may each be placed in a large quantity of the fluid, which must be frequently changed, until the bony part is softened, which may be known by the needle test. The parts are washed free of the acids by frequent changes of water, then placed in weak, afterwards in strong, alcohol for preservation. Permanent preparations of elastic tissue may be made thus : Soak half-inch cubes of ligamentum nuchae of ox or horse in this fluid for a week; wash free of acids and preserve in spirit, and make trans- verse and longitudinal sections in the usual way. (Stirling). Silver Nitrate. All animal tissues which are to be subjected to nitrate of silver and chloride of gold processes, must be perfectly fresh and carefully washed in 56 METHODS OF MICROSCOPICAL RESEARCH. distilled water before being treated by either of these re-agents. Make a 1 per cent, solution of nitrate of silver in distilled water, keep it in a bottle carefully covered all over with black paper. This solution is to be further diluted to a -J, a ^, or a J per cent, as required. Nitrate of silver is used to intensify and demonstrate the outlines and features of endothelium and cartilage cells. The cement substance between the cells absorbs the solution all not absorbed must be carefully washed away with distilled water, and the tissue exposed to direct sunlight. Not the slightest taint of the unabsorbed silver solution must be allowed to re- main upon the surface of the tissue, the staining of which would be spoiled by the black oxide granules of decomposed nitrate of silver. The tissues must be treated within a few minutes after death. Omentum of rabbit. Kill a rabbit by bleeding. Remove the omentum and wash it in distilled water; place it in a J per cent, solution ten minutes ; wash in ordinary water thoroughly ; place the whole in a saucer containing water, and expose to diffuse daylight till slightly brown. Small pieces may now be stained with logwood and mounted in glycerine or glycerine jelly, and the logwood stain may be dispensed with if desired. Septum Cysternce Lymphaticce magnce of a frog. Kill a frog and immediately open the abdomen. Gently push on one side the stomach, bowels, &c., and pour distilled water on the part behind the stomach, when a delicate membrane floats up. Now pour a -J per cent, solution, drop by drop, over this till it becomes milky, and treat as above. THE PREPARATION OF ANIMAL TISSUES. 57 Lungs of a kitten, or puppy. Distend these with a \ per cent, solution immediately after death, then ligature the trachea, and sink them in alcohol till required. Tendon. Tendons and their sbeaths are covered by endothelium. Pinch the tail end off a recently killed mouse, and wash the fine tendon fibres thus drawn out in distilled water, and treat with a ^ per cent, solution. Take another set of these fibres, pencil off their endothelium with a camel-hair brush dipped in distilled water, then silver. This shows the cell spaces in tendon. Cornea. The cell spaces of cornea may be shown by scraping away the epithelium from the anterior surface of the cornea of a pithed frog, then applying a 1 per cent, solution till greyish white. The cornea must be snipped at its edges, after being silvered, to make it lie flat on the slip. A rat's cornea may be likewise treated and stained with logwood. Blood Vessels. Kill a small quadruped by bleed- ing. Syringe out the blood vessels with distilled water; then inject a % per cent, solution. Use the spleen, mesentery, and intestines. The spleen must be hardened in alcohol, and sections cut, exposed to light, after staining with logwood, or not, as desired. This shows the endothelium of the venous sinuses. The intestines must be cleared of their contents by distilled water ; exposed to light in a saucer containing water ; a piece of small or large intestine is snipped out, laid on a slide with its mucous membrane upwards, which is gently scraped away, the muscular and serous coats remain, and are to be mounted in balsam. 58 METHODS OF MICROSCOPICAL RESEARCH. Membranous Connective Tissue. The omenta of an adult cat and rabbit, also of a young rabbit, are to be treated like the omentum of rabbit as already described, to show the membranous connective tissue. The omentum of the young rabbit shows developing fat cells and blood vessels. Some of the above should be stained with logwood. Adenoid Tissue. "With a hypodermic syringe in- ject a fresh lymphatic gland with a J per cent, solution, and after placing it for twenty-four hours in alcohol, make sections. These are stained with logwood, and exposed to light till brownish. Cement Substance of Non-striped Muscle. Washout a small length of intestine of a rabbit with distilled water ; then fill it with a per cent, solution, and tie both ends ; then place it in a J per cent, solution for a quarter of an hour. Wash away all the silver, and cover over with water in a saucer. Thin Iamina3 of the outer muscular fibres are to be stripped off with broad nibbed forceps. Mount some stained with logwood in balsam ; others un- stained, as desired. These also show the lymphatics. Sciatic Nerve of a Frog. To show Ranvier's- crosses, kill a frog, and dissect out the sciatic nerve, wash it in distilled water, then place a piece a line in length in a J per cent, solution for five minutes. Wash it now thoroughly in water and tease it care- fully in glycerine, cover, and expose to light till brownish. Mount a small length of an entire thick- ness of an intercostal nerve of a rat or mouse, to show its endothelial covering. Of course it is not to be teased, and it must be very carefully washed or the endothelium will be destroyed. Lymphatics of the Diaphragm. Expose the THE PREPABATION OF ANIMAL TISSUES. 59 posterior (ventral) surface of a rabbit or guinea- pig, immediately after death by bleeding, thus : Ligature the gullet and the posterior vena cava, then remove all the abdominal viscera. Tie up by the hind legs, then with a brush dipped in distilled water brush away all the epithelium covering the centrum tendinuin of the diaphragm. "Wash silver solution over it, and treat as already explained. This silvers the endothelia lining the lymphatics. Chloride of Gold. This salt is sold in small tubes containing 15 grains. A 2 per cent, solution is to be made with very pure distilled water, and kept in a black bottle, like the silver solution. The operator must also provide himself with formic acid and two or three fresh lemons. Development of Capillaries. Snip off the tail of a half-grown tadpole, place it in a watch glass, and squeeze the juice from a fresh lemon over it, and let it remain immersed for five or ten minutes ; wash it in distilled water to remove all the juice ; then steep it for half-an-hour in a 1 per cent, solu- tion. Wash away the surplus gold, then place in a mixture of one part of formic acid and three parts of water for twenty-four hours in a cool place and away from the light. The gold chloride will then be reduced. The following are to be treated in exactly the same manner : A whole Cornea. That of a pig, dog, or cat, to show the nerves of the cornea, also the cornea corpuscles. 60 METHODS OF MICROSCOPICAL RESEARCH. A Piece of Skin. This should be taken from the snout of a pig or a mole, or both ; also the soft part of a duck's bill. Sections show the nerves of skin. Striped Muscle. The recti muscles of a rabbit's eye are to be taken, a strip cut off lengthwise. These show nerve terminations in striped muscle. Muscle ivith its Tendon. Take a piece of the diaphragm of a rabbit ; let the piece be part of the centrum tendinum with its attached muscle. Sec- tions made in the long axis of the muscle fibres show the terminations of muscle in tendon; that is to say, the connecting links are shown. Tendon from the Tail of a Mouse. After treating a few leashes as above, snip off a piece a line in length; tease in glycerine and cover, or simply press the cover glass upon it till it flattens out. This preparation shows the relations of the cells and fibres in tendon. Tail of Rat. After treating as above, with the exception that it must remain an hour in the gold solution, decalcify, by placing in chromic and nitric solution, then make transverse sections and stain with logwood. Nerve Ganglia. Treat the heart of a frog, in small pieces, as above ; dissect out the nerves and ganglia. These are also found lying along the course of the abdominal aorta of the frog, and may be taken from there if preferred. The nerve ganglia of the bladder and ureter of a small mammal may likewise be thus demonstrated. THE PREPARATION OF ANIMAL TISSUES. 61 The Injection of Blood Vessels. Blood and lymphatic vessels are more satisfac- torily demonstrated when filled. The former are better injected with a fluid which becomes solid in ordinary temperatures. The vessels must be fully distended at the time and this distention must remain. Gelatine forms the foundation of injection masses for the above purposes, because it can be liquefied at blood heat and it solidifies in a little lower temperature; then, again, it is capable of being easily cut, and does not become brittle, but remains tough and resistant, though sufficiently soft. Injections of different colours are employed in order that the arteries may be distinguished from the veins, and these again from other channels, such as bile ducts, lymphatic vessels, capillaries, &c. The distribution of the capillary vessels, indeed, in many cases cannot be satisfactorily studied or demonstrated without injection or other special modes of preparation. Injections of single organs, or parts carefully dissected from the body of an animal, all the larger arteries and vessels being carefully tied, can be made ; whilst the entire blood system of mammalian animals, including man, of birds and reptiles, can be injected from an arterial trunk, so that the finest capillary vessels shall be perfectly filled. Red Mass. This is made of gelatine coloured with carmine. In making it the greatest care must be exercised in 62 METHODS OF MICROSCOPICAL BESEARCK. order to ensure a neutral or at least a slightly acid mass ; because if it should have any alkaline re- action, it will diffuse through the vessels, stain the adjacent tissues, and thus render the preparation altogether worthless. It is preferable that the mass should be slightly acid, but if too much so, granulation of the carmine will ensue, and the fluid will not be driven into the arterioles far less into the capillaries. Parts injected with a carmine and gelatine mass must be immersed in equal parts of water and methylated spirit, with 1 per cent, of acid added thereto. DR. CARTER'S CARMINE MASS. Take of Carmine, 60 grains. Strong Ammonia, 120 minims. Glacial Acetic Acid, 86 minims. Solution of French Gelatine (gold label), 1 part to 6 parts of Water, 2 ounces. Water, 1^ ounce. Dissolve the carmine in the ammonia and water, filter if necessary. With this mix 1J ounce of the hot solution of gelatine. Mix the acetic acid with the remaining half ounce of gelatine, and drop this mixture, very slowly, into the carmine and gelatine solution, with constant stirring. DR. STIRLING'S CARMINE MASS. Take of Carmine, 60 grains. Strong Ammonia, 60 minims. Glacial Acetic Acid, 80 minims. Gelatine, 1 ounce. Water, q.s. Soak the gelatine in water for several hours ; pour off the water, which is not absorbed, when the THE PEEPAEATION OF ANIMAL TISSUES. 63 gelatine is completely saturated and swollen, and melt it in a water bath. Strain, whilst hot, through flannel and make up the solution to 2 ounces. Place the carmine in a mortar, add to it the ammonia and 2 ounces of water, and leave it for twelve hours. Filter, and add the acetic acid, drop by drop, stirring all the while, until the ammonia is completely neutralised. As the odour of the ammonia becomes faint, the acid must be added very cautiously. As long as there is free ammonia, the fluid is dull red, but it becomes of a brilliant colour as soon as the ammonia is neutralised. Now mix the two solutions at a temperature of 40 C. DR. G-. SIMS WOODHEAD'S MASS. Take of Carmine, 4 parts by weight. Liq. Ammonia, 8 parts by measure. Gelatine (Cox or Coignet's), 10 parts by weight. Distilled Water, 100 parts by measure. Put the carmine in a mortar, and pour on the ammonia, when an almost black paste will be formed if the carmine is pure ; pour in the water, and set the solution aside to be filtered. Place the gelatine in a narrow glass jar, and add sufficient distilled water to cover it, and allow it to stand until the gelatine is thoroughly softened. Warm the carmine solution in a pan of water, kept at nearly boiling point over a Bunsen gas burner, and add the gelatine; stir thoroughly, add a 10 per cent. solution of acetic acid, drop by drop, until the alkalinity of the ammonia is neutralised, and the fluid shall be even slightly acid. The point at which this takes place will be recognised by the pungent odour of the ammonia becoming fainter and 64 METHODS OF MICROSCOPICAL RESEARCH. fainter, and that of the acid substituted, whilst the fluid loses its bright carmine, transparent colour, and turns a dull brownish-red. BLUE INJECTION MASS. Take of Soluble Prussian Blue, 4 drachms. Gelatine, 4 ounces. Distilled Water, 20 ounces. Thoroughly mix the blue powder in a mortar with half the water. Treat the gelatine as directed in the case of the red mass, and add the blue solu- tion very gradually, and with constant stirring, to the liquefied gelatine. Filter through fine flannel. The greatest care must be exercised that no trace of alkali shall enter into the composition of this mass all the vessels used in making it should be carefully washed with acidulated water, and the purest distilled water must be used in its prepara- tion. Its colour is liable to fade and sections injected with it, especially when mounted in balsam, lose their colour after a time. It is better, therefore, to mount all specimens injected with Prussian blue in a slightly acid medium glycerine jelly prepared with boracic acid pure glycerine with a small proportion of acetic acid, or Farrant's medium are the best preservatives for these injec- tions. BLUE INJECTION MASS. (Robin's modification of Dr. Beetle's Prussian Blue) is made as follows : Take (a) Sulphocyanide of Potassium, saturated solution 90 c.c. Glycerine, 50 c.c. (b) Liquid perchloride of Iron at 30, 3 c.c. Glycerine, 50 c.c. THE PREPARATION OF ANIMAL TISSUES. 65 Mix slowly, and combine the mixture with three parts of a glycerine-gelatine injection mass made as follows (Robin's formula) : Dissolve, in a water bath, 50 grammes of French gelatine (gold label) in 300 grammes of distilled water in which some arsenious acid has been dis- solved ; add 150 grammes of glycerine and a few drops of carbolic acid. SCHEELE'S G-REEN INJECTION MASS (Robin's formula). Take of Arseniate of Potash (saturated solution), 80 c.c. Glycerine, 50 c.c. and of Sulphate of Copper (saturated solution), 40 c.c. Glycerine, 50 c.c. Mix and combine with three parts of the gly- cerine gelatine injection mass as in the preceding formula. Aqueous Injection Fluids. CARMINE. (Emery's formula.) To a 10 per cent, ammoniacal solution of carmine add acetic acid, with constant stirring, until the colour of the solution becomes brilliant red. After the precipitation of the carmine, pour off the super- natant fluid and inject the clear solution. Place the injected specimens in strong alcohol to fix the carmine. CARMINE. (Gerlach's formula.) Carmine, 77 grains. Distilled water, 70 grains. Liquor ammoniae, 8 minims. Dissolve the carmine in the ammonia and water, and leave the solution exposed to the air until all 5 66 METHODS OF MICROSCOPICAL RESEARCH. the ammonia has evaporated. Dissolve a drachm and a half of best gelatine in a drachm and three- quarters of water ; add a few drops of glacial acetic acid. This fluid is to be injected warm. ACID CARMINE FLUID. (Dr. Scale's formula.) Carmine, 5 grains. Glycerine, with 8 to 10 minims of acetic acid, ^ ounce. Glycerine, 1 ounce. Alcohol, 2 drachms. Distilled water, 6 drachms. Ammonia, a few drops. Mix the carmine with a few drops of water, and when thoroughly incorporated, add 5 minims of liquor ammonia. Add about half an ounce of the glycerine, and shake the mixture well in a bottle. Next, pour in very gradually the acid glycerine, frequently shaking the bottle during admixture. Test the fluid from time to time with blue litmus paper, and if not of a very decidedly acid reaction, add a few drops more acid to the remainder of the glycerine and mix as before. Lastly, add the alcohol and water, very gradually, shaking the bottle thoroughly after the addition of each succes- sive portion, until the whole is well mixed. PRUSSIAN BLUE FLUID. (Dr. Beale's formula.) Wood-naphtha, or pyro-acetic spirit, 1| drachm. Glycerine, 2 ounces. Alcohol, 1 ounce. Ferrocyanide of Potassium, 12 grains. Tincture of Perchloride of Iron, 1 drachm. Distilled Water, 3 ounces. Dissolve the ferrocyanide of potassium in 1 ounce of the glycerine, mix the tincture of iron THE PREPAKATION OF ANIMAL TISSUES. 67 with the other ounce. Put this into a bottle. Add the iron mixture, drop by drop, to the ferrocyanide solution, shaking all the time. Mix the naphtha with the alcohol, add the water very gradually, and thoroughly shake the whole. PRUSSIAN BLUE ACIDULATED GLYCERINE FLUID. (Dr. Beetle's formula.) Price's Glycerine, 2 fluid ounces. Tinct. of Sesquichloride of Iron, 10 minims. Ferrocyanide of Potassium, 3 grains. Strong Hydrochloric acid, 3 minims. Distilled Water, 1 ounce. Dissolve the ferrocyanide in one half of the glycerine, and the sesquichloride of iron in the other, add the latter drop by drop to the former. Finally add the water and the acid, and to the whole add 2 drachms of alcohol. NEUTRAL GLYCERINE PRUSSIAN BLUE FLUID. (Dr. Beale's formula.) Glycerine, 1 ounce. Alcohol, 1 ounce. Ferrocyanide of Potassium, 12 grains. Tincture of Perchloride of Iron, 1 drachm. Distilled Water, 4 ounces. Dissolve the ferrocyanide in one ounce of the water and glycerine, add the tincture of iron to another ounce. Mix these solutions very gradually, in a bottle, and shake thoroughly. The iron is to be added to the ferrocyanide solution. Lastly, the alcohol and the remaining water are to be gradually added, with constant shaking of the bottle. Specimens injected with this fluid should be preserved, and mounted, in acidulated glycerine. 68 METHODS OF MICROSCOPICAL RESEARCH. NEUTRAL GLYCERINE TURNBULL'S BLUE FLUID. (Richardson's formula.) Ferridcyanide of Potassium, 10 grains. Sulphate of Iron, 5 grains. Distilled Water, 1 ounce. Price's Glycerine, 2 ounces. Alcohol, 1 drachm. Proceed as in the last formula. Mix the sulphate of iron in 1 ounce of the glycerine, and the ferrid- cyanide in a small quantity of water, and mix this with the other ounce of glycerine. Gradually mix these two solutions., with constant shaking, and add the alcohol. Turnbull's blue is not so liable to fade as Prussian blue. 69 CHAPTER IV. On Injecting Blood Vessels, &c. IT has been generally, though erroneously, con- sidered that a perfect injection of an animal cannot be secured until the rigor mortis has passed away; it is, however, indubitable that the most successful injections have been made immediately after death. Dr. Beale 1 says: " I have found that most perfect injections may be made before the muscular rigidity begins, that is, within a few minutes after the death of the animal. Most of my fine injections have been made less than five minutes after death, and in the case of very young animals, so complete has been the injection of the capillary vessels, that where the capillary has not been fully developed, the injection has filled the pervious portion, and has penetrated to the very spot where the tube was commencing to be formed." This the author can entirely corroborate. The ordinary process for injecting blood vessels is with the syringe ; as this is a very delicate opera- " How to Work with the Microscope," fifth ed., 1880, p. 114. 70 METHODS OF MICROSCOPICAL RESEARCH. tion requiring much dexterity of manipulation, considerable practice, some experience and great care, the student must not be discouraged by disappointments and failures, but persevere until success is attained. It is advisable to practise with cold aqueous fluid media, and this operation being successfully accomplished, to go on to injections with the warm gelatine "mass." It will be found best, also, to commence by injecting the organs of animals, such as the kidney, liver, lung, large and small intestines, &c., of the cat, the sheep, or the pig, the eye of an ox (from the artery), and by complete injections (from the aorta), of small animals, e.g., rat, guinea-pig, rabbit and frog, before attempting to inject a whole animal. The kidney should be singly injected with carmine mass through the artery, and a double injection should also be made with carmine from the artery, and with blue from the vein. The liver in like manner may be injected from the portal and hepatic veins and from the bile ducts (thus ligature the common bile duct, remove the fundus of the gall bladder, remove as much bile as gentle pressure will force out, affix the cannula, and inject). In injecting isolated organs removed from the body of an animal, the greatest care must be taken to tie all divided vessels, and to use no more than the necessary pressure upon the piston of the syringe. The slower and more gradual the flow of the fluid, the more perfect will be the injection. All animals, or " parts " to be injected with a gelatine mass must be placed in warm water of a temperature from 40 to 45 C. during the process. ON INJECTING BLOOD VESSELS, ETC. 71 To INJECT AN ANIMAL WITH A GELATINE " MASS " BY THE SYEINGE. Arrange so that they may be ready to hand the necessary instruments and apparatus, viz., the syringe, thoroughly cleaned and tested for work, with its stopcocks and cannulae, scalpels of several sizes, a large and a small pair of scissors, dissecting forceps, " bull-dog " forceps (for clamping leaking vessels), a curved needle threaded with silk or thread for tying vessels, a wash-bottle of a capacity of from half a pint to a pint. The injection mass is to be melted by standing the vessel containing it in hot water, and it is to be kept fluid in a water-bath of a temperature from 40 to 45 C. Chloroform the animal (say a rabbit) which is to be injected, and make an L-shaped incision into the thorax so as to expose the heart and aorta. This is done by cutting upwards along the middle line of the sternum nearly as far as the root of the neck, then making a second incision at right angles to this, to the rabbit's left. A triangular flap is thus made and the heart, enclosed in the pericardium, exposed. Having cut through the pericardium, seize the apex of the heart with a pair of forceps and snip it off. The right and left ventricles are thus opened, and the animal instantly bleeds to death. The opening in the right ventricle, leading to the pulmonary artery, has a crescentic, slit-like appearance, whilst the opening in the left ventricle, leading to the aorta, is circular. Insert the cannula into the aorta and tie it in securely with a silk ligature. Wash all blood from the cavity of the thorax in order to ensure the cleanliness of the 72 METHODS OF MICROSCOPICAL RESEARCH. water-bath. Now place the rabbit into a warm water-bath of a temperature of from 40 to 45 C., and let it remain for from 10 to 15 minutes, i.e., until thorougly warmed. The entire abdomen may be carefully opened along the middle line to allow the warm water to surround the internal organs, and to ensure the equable flow of the injected fluid into all parts of the body. When the animal is thoroughly warm the syringe, which should also have lain for some time in warm water is to be carefully filled with the injecting fluid, in which its nozzle is to be entirely immersed, whilst the piston is worked several times to ensure the expulsion of all air. It is then placed in the stopcock, which should, however, first be filled with fluid from the syringe to prevent the introduction of any air, and which has already been inserted into the neck of the cannula to receive the nozzle of the syringe, and which, stopcock, is to be turned off each time it may be necessary to refill the syringe ; now with a gentle equable pressure the fluid is to be injected into the animal until the entire system of blood vessels shall be filled, but not distended. The operator from time to time examines the head, opens the eyes and parts the lips of the animal to watch the progress of the work. If the fluid is flowing properly, the mouth will almost immediately exhibit signs of the colour with which the injection is being made, and which will first be seen in the gums of the lower incisor teeth. When the eyeballs show the finest lines of colour traversing them, the capillaries may be considered to be filled and the injection complete and perfect. The animal is now to be put into cold water in a ON INJECTING BLOOD VESSELS, ETC. 73 large vessel, and placed under a tap which is to be allowed to drip slowly upon it for two hours; it may then be dissected and the organs placed in 70 per cent, methylated spirit for twenty-four hours and then transferred to strong methylated spirit in which they are to be preserved. If it is desired to inflate the lungs, remove them entire; very carefully tie all vessels and let them remain in the warm water. Melt together and keep fluid in a warm water-bath, two parts of cacao butter and one part of lard, suspend the lungs in the water-bath, insert a funnel into the trachea, and pour the butter and lard gradually into the lungs until they are fully inflated but no more great care must be exercised not to over-distend them. Now place them in cold water and after- wards into spirit as already described. Dr. Fearnley has devised an elaborate and most effective modification of Ludwig's constant pressure apparatus for making injections, which he fully describes in his " Practical Histology," 1 and which ensures such constancy, accuracy and delicacy in the pressure upon, and flow of, the injecting fluid, that there can be no comparison between its almost automatic action and that of the syringe worked by a piston. This apparatus consists of a bath, having a shallow part to receive the animal to be injected, and a deeper part for the vessel containing the injection fluid to stand in. A large (40 ounce) Wolff's bottle, with three necks, is fitted with three 1 " A Course of Elementary Practical Histology," by William Fearnley, p. 70. 74 METHODS OF MICROSCOPICAL RESEARCH. perforated india-rubber stoppers. A glass tube reaching nearly to the bottom of the bottle passes through the central stopper; each of the other stoppers is fitted with a glass tube projecting but little beyond the stopper into the bottle, but pro- jecting sufficiently above the stopper to admit of a piece of india-rubber tubing, of the diameter used for infant's feeding bottles, being fixed upon it. A basin, or still better, a water-bottle, of a ON INJECTING BLOOD VESSELS, ETC. 75 capacity of a litre or more, a mercurial manometer, a Higginson's syringe, a spirit lamp, or a Bunsen's gas burner, some glass and brass cannulae, and some india-rubber tubing, two clamps (one for the pressure tube, the other for the delivery tube), will also be required. To make an injection of an animal with a gelatine mass, proceed as follows : Fill the water-bath with water of a temperature of from 40 to 45 C. Keep it at this temperature by placing a spirit lamp or a Bunsen gas burner under the bath. Melt the gelatine mass and pour it, through flannel, into the two-stoppered Wolffs bottle. Place this bottle in the place provided for it in the bath the bottle should be weighted to prevent its floating as the fluid is withdrawn from it. Arrange the manometer, pressure bottle, syringe and basin (or water-bottle), as shown in illustration. Now chloroform the animal to be injected, and pro- ceed as already described ; affix the cannula, tie it securely into the aorta, and after washing away all blood from the thorax, &c., place the animal in the water-bath. The animal must be submerged in the water, but not too deeply. Clamp the delivery tube of the bottle containing the injection fluid and, with the syringe, pump into the pressure-bottle from the basin or water-bottle sufficient water to raise the mercury in the manometer one inch; remove the clamp from the delivery tube, allow the air to escape from it, and immediately connect it with the canuula. The tube must remain submerged whilst the connection is being made. Now keep up an equable pressure by means of the syringe, and when the mercury in the manometer shall have risen to 76 METHODS OF MICROSCOPICAL RESEARCH. from 4 to 5 inches, it will be found that the animal is completely injected. The head must be examined from time to time, as already described, so that the rising colour of the lips, gums, tongue and eyes may be watched. The animal is now removed to cold water, and treated as directed above. This method of applying pressure is wonderfully delicate and equable; it will be found that whilst the mercury in the manometer can be raised almost imperceptibly, one entire compression of the barrel of the syringe raises it 1 inch. 77 CHAPTER V. On Staining Fluids, and Staining. THE value of the various staining processes consists in the perfection with which the differen- tiation of the tissues is accomplished, and their features and structure brought out. In the stain- ing of tissues it is all important that the nuclei of the cells shall be deeply stained, whilst the cellular structure is clearly defined. Staining is an art, requiring much experience and great delicacy of manipulation. It is one thing to colour a section or a tissue, quite another to stain, and especially to double-stain and differen- tiate it. In double and triple staining the great desideratum is to secure a thoroughly good nuclear stain as the ground colour, and to supplement this with a delicate tint, or tints, which shall prove a good contrast to the first stain and thoroughly differentiate the tissue without diffuseness. For staining the nuclei of the cells, for beauty and permanence, no staining fluids have yet been discovered to surpass haematoxylin, carmine and picro-carmine. The brilliancy of carmine has its advantages in many cases, whilst the delicacy, the 78 METHODS OP MICROSCOPICAL RESEARCH. beautiful gradations of tints, from the deepest violet to the most delicate blue, which by careful treat- ment are produced by a good logwood stain, give a sense of relief to the eye during prolonged observa- tions which no other tints, perhaps, afford. No more beautiful stainings have been produced than those obtained by a prolonged immersion (say for twelve hours) in a logwood solution, made according to the first formula given below, and diluted to a light violet colour, which after the sections so stained have lain in tap water for twenty- four hours will result in a most beautiful blue ground colour with the nuclei of a darker blue. Let them then be placed in spirit for half an hour, and then in a stain made from 2 parts of a saturated alcoholic solution of rubin and 1 part of a saturated alcoholic solution of yellow eosin diluted with alcohol, until it becomes of a delicate pink shade, and in which the sections may be kept until they are " cleared " and mounted. The sections thus treated will be found to be distinctly triple- stained in blue, crimson and orange colour without diffuseness and with perfect differentiation, and if mounted in " xylol balsam' 9 they will prove quite permanent. Carmine and picro-carmine also are invaluable as nuclear stains, whilst for many tissues, and especially for most vegetable tissues, they are preferable to logwood, and, when mounted in a suitable medium, sections stained with them prove equally beautiful and permanent preparations. A carefully selected list of formulas for the most important and reliable staining re-agents is here appended, the proper media in which sections ON STAINING FLUIDS, AND STAINING. 79 stained by them should be mounted being given with each formula. HJIMATOXYLIN STAINING FLUIDS. 1. Haematoxylin (Crystals), 1 gramme. Alum powdered, 10 grammes. Distilled Water, 100 c.c. Alcohol, 5 c.c. A small piece of Camphor. This is a splendid nuclear stain. It does not become active until from two to three months after it is made. It improves by keeping, and remains perfectly good for two or three years. It must be filtered each time it is used, and may be used over and over again. Its full strength is too strong for use ; lor rapid staining, i.e., in from five to ten minutes, add to it an equal volume of distilled water. For slow staining, dilute it until its colour becomes a light violet, and leave the section in the stain thus diluted for six to twelve hours. Over- stained sections can be decolourised to any desired tint in distilled water 5 parts, acetic acid 1 part. After staining and, if necessary, decolourising the sections, place them in tap water for twenty- four hours, and they will be found of a beautiful blue tint. Mount in xylol balsam. 2. EHRLICH'S FOEMULA. (a) Haematoxylin, 1 to 2 grammes. Absolute Alcohol, 100 c.c.m. (6) Glycerine, 100 c.c.m. Distilled Water, 100 c.c.m. Glacial Acetic Acid, 5 c.c.m. Alum Sulph. (powdered), to saturation. Mix the two solutions. 80 METHODS OF MICROSCOPICAL RESEARCH. Allow it to stand for about six weeks, occasionally shake it, when it will have obtained its greatest depth of colour, then filter it once for all. It will then stain in about half an hour or less and does not overstain. But better results are obtained by adding a few drops to an ounce of distilled water and leaving the sections therein for from three to four hours. It keeps well, and as it gets older, stains more rapidly. If its action becomes too rapid or diffuse, add more acetic acid. It may be used over and over again. Mount in xylol balsam. 3. WEIGERT'S FORMULA. (a) Hsematoxylin (Crystals), 1 gramme. Absolute Alcohol, 10 c.c. Distilled Water, 100 c.c. (b) Ferridcyanide of Potassium, 2| grammes. Borax (powdered), 2 grammes. Distilled Water, 100 c.c. Place the sections in absolute alcohol for one hour then in solution (a) until they become quite black then place them in distilled water for a few minutes. Decolourise in solution (6) until they are of a dark brown. This does not keep good and active beyond a few months. Filter each solution every time it is used, and it may be used several times. Mount in xylol balsam. This is a very fine nuclear stain, and brain, spinal cord, nerves, &c., are superbly stained by it. It is also a splendid stain for micro-photographic purposes, as the beautiful shades of brown which carefully stained sections exhibit give very fine photographic results. Any animal or vegetable ON STAINING FLUIDS, AND STAINING. 81 sections, of which it is desired to produce lantern slides or micro -photographs, may be stained by this process. Mount in xylol balsam. 4. KLEINENBERG'S FORMULA. (a) Prepare a saturated solution of chloride of calcium in alcohol (of 70 per cent.), add a small quantity of powdered alum, filter, mix 1 part of this with 8 parts of 70 per cent, alcohol. (&) Make a saturated solution of haematoxylin crystals in absolute alcohol. When using this for staining purposes add to a small quantity of solution (a), in a watch glass or saucer, sufficient of (b) to give it the desired tint. This is a very fine nuclear stain, and permanent, but the stain itself will not keep very long ; all sections or tissues to be stained in it must be thoroughly soaked in distilled water so that no trace of acid may remain in them. Slow staining in a very dilute solution gives the best results. Mount in xylol balsam. 5. MITCHELL'S FORMULA. Take of finely ground Logwood, gii. "Alum Potash, 5ix. Glycerine, fl. %iv. Distilled Water, quant, sun . Moisten the logwood with water until it is quite damp. Place it in a large funnel with filter paper and then pass ordinary water through it untilj the liquid runs away almost colourless. When all the water has drained from it, place the logwood on a large dish to dry. Dissolve the alum in 8 ounces of distilled water, 6 82 METHODS OP MICROSCOPICAL RESEARCH. place the logwood in a vessel of sufficient size and pour the alum solution upon it, allow it to remain for forty-eight hours, stir it occasionally, strain it off; add the glycerine and filter. Add 5 per cent, of absolute alcohol to it, and keep it in a thoroughly well stoppered bottle. For rapid staining this fluid may be used full strength, but better results will be obtained from prolonged immersion, say for twelve hours, in a weak solution. This stain will give a delicate violet tint to the sections with beautiful clearness and differentiation. Mount in xylol balsam. Carmine Staining Fluids. 1. AMMONIA CARMINE. (Dr. Heale's Formula.) Carmine, 10 grains. Liquor Ammonia for t., B.P., | drachm. Price's Glycerine, 2 ounces. Distilled Water, 2 ounces. Alcohol, | ounce. Dissolve the carmine in the ammonia by means of heat. Boil for some seconds and allow it to cool. Let the ammonia evaporate for an hour or more, and when the odour is nearly gone, add the glycerine, water and alcohol, and filter. When, from keeping, the carmine is precipitated, add one or two drops of ammonia. It may be used full strength or diluted with alcohol, which will increase its penetrative power. This is a fine, nuclear stain. Mount in xylol balsam. 2. RANVIER'S FORMULA. Distilled Water, 100 parts. Ammonia, 1 part. Carmine, 1 part. ON STAINING FLUIDS, AND STAINING. 83 Rub up the carmine in a mortar with a small quantity of the water, add the ammonia, and when the carmine is dissolved, add the remainder of the water. If there is an excess of ammonia, use heat until the carmine begins to be precipitated. Mount in xylol balsam. 3. HUXLEY AND MAETIN'S FORMULA. Carmine, 2 grammes. Liquor Ammoniae/or., B.P., 4 c.c. Distilled Water, 48 c.c. Dissolve the carmine in the ammonia and water. Place it in a bottle without a stopper until all odour from the ammonia has nearly passed away. Keep it in a thoroughly good stoppered bottle. When used it should be diluted with from fifteen to twenty times its bulk of water. Mount in xylol balsam. 4. BORAX CAEMINE FLUID. (Foster and Balfour's Formula.) Carmine, 3 grammes. Borax, 4 grammes. Distilled Water, 97 grammes. Mix, apply heat with constant stirring until the liquid nearly boils, set it aside to cool and when cold, add Alcohol (Eectif.), 70 c.c. Distilled Water, 30 c.c. Shake it up thoroughly, let it stand for thirty-six to forty-eight hours, and filter it. Mount in xylol balsam. Specimens stained in these carmine fluids may be 84 METHODS OF MICROSCOPICAL EESEAECH. rendered more brilliant, and any diffuseness re- moved by immersion in Hydrochloric Acid, 1 per cent, in Distilled Water, 1 part. Alcohol (Rectif.), 2 parts. PICEO-CAEMINE STAINING FLUIDS. (Gage's Formula.) 1. Take of carmine and of picric acid equal parts by weight. Dissolve the picric acid in 100 times its weight of distilled water. Heat may be used if necessary. Dissolve the carmine in 50 times its weight of liquor ammonia fort., B.P. Mix the two solutions, filter through thick filter- paper, evaporate the filtered liquid to dryness, dissolve the filtrate in 100 times its weight of distilled water. Filter until a perfectly clear solu- tion results. Then add to each 100 c.c. of the solution, 25 c.c. of Glycerine (Price's). 10 c.c. of Absolute Alcohol. This will keep perfectly well, but should be filtered at intervals of a few months. 2. WEIGEET'S FOEMULA. Soak 2 grammes of carmine for twenty-four hours in 4 grammes of ammonia. Place it under a glass shade to prevent evaporation, add 200 grammes of a very strong solution of picric acid, and replace it under the glass shade for a second period of twenty-four hours. Then add, very gradually, acetic acid until precipitation com- mences. Again put it aside for twenty-four hours. Filter, then add ammonia in small quantities drop by drop at intervals of twenty-four hours until the ON STAINING FLUIDS, AND STAINING. 85 solution becomes clear. If this fluid when used overstains yellow, add acetic acid, if red, add ammonia. 3. PiCRO-LiTHiuM CARMINE. Dissolve 2J grammes of carmine in 100 c.c. of a saturated solution of lithium carbonate, add 2 to 3 c.c. of a saturated solution of picric acid. Filter. All preparations stained with picro-carmine may be mounted in xylol balsam, but Farrant's medium is preferable. PICRO-CARMINE AND EOSIN. (Lang's Formula.) Mix 50 parts of a 1 per cent, picro-carmine solution with 50 parts of a 2 per cent, aqueous solution of eosin ; after prolonged immersion for from twelve hours to forty-eight, or even seventy- two hours wash out the picrin in repeated changes of alcohol, until the eosin ceases to colour the spirit. This is a very fine double stain. Mount in xylol balsam. STJLPH-INDIGOTATE OF SODA. Make a saturated solution of sulph-indigotate of soda in distilled water, and to each ounce thereof add from 3 to 6 minims of rectified alcohol, filter, and keep in a closely stoppered bottle. It is better to make small quantities of this fluid as required, as it does not keep very long. This stain is suit- able only for fibrous tissues skin, scalp, hair, and for epitheliomatous growths. Mount in xylol balsam. 86 METHODS OP MICROSCOPICAL RESEARCH. Cochineal Staining Fluid. ALCOHOLIC COCHINEAL. (Mayer's Formula.) Macerate cochineal powder in 70 per cent, alcohol for some days, in the proportion of 10 c.c. alcohol to 1 gramme of the cochineal. Stir frequently and filter. Saturate the specimens before staining them with 70 per cent, alcohol, and use alcohol of that strength for all the processes connected with the use of this stain, and for diluting it. After staining the speci- mens, wash them in repeated changes of the alcohol until they cease to tint it. ALUM COCHINEAL. (Czoker's Formula.) Pulverise in a mortar, cochineal 7 grammes, and dried alum 7 grammes; add 700 c.c. of distilled water, and boil down to 400 c.c. When cool add 1 per cent, of carbolic acid crystals ; filter several times. It will remain fit for use for about six months, when it must again be filtered, and a small additional quantity of carbolic acid added to it. Use it full strength, it does not over stain how- ever long the specimens are left in it. The specimens must be immersed in it for at least two hours, and may require to remain immersed from three to six hours. It acts as a double stain, colouring the nuclei violet and the tissues red. ANILIN BLUE BLACK. Dissolve 1 decigramme in 4 c.c. of distilled water, to this add 100 c.c. of rectified alcohol, and keep the solution in a well- stoppered bottle. ON STAINING FLUIDS, AND STAINING. 87 Stain the specimens in this fluid much diluted with spirit. The stained specimens may be kept, to be mounted as required, in spirit to which a small quantity of this fluid has been added. This is a most valuable stain for brain, spinal cord, nerve centres, and the nervous system gener- ally, also for bone and cartilage. Mount in xylol balsam. Anilin Dyes as Staining Fluids. These are valuable for many purposes, and have many useful properties. They stain rapidly, with great clearness and brilliancy, and are especially useful as double and triple stains in conjunction with haematoxylin or carmine. No more beautiful or effective staining process has been discovered than that in which the speci- mens, having been stained in hsematoxylin fluid subjected to the action of " tap " water for twenty- four hours, until they have acquired a beautiful blue tint, are afterwards stained in a weak solution of 2 parts f uchsin and 1 part yellow eosin. This process gives a clear and pure triple stain. The student will find by experiment many equally useful and beautiful combinations of these dyes. It has always been affirmed that most of the anilin colours are more or less, and indeed, more rather than less, fugitive, but the author ventures to affirm as the result of his long experience that if the necessary precautions are taken there is no reason whatever why specimens stained with anilin colours should not remain as permanent as any others, and he has in his collection preparations so 88 METHODS OF MICROSCOPICAL RESEARCH. stained, made twenty years ago, which are as per- fect as on the day they were mounted. Since xylol has been introduced anilin stains may be used with even greater confidence, as that pre- paration tends rather to preserve than to destroy the anilin colours, which are undoubtedly adversely affected by benzol. Xylol, therefore, should be used to clear the specimens before mounting them, and Canada balsam, which has been thoroughly hardened and re- dissolved in xylol, should be used as the mounting medium. If the specimens, having been cleared in a mixture of equal parts of xylol and absolute phenol, are mounted in " xylol balsam, 5 ' brilliant clearness will result, and the preparations will prove absolutely permanent. The author has found it most convenient to make saturated solutions in alcohol of the anilin colours given in the appended list, and to stain the specimens slowly in weak solutions, either alcoholic, made by adding a few drops of the saturated solution to alcohol, or aqueous, made by adding a small quantity of the stain to distilled water. The advantage of using alcoholic solutions is that when the dilute stain has been found to give the desired tint it may be preserved for use over and over again. It must be filtered each time, and as its colour becomes lighter, must be restored to its original tint by the addition of a few drops of the " stock " solution. The following will be found the most generally useful of the anilin colours and sufficient for all purposes. Anilin Blue. Nicholson's Blue. ON STAINING FLUIDS, AND STAINING. 89 Soluble Blue. Bismarck Brown. Chrysoidin. Eosin (Red). Eosin (Yellow). Fuchsin. Gentian Violet. Acid Green. Iodine Green. Magenta. Methyl Green. Methyl Violet. Orange. Rubin. Saffranin. Spiller's Purple. Vesuvin. Bismarck Brown. Make of this (and of all the above) a saturated solution in alcohol (all these solutions should be filtered at intervals of a few weeks). Add a few drops to a small staining saucer full of spirit, and place the specimens in it for a few minutes. Remove them to alcohol, clear in xylol and phenol and mount in xylol balsam. If it is desired to mount the specimens in Farrant's medium, or glycerine, wash them first in alcohol, then in distilled water. Bismarck Brown does not over- stain, nor does it wash out readily either in water or alcohol. It is suitable for bacteria and micrococci, and forms a very good double stain in conjunction with other colours : its brown tints render it a good stain for preparations intended for micro-photo- graphy. Eosin. Both red and yellow eosin are most use- 90 METHODS OF MICROSCOPICAL RESEARCH. fill stains either singly or in conjunction with other staining fluids, and especially with hgematoxylin ; whilst, as before stated, yellow eosin combined with fuchsin gives, with hsematoxylin as the ground stain, a precise and beautiful triple stain. Eosin has great staining and especially penetrat- ing powers, and specimens may remain in a weak alcoholic solution for an indefinite time without fear of their becoming over-stained. No trace of acid must remain in any medium in which eosin stainings are mounted ; but preparations mounted in xylol balsam are practically permanent. Epithelium is beautifully stained by eosin and the blood of the frog or triton double stained with hsematoxylin and eosin forms a superb preparation. Iodine Green. Add to distilled water, in a staining saucer, sufficient of the saturated solution in alcohol to give it a fairly deep tint. Place the specimens in distilled water for a short time before staining them. Iodine green stains very rapidly, and in many cases instantaneously. Wash them in water, dehydrate in absolute alcohol, clear in xylol and phenol, and mount in xylol balsam. Stained specimens may remain in a weak alcoholic solution of this dye indefinitely and be mounted as required. Methyl Green. This is an intense nuclear stain, and Dr. Curschmann states that it has "a peculiar affinity for amyloid substance, colouring it an intense violet; whilst surrounding tissues that have not undergone degeneration are stained green or bluish green." Methyl Violet is also a valuable stain for tissues which have undergone amyloid degenera- tion : the amyloid substance is stained red, and the unaffected parts violet. ON STAINING FLUIDS, AND STAINING. 91 Orange and Rubin. These combined, in about the proportion of 1 part of the former to 2 parts of the latter, give, in conjunction with haema- toxylin as the ground stain, a beautiful triple stain. Saffranin is a good nuclear stain, imparting a beautiful rose tint to the tissues, and staining the nuclei a very deep red. Spiller's Purple is also a good nuclear stain, and is a very restful colour for the eyes. Vesuvin, in conjunction with anilin blue, is very effective as a stain for bacilli and bacteria. Specimens stained with anilin dyes must not be cleared in oil of cloves or cedarwood oil, but exclu- sively in xylol and absolute phenol in exactly equal proportions. This medium clears almost instantane- ously and perfectly, having also a preservative rather than a destructive effect upon the anilin colours of which it increases the brilliancy ; whilst, moreover, the xylol balsam, in which they should be mounted, exercises a preservative effect upon the preparations, and which it renders practically permanent. (Haematoxylin crystals, and the anilin dyes, are supplied of the finest quality by Dr. G. Griibler, of Leipzig, and can be obtained from his agent, Mr. C. Baker, 244, High Holborn, London, W.C.) The Staining of Bacilli and Bacteria. To stain bacillus tuberculosis : FORMULA No. 1. 1. 1^ Fuchsin, 1 gramme. (a) Absolute Alcohol, 10 c.c. Carbolic Acid, 5 grammes. (b) Distilled Water, 100 c.c. 92 METHODS OF MICROSCOPICAL RESEARCH. Mix the two solutions. (This remains active for about two months.) 2. Anilin (or Methylene) Blue, 2 grammes. Absolute Alcohol, 15 c.c. Distilled Water, 85 c.c. (This will keep a long time.) Spread a thin layer of sputum on a thin glass cover, dry, and fix it over a spirit lamp or Bun sen's gas burner until it commences to give off vapour; then float the cover, sputum downwards, upon the surface of a small quantity of fluid (1) in a staining saucer ; leave it for fifteen minutes. In the case of sections, immerse them in the fluid for twenty minutes. Now decolourise in 1 part of sulphuric acid to 7 parts of distilled water, until all colour has dis- appeared. Wash away all trace of the acid in distilled water, when a slight tinge of colour will become perceptible. Float the cover upon, or immerse the sections for a few minutes in, solution 2. Wash in distilled water, place in absolute alcohol for three or four minutes. Let the cover become perfectly dry, and mount in xylol balsam. In the case of sections dehydrate them in absolute alcohol, clear them in xylol and phenol, and mount them in xylol balsam. (The xylol in which the balsam is dissolved will clear the sputum on the cover.) 2. DR. HENEAGE GIBBES' FORMULA. 1^ Magenta Crystals, 2 grammes. Pure Anilin, 3 grammes. Absolute Alcohol, 20 c.c. Distilled Water, 20 c.c. ON STAINING FLUIDS, AND STAINING. 93 Float a thin glass cover upon which sputum has been spread or immerse sections, as in the former process, in this fluid, for twenty minutes. Decolorise in a 33 per cent, solution of nitric acid in distilled water, wash free from acid and float the cover upon, or immerse the sections for a few minutes in, a saturated solution of chrysoidin in distilled water ; when they are stained brown, wash them in distilled water, place them in absolute alcohol for a short time. Clear the sections in xylol and phenol, and mount in xylol balsam. If a small crystal of thymol is dissolved in absolute alcohol and added to each of the solutions in the above two formulas it will tend to their preservation. Each of these solutions should be filtered into the staining saucers when used. EHELICH'S PROCESS. Make a saturated solution of phenylamin in distilled water. This is done by prolonged shaking up the water with the floating anilin and filtering ; add to the filtered mixture a saturated solution of fuchsin in absolute alcohol until a brilliant red colour is obtained. Now, as in the former processes, spread a thin layer of sputum upon a thin glass cover and dry, and fix it over a spirit lamp or Bunsen's gas burner. Float the cover upon the fuchsin solution for from fifteen to twenty minutes, wash it in distilled water and float it upon a 33 per cent, solution of nitric acid in distilled water for a few seconds, when it will be found quite colourless. Now float the cover for a few minutes, on solution No. 2, of formula No. 1. Wash in distilled water, dehydrate in absolute alcohol, mount in xylol balsam. 94 METHODS OF MICROSCOPICAL RESEARCH. WEIGERT'S PROCESS for Schizomycetes. Bacilli, Bacteria and Micrococci. 5t A 2 per cent, solution of Gentian Violet in Distilled Water, 12 c.c., and a Saturated Aqueous Solution of Anilin oil (filtered) 100 c.c. Stain the sections in this. Make a Solution of Vesuvin, 1 gramme. Eectified Alcohol, sp. gr. 830, 10 c.c. Distilled Water, 100 c.c. Stain in this for fifteen minutes. Clear in xylol and phenol. Mount in xylol balsam. REV. GORDON THOMPSON'S PROCESS. The special feature of this valuable process con- sists in fixing the bacilli, bacteria or micrococci, after staining them with the ground colour, by subjecting them to the action of a solution of gold made by dissolving 15 grains of gold chloride in 4 ounces of distilled water. This solution is to be poured upon the cover, on which the bacilli have been spread and allowed to remain upon it for two or three seconds and then poured off again, the cover is then to be washed in distilled water. The gold appears to form a tube round the bacteria and so encased, they appear to be rendered absolutely permanent ; preparations, in the author's possession mounted by this process twelve years ago, remaining as brilliant in colour as the day they were made. For the ground stains Methyl violet, fuchsin and acid green are used, the covers being floated upon the solution, washed in distilled water, then treated with the gold solution, and again washed in distilled water. ON STAINING FLUIDS, AND STAINING. 95 For the second stains use Acid green, Bismarck brown or chrysoidin ; wash in distilled water. Mount in pure glycerine jelly containing no trace of acid. The best contrasts of colour will be found to be Fuchsin followed by acid green. Fuchsin followed by anilin (or soluble) blue. Bismarck brown followed by anilin (or soluble) blue. Methyl violet followed by acid green. Clearing Media. Specimens which are to be mounted in Canada balsam or any other resinous media must be pene- trated by a " clearing " agent, which shall render them perfectly transparent and remove from them all traces of the alcohol in which they have been dehydrated or kept. It is now generally admitted that hardened balsam, which has been re-dissolved in xylol is the best and most reliable preparation of that most useful, valuable and permanent mount- ing medium. It is obvious that a clearing agent which consists in great measure of the solvent of the balsam must be peculiarly adapted to precede the balsam, and it may at once be said that the best, the cleanest in its application, and the easiest to use, consists of a mixture of exactly equal parts of xylol and absolute phenol. The manner of its use is simplicity itself, whilst its penetrative and " clearing " actions are practically instantaneous. Much has been said and written about elaborate processes for running the 96 METHODS OF MICROSCOPICAL RESEARCH. clearing agent under the sections, letting them sink through alcohol into the clearing agent placed at the bottom of a test tube or saucer, with alcohol above it, raising the sections with " lifters," &c., but this complicated process tends to the destruc- tion of the sections, which cannot be too little manipulated. Now, in xylol and phenol we have a medium which requires no such treatment, all that is necessary being to place the fluid in a saucer to the depth of about a quarter of an inch. The saucer should be a large one, so that there shall be no difficulty in removing the sections, or any necessity for dragging them up the side of the saucer. Place the sections for five minutes in absolute alcohol, transfer them, with a thick curved needle which has a blunt point, to the surface of the clearing fluid. The alcohol is almost instantly removed and the sections sink to the bottom. Then take a perfectly clean cover, and with the curved needle float a section carefully on to the cover and spread it perfectly flat, drain 'the excess of the fluid on to a piece of blotting paper, by holding the cover in forceps and placing its edge upon the blotting paper, then lay the cover section upwards upon the blotting paper to remove the clearing fluid from its under surface, and before the clearing fluid evaporates apply xylol balsam to the section. The balsam being miscible with the clearing fluid, there is nothing antagonistic or greasy to remove, which is the case when oil or turpentine, or both, are em- ployed as clearing agents. This medium is the best that can be used for clearing sections cut from specimens which have been embedded in celloidin, upon which it has no solvent effect. ON STAINING FLUIDS, AND STAINING. 97 When, however, it is not desired to mount with the section its surrounding and supporting celloidin, Turpentine will be found the best solvent of that material as well as of paraffin when that has been used as the embedding agent. Turpentine should not be used for specimens or sections which have been hardened or preserved in alcohol or methylated spirit, as it has great shrink- ing power. It will be found very useful for entomo- logical preparations, and especially for insects with very hard integuments ; these must be left to soak in turpentine until they are cleared, but small and delicate insects can be better cleared in xylol and phenol. Turpentine is also a good clearing agent for vegetable specimens of a woody nature. All specimens or sections cleared by turpentine should be mounted in Canada balsam, hardened and re- dissolved in turpentine, and carefully filtered. Oil of Cloves has some advantages over all other clearing agents (xylol and phenol excepted), and is, perhaps, the most universally used. It combines well with both alcohol and balsam and does not shrink the specimens as turpentine does ; but "clearing" with this oil involves its removal by placing the sections in turpentine after they have been cleared, because if the clove oil remains in the specimens or sections, although it is miscible with the balsam, it prevents the balsam from hardening and so renders the preparations unsafe. Sections cut from specimens which have been embedded in celloidin and which it is desired to mount so embedded must not be cleared in oil of cloves, as it dissolves the celloidin at once ; it is therefore a good clearing agent when it is desired to 7 98 METHODS OF MICROSCOPICAL EESEARCH. remove celloidin. It must be remembered that oil of cloves renders specimens which have been allowed to remain in it very brittle. Oil of Bergamot is a valuable, though costly agent ; it clears the celloidin embedded specimens and does not affect anilin stains to any great extent. It is fairly rapid in its action and clears well. Cedar Wood Oil. This oil is not expensive, but is very slow in its action, it also has the valuable properties of clearing perfectly, however slowly, and of not injuriously affecting anilin dye's, or of shrinking specimens which have been hardened or preserved in alcohol. It is very useful for clearing vegetable sections, though extremely slow in doing so, and celloidin sections may be cleared in it, but it takes several hours to render them transparent. Mounting Media and Cements. Many media in which specimens may be examined and delicate dissections made for examination, are not in the true sense of the term preservative and therefore serve only a temporary purpose. Many solutions again which re-act upon tissues, and elucidate their features and composition serve only, in like manner, for purposes of temporary examina- tion and study. It is intended, in the following list of media in which specimens and sections can be mounted, to give the formulae and methods of using and applying the best preservative media so as to secure the permanence and safety of the prepara- tions which are made with them. The list will be supplemented with formulae for the most useful fluids in which to make dissections and to place ON STAINING FLUIDS, AND STAINING. 99 tissues or specimens for temporary observation and examination only. Canada Balsam. The most universally used and useful of all mounting media is the oleo-resin obtained from Abies Balsamea and Pimis Canadensis and is mostly imported from Quebec. The balsam is obtained from the tree by puncturing the blisters, or vesicles, which form under the bark of the trunk or its branches, and collecting their fluid-contents in a bottle. It is at first opaque, but gradually clarifies and becomes transparent. It consists of 24 per cent, of volatile oil, 60 per cent, of resins soluble in boiling alcohol, and of 16 per cent, of resins soluble in ether, but insoluble in alcohol. As it hardens very slowly, it is, for histological purposes and for use as a mounting medium, generally evaporated to dryness and re-dissolved in either benzol or xylol, preferably in the latter. These solutions harden rapidly, are perfectly transparent, practically permanent and preservative. For some exceptional purposes, e.g., for specimens which require to be mounted in deep cells, pure, undiluted balsam will be found useful. In order to prepare Canada Balsam for histo- logical purposes it should be gently heated over a sand bath and thus slowly evaporated until it be- comes quite hard. Great care must be taken that the heat does not become too great so that the balsam is burned, in which case it becomes of a dark brown colour and is consequently spoiled. When quite dry and brittle it is to be reduced to a coarse powder and re-dissolved in an equal quantity of one or other of the solvents above named. For delicate entomological and some vegetable prepara- 100 METHODS OF MICROSCOPICAL EESEAECH. tions hardened balsam may be re-dissolved in turpentine all these solutions of balsam, whether in xylol, benzol or turpentine must be carefully filtered for use, and should be kept in wide-mouthed capped bottles. 1. GLYCERINE JELLY. 1^ Glycerine (Price's), 50 c.c. Distilled Water, 42 c.c. 1 Boracic Acid (Crystals), 2 grammes. Gelatine (Gold label), 6 grammes. 1 new laid Egg. Soak the gelatine in water for at least twelve hours. Pour off the water, and place it in a vessel with the distilled water. Melt it in a water bath. When melted, add the glycerine and acid. Remove the vessel from the water bath, beat up the white (only) of the egg, and thoroughly incorporate it with the fluid at a temperature of from 30 to 35 C. (not more). Replace the mixture in the water bath, and gently boil it until it has assumed a flaky appearance. Now filter it through the finest filtering paper, which must be done either in a warm oven, or with a hot filter. Keep it in a well stoppered stock bottle, and place some, for use, in a wide mouthed capped bottle. When used, the bottle should be placed in a bath of water sufficiently heated, and kept so, to thoroughly liquify the " Jelly." 1 Carbolic Acid (Crystals), or Formic Acid may be substi- tuted for this ingredient the former, indeed, is generally used but Boracic Acid does not affect hsematoxylin stainings, which are unquestionably sooner or later much injured by the other acids. ON STAINING FLUIDS, AND STAINING. 101 2. LAWRENCE'S FORMULA. Soak J Ib of best gelatine for some hours in cold water, pour off the water, and heat the gelatine until it melts. Let it cool, but whilst it still remains liquid, add to each (fluid) ounce a (fluid) drachm of white of egg. Boil gently until it becomes clear, filter it through fine flannel, and add to each ounce of the solution 6 drachms of a mixture of 1 part (Price's) Glycerine and 2 parts of Camphor water. 3. KAISER'S FORMULA. Place one part of finest " gold label " French gelatine in 6 parts of distilled water, let it soak for two or three hours, then add 7 parts of best glyce- rine, and for every 100 grammes of this mixture, L gramme of strong Carbolic acid. Warm for from ten to fifteen minutes, stirring all the time ; when all the flakiness has been removed, filter it. GELATINE AND HONEY. (JJeane's medium). fy Gelatine (Best " gold label "), 1 ounce. Honey, 5 ounces. Distilled water, 5 ounces. Eectified alcohol, | ounce. Creasote, 6 minims. Soak the gelatine in the water until it is quite softened. Boil the honey in a water bath, and when it is boiling add the gelatine and water. Now boil the mixture, let it cool slightly, then mix the alchohol and creasote together and add them. Filter through fine flannel. 102 METHODS OF MICROSCOPICAL RESEARCH. GUM AND GLYCERINE. (Warrant's medium). 1 1. Gum arable (Best picked), 4 ounces. Distilled water, 4 ounces. Glycerine, 2 ounces. Keep it in a well stoppered bottle with a lump of camphor in it. 2. Gum arable, 1 ounce. 2 Glycerine, 1 ounce. Distilled water, 1 ounce. Arsenious acid, 1| grains. Dissolve the arsenious acid in the water, and the gum in this mixture, without heat, add the glycerine and incorporate with great care to avoid the forma- tion of air-bubbles. Glycerine, mixed with distilled water, forms an admirable medium in which to examine tissues and specimens and make minute dissections. It is sometimes used thus diluted as a mounting fluid, but is not to be relied upon as a preservative medium, whereas undiluted glycerine, when once the crucial difficulty of sealing it up and preventing leakage from the preparation has been successfully achieved, may be considered not only unsurpassable as a mounting fluid in cases for which it is suitable, but entirely reliable as a preservative. The her- metically sealing of the glycerine under the cover, or in the cell, must be most carefully done by applying at least three coats of shellac cement to be followed by two or three coats of white zinc cement, the formulae for making which will be given in their places, and the method of applying them 1 Beale, page 68. 2 Micrographic Dictionary. ON STAINING FLUIDS, AND STAINING. 103 described in the chapter on Mounting. All tissues, sections, and specimens, to be mounted in pure glycerine, should be subjected to a prolonged immersion in glycerine and distilled water, the pro- portion of the glycerine to that of the water being gradually increased every day or two until pure has been substituted for diluted glycerine. Great care must be taken that the specimen shall be thoroughly saturated with the glycerine before mounting it therein. This soaking may be done either in watch-glasses or very small staining saucers, and many specimens or sections can thus be immersed together. In order to prevent the glycerine from absorbing moisture from the atmosphere, and to ensure clean- liness, the saucers should be placed under bell- glasses whilst the soaking is in progress. This process, moreover, prevents the serious shrinkage which too frequently results, especially with delicate tissues and specimens, which have been mounted in glycerine without previous saturation therewith. Tissues, sections, delicate marine animals, insects, &c., which are to be mounted in glycerine may be preserved therein until required. Glycerine undoubtedly affects logwood stainings adversely, but if slightly acidulated with acetic acid it does not attack 1 carmine or picro-carmine. Acetate of Potash. Make a saturated solution in distilled water, it may be so used or diluted if 1 Farrant's medium is preferable for the mounting of speci- mens stained by either of these. It is advisable to rather overstain and to soak the specimens in the medium for two or three days, in order to extract the superfluous stain before mounting them. 104 METHODS OP MICROSCOPICAL RESEARCH. necessary. This fluid should be allowed to flow under the cover by capillary attraction, and the mount be sealed up in from twenty-four to thirty- six hours afterwards. NAPTHA AND CREASOTE. (Dr. Beale's formula.) Creasote, 3 drachms. Wood-naptha, 6 ounces. Distilled Water, 64 ounces. Chalk, quant, suff. Mix the naptha and creasote together, and add sufficient chalk to produce a thick paste. When this has been mixed smooth, add gradually the water, thoroughly incorporating all the ingredients in a mortar, and let it stand in a lightly covered vessel for two or three weeks. Stir it occasionally. Then pour off the super-natant fluid, which, if necessary, filter. Finally, add some small lumps of camphor, and keep it in a well-stoppered bottle. GOADBF'S FLUID. Bay Salt, 4 ounces. Alum (powdered), 2 ounces. Corrosive Sublimate, 4 ounces. Distilled Water, 2 quarts. This is an admirable preservative for delicate marine specimens, all entoinostraca and allied organisms ; for larvae and some entomological specimens. Mounting Fluid for Algfie (of which it preserves the colour) and for fresh gatherings of diatomacese, desmidiaceae, &c. Acetate of Copper, 15 grains. Camphor Water, 4 ounces. Distilled Water, 4 ounces. Acetic Acid (glacial), 20 minims. ON STAINING FLUIDS, AND STAINING. 105 Mix and add Glycerine (Price's best), 8 ounces. Bichloride of Mercury, 1 grain. Mix thoroughly, and filter very carefully, Sty rax. A liquid gum which exudes from the bark of the Liquidambar orientate. This is used specially for the mounting of diatomaceae, and the method of its use will be described when, in the chapter on " Mounting," full instructions are given for cleaning and mounting these organisms. Styrax is soluble in alcohol, ether, benzol, and xylol, which last is its best solvent for microscopical purposes. The solution should not be too thick, and must be carefully filtered. Styrax is preferable to Canada Balsam as a medium in which to mount diatoms, its refractive index being greatly higher than that of Balsam, whilst it is equally safe and durable. Monobromide of Naphthalin is soluble in alcohol and ether ; its high refractive index- double that of Canada balsam renders it a valu- able mounting medium for diatomacese. The cover should be " ringed " with wax before any cement is applied to secure it. Then two coats of shellac varnish, followed by white zinc cement, or, if the slides are to be used with oil immersion objectives, by the special cement of which the formula is given amongst the CEMENTS. Dammar Varnish. Gum dammar, dissolved in benzol, has been much used as a mounting medium ; its only recommendation, however, is that it is easy to apply. It is not safe, neither is it durable, and the author strenuously advises that 106 METHODS OF MICROSCOPICAL RESEARCH. it shall be entirely discarded for all purposes of mounting, and used only as a cement, in which character it has its uses, and especially it is good for " ringing " glycerine jelly and glycerine " mounts " before the application of the shellac varnish, and the finishing white zinc cement. Dammar varnish is made thus : 1^ Indian Gum Dammar, 4 drachms. Benzol (best) 8 (fluid). Let it stand for two or three days, then pour off the super-natant liquid, and add thereto 1 drachm of spirits of turpentine. Filter if necessary. Bell's Cement. 1 This is a thoroughly reliable cement for many purposes ; it is easy to use, as it flows readily from the brush and hardens rapidly. Shallow cells can be made with it, and it may be used for sealing up preparations mounted in fluids, glycerine, or glycerine jelly, in place of shellac varnish, the cover being previously " ringed" with the following : GELATINE CEMENT. (Marsh's formula.) Soak half an ounce of gelatine in water until it is thoroughly soft. Pour off nearly all the water, and melt the gelatine in a hot water bath ; add 3 or 4 minims of creasote and keep it in a capped bottle. It must be used warm, it sets very quickly, and may be followed by Bell's cement, or shellac varnish^ and the slides be finished with white zinc cement. 1 This cement must be purchased from its makers, Messrs. J. Bell & Co., 338, Oxford Street, London, or from the opticians ; its formula being a secret. ON STAINING FLUIDS, AND STAINING. 107 Asphalt Varnish. It is better to purchase this also. Mr. Kitton, however, finds that asphalt dissolved in benzol, and to which is added some gold size, answers its purpose well. Qold Size. This, again, should be purchased, as also should Marine Glue, which, as well as the following cement, is used for affixing cells to slips. G-UTTA PEECHA CEMENT. (Harting's formula.) Divide gutta percha into small pieces, and dis- solve it, by means of moderate heat, in 1 5 parts of oil of turpentine ; when dissolved, strain it through linen, and whilst the solution is kept at a gentle heat, stir into it one part of shellac. For affixing cells to the slips, heat the cement, make a ring on the slip the size of the cell, attach the cell and heat the slip until the cell is affixed by an even layer of the cement, so that no leakage may occur. Shellac Cement or Varnish. Break up shellac into very small flakes, and put these into a bottle with strong methylated spirit ; shake it up frequently, until there results a solution sufficiently thick to be used easily with a brush. When from keeping it becomes too thick, add more spirit. WHITE ZINC CEMENT. (Walmsley's formula), and given by him to the author. In order to ensure that the white oxide of zinc which is the foundation of this valuable and per- manent cement shall be of the best quality, and well ground, it is advisable to purchase a large tube of artist's "zinc white," rather than to grind for 108 METHODS OF MICROSCOPICAL RESEARCH. oneself, the ordinary white oxide of zinc in drying oil. The tube of zinc white is to be emptied into a large bottle, and as much as possible of the oil to be removed, by shaking up the white zinc in a con- siderable quantity of benzol, and when the zinc has settled to the bottom of the bottle, pouring it off. A saturated solution of gum dammar in benzol is to be made in another bottle, which should be placed in a water bath, kept hot, until the gum dammar is dissolved. "When this is accomplished the solution of gum dammar is to be poured upon the white zinc, the bottle thoroughly shaken up, and the mixture strained through fine muslin. Now add about half a drachm of best gold size to each ounce of the solution. If too thin, allow the benzol to evaporate until the cement becomes of a con- sistency to flow smoothly and readily from the brush if too thick, add benzol. This cement should be kept for use in a capped bottle with a wide mouth, and thoroughly stirred (not merely shaken) up each time it is used. White zinc cement properly made according to this formula, is tho- roughly reliable and safe. It never cracks, will stand ordinary wear and tear well, but not, of course, rough usage. The author's experience is, that no other white zinc cement than this is of any value or permanency. Preparations intended for use with oil immersion objectives must not be finished with this cement, as the essential oils used for immersion purposes at once attack and soon destroy it. Cement for finishing preparations to be examined with immersion objectives. Place five or six pieces of gum mastic, about the ON STAINING FLUIDS, AND STAINING. 109 size of a pea, in sufficient strong methylated spirit to dissolve them thoroughly. Soften some isinglass in water, remove all the water and dissolve it in two ounces of absolute alcohol. To this add two small pieces of gum ammoniacum rubbing them up in a mortar until they are dissolved. Mix the two solutions, and keep the cement in a wide mouthed stoppered bottle. When using it place the bottle in hot water until the cement is liquefied. Embedding and Freezing. The great desideratum in embedding specimens, for the purpose of making sections of them, is, that they shall be firmly and equably supported in all directions and that no distortion, displacement or injury to the tissues, organs or organisms embedded shall result. It is therefore most important that the student should acquire the knowledge and experience necessary to enable him to decide which process will afford and ensure the delicacy combined with solidity required in special cases. The methods of embedding are twofold, " simple " and " interstitial," the former consisting in enclosing the specimen to be sectionised in a soft substance which will consistently and equably support it in all directions ; whilst the latter involves the careful infiltration of the specimen with, and its thorough permeation by, a " mass " which, whilst warm, is liquid, and which solidifies as it cools ; or, as in the case of celloidin, the saturation of the specimen with that substance, held in solution by ether, 110 METHODS OF MICROSCOPICAL RESEARCH. which evaporating leaves the specimen perfectly embedded in a transparent and firmly gelatinous matrix. Lee, in his invaluable Text-Book 1 treats of the "theory of embedding" in a manner so exhaustive and practical that the author ventures to quote largely from his clever pages and tendering his best acknowledgments. Lee, then, says, after referring to the simplest methods of embedding : " A further object is proposed in the case of the other (i.e., the more elaborate) class of methods, which may be designated methods of interstitial embedding or infiltration methods. In these it is proposed to fill out with the imbedding mass the natural cavities of the object, in order that their lining membranes, or other structures contained in them, may be duly cut in situ, or, going a step further, it is proposed to surround with the supporting mass not only each individual organ, or part of any organ that may be present in the interior of the object, but each separate cell, or other anatomical element, thus giving to the tissues a consistency they could not otherwise possess, and ensuring that in the thin slices cut from the mass all the details of structure will precisely retain their natural relations of position. Such a process of embedding is at the same time practically a process of hardening, in so far as it enables us to give to tissues a degree of firmness that could otherwise only be obtained by the em- ployment of chemical processes, such as prolonged treatment with chromic acid and the like. The 1 "The Microtomist's Vade Mecum " (page 162). J. & A. Churchill. ON STAINING FLUIDS, AND STAINING. Ill principle of the methods of this second class is either, (like that of the first,) that by the immersion of the object to be cut in some material that is liquid whilst warm, and solid when cold, all the parts of the object may be duly surrounded by the supporting mass (the second class differing from the first chiefly in the employment of materials possess- ing greater power of penetration whilst liquid, in longer immersion in the liquid mass, and in such previous preparation of the object, by soaking in some liquid that is a solvent of the embedding material, as makes it more readily susceptible of infiltration by the latter) ; or the processes may be based on another principle, namely, that of the employment of substances which whilst in solution are suniciently fluid to penetrate the object to be embedded, whilst at the same time after the evaporation or removal by other means of their solvent, they acquire and impart to the embedded object sufficient firmness for the purpose of cutting. The collodion process suniciently exemplifies this principle. If a piece of soft tissue be dehydrated, and soaked first in ether and then in collodion, and if the ether be allowed slowly to evaporate, the tissue and surrounding mass of collodion will acquire a consistency such as to admit of thin sections being cut from them." Embedding in Carrot. This is a very primitive and almost exploded process, but it has its uses and advantages when cylindrical specimens, for instance, vegetable stems, are to be embedded ; it is also available for such tissues as can be rolled up and so enclosed in the centre of the carrot. A " well " microtome is, of course, neces- 112 METHODS OF MICROSCOPICAL RESEARCH. sary to this process, whilst a set of cork cutters, or of brass tubes sharpened at one end, must be provided ; one of these tubes must be of the exact diameter of the well of the microtome, and the others, progressively, of smaller diameters, so that the various diameters of the specimens may be matched. A sound carrot must be chosen and a cylinder cut out of its centre, with the largest tube, precisely to fit the well of the microtome, and of the same length as the depth of the well ; now, with a knife divide this cylinder into two equal halves, place the two halves together, and with the tube nearest in size to, but slightly smaller than, the specimen to be embedded cut out the exact centre of the cylinder; this will give two semi-circular pieces of carrot each with a semicircular groove, in one of which place the specimen, lay the other piece upon it, and the specimen is embedded in a cylinder fitting tightly into the well of the microtome, and sections can be cut, after practice, of a great degree of thinness, and without difficulty. Embedding in Pith. This substance is used in the same manner as the carrot. A cylinder of pith exactly the size of the well of the microtome is divided longitudinally into two equal halves, and the specimens embedded between the two halves, and if possible without the removal of any of the central pith. Pressure and "kneading" will generally be found sufficient to form a central tube, in which the specimen can be enclosed, with the advantage that no portion of the pith having been actually removed, the cells displaced by pressure will to a great extent recover their original position and exert the desired central pressure and give ON STAINING FLUIDS, AND STAINING. 113 the necessary " all round" support to the specimen. The embedding being accomplished, the cylinder is to be driven into the well of the microtome and moistened with alcohol, which will cause the pith cells to swell and firmly enclose and support the specimen, whilst if the cylinder should not already fit into the well with the necessary exactitude this swelling will ensure perfect tightness. Wax and Oil. Equal parts of white wax and olive oil are to be melted by heat over a sand bath. In warm weather, or in a hot room, the proportion of wax should be slightly increased ; in colder weather, or in a cool room, increase the proportion of oil. The mass must, of course, be melted when used. The specimens to be embedded should be immersed in alcohol and thoroughly dehydrated, and then placed for a few seconds in collodion, which being allowed to evaporate to dryness, the specimen is then embedded either in the well of the microtome or in a paper tray. The ring of collodion surround- ing the sections will be dissolved away by the clear- ing medium. Embedding in Paraffin. The infiltration with paraffin, and subsequent embedding of speci- mens therein, is a process of much nicety and requiring great care, and first, the selection of a suitable paraffin and the composition of the "mass" are all-important. Two paraffins with the different melting-points of 110 F. and 140 F. should be combined in such proportions as shall give a firm and homogeneous mass which, when after melting it has solidified, will cut perfectly smoothly in a tem- perature of from 55 to 70 F. It will be found in practice that two parts of the paraffin of the 140 8 114 METHODS OF MICROSCOPICAL RESEARCH. melting point, and one part of that melting at 110, will afford a mass which will answer perfectly well when cut in a temperature of 70 D . That with the lower melting point being combined in the propor- tion of one-fifth of its bulk with four- fifths of that of the higher melting point, will cut well in a tem- perature of 60 ; whilst equal parts of the two paraffins at 110 and 140 melting points respectively will give the most satisfactory results when cut in a temperature of 55 F. The paraffin is to be melted, in a water oven, in porcelain pipkins (having covers and handles), and the embedding may be done in paper or cardboard trays, but it will be found more convenient and much safer to use, according to the size of the specimen to be embedded, the small porcelain pans in which moist water colours are sold (these give nice little firm blocks requiring no trimming) ; for larger specimens, small porcelain vessels used in various chemical processes are readily procurable, whilst small glass vessels, or large (flat-bottomed) test-tubes will answer admirably for specimens of any considerable size. The specimens must be thoroughly hardened and perfectly dehydrated, and when about to be embedded are to be placed in strong alcohol, or, better still, in absolute alcohol, for a short time ; from this they are removed into creasote and left therein until they are well impregnated (say for six hours). Having removed as far as possible all creasote from their surfaces, the specimens are placed in the melted paraffin, in which, kept at a temperature slightly above its melting-point, they are to remain from one to four hours according to their size, and to ON STAINING FLUIDS, AND STAINING. 115 ensure absolute permeation by, and saturation with, the paraffin. The blocks must be allowed to cool thoroughly before they are removed from the moulds; when removed, they may be pared down to any size desired, and in such a manner that the face of the embedded specimen, from which it is intended to cut sections, shall be at the surface. A sufficient body of paraffin must be left surround- ing the specimen to ensure the necessary solidity and equable support. Specimens thus embedded and prepared for sectionising may be kept for an indefinite time without alcohol, and used when required. As they are perfectly embalmed and preserved, the only necessary precaution is to ensure absolute freedom from moisture in the bottles or boxes in which the blocks are stored. The paraffin can be dissolved out of the sections by means of chloroform, turpentine, turpentine and oil of cloves mixed in equal proportions (this will also clear the sections), or xylol; the latter being of course preferable when the sections are to be " cleared" in xylol and absolute phenol, and mounted in xylol-balsam. When turpentine (only) is used, and the sections are to be cleared in xylol and phenol, they must be placed in absolute alcohol for a short time to remove the turpentine, before being immersed in the clearing fluid. Specimens can also be satisfactorily embedded in paraffin by the following method (Giesbrecht* s) , founded upon the rapid evaporation of chloroform, and which, at the same time, is a speedy solvent of paraffin. The specimens to be embedded having been immersed to saturation in absolute alcohol are next placed in chloroform ; a short time having 116 METHODS OF MICROSCOPICAL RESEARCH. been allowed for saturation therewith, the chloro- form and the specimens are together heated to the melting point of the paraffin, small pieces of paraffin being gradually added during the heating ; air bubbles will arise from the specimens, and when these cease no further addition of paraffin will be necessary. The " mass " must be kept at its melting-point in a dry oven for some considerable time after the addition of the paraffin, in order to ensure the entire evaporation of the chloroform, any remains of which in the mass would prevent its becoming sufficiently hard to cut satisfactorily. The embedding is of course done as in the ordinary paraffin process. EMBEDDING IN GUM AND GLYCERINE. Iy Gum Acacise Mucilage, B.P., 90 parts. Price's Glycerine 10 ,, Add 1 per cent, of carbolic acid crystals. Place the specimens to be embedded in this mix- ture in watch glasses, or small (water colour) saucers, and let them remain exposed to the air of the labo- ratory for four days. In that time the mixture will have set into a firm, gelatinous mass. Blocks, in each of which one specimen is enclosed, are then to be cut out, and again left exposed for three days to hardeo, being turned over from time to time that they may dry equally on all sides. When dry, the blocks may be pared down to suitable size, and affixed to corks with strong gum water, and on these they may be preserved, to be sectionised as required. The gum can be removed from the sections by immersion in distilled water before staining them. ON STAINING FLUIDS, AND STAINING. 117 Embedding in Celloidin. 1 This delicate and cleanly and most effective process is pursued as follows : A thin solution of the celloidin is made in equal parts of absolute alcohol and ether. A thick solution is also made in the same manner, by increasing the proportion of the celloidin. The specimens perfectly dehydrated are to be placed in absolute alcohol for twenty-four hours, and then transferred into a mixture of equal parts of absolute alcohol and ether for a second period of twenty-four hours. They are now to be immersed in the thin solu- tion of celloidin for twenty-four hours, and after- wards in the thicker solution for three days. They are then to be placed in paper trays, so made as to admit of a good margin of celloidin sur- rounding the specimen occupying the centre of the tray. The tray is then to be filled with the thick solution of celloidin, which is to be allowed to evaporate, the tray being filled up from time to time, as evaporation goes on, until the specimen is covered, as well as surrounded, by a layer of celloidin in other words, embedded in a block of celloidin, of the size and shape of the tray. When the celloidin begins to set firmly, and before any shrinkage sets in, the tray, with the specimen, is to be plunged into a vessel with an air-tight cover, containing strong methylated spirit, and in this the 1 " Schering's Celloidin," which is patented, can be obtained at Mr. C. Baker's, 244, High Holborn, London, W.C. The celloidin shavings, recently introduced, will be found much more convenient to use than the tablets. 118 METHODS OP MICROSCOPICAL RESEARCH. blocks may be preserved until required for section- ising. The purpose, in embedding in celloidin, is to secure the normal position (in situ) of all the component parts of an organism or specimen. The film of celloidin surrounding all the organs and parts, and preserving their respective features and positions, must be retained, and mounted with the section. When these sections are to be mounted in Canada balsam, they must be cleared in xylol and phenol (as oil of cloves destroys the celloidin) and mounted in xylol balsam. Celloidin sections may be mounted in glycerine, glycerine jelly, or Farrant's medium, all of which render the celloidin nearly as transparent as does balsam. These sections may be stained by means of all the ordinary staining fluids, except, perhaps, one or two of the more powerful anilin dyes which sometimes, though curiously enough not invariably, colour the cel- loidin indelibly. If stained by logwood they should be strongly stained in order to admit of the extrac- tion of the colour from the celloidin by the acetic acid process, whilst the tissues remain sufficiently stained. Specimens embedded in celloidin should be cut by the ether freezing process. Gum and Syrup. For ordinary tissues make a mixture of 5 parts of gum mucilage B.P. and 3 parts of simple syrup B.P. For nerve, brain, spinal cord, and any brittle tissues the com- bination should be 5 parts of gum mucilage to 4 parts of syrup. Tissues and specimens to be cut upon the ether freezing microtome are to be immersed in this fluid for three or four days. They may be preserved for ON STAINING FLUIDS, AND STAINING. 119 an indefinite time in the mixture, indeed, perma- nently. The specimens must have been carefully hardened and thoroughly freed, by prolonged soak- ing in water, from all traces of the hardening reagents, before placing them in the gum and syrup. The ether freezing process is conducted thus. The gum and syrup is wiped off the specimens with a cloth, and they are placed in the strongest possible solution of gum acacias for a few hours before being frozen and cut. The microtome and freezing ap- paratus having been prepared for use, a specimen is placed upon the freezing plate and painted round the edges and over the surface by means of a brush with the strong, pure gum-water from which it was taken. It is then frozen by means of the ether spray until it can be easily and smoothly cut. Practice and experience will soon enable the operator to stop the freezing at the right moment. Should the ice, however, become too hard, and therefore brittle, a few seconds' delay before com- mencing to cut the sections will ensure its being in the right condition for cutting. On Section Cutting and Microtomes. Section cutting, like staining, is an art requiring not only practice and experience, but knowledge and judgment. Thorough knowledge of the struc- ture and functions of the organisms, organs and specimens to be sectionised is equally necessary with the judgment enabling one to decide as to the thickness or thinness of the sections of, say, an organ which shall most satisfactorily display its structure. It is quite possible to cut sections too 120 METHODS OP MICROSCOPICAL RESEARCH. thin so thin, indeed, as to obliterate all the features and structure which it is necessary to study in order to arrive at the physiological or biological elements of the specimen. Brain, spinal cord, and the like tissues, for example, cannot be cut too thin kidney and several other organs can easily be so, and thus be rendered worthless for purposes of study. It is well, therefore, to provide one's self with three sections of such specimens, as it may be found possible, by experience, to cut too thin ; so that for general observation and study, with low powers, a thin section is at hand, for medium powers a thinner section, and the thinnest for use with high and the highest powers. Some writers and practical workers assert that it is not possible to cut the thinnest sections from a celloidin-embedded specimen by the ether freezing process ; the author can only say that he has cut sections of entire human eyes, so embedded and frozen, sireoths of an inch thick (or rather thin), and of brain and spinal cord, &c., jcfeoth of an inch thin. For cutting sections by the ether freezing process there is (with the exception of the automatic Minot microtome recently produced and fully described in later pages) no better microtome than the Cathcart, as perfected and supplied by Mr. Alexander Fraser, of Edinburgh (which is fully described and illustrated on pages 127-8), with admirable arrangements and apparatus for embedding specimens in paraffin and for cutting those also, the freezing and this arrange- ment being removable at will and interchangeable. ON STAINING FLUIDS, AND STAINING. 121 Professor Rutherford's Microtome. This is Stirling's original well and screw- microtome, plus an ice box arrangement. It is used by filling the ice box with well-powdered and mixed ice and bay salt in equal quantities. Whilst freezing is going forward the well may be covered with felt. One or several tissues can be cut at once, either with a Rutherford's knife, or, what is now found to be far better, a plane iron. By a plane iron, we mean the iron taken out of a car- penter's smoothing plane. These irons must be 2f inches broad, and are used either with or without a covering of wood. When double-faced with wood, the handling of them is more pleasant, but it does not add to efficiency. The iron must be sharpened on a very smooth hone, and then well stropped. In sharpening, care must be taken to rub the entire 122 METHODS OF MICROSCOPICAL RESEARCH. length of the cutting edge evenly. This is best done by taking care to rub the edge on the stone in such a way that every portion of its length is upon the stone at the same time. In stropping, it will be found most convenient to rest the plane iron against a table edge, and push or pull the strop over it. The advantage of the Rutherford freezing micro- tome is that it can be also used as an ordinary Stirling's microtome for imbedding in carrot, paraffin or wax. Its disadvantage is the same as that of ice freezing machines of whatever description that it is cold, disagreeable work filling in the ice and salt, and the former is not always to be obtained, and when once charged, cutting must follow, or the ice melts. Mr. C. Baker, 244, High Holborn, W.C., sells an excellent Rutherford's freezing microtome, which, of course, must have its cutting face covered with plate glass. ON STAINING FLUIDS, AND STAINING. 123 Williams' Freezing Microtome. This is made by Messrs. Swift & Son, 81, Totten- ham Court Road, London, W.C., who also make a registered knife carrier, which carries^the blade of a razor : the whole goes under the name of " Swift's Knife." Until the plane iron was thought of, Swift's Knife had the field all to itself, and there will still be those who will prefer it to the plane iron. This combination has this great advantage : that in winter, when ice is plentiful, the machine is charged, and freezes strongly for an liour or more. For class work this is excellent : the attendant charges the machine just before the class meets, and the demonstrator cuts a hundred sections of any prepared tissue in a few minutes. If he wishes to cut another specimen, he removes the first, and 124 METHODS OF MICROSCOPICAL RESEARCH. clears out the grooves on the top of the section carrier with the back of a knife ; then he places his specimen upon the machine, and paints it round with gum solution, and the whole freezes fit for cutting in one minute or so. With very little prac- tice it is quite easy to cut fifty excellent sections of each of fifteen or twenty specimens with one charge of ice and salt. To manage Swift's knife, we proceed thus : First take the blade away, and lay it flat upon a smooth hone well oiled, and rub it first on one side, then the other, until the entire edge on either side is ground by the hone at once. The writer rubbed over twenty hours before this was accomplished with the razor blade of his Swift's knife. Both sides of the blade are ground quite flat, to begin with, not hollowed ; but cutlers either cannot or will not grind a blade with its back and its edge perfectly parallel, so that this has to be done by one's self. Having ground the knife blade, we fix it in the handle and strop it. We then take off the handle and fix the blade in the carrier in such a way that the edge must be perfectly level with the top of the microtome, and on a much lower level than the back. This position of the knife in the frame is to the last degree essential, and we effect it thus : First place the knife-blade in the frame, and lower the screws which the edge of the blade rests upon, so that the edge is lower than the back a good deal. After making it firm, we complete the levelling by freezing a piece of tissue, and taking away a thick section, cutting away from us, with the knife in the frame just as we have put it roughly. We now place each end of the blade alternately ON STAINING FLUIDS, AND STAINING. 125 against the same part or bit of the frozen tissue, and raise or lower either end by screwing either of the two screws nearest to us. All is now complete, and we cut section after section by lowering the knife edge for each fresh cut by giving the screw furthest away from us a part of a revolution. To Fill an Ice Microtome. Get a piece, or pieces, of ice the size of two closed fists, and the same bulk of either common table salt or, even better, of bay salt. Powder both, and thoroughly mix them. The ice is most readily reduced to fine powder by being surrounded with flannel and bruised with a broad-faced hammer, or a carpenter's mallet. Fill in the mixed ice and salt and press it well down, but take care that the trough of the microtome is not so full that the top touches the ice and salt. The top is to be placed on the microtome and screwed fast. In winter the machine, after being charged, is ready for freezing in five minutes or so ; in summer it may be ten minutes or more. Freezing proceeds, as we have before said, for an hour or two. The waste pipe at the bottom of the microtome is to be connected by caoutchouc tubing, with a basin to receive the melted ice. The disadvantage of Williams' microtome is, that, like all ice microtomes, it is disagreeable to charge even when ice is plentiful, and cutting must take place at the time the machine is charged ; that is to say, if the operator is called away, as in medical practice often happens, his ice charge melts and has to be renewed. 126 METHODS OF MICROSCOPICAL RESEARCH. Dr. Fearnley's Ether Freezing Microtome. Dr. Groves suggested the conversion of Williams' ice freezing microtome into an ether freezer. This is now called the Grove- Williams microtome, and is made by Messrs. Swift & Son, and used with Swift's knife. For those who are not affected by the inhalation of atmospheric air charged with ether, the best and simplest ether freezer for use with Swift's knife is ON STAINING FLUIDS, AND STAINING. 127 Dr. Fearnley's ether freezing microtome. This is simply the top of the Grove- Williams' microtome, fitted with a clamp and screw arrangement for attaching it to the work-bench. The ether nozzle is immediately under the frame of the glass plate, and the bottle of ether stands under the machine on the table as shown in wood-cut. Fearnley's microtome is also made by Messrs. Swift & Son. Cathcart's Microtome. This is an ether freezing microtome for use either with a stout razor, or with a plane iron. Cathcart's invention, used with a plane iron, for cheapness, sim- plicity, and efficacy cannot be surpassed, if equalled, by any ether or ice microtome we know of. With the expenditure of two drachms of ether, the operator can cut sixty or seventy sections in almost as many seconds, every section being of exquisite thinoess. The ether used is methylated ether of 720 s.g. The freezing process has already been described. This being accomplished, the tissue is elevated to the knife by a very small fraction of a revolution of the screw with the left hand, whilst the right drives the plane iron. The iron must be held with the edge far below the level of the rest of the iron, and the screw movement and the push of the iron move- ment must take place alternately. When a mass of sections has accumulated on the iron, it must be floated off into a saucer of water. (See illustration on page 128.) An admirable and most useful microtome is made by Zeiss. It can be seen at the depot of, and purchased from, his London agent, Mr. C. Baker. 128 METHODS OF MICROSCOPICAL RESEARCH. The " Kecking Microtome " manufactured and supplied by the Cambridge Scientific Instrument Company, is in all respects an admirable and most THE CATHCART MICROTOME. effective machine, and especially for the cutting of serial (or ribbon) sections. A full illustrated description, as well as directions for its use, can be obtained on application to its makers. ON STAINING FLUIDS, AND STAINING. 129 The " Thoma Microtome " adapted equally for the paraffin embedding and ether freezing processes, is an exquisitely beautiful and reliable instrument. The preferable size for ordinary use is that named the Thoma B. 2. A verbal description would be of but little use, and the author advises the student to see one, and this he can (generally) do on applica- tion to Mr. C. Baker. Its manufacturer, Mr. R. Jung Mekaniker, Heidelberg, will send a full (illustrated) description on application. Minot's Microtome. This beautiful instrument is made in three forms (or to use the inventor's expression, " models "). In its construction the chief endeavour has been to render its various parts as " massive " as possible, and to make the adjustments for the various thick- nesses of the sections perfectly automatic, whilst, at the same time, portability has been considered. The thickness of the section is regulated by a special toothed wheel arrangement. On the ascent of a vertical slide a lever strikes one of the six spokes of a wheel, and the ratchet turns the toothed wheel of the micrometer screw so that the horizontal slide, to which the preparation is attached, is brought into contact with the edge of the knife. The six spokes of the controlling wheel are of different lengths, and are marked 1 to 6, which numbers correspond with the number of teeth on the wheel moved at each revolution of the driver. In Model I. the total number of teeth is 150, and as one complete turn of the micrometer screw equals 0*5 m.m., by means of the spoked wheel sections 9 130 METHODS OF MICROSCOPICAL RESEARCH. can be obtained of the respective thicknesses of s-io, rio, rib, Ts 9 ea, ^an6, or PHOTOGRAPHIC APPARATUS. SPECTACLES, EYE-GLASSES, And every Eequisite for MICROSCOPIC & PHOTOGRAPHIC WORK Can be supplied by us. Plea.se Note JLcLd.i?ess : 35, LUDGATE HILL. MASON'S MICROSCOPES . Height when closed, ^*|LdH^ llL ^ Spread of foot, 10 in. The S.W. Histological and Botanists' Microscope as above Fig. With 1 in. 23 and th 80 in Polished Mahogany Case with 1 B Eye-piece. 4 17s. 6d. This instrument is confidently recommended to Students as thoroughly reliable. The fine Motion is equal to the most expensive, Coarse ditto slides through velvet-lined fitting ensuring a perfectly smooth motion. All fittings are compensated for wear, making it the cheapest and most efficient Microscope ever offered at the price. IR,. GK IMI.A_SO:Er (From J. SWIFT), 69, PARK ROAD, CLAPHAM, LONDON, S.W. ESTABLISHED 1880. Living Specimens for the Microscope. JOHN HOOD, F.E.M.S., BEGS to intimate to Teachers and Students of Biology, and those studying Natural History with the aid of the Microscope, that he has always at hand the following list of Pond Life: PROTOPHYTA, PROTOZOA, BOTIFERA, HYDROZOA, POLYZOA, &c. Specimen Tube, with drawings and descriptions, Is. post free. A Series of 26 Tubes, 21s. Address: 50, DALLFIELD WALK, DUNDEE. PATENT PROJECTION MICROSCOPE. Silver Medal awarded, Cornwall Exhibition, 1894. **** Hlwwft COMPLETE PROJECTION. FOR JB4 17s. 6d. Has the following advantages over any Projection Microscope extant: 1. May be used with any ordinary lantern either with lime or electric light. 2. On the table as an ordinary stand. 3. Any suitable microscopic objective may be used. 4. No loose parts, and so cannot get out of order. 5. Last, but not least, it is the cheapest efficient Projection Microscope in the market , equals any instrument in performance at three times its cost. As used by the School Board and Plymouth Technical Schools. ILLUSTRATED CATALOGUE FOR STAMP. ALL APPAEATUS FOE PHOTO-MICEOGEAPHY. GK :M:.A.SO:ET (From J. SWIFT), 69, PARK ROAD, CLAPHAM, LONDON, S.W Established 188O. MICROSCOPE Stanley's New Histological, With first-class &th and rd Object Glasses, B Eyepiece, Indicator, &c., in polished Mahogany Case, 5 5s. Improved Complete MOUNTING CABINET, for Practical Physiology, 1 5s. Specially Thin 3 by 1 GLASS SLIPS, 4s. per Gross. Thin Glass Covers, Labels, and all the Necessary Requisites for Students' use, at most Moderate Prices. A large and well-assorted Stock of Special Objects for the microscope, Is. each. PRICE LIST POST FREE. STANLEY, Optician to H.M. Government, Science and Art Department, Council of India, Admiralty, &c. ; Guy's, St. Bartholomew's, St. Thomas's, and other Hospitals. CLINICAL THERMOMETERS, with Indestructible Index, in German Silver Cases, 3s. 3d. each. Railway Aj3i3i?oion, London Bridge, S.E. ZEISS MICROSCOPES AND APPARATUS KEPT IN STOCK. Telephone 4871. C. BAKER'S OPTICAL AND SURGICAL INSTRUMENT WAREHOUSE, 244, HIGH HOLBORN, LONDON. Agent for C. ZEISS, E. LEITZ, C. REICHERT, and other foreign manu- facturers. H- Stains and Reagents a speciality. A LARGE COLLECTION OF STUDENTS' MICROSCOPES. C. BAKER'S MODEL HISTOLOGICAL MICROSCOPE, as figured, with 1 Eye-piece, 2 Objectives by Leitz, No. 3 (f in.) and No. 7 (| in.), in Case ' . . . . ' 6 10 Ditto, ditto, with Zeiss Objectives, A (j* in.) and D ( in -)> in Case 700 ZEISS'S STUDENT'S MICROSCOPE, with Eye-piece, 2 Objectives, A (| in.) and D ( in.), in Case 6 13 LEITZ'S STUDENT'S MICROSCOPE, with 2 Eye-pieces, 2 Objec- tives, No. 3 ($ in.) and No. 7 (i in.), in Case . . 3 12s. 6d., 4 10s. Od., 650 BACTERIOLOGICAL MICROSCOPES, with 2 Eye pieces, 3 Objec- tives (| in., J in., and T V in. Oil), Abbe Condenser, Triple Nose- piece, in Case . . . ." 15, 16, 18 CATALOGUES POST FREE. ESTABLISHED 1765. SWIFT & SON'S Patent 4-Legged Microscope, \Yhich will accommodate itself to any uneven surface, therefore has more lateral resistance than any other instrument made. o Price, with 3 and : in. objectives, 1 eyepiece, and iris diaphragm, in mahogany cabinet, 7. Double nosepiece, 15s. Patent roller clip, as shown, 10s. 6d. Fully Illustrated Circular on application. UNIVERSITY OPTICAL WORKS, 81, TOTTENHAM COURT ROAD, W. C. BAKER'S Optical and Surgical Instrument Warehouse 244, HIGH HOLBORN, LOHDON, Nelson Model Microscope, No. la, as figure Ditto No. 1 Ditto 2 Ditto 3 35 30 16 16 880 CATALOGUES POST FREE, CATALOGUE OF THE PUBLICATIONS or BAILLIERE, TINDALL, & COX, IN MEDICHSTE, SCIENCE AND ART. CONTENTS. PERIODICAL PUBLICATIONS . DIRECTORIES PAGE I Back of Title ) ANATOMY 9, etc. ART, ARTISTIC ANATOMY, ETC. 11, etc. CHEMISTRY 14, etc. MEDICINE, SURGERY, AND ALLIED SCI ENCES 26, etc. PHARMACY 30, etc. STUDENTS' AIDS SERIES 36, etc. VETERINARY MEDICINE AND SURGERY 39, etc. WHITE'S PHYSIOLOGICAL MANIKIN 42 LONDON : 20, 21, KING WILLIAM STREET, [PARIS AND MADRID.] 1895. STRAND Bailliere, Tindall, and Cox have special facilities for the disposal of authors' works in the United States and abroad ; being in almost daily com- munication with the principal houses and agents. PERIODICAL PUBLICATIONS. The Medical Press and Circular. Established 1838. Published every Wednesday in London, Dublin, and Edinburgh. Price 5d. ; 1 Is. per annum, post free, in advance. Journal of the British Dental Association. A Monthly Review of Dental Surgery. Published on the 15th of each month. Price 6d., or 7s. per annum, post free. The Analyst. The Official Organ of "The Society of Public Analysts." Monthly, price Is. ; 10s. 6d. per annum. The Veterinary Journal, and Annals of Comparative Patho- logy. Monthly, price Is. 6d. ; 18s. per annum. The Australasian Medical Gazette. Monthly, 2s., or yearly post free, price 21s. Indian Medico-Chirurgical Review. Monthly, Is. 6d. ; yearly subscription, 16s., post free. Pathology. A series of illustrations of Pathological Anatomy issued in parts, each containing 4 plates in colours, with accompanying descriptive text by PROFESSORS KAST, of Breslau, and RUMPEL, of Hamburg. Eevised and edited by M. ARMAND KUFFER, M.D. Oxon. Twelve parts by subscription, post free, 2 8s. Single parts, 6s. each. Single plates, Is. 6d. each. International Journal of Microscopy and Natural Science. Edited by Mr. ALFRED ALLEN. Price 2s. 6d. West London Medical Chirurgical Reports. Vol. III., 1886-7 and 1887-8 \ IV., 1888-9 1889-90 Each 5s. V., 1890-1 1891-2 J Transactions of the Royal Academy of Medicine in Ireland. Annual volumes, 14s. Foreign postage extra. DIRECTORIES. The Register of the Royal College of Veterinary Surgeons ; published annually in accordance with the Act of Parliament. Price 2s. 6d., post free in the United Kingdom. Australasian Medical Directory and Handbook. Edited by LUDWIG BRUCK. Corrected to August, 1892. Price 12s. 6d. Commercial Directory for Spain, her Colonies and South America, containing 500,000 Names and Addresses of the Commercial Houses, Public Officers, Offices, etc., etc. Annual, price 25s. ALPHABETICAL INDEX OF AUTHORS. ABERCROMBIE (J.) On Tetany in Young Children 15 ADAMS (W.) Surgical Treatmeni of Deformities 17 ALLAN (F. J.) Aids to Sanitary Science 31 ALLAN (J. H.) Tables of Doses 25 ALLEN (Alfred) Microscopical Science : wrapper ALLINGHAM (H. W.) Colotomy 8 ATTENDANTS. Handbook for Attendants on the Insane 24 BAKER (Benson) How to Feed an Infant 28 BALL Nose and Pharynx 28 BAN HAM Veterinary Posological Tables 39 BANNATYNE (A.) Aids to Pathology 29 BEACH (Fletcher) Psychological Medicine 31 BERNARD (Claude) and HUETTE'S Text-book of Operative Surgery 33 BLACK (C). Atlas of the Male Organs of Generation 10 BLACKLEY (C. H.) Hay Fever, its Causes and Treatment 22 BODDY (E. M.) History of Salt 32 Hydropathy 23 BOWDICH (Mrs.) Confidential Chats with Mothers 15 BOWLES (R. L. ) On Stertor and Apoplexy u BO YD (Stanley) Movable Atlas of the Foot, its Bones and Muscles 21 BRAND (A. T.) Pocket Case Book 14 BROCHARD (J.) Practical Guide for the Young Mother 28 BROWN (George) The Student's Case-book 14 Aids to Anatomy 9 Aids to Surgery 33 BROWNE (Lennox) The Throat and Nose, and their Diseases 34 Diphtheria 18 BROWNE ( W. J.) The Moon, its Influence on Weather 27 BURTON (J. E.) Translation of Ebstein's Gout 22 CAMERON (Chas.) Microbes in Fermentation, Putrefaction, and Disease ... 12 The Cholera Microbe and How to Meet It 15 CAMERON (Sir C. A.) History of the Royal College of Surgeons in Ireland 23 CAMPBELL (C. M.) Skin Diseases of Infancy and Early Life 32 CANTLIE (Jas.) Atlas of the Hand 10 Text-book of Naked- Eye Anatomy 9 C ARD WELL (B.) Translation of Hygiene of Beauty 24 CASSFLLLS (J. Patterson) Deaf-mutism and the Education of the Deaf-mute 17 CHARCOT (J. M.) Bright's Disease of the Kidneys 25 CHRISTY (T.) Dictionary of Materia Medica 25 CHURCHILL (Fleetwood) Obstetrical and Gynaecological Nursing 28 CLARKE (J. Jackson) Cancer, Sarcoma and other Morbid Growths 14 CLARKE (Percy) Medical Laws 26 CLARKE (E. H.) The Building of a Brain 13 CLARKE (Ernest) Atlas of Eye 20 COFFIN (R. J. Maitland) Obstetrics 28 COLE (M. J.) Modern Microscopy ... 27 COOMBE (Russell) Epitome of B. P. 30 CO O PER (R. T.) On Vascular Deafness 18 COTTERELL (Ed.) The Pocket Gray, or Anatomist's Vade Mecum 9 COURTENAY (E.) Practice of Veterinary Medicine 39 COZZOLINO (V.) The Hygiene of the Ear 18 CRAWFORD (W. S.) Ulcers and their Treatment 35 CROOKE (G. F.) The Pathology of Tuberculosis 17 CROSS (M. J.) Modern Microscopy 27 CRUISE (F. R.) Hydropathy 23 CULLIMORE (D. H.) Consumption as a Contagious Disease 16 2 Baillifere, Tindall, and Cox's Books. PAGE CULLIMORE (D. H.) The Book of Climates 16 DARLING (W.) Anatomography, or Graphic Anatomy 9 The Essentials of Anatomy 9 DAWSON (W. E.) Guide to the Examinations of the Apothecaries' Society 19 DAY (W. H.) Irritable Brain in Children 13 DENNIS ( Hy. J. ) Second-Grade Perspective Drawing 1 1 Third-Grade Perspective Drawing 12 DESSAR (L. A.) Catarrhs and Colds 14 DOLAN (T. M.) Whooping Cough, its Pathology and Treatment 35 DOWSE (T. Stretch) Apoplexy 11 Syphilis of the Brain and Spinal Cord 13 Skin Diseases from Nervous Affections 32 The Brain and the Nerves and Influenza 13 DRAGENDORFF (Prof. G.) Plant Analysis 15 DRYSDALE (C. R.) Nature and Treatment of Syphilis 34 DRYSDALE (John) The Protoplasmic Theory of Life 34 DUDGEON (R. E.) The Sphygmograph 32 DUFFEY (G. F.) Note-taking 14 EBSTEIN (Prof.) The Treatment of Gout 22 EDWARDS (F. Swinford) Urinary Surgery 35 ERSKINE (T.) Hygiene of the Ear 19 EVANS (C.'W. De Lacy) How to Prolong Life? 18 Consumption : its Causes, Treatment, etc 16 E WART (W.) Cardiac Outlines 14 Heart-Studies, Chiefly Clinical 22 How to Feel the Pulse 32 Symptoms and Physical Signs 14 FAU (}.) Artistic Anatomy of the Human Body II Anatomy of the External Form of Man II FIELD (G. P.) Diseases of the Ear 18 FINNY (F. M.) Clinical Fever Chart 21 FITZGERALD (H. P.) Dictionary of British Plants and Flowers 13 FLAXMAN (J.) Elementary Anatomical Studies for Artists H FLEMING (G.) Text-book of Veterinary Obstetrics 39 Neumann's Parasites of Domestic Animals 39 Text-book of Veterinary Surgery 39 Roaring in Horses 40 Practical Horse-Shoeing 40 Animal Plagues, their History, Nature and Treatment 40 Contagious Diseases of Animals 40 Tuberculosis 40 Human and Animal Variolse 40 Heredity and Contagion in the Propagation of Tuberculosis 40 FORD Ophthalmic Notes 20 FOTHERGILL (J. Milner) Chronic Bronchitis 13 The Physiological Factor in Diagnosis 17 Aids to Diagnosis 18 The Physiologist in the Household 31 Diseases of Sedentary and Advanced Life 29 Aids to Rational Therapeutics 34 Vaso-Renal Changes 24 FOY (Geo.) Anaesthetics : Ancient and Modern 9 FUCHS (Dr.) The Causes and Prevention of Blindness 20 GANT (F. J.) Text-book of the Science and Practice of Surgery 33 Baillifere, Tindall, and Cox's Books. GANT (F. J.) Diseases of the Bladder, Prostate Gland, and Urethra 13 Examinations by the Conjoint Board 19 Students' Surgery 33 GARMANY (J. J.) Surgery on the Cadaver 33 GARROD (A. E.) Handbook of Medical Pathology 29 GEMMELL (Wm.) Derraic Memoranda 32 GERSTER Aseptic and Antiseptic Surgery 33 GIRAUD-TEULON Anomalies of Vision 20 GLASGOW-PATTESON (R.) Skin and Hair 32 GOOD ALL (E.) Microscopical Examinations of the Brain 23 GORDON (Chas. A.) Our Trip to Burmah 13 Aids to Psychological Medicine 38 Life on the Gold Coast 8 Lessons in Military Hygiene and Surgery 23 A Manual of Sanitation 23 Rabies and Hydrophobia 23 Island of Madeira 16 GORDON (T. Hurd) Aids to Practical Chemistry 36 GORE (Albert A.) Our Services Under the Crown 27 Medical History of African Campaigns 8 GOULD Illustrated Dictionary of Medicine, Biology, etc 18 GOW (W. J.) Handbook of Medical Pathology 29 GRANVILLE (Mortimer) Gout 22 GREEN (F. W. Edridge) Memory 27 Detection of Colour Blindness.. 20 GREENWOOD (J.) Laws Affecting Medical Men 26 GREENWOOD (Major) Aids to Zoology 35 GRESSWELL (J. B. and A. G. ) Manual of Equine Medicine and other works 40 GREVILLE (H. Leicester) Student's Hand-book of Chemistry 15 GRIFFITHS (A. B.) Micro-Organisms 12 GRIFFITHS (W. H.) Text-book of Materia Medica and Pharmacy 25 Notes for Pharmacopoeial Preparation . 30 Posological Tables 31 GUBB (Alfred S.) Aids to Gynaecology 22 GUILLEMARD (F. H. H.) Endemic Hsematuria 20 HAIG-BROWN Tonsillitis 35 HALTON (R. J.) Short Lectures on Sanitary Subjects 24 HANDBOOK for Attendants on the Insane 28 HARRIS (Vincent) Manual for the Physiological Laboratory 23 HARRIS (V. D.) Kuhne's Guide to the Demonstration of Bacteria 12 HARTMANN (Prof.) On Deaf-mutism, Translation by Dr. Cassells 17 HAYNES (Stanley) Healthy Homes 24 HAZARD (W. P.) Diseases of Live Stock 41 HE1BERG (Jacob) Atlas of Cutaneous Nerve Supply 27 HEPPEL Analytical Conic Sections 38 HERRINGHAM (W. P.) Handbook of Medical Pathology 29 HERSCHELL (Geo.) Indigestion 24 Heart Diagrams and Case-book 22 HEWITT (Frederic) Anaesthetics 9 HILL (J. W.) Principles and Practice of Bovine Medicine 40 Management and Diseases of the Dog 40 HIME (T. W.) Cholera: Ho\v to Prevent and Resist It 15 The Practical Guide to the Public Health Acts 31 HOARE Veterinary Therapeutics 40 HOGG (Jabez) The Cure of Cataract 20 4 Bailliere, Tindall, and Cox's Books. PAGE HOGG (Jabez) The Impairment of Vision from Shock 20 Parasitic or Germ Theory of Disease 12 HOPGOOD (T. F.) Notes on Surgical Treatment 33 HORNER (Professor) On Spectacles 20 HOWAT (G. R.) How to Prevent and Treat Consumption 16 HUNTER (Ch.) Manual for Dental Laboratory 17 HUSBAND (H. Aubrey) Handbook of Forensic Medicine 21 Handbook of the Practice of Medicine 26 Student's Pocket Prescriber 31 Urine 35 HUTCH INSON (Jonathan) Aids to Ophthalmic Medicine and Surgery 20 ILLINGWORTH Hydrophobia 23 INCE (J.) Latin Grammar of Pharmacy 30 INTERNATIONAL MEDICAL CONGRESS 24 JAMES (Brindley) Replies to Questions in Therapeutics 38 JAMES (M. P.) Therapeutics of the Respiratory Passages 34 Vichy and its Therapeutical Resources 35 JENNINGS (C. E.) On Transfusion of the Blood and Saline Fluids 35 Cancer and its Complications 14 JENNINGS (Oscar) On the Cure of the Morphia Habit 27 JESSETT (F. B.) Surgical Diseases of Stomach and Intestines 8 Cancer of the Mouth and Tongue 14 Cancer of the Uterus 14 JONES (H. Macnaughton) The Diseases of Women 22 Subjective Noises in the Head and Ears 18 Hints for Midwives 28 and STEWART Handbook of Diseases of the Ear and Naso- Pharynx 19 JONES (H.) Guide to Sanitary Science Exams 32 JONES (T. Wharton) Blood in Inflammation 24 JUKES-BROWNE (A. J.) Palaeontology (in Penning's Field Geology) 21 KAST AND RUMPEL Illustrations of Pathological Anatomy 29 KEETLEY(C.R. B.) Guide to the Medical Profession 26 Surgery of Knee Joint 33 KENNEDY (Hy.) An Essay on Fatty Heart , 22 KINGZETT Nature's Hygiene 23 KNIGHT (G. D.) Movable Kidney 25 KUHNE Demonstration of Bacteria 12 LAMBERT (J.) The Germ Theory of Disease 40 LEASK (J. G.) Questions at Medical Science Examinations 19 LEDWICH (J.) Anatomy of Inguinal and Femoral Regions 9 LEONARD (H.) The Pocket Anatomist 9 Bandaging 13 Hair 22 and CHRISTY Dictionary of Materia Medica 25 LE SUEUR Analytical Geometry, Straight Line and Circle 38 LETHEBY(Hy.) The Sewage Question 32 LIAUTARD (A.) Animal Castration 40 Diseases of Live Stock 41 Lameness of Horses 40 Operative Veterinary Surgery 40 LITHGOW (R. A . Douglas) From Generation to Generation 22 LOWNE (B. T.) Aids to Physiology ., 37 LUNN(C.) The Philosophy of Voice 35 Bailli&re, Tindall, and Cox's Books. 5 PAGE LUNN (C.) Artistic Voice in Speech and Song 35 LUPTON (J. I.) Horses : Sound and Unsound 40 MACDOUGALL (A. M.) The Maybrick Case 21 MACKENZIE (Sir M.) Diseases of the Throat (in Gant's Surgery) 33 McCAW (John) Aids to the Diagnosis and Treatment of Diseases of Children 15 MADDEN (T. More) Clinical Gynaecology 22 Churchill's Obstetrical Nursing 28 MADDICK (Distin) Stricture of the Urethra 33 MAGNE (Dr.) How to Preserve the Sight 20 MARTIN (B. R.) Diphtheria 18 MARTIN (J. W. & J.) Ambulance Work (Questions and Answers) 8 Nursing (Questions and Answers) 28 MASSE (J. N.) Text-book of Naked-Eye Anatomy 9 MAYBURY Student's Chemistry 15 McARDLE (J. S.) Notes on Materia Medica 26 McBRIDE Anatomical Outlines of the Horse 41 McLACHLAN (John) Anatomy of Surgery 33 MEARS (W. P.) Schematic Anatomy ...'. 9 MELDON (Austin) A Treatise on Gout 22 MEYRICK (J. J.) Stable Management in India .' 41 MILLARD (H. B.) Bright's Disease of the Kidneys 25 MILLER (B. E.) Diseases of Live Stock 41 MOLONY (M. J.) Rupture of the Perineum 32 MONIN (E.) Hygiene of Beauty 24 MOORE (E. H.) Clinical Chart for Hospital and Private Practice 34 MOORE (J. W.) Text Book of Eruptive and Continued Fevers 21 MORDHORST (Carl) Rheumatism. Its Treatment by Electric Massage ... 32 MORGAN (John) The Dangers of Chloroform and Safety of Ether 8 MORRIS (Malcolm) The Skin (in Gant's Surgery) 33 MUCKLEY (W. J.) Student's Manual of Artistic Anatomy II A Handbook for Painters and Art Students on the Use of Colours 16 MURRAY (R. Milne) Pregnancy 10 MURRELL (W.) Aids to Forensic Medicine and Toxicology 21 Prevention of Consumption 16 MUTER (J.) Manual of Analytical Chemistry 15 NALL (S.) Aids to Obstetrics 28 NAPHEYS (G. H.) Handbook of Popular Medicine 18 Modern Therapeutics 34 NATIONAL SOCIETY FOR PREVENTION OF BLINDNESS 20 NEUMANN (L. G.) Treatise on Parasites and Parasitic Diseases of Domes- ticated Animals 39 NORTON (A. T.) Text-book of Operative Surgery 33 Osteology for Students 23 Affections of the Throat and Larynx 35 Movable Atlas of the Skeleton 10 Clinical Lectures on Recent Surgery 33 OGSTON On Unrecognised Lesions of the Labyrinth 18 ORMSBY (L. H.) Deformities of the Human Body 17 Phimosis and Paraphimosis 30 PALFREY (J.) Atlas of the Female Organs of Generation 18 PALMER (J. F.) How to Bring up Children by Hand 28 PARKE (Surgeon) Climate of Africa (in Cullimore's Book of Climates) 16 PEDDIE (W.) Manual of Physics 30 PENNING (W. H.) Text-book of Field Geology 21 6 Bailliere, Tindall, and Cox's Books. PENNING (W. H.) Engineering Geology 21 Notes on Nuisances, Drains, and Dwellings ... 24 PETTENKOFER (Von) Cholera : How to Prevent and Resist It 15 PIERSOL (G. A.) Text-book of Normal Histology 23 POLITZER (Prof.) Dissections of the Human Ear 19 Text-Book of Diseases of the Ear 19 POWER (Hy.) Movable Atlas of the Eye, and the Mechanism of Vision 10 Diseases of the Eye (in Gant's Surgery) 33 POWER (D'Arcy) Handbook for the Physiological Laboratory 23 POYSER (R.) Stable Management of Troop Horses in India 41 PRATT (W.) A Physician's Sermon to Young Men 27 PROCTOR (Richd.) The Stars and the Earth 12 PSYCHOLOGICAL ASSOCIATION'S Handbook for Attendants on the Insane 28 PURVES (L.) Aural Diseases (in Gant's Surgery) 33 RABAGLIATI (A.) Muscular Affections which Simulate Diseases of the Pelvic Organs in Women 22 The Classification and Nomenclature of Diseases 1 8 REGIS Mental Medicine 27 REMSEN (Ira) Principles of Theoretical Chemistry 15 RENTOUL Reform of Medical Charities 26 REYNOLDS (R. S.) The Breeding and Management of Draught Horses 41 RICHARDS (J. M.) A Chronology of Medicine 26 RICHARDSON (B. W.) The Healthy Manufacture of Bread 21 RI VINGTON ( W. ) Medical Education and Organization 26 ROBERTSON (William) A Handbook of the Practice of Equine Medicine... 41 ROCHE (J.) Hernia and Intestinal Obstruction 23 ROCHET (Chas.) The Prototype of Man, for Artists 12 ROSE (W.) Neuralgia 28 ROTH (W. E.) Elements of School Hygiene 23 Theatre Hygiene 23 ROUTH (C. H . F. ) Overwork and Premature Mental Decay 29 On Checks to Population 31 RUFFER (Armand) Illustrations of Pathological Anatomy 29 SARCEY (F.) Mind your Eyes 20 SCHOFIELD (A. T.) Examination Cards Pathology 20 Minor Surgery and Bandaging 33 SEMPLE (C. E. A.) Aids to Botany 13 Aids to Chemistry 14 Aids to Materia Medica 25 Aids to Medicine 26 Aids to Pharmacy 30 - Diseases of Children 15 The Voice Musically and Medically Considered 35 The Pocket Pharmacopoeia 30 SEWILL(Hy.) Manual of Dental Surgery 17 Dental Caries and the Prevention of Dental Caries 17 SHARMAN (J. S.) Notes on Inorganic Materia Medica 26 SIMON (W.) A Manual of Chemistry 15 SMITH (F. A. A.) Keep your Mouth Shut 32 SMITH (F.) Manual of Veterinary Hygiene 41 Manual of Veterinary Physiology 41 SOHN (C. E. ) Dictionary of the Active Principles of Plants 15 SPARKES (John C. L.) Artistic Anatomy n SQUIRE (P. W.) Posological Tables 31 Bailliere, Tindall, and Cox's Books. 7 PAGE STARK (A. Campbell) Practical Pharmacy 20, 30 STARR (M. Allen) Brain Surgery 33 STEPHENSON (J. B.) Medicinal Remedies 26 STEVENS (Geo. T.) Nervous Diseases 27 STEWART (W. R. H.) Practitioner's Handbook of Diseases of the Ear 19 Aids to Otology 18 STONE (G.) Translation of Politzer's Dissections of the Human Ear 19 STRAHAN (J.) Extra-Uterine Pregnancy 28 STUDENTS' AIDS SERIES 36 SUTTON (H. G.) Lectures on Medical Pathology 29 SUTTON (Bland) Dermoids 17 SWEETING (R. D. R.) The Sanitation of Public Institutions 24 SYMINGTON (J.) Anatomy of the Child 9 TELLOR(L. V.) Diseases of Live Stock 41 TEULON (G.) The Functions of Vision 20 THIN (George) Introduction to Practical Histology 23 THOMAS Hydatid Disease 23 THOROWGOOD ( J. C.) Consumption ; its Treatment by the Hypophosphites 16 The Treatment of Bronchial Asthma 12 Aids to Physical Diagnosis 18 THUDICHUM (J. L. W.) The Physiological Chemistry of the Brain 13 Aids to Physiological Chemistry 38 Aids to Public Health 31 Polypus in the Nose 31 The Coca of Peru, and its Remedial Principles 16 The Spirit of Cookery 21 TICHBORNE (Professor) The Mineral Waters of Europe 27 TIDY (Meymott) and CLARKE (Percy) Medical Laws 26 TIM MS (G.) Consumption ; its Nature and Treatment 16 Alcohol in some Clinical Aspects, a Remedy, a Poison 8 TOMSON Medical Electricity 19 TRANSACTIONS of Royal Academy of Medicine in Ireland Inside cover TUCKEY (C. Lloyd) Psycho-Therapeutics 24 TURNER (Dawson) Manual of Medical Electricity 19 TYSON (J.) The Urine, a Guide to its Practical Examination 35 UNDERWOOD (Arthur S.) Aids to Dental Surgery 17 Aids to Dental Histology 17 USHER (J. E.) Alcoholism 8 VINTRAS Diabetes 18 WAGSTAFFE (W. W.) Atlas of Cutaneous Nerve Supply 27 WALDO and WALSH Bread, Bakehouses 12 WALLACE (J.) Localised Peritonitis 29 WALSH (D.) Aids to Examinations 19 WALSHAM Deformities of the Foot 21 WALSHAM and POWER Surgical Pathology 33 WELPLY (J. J. ) Creameries and Infectious Diseases 24 WHERRY (Geo.) Clinical Notes on Nerve Disorders 27 WILLIAMS (J. W.) Aids to Biology 13 WILLIAMSON (J. M.) Ventnor and the Undercliff 16 WILLSON (A. Rivers) Chemical Notes for Pharmaceutical Students i; WILSON (J.) A Manual of Naval Hygiene 24 WINDLE (B. C. A.) Proportions of the Human Body 12 WINSLOW (L. S. Forbes) Fasting and Feeding 20 WITKOWSKI (G. J.) Movable Atlases of the Human Body 10 YONGE (E. S.) Aids to Surgical Anatomy 36 YOUNG (J. K.) Orthopedic Surgery 21 Baillifere, Tindall, and Cox's Books. AN ALPHABETICAL INDEX OF WORKS, IN MEDICINE, SURGERY, SCIENCE AND ART, PUBLISHED BY BAILLIERE, TINDALL, & COX, Abdominal Surgery. Colotomy, Inguinal, Lumbar or Transverse ; for Cancer, or Stricture with Ulceration, of the large Intestine. By HERBERT W. 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Size of sheet 28 J by 22 inches. No. 1. The External Form and Elementary Anatomy of the Horse. Price 3s. 6d., or mounted on roller and varnished, 6s. 6d. No. 2. The Age of Domestic Animals. Price 2s. 6d., or mounted, 5s. 6d. No. 3. The Unsoundnesses and Defects of the Horse. Price 2s. 6d., or mounted, 5s. 6d. No. 4. The Shoeing of the Horse, Mule and Ox. Price 2s. 6d., or mounted, 5s. 6d. No. 5. The Elementary Anatomy, Points and Butcher's Joints of the Ox. Price 3s. 6d., or mounted, 6s. 6d. Price per set of Five, 12s. ; or mounted, 27s. NOW READY. Price 6 nett. A NEW AND GREATLY IMPROVED EDITION WHITE'S PHYSIOLOGICAL MANIKIN. DESIGNED UNDER THE DIRECTION OF FRANK H. HAMILTON, M.D., LL.D. 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