(?otitell IninerHitg Cibrarg Jltt;aca, Nrm ^nrh THE CHARLES EDWARD VANCLEEF MEMORIAL LIBRARY BOUGHT WITH THE INCOME OF A FUND GIVEN FOR THE USE OF THE ITHACA DIVISION OF THE CORNELL UNIVERSITY MEDICAL COLLEGE BY MYNDERSE VAN CLEEF CLASS OF 1674 1921 Cornell University Library QH 205.C95 1922 Modern microscopy; a handboolc for beginne 3 1924 003 074 303 Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003074303 MODERN MICROSCOPY PLATE I. #--.- X ^AnW^V-- // U - *V.;;.,. t>.^^*^ (A) ASTEKOMPHALUS AKACHNE. ^p' ^^^ (C) Section of Nummulite. ft: r (B) Tryi'Anosome brucei. A^^o^il > -V (D) Cemekt Surfaces foe Comparison. (E) Structure of Electric "Weld. (F) Egss of Spider. All the above by Members of the Photomicrographic Society. I^Tontispiece {See pp. 89 and 90.) MODERN MICROSCOPY A HANDBOOK FOR BEGINNERS AND STUDENTS BY M. I. CROSS AND MARTIN J. COLE LECTURER IN HISTOLOGY AT COOKE'S SCHOOL OF ANATOMY FIFTH EDITION REVISED AND REARRANGED BY HERBERT F. ANGUS WITH CHAPTERS ON SPECIAL SUBJECTS BY VARIOUS WRITERS CHICAGO CHICAGO MEDICAL BOOK COMPANY 1922 IPrinted in Great Britain PREFACE TO FIFTH EDITION In the preparation of this new edition the original intention that it should be for beginners and students has been steadily kept in view. The first part has been entirely rewritten to bring the book into line with present-day knowledge and methods, and Parts II. and III. have been added to and rearranged with the help of numerous friends and contributors, to whom our most cordial thanks are due, so as to present to the reader as complete a picture as possible, in the space available, of the Microscope in two widely dissimilar aspects — viz., as the handmaid to Science and as the key to a new and, for many, unsuspected world full of interest and beauty. M. I. C. London, April, 1922. PREFACE TO FIRST EDITION This handbook is not intended to be an exhaustive treatise on the microscope, nor to give particulars of the various patterns of instruments that are made, of which details can be seen in the makers' catalogues, but to afford such information and advice as will assist the novice in choosing his microscope and accessories, and direct him in his initial acquaintance with the way to use it. The directions for preparing microscopic objects by Mr. Martin J. Cole are the outcome of a very long experience as a preparer of microscopic objects of the highest class, and cannot fail to be of the greatest service to the working microscopist. M. I. CROSS. London, 1893. CONTENTS PART I THE MICROSCOPE— ITS CONSTRUCTION AND USE OHAPTEK p^Qj,a Introduction . ix_x I. Definitions 1_6 II. Optical Elements 7_10 III. Optical Systems 11-15 IV. Illumination — General 16-28 v. Illumination — Special 24-28 VI. Stands . 29-39 VII. Artificial Illuminants 40-46 VIII. Method of Use — Elementary - 47-51 IX. Method op Use — Advanced 52-57 X. Recording Apparatus - 58-67 XI. Tests- - 68-77 Appendices I. G-lossahy of Technical Terms - 78-80 II. Official Gauges and Specifications 81-84 III, Microscopical Societies and Clubs 85-91 PART II THE MICROSCOPE AND THE SCIENTIST CHAPTER XII. The Microscope in Medicine — Introduction 92-102 XIII. The Microscope in Medicine — Public Health 103-111 XIV. The Microscope in Medicine — Tropical Diseases 112-120 XV. The Microscope in Histology 121-167 XVI. The Microscope in Geology • - 168-185 XVII. The Microscope in Engineering 186-198 XVIII. The Microscope in Agriculture - 199-215 Vlll CONTENTS PART III THE MICROSCOPE AND THE NATURALIST CHAPTER Inteoduction XIX. Pond Life XX. Frbsh-watbr Mites XXI. FOKAMINIFERA XXII. Mosses and Livbewobts XXIII. Mycbtozoa XXIV. Mounting Common Objects PAGES 216-223 - 224-248 249-253 - 254-266 267-276 277-289 290-309 Indbx 310-315 INTRODUCTION The development of the microscope to its present state of per- fection has been contemporaneous with the growth of scientific knowledge during the past century. It was only in 1824 that TuUy made his first Achromatic Microscope, and the records of Medical Eesearch, the Eoyal Microscopical and other Societies, and numerous departments of industry, bear witness to the unceasing efforts that have been made to improve the optical portion, and to render the mechanical means for using the lenses convenient and efficient for the purpose. As the handmaid of science, it has always kept pace with the needs of the workers in the new avenues which have opened up, frequently making a stride forward, either in theory or con- struction, and assisting in the elucidation of new facts or the clearer perception of fine structure. The assured position of the microscope in relation to scientific work will be evident by reference to the second part of this book, but microscopy has a wider sphere as a minister to pleasure and recreation. In this capacity it offers to its users the power to see into a world teeming with life that is invisible to the unaided eye. It is unfortunate that the interest of the amateur is less active than formerly, for much of the improve- ment in the microscope has been due to the criticism and encouragement of amateur microscopists. The third part of this book will indicate directions in which profitable work may be done by any intelligent observer. X INTKODUOTION It is not possible, without instruction and considerable ■ practice, to make the best use of the microscope, and it is only by knowing fully what can be done and how to do it that the worker will be enabled to wrest from his instrument all that it is capable of yielding. There is a tendency for work to be less well done than it need be, through insufficient teaching and appreciation of elementary facts, and its value to the individual user depends very largely on the methods of manipulation adopted. It is hoped that the directions given in this book will assist beginners and all who work with the microscope to a clear understanding of the best way of using their instruments. The due presentation of Parts II. and III. has somewhat encroached on the space originally allotted to Part I., but nothing essential has been omitted, and, studied in conjunction with the various makers' catalogues, it is hoped that a more complete understanding will be obtained of the construction and use of the instrument than if more space had been allotted to the subject in an endeavour to make it complete in itself. The four makers whose catalogues should be consulted are : Charles Baker, 244, High Holborn, London, W.C. 1. E. and J. Beck, Ltd., 68, Oornhill, London, E.G. 3. J. Swift and Son, Ltd., 81, Tottenham Court Koad, London, W. 1. W. Watson and Sons, Ltd., 313, High Holborn, London, W.C. 1. MODERN MICROSCOPY PART I CHAPTEE I DEFINITIONS Glass — its Physical Properties. Many people think of a microscope as a more or less complex mechanism of brass, beautifully lacquered, to which one or two lenses are incidentally attached, but actually the lenses are the essential part, the mechanism being only incidental. These lenses might be made of any transparent substance, which could be worked with accuracy to predetermined curves, and in bygone days, when the glass-makers' art bad not reached the degree of refinement it has to-day, attempts were made to use natural crystals, including precious stones ; but the varieties of glass now obtainable render recourse to such material un- necessary, excepting perhaps in the case of fluorite, which in some optical combinations gives a correction as yet unobtainable with any variety of artificial glass, and quartz, which must be used when it is essential (see Chapter XII.) that ultra-violet light should be employed, as in some research work. It is common knowledge that a convex lens increases the apparent size when the object looked at and the eye are placed in a correct position relative to it. This it does by altering the path of the light rays passing through it in accordance with the law of refraction (Fig. 1). But it is not generally known that the apparent size of the object, due primarily to the curved surfaces of the lens, is also influenced by the density of the glass — that is to say, that two 1 2 MODEEN MICKOSCOPY lenses having the same curvature, but of different densities, would not give images of equal size : the lens made from the denser glass would refract or bend the light rays more, and the image would be larger. The numerical expression of this bending power as compared with air, taken as unity, is called the refractive index, or E. 1. — -- Fig. 1. — Enlakgbd Image of an Object seen theotjgh a Bi-Convex Lens. The refractive index of water is 1"33, while that of glass ranges from 1'4785 to 1-7566. It is also generally known that the various colours occurring in nature indicate as many different wave-lengths of the vibra- tions which constitute light. These colours or vibrations of varying wave-lengths are unequally affected when passing through SCREEN WITH SLIT THROUGH WHIOH A NARROW BEAM OF LIGHT PA55ELS Fig. 2.— Beam of Light split up into its Component Colours by^Means of A Prism. a lens, but the effect is especially noticeable when a well-defined beam of light is passed through a prism, as diagram (Fig. 2). It is also observable when looking through a powerful single lens, such as a burning-glass or a cheap field-glass, the object being fringed with colour : the more powerful the uncorrected lens the more marked the colour. DEFINITIONS 3 This is known as dispersion, and, like density or power of refraction, is another variable property of glass altogether in- dependent of the density. It would be possible, therefore, to have two lenses of the same curvature and the same density or refractive power, one of which would give a more pronounced colour fringe to the image than the other. Hence arises the possibility of constructing a complete range of special glasses known as optical glass, which, by the admixture of minute quantities of various elements, can be made to give varying proportions of refractive and dispersive power. With such a series of glasses it is possible to correct the faults of one lens, or series of lenses, by another or second series, or even a third. See Figs. 7, 8, and 9, p. 8, for the means of correction ; and Fig. 28, p. 23, for one of the chief faults which, in addition to that noted above, must be corrected. Magnification. To examine an object in which there is fine structure one instinctively approaches it and, if it is portable, lifts it up. The nearer it is brought to the eye the larger the image on the retina and the more visible the fine detail becomes, until a point is reached where the internal lens of the eye, which automatically adjusts itself for objects at different distances, is at its extreme limit of adjustment. For the greater part of adult life this limit for the normal eye is set at 10 inches, and if an object is brought closer than this, the gain in size of the image is counterbalanced by the loss of definition. The Standard of Comparison. This, then, is the standard of comparison when speaking of magnification, the maximum size of image obtainable with the unaided eye, and on this the nomenclature of microscope lenses is based. If it wore possible to see a small disc sharply defined at 1 inch distance from the eye, it would be found ten times the width of the same disc held at 10 inches, the apparent diameter of an object being proportionate to the distance from the eye. A lens, therefore, which will give at 10 inches on an optical bench an image ten times the width of the object upon which it is focussQd 4 MODEEN MICEOSCOPY is called a 1 inch (a lens of 1 inch focus producing approximately this effect), and is said to magnify 10 diameters, or simply 10 X . Similarly, one giving 20 x is called J inch, 60 X |^ inch, and so on throughout the whole range. Diffraction and the Abbe Theory. Magnification looked at from this point of view is simple enough, but it was found, before the present-day theory of microscopical vision was propounded, that lenses giving the same magnification did not necessarily show the same amount of detail, or, to use the microscopical expression, give the same resolution. This remained a puzzle until Professor Abbe solved the problem by pointing out that the very fine detail of microscopic objects, acting as a grating, sets up interference phenomena, the vibrations of light damping one another out in such a way that, having passed the object, the rays, no longer vibrating in every azimuth and at all angles of inclination, are broken up into a central beam and a number of subsidiary or diffracted beams spaced, according to the nature of the structure, about the central beam in various patterns, which are repeated at definite distances from the central beam outwards, according to the fineness of the structure under examination. The finer the structure the greater the distance of the primary series of diffracted beams from the centre and the greater the spacing between the primary, secondary, and succeeding spectra, as the diffracted beams are called, the light being broken up in the process so that each diffracted beam appears as a miniature spectrum. He proved by a simple series of diaphragms that, unless a lens could receive light at a sufficiently wide angle to take in at least a portion of the first diffracted beam, as well as the central beam, no detail could be observed in the object, and that the greater the number of spectra received by the lens, the greater the accuracy of the image, although for all practical purposes the primary, or even a portion of the primary, was sufficient to give correct information as to the number of lines or dots in any given structure, but not necessarily the correct relative spacing of such detail. DEFINITIONS 5 Numerical Aperture. Professor Abbe also suggested a method of expressing numeri- cally the resolving power of a lens, whether used dry or with the front immersed. It had already been observed that the elimination of the air space between the lens front and the thin glass covering the object by the interposition of a fluid medium gave increased resolution, owing to the reflection and refraction of the light rays at the surface of the cover-glass being avoided ; but the importance of thus passing on to the lens the rays making the COVER GLASS UPPEIR SURTACE / OF OBJECT SLIP Fio. 3. — Lenses with Fbonts Dry and Immersed, showing Eats received BY Each. greatest angle with the central beam was not understood until Professor Abbe pointed out that these wide angle rays alone resolved the detail of the very finest structures. There was no accepted method of comparing the performance of dry and immersed lenses, and the whole question was confused until Professor Abbe suggested compounding the angle of light which the lens passed (angular aperture) with the medium in which the lens was used by multiplying the sine of half the angle of aperture by the refractive index of the medium ; this measure of the resolving power of an objective, known as ' numerical aperture,' is usually abbreviated to N.A. Optical Index.* Obviously the N.A. and magnification must bear some intimate relation, as it would be equally useless to combine high magnifi- cation with too low an N.A., or high N.A. with a magnification so low as to leave the detail resolved still invisible ; the extremes * Owing to the use of unavoidable technicalities this definition will hardly be intelligible to the beginner until he has digested the information given in Chapters II. and III. 6 MODERN MICROSCOPY are useless, and even a marked tendency in either direction renders manipulation extremely difficult. Nelson, with whom the study of the possibilities of the micro- scope has been a life's work, suggested, therefore, another less well-known but valuable term, called the ' optical index,' abbre- viated to O.I., which defines the N.A. necessary for every 100 X of magnification (10 initial X 10 eyepiece) as N.A. 0-26, expressed numerically by multiplying the N.A. by 1,000 and dividing by the initial magnification. The O.I. therefore of the ideal lens is 26, and the nearer the O.I. of any lens approaches this figure the greater its efficiency ; but this is the limit when the microscope is used by an expert under the most favourable conditions. In practice, for routine work, in the execution of which it is impossible to exercise the same care, the ratio of magnification to N.A. is increased in the proportion of approximately 2 : 1. Thus a lens of initial magnification 60 on the long tube that is I inch would supply, used in conjunction with an eyepiece 10 X , all the magnification required to utilize the resolving power of N.A. 1'30 which is the N.A. usually associated with a ■^ inch. The O.I. of such a lens would be : 1-30x1,000 -„ 50 ^^' which corresponds exactly with the definition given above. Allowing for difference of tube length, which necessitates a somewhat higher initial magnification to compensate for the shorter tube now almost universally used, a ^ inch of N.A. I'SO would approximate very closely to it, which does but confirm the view expressed by Barnard on p. 95 under subhead ' Diagnostic Apparatus.' For minor definitions see Glossary of Technical Terms, p. 78. CHAPTER II OPTICAL ELEMENTS Magnifiers, Objectives, Eyepieces, Tube-Length. In the preceding chapter we purposely used the more general term ' lens ' to designate the instrument of magnification. We must now differentiate between those low-power lenses, which are used by themselves alone, usually called 'magnifiers,' and those which form part of an optical system. These Magnifiers may be simple or compound. Fig. 4.— Simple Fig. 5.— Compound Fig. 6.— Simple Magnifier Magnifier. Magnifier (Plano-Oonvbx). (Aplanatic). (Bi-Oonvex). The simple magnifier, as indicated in above diagram (Figs. 4 and 6) is merely one piece of glass ground to a convex curve on one or both sides. The usefulness of such a lens is limited to approximately 2-inch 5 x . In higher powers the want of flatness in the field of view, due to spherical aberration (see Glossary of Technical Terms, p. 78, and diagram illustrating this fault, Fig. 28, p. 23), also colour fringes round the object, become very marked. The most usual form of compound magnifier is composed of three lenses cemented together after Steinheil's formula (Pig. 5). The corrections thus introduced enable one to use this magnifier up to a power of J-inch 20 x or even ^-inch 30 x ; but the field of view in the latter is very limited, and the working distance very small. These lenses are known as Aplanats. 8 MODEEN MICEOSCOPY The usual powers in this type are : 6x 10 X 12 X 16 X 20 X. The image given by both these types of magnifier is erect (see Pig. 1, p. 2) — that is to say, the image is seen the same way up as the object, and the movement of any tool used while the object is being manipulated is not reversed ; but, as already indicated, the higher powers are difiScult to use owing to close working distance, close eyepoint, and limitation of field of view. All these disadvantages can be overcome, however, by picking up the image with another magnifier, called an ' eyepiece,' and a lens used in this way with an eyepiece is called an ' objective.' Objectives. Owing to the fact that the image given by an objective is magnified by an eyepiece, it must be still better corrected. This Fig. 7. — Low Fig. 8. — Medium Power. Power. Fig. 9.— High Power. is effected by mounting one, two, or three combinations behind the front lens, which may itself be simple, as in Figs. 8 and 9, or compound, as in Fig. 7, all accurately centred with it, every detail of these additional lenses — the glasses of which they are composed, the curves to which they are ground, and the distances apart at which they are set — being carefully computed to correct the faults in the image given by the front lens, which latter is chiefly responsible for the magnification, before it is passed on to the eyepiece. OPTICAL ELEMENTS 9 In the accompanying diagram the train of lenses required for the correction of a low, medium, and high-power objective are shown, each element of the train usually consisting of two simple lenses cemented together, forming a so-called doublet. Eyepieces or Oculars. Eyepieces used in conjunction with above magnify the initial image given by the objective from 5 x to 20 x . Higher powers are sometimes used for testing and comparing the performance of objectives. They are, however, testing appliances rather than parts of a microscope, however complete, and as such would be out of place if included here. Many patterns have been devised from time to time. There are, however, only three in general use, as figured below. Of ; sssssy- FiG. 10. HUYGHENIAN. Fig. 11. Compensating. Fig. 12. Adjustable. these, the Huyghenian is the type supplied with 99 per cent, of present-day outfits, the most useful magnifications being 6x and 10 x; but the other two types are extremely useful in research work, more particularly in the higher powers (see Chapter IX.). Tube-Length. As the total magnification of the objective and eyepiece is dependent on the distance separating them, its influence must now be considered. 10 MODEEN MICROSCOPY We have seen in Chapter I. that a magnified image is always compared with the size of the object, as seen by the unaided eye, at the nearest point at which distinct vision can be maintained — viz., 10 inches— the degree of magnification being thus determined. Based on this standard, the initial power, or power before applying the eyepiece of an objective 1 inch, is lOx, and if the eyepiece is so adjusted as to pick up the image at this point, the power of the eyepiece being 10 x, the total magnification will be 100 X . The earlier microscopes were constructed on this principle, the distance between objective and eyepiece being 10 inches, but the extended use of the instrument by students created a demand for a more portable form, resulting in the reduction of the length of tube to approximately 6^ inches, or two-thirds of the standard. Therefore the initial magnification of an objective 1 inch on such a microscope is 10 X f = 6f , and the total magnifi- cation with an eyepiece 10 X is 66 X . For all ordinary purposes the tube-length may be reckoned from the two ends of the adjustable tube, into one end of which the objective screws, and upon the other end of which the eyepiece rests ; but for very critical work, comparison of perform- ance of objectives, etc., a more accurate method must be em- ployed, as the higher power objectives can only be used efficiently at the exact optical tube-length for which they have been adjusted in the course of manufacture, after an allowance has been made for possible variation of conditions (see Chapter XL). CHAPTER III OPTICAL SYSTEMS Erect and Inverted Image: Monocular and Binocular. As we have seen in the foregoing chapter, the addition of an eyepiece increases working distance and power, but the image seen by means of such an optical system, owing to the crossing of the rays within it, becomes inverted. This is no disadvantage in observation microscopes, but the erect image is essential for Fig. 13. selecting, preparing, and mounting specimens for subsequent examination with the observation microscope, as all movements of the necessary tools are reversed if the image is inverted. Erectors. The inverted image can, however, be reinverted or erected by interposing a series of reflecting surfaces between the objective 11 12 MODEEN MICROSCOPY and the eyepiece. The two most satisfactory methods of doing this are shown in the accompanying diagrams. Both are efficient, the train of Porro prisms (Fig. 15), similar to those used in a prismatic field-glass, as suggested by Greenough, being more generally used, possibly on account of the pronounced stereoscopic effect obtained, by means of paired objectives, when used binocularly, and to the simplicity of the Fig. 14. Fig. 15. monocular form, which can be had merely as an attachment ; but the Stephenson (Pig. 14) is far superior for prolonged work, because it not only erects the image, but enables the body of the microscope to be set at an angle in relation to the object. It is generally necessary to keep an object in course of pre- paration horizontal, and the possibility of doing so while looking down a tube at a convenient angle will be appreciated by all who have attempted exacting work of a fine nature for a prolonged period (see p. 263). In the Porro type of erector the tube jmust be at right angles to the object stage. OPTICAL SYSTEMS 13 Erect Image Systems. We have, therefore, available for preparation microscopeB, two types of magnifier used without eyepiece, and two types of an objective-eyepiece system with interposed erector — viz. : (1) Simple magnifier without eyepiece, as Figs. 4 and 6. (2) Compound magnifier without eyepiece, as Fig. 5. (3) Stephenson with eyepiece and erector, as Fig. 14. (4) Porro-Greenough with eyepiece and erector, as Fig. 15. The maximum useful magnification of No. 1 is 5 x , of No. 2 20 X , of No. 4 100 X , while that of No. 3 is limited only by the working distance of the objective — that is, the distance between the front lens and the object — which must, however, be sufidcient to allow of the necessary manipulation. Nos. 3 and 4 can be had either monocular, which is unusual, or binocular. A series of illustrations showing the usual method of mounting each of the above will be found in Chapter VI. Inverted Image Systems. Observation microscopes are all of this type ; they may be monocular, as is the case with 99 per cent, of the instruments in use, or binocular. Monocular. The practical limitation of magnification of the monocular form of this type is 1,000 x , such magnification being made effec- tively possible by the immersion of the front of the objective in a medium (known as cedar-wood or immersion oil) of the same refractive index as an average crown glass, 1"52. The most useful objectives are : 2 inch, 1 inch, f inch, ^ inch, used dry, and -^ inch used immersed in oil ; although weaker, intermediate, and even higher powers are made, the last, how- ever, present no advantage in resolution, and little, if any, in magnification. These, with the two most useful Huyghenian eyepieces, 6 x and 10 x , give on the 6f-inch tube a range of magnifications as under : 2 in. 1 in. | in. J in. A i"- Eyepiece 6x ... 20 x 40 x 60 x 240 x 480 x lOx ... 33x 66x lOOx 400x 800x 14 MODERN MICROSCOPY The highest objective, although universally called a xV inch, is about j-T inch by actual measurement ; consequently, the magnifications obtained are somewhat higher than those shown in this table, which are calculated for a true ^^ inch, thus bringing the total maximum magnification up to 1,000 X, as referred to repeatedly throughout these pages. BiNOCXJLAB. Of the binocular observation microscopes there two types : the Wenham, which is the older and less efficient, seeing that it is useless as a binocular for powers exceeding 200 to 300 x , and Fig. 17. the comparatively recent high-power binocular, which, as its name implies, has no such limitation. The above diagrams show the means employed in either pattern to split up the light after it has passed through the objective into two beams. In the Wenham (Fig. 16) half the beam passes direct to the eye, the other half being diverted by means of the prism P, the edge of which bisects the back lens of the objective. In the more modern pattern (Fig. ] 7) the separation is effected by a lightly silvered reflecting surface, which allows the whole beam to pass directly through it (the prism G being added to maintain direction), reduced in intensity by one-half, the remaining half being reflected from the silvered surface to the second tube, up which it passes, after being again reflected at H. Both patterns can be used as a monocular by sliding the prism out of the optic axis of the instrument. To these two types may be added the Grcenough, described in OPTICAL SYSTEMS 15 the earlier pages of this chapter as a preparation microscope, owing to the fact that it gives an erect image ; but it is often used as an observation instrument, on account of the wonderful plasticity of the image obtained with it. Therefore we have : For objects requiring a magnification of not more than 100 x , such as botanical and entomological objects and the larger forms of pond life, the Greenough. For the above, together with the whole range of pond life, the Wenham (see p. 235). For objects requiring greater magnification than 200 to 300 x , the high-power binocular. CHAPTER IV ILLUMINATION— GENEEAL For Opaque and Transparent Objects. HiTHEETo we have presupposed the efficient illumination of the object examined, for the magnifications specified would be quite unusable if such were not the case. We will now examine the means available for illuminating an object, firstly as regards the source of the light, and secondly as to the optical equipment necessary to utilize it to the best advantage. The Source of Light. This may be natural or artificial. If a north window were available looking out on to a huge cumulus cloud brilliantly lighted by the summer sun, and it were possible for these conditions to be constant whenever the microscope was in use, nothing more would be required, but unfortunately such is not the case. An artificial source is therefore to be preferred. Of artificial sources of light there are in these days an almost embarrassing variety, and one of the best, if not the best, for all-round research work is still an oil lamp ; yet, if the best, it is certainly not the most convenient, and it is necessary, when recommending appliances, to draw a sharp distinction between the microscope used as an instrument for investigating the unknown and the microscope used as a tool for routine work, such as demonstrating well-known histological detail to students, or for determining the presence or absence of certain organisms or constituents by which the medical officer of health, the engineer, or the chemist may be guided. For the former, no refinement which will add a fraction of 16 ILLUMINATION— GENERAL 17 1 per cent, to the efficiency of the outfit can be thought un- necessary, but if, with misguided zeal, these refinements are recommended to the routine worker, he is not helped, but merely hampered thereby. We have therefore devoted a chapter to the consideration of artificial illuminants suitable for the research worker (see Chapter VII.), and will proceed to lay down the requirements for a suitable universal lamp for the routine worker, merely remarking that, if the microscope so illuminated does not give the best of which it is capable, the loss in efficiency is negligible where known structure is concerned. The essentials are : An electric lamp of, say, sixteen candle- power with frosted bulb, adjustable for height throughout a range of, say, 12 inches, the lowest point from the table to the centre of the bulb being about 2^ inches, hooded in such a way that no direct light reaches the eye, but yet allows a condensing lens to be placed in close proximity to the bulb. For the examination of transparent objects only the adjustment for height could be dispensed with, but the above specification will be found universally useful. Having decided upon the source of light, we will now consider how best it can be applied to illuminate efficiently an object under examination. From the illumination point of view objects are of two classes, transparent and opaque, the former preponderating, but as the illumination of an opaque object is the simpler problem, we will take this class first. Opaque Objects. For objects requiring but low magnification, where there is plenty of working distance — that is, distance between the front of the objective and the object— a condensing lens, called a bull's-eye, will usually be found sufficient. Such lenses range from 1| to 2^ inches in diameter, and the usual method of mount- ing is shown in Fig. 52a, p. 45. Fig. 18, showing the path of rays when the lens is placed at different distances from the light source L, will enable one to adjust such a lens to better advantage than pages of description. For somewhat closer working distances— say f to J inch — or 18 MODERN MICROSCOPY where the greatest possible brilliance is required, a silver side reflector used in conjunction with the bull's-eye will be found useful. The essential part of this reflector is a highly polished para- boloidal surface cut out at the top in order that it may fit closely to the objective, as shown in Fig. 19. ^.^. Fio. 18. Objects necessitating the use of objectives with still closer working distance — say i to ^ inch — can often be efficiently illuminated by using the condensing lens in the position shown in Fig. 20, but this depends somewhat on the nature of the object and the skill of the operator. As a general rule, such objects are best illuminated by means of a vertical illuminator, which is used in conjunction with the condensing lens. ILLUMINATION— GENEEAL 19 There are two patterns of vertical illuminator, both dependent on the same idea — namely, that of using the objective itself as a condensing system, by means of which light is focussed upon the object, the magnified image of the object so illuminated being transmitted to the eyepiece through the same objective. To do this it is necessary to interpose between the objective and the tube of the microscope a reflector, the so-called vertical Fig 19. II MI I I I I I M «iimiQS]]» Fio. 9.0. illuminator, which shall throw a beam of light, projected upon it by the condensing lens, downwards through the objective, as shown in diagram (Fig. 21), of which the reflector is a prism bisecting the objective, one half being used for illumination and the other for observation. In Fig. 22 the reflector consists of a plate of thin glass, the whole of the objective being used for condensing the light and also for observation. 20 MODEEN MICEOSCOPY For low powers this transparent reflector is sometimes placed between objective-front and object (see Chapter XVII.)- Flo. 21. Fig. 22. Transparent Objects. These, as already stated, owing to the extreme tenuity of microscopic objects proper, and to the fact that the larger objects are cut or ground into slices, constitute the bulk of objects with which a microscopist has to deal. Mirrors. — Every microscope, therefore, whether it be of the preparation or observation type, is fitted with a mirror or mirrors, usually two, of silvered glass, one plane and the other concave. In the highest critical work, in which it is necessary to focus the image of the illuminant, or source of light, in the same plane as the object, the new stainless steel is sometimes used instead of silvered glass for the plane mirror, a first surface image being thus obtained free from duplication of image always present in a glass mirror of the ordinary type, often to such an extent, owing to want of parallelism between the two surfaces of the glass, as to render the mirror useless except for routine work. White opal glass, or white card, is also useful either in place of, or mounted in a cap fitting over, the usual plane glass mirror of a preparation microscope. ILLUMINATION— GENEEAL 21 The purpose of these mirrors or reflectors is to receive the light from the light source, and reflect it upwards parallel to and coincident with the optic axis of the instrument. The plane mirror does nothing more ; it merely alters the direction of the light rays without altering their character in any way. If parallel before, they remain parallel after reflection ; if divergent, divergent. It / Fig. 23. is immaterial, therefore, at what distance this mirror is fixed below the object, as shown in diagram, but the concave mirror condenses the light, bringing parallel rays, also slightly divergent and convergent rays, to a focus on the object, according to the distance at which it is set beneath it, as shown in diagram below. In low-power microscopes, therefore, where no other means of concentrating light on the object is provided, this mirror should Fig. 24. be adjustable as to distance from the object, but very few modern microscopes are so poorly equipped as not to have a sub-stage condenser, and, as the concave mirror is never used with any form of lenticular condenser, its retention, in all but the cheapest patterns, is somewhat of an anachronism. Condensers. — The concave mirror as shown above concen- 22 MODERN MICROSCOPY trates light on the object, however inefficiently, but what the concave mirror does inefficiently a series of lenses used with the plane mirror will do far more efficiently, although it is as true of the lenses as of the mirror that they cannot increase the amount of light given by the illuminant ; they can only concentrate it, which, however, the better class condensers do to such good purpose that a tinted glass screen is grateful to the eye, even when the source of light is only an oil lamp. Optical Systems. — The chief types are shown below, from which it will be seen that the same method of correction is applied to these sub-stage systems as to objectives (see Figs. 7, 8, 9, p. 8) ; but the need of correction is not nearly so great in the case of the condenser, consequently the majority of the instruments in use are fitted with the Abbe type (Pig. 25), Fig. 25. — Abbb. Fis. 26. Fig. 27 Oil Immersion. because it is inexpensive, easy to use, and gives reasonable efficiency for routine work — at any rate, up to the extreme limit of useful magnification, 1,000 x . Relative Efficiency. — It will not, however, give a sufficiently wide homogeneous or solid cone of light as to allow an objective over N.A. 0"70 to give the best result of which it would otherwise be capable ; the reason is at once apparent if we trace the path of rays, axial and peripheral, through such a system (see Fig. 28). Owing to the spherical aberration, as it is called, one can only illuminate the object with a solid cone of light of N.A. 0'50, which is about the amount that an objective of N.A. 0'70 will pass without loss of definition (see p. 54), or with a wide angle hollow cone (Fig. 29), not with both ; the focal planes of the marginal and central rays are so widely different that both cannot be brought coincident with the plane of the object at the same time. ILLUMINATION— GENEEAL 23 For research work, therefore, an aplanatic (or achromatic, as it is sometimes called) condenser should be chosen (Pig. 26), or even one of the oil immersion type (Fig. 27) ; but such condensers require very careful adjustment to the optic axis of Fie. 28. the microscope, which varies to an appreciable extent with each objective ; from which it follows that they can only be used to advantage on the finest stands. The numerical apertures of these types are approximately as under : Type. Total Aperture. Aplanatic Aperture. Dry. Immersed. Dry. Immersed. Abbe Aplanatic Aplanatic, front lens removed Immersion Immersion, used dry Immersion, front lens removed 10 1-0 0-50 1-0 0-60 1-20 1-30 0-50 0-90 0-40 0-90 0-55 1-30 All these condensers are usually fitted with an adjustable aperture diaphragm called an 'iris' diaphragm, by which the aperture can be varied at will — see p. 54 (3) ; also a carrier for stops, as Pig. 30, p. 26, or light modifers (see pp. 45, 46, and 97). CHAPTEE V ILLUMINATION— SPECIAL Monochromatic ; Polarized ; Dark-Ground. The efficiency of the means of illuminating objects described in the preceding chapter can be increased for special work by — 1. Altering the character of the light itself. 2. Altering the homogeneousness of the beam, the character of the light remaining unaltered. Monochromatic Illumination. The character of the light can be altered by selecting vibrations of specified wave-lengths, or colour, and also by polarization. The illumination of the object by light of approximately one wave-length, or colour, eliminates the chromatic aberration due to the dispersion of the glass used in making the objective (see Chapter I., pp. 2 and 3), which is never entirely corrected. This can be effected by using an illuminant giving only light of the particular wave-length required, by using an ordinary illuminant and stopping out by a light filter the wave-lengths not required, or by a combination of both methods. The sharpening-up of the image thus effected is particularly useful in research work and photomicrography, but, as can readily be imagined, there is a very considerable loss of light involved whichever method be adopted, and the more nearly the light used approaches to a true monochrome the greater the loss. The fullest possible advantage of this method, therefore, can only be obtained by using a powerful illuminant, but for ordinary routine work a blue, or preferably green, glass screen, although it passes light throughout a series of wave-lengths other than the 24 ILLUMINATION— SPECIAL 25 dominant group which gives the glass its colour, will be found advantageous ; the reduction in intensity of illumination when used with an ordinary illuminant, such as described in the preceding chapter, being more than compensated for by the increased sharpness of the image. Polarized Illumination. The alteration of the character of the light by polarization is of primary importance to the geologist, as our knowledge of the constituents of the crystalline rocks is dependent on the differences of constitution revealed by this means. For full explanation of what is meant by polarisation, of the means by which such illumination can be effected, and of the results thereby obtainable, see Chapter XVI. (' The Microscope in Geology'). Dark-Ground Illumination. The alteration of the homogeneousness of the illuminating beam without altering the character of the light itself is par- ticularly useful — in the lower powers to the naturalist, and in higher powers to the medical officer of health — as by such means it is possible to show the almost invisible transparent organisms of the pond and the altogether invisible disease germs — invisible, that is, by ordinary illumination in the living state — brilliantly illuminated in a field of inky blackness. This is the so-called dark-ground, or dark-field, illumination ; it is effected for the Low POWKE objectives by stopping out the central beam of the condenser with an opaque disc. Ordinary Condenser.— The stop (Fig. 30) for the Abbe should be half as wide again as the diaphragm opening, the edge of which is just visible at the back of the objective when the eyepiece is removed. This margin allows for stray light, want of perfect centration, influence of water when examining pond life., etc., and can be reduced in the better corrected condensers, more particularly if means are available for centring the fitting 26 MODERN MICROSCOPY carrying the condenser to the optic axis of the objective in use. For very low powers the front lens of the condenser can be removed, as shown in Fig. 31, from which it will be seen that in neither case does any direct light enter the objective, and that an object placed at the point of intersection of the annular beam would be brilliantly illuminated, and therefore seen very distinctly against the black background. The removal of the front lens gives working distance enough to focus through a considerable depth of water. Special Condenser. — For microscopes without condenser a lens with ground and blackened centre disc can be obtained, called Fig. 29. FxG. 30. Fig. 31. a ' spot lens ' ; this lens is generally of sufficiently long focus to work through a fair depth of water, but the spot, being fixed, cannot be adjusted to give the best results with any one objective. It will not, therefore, give such good results as a separate stop used in conjunction with an ordinary condenser. The stops usually sold for the purpose are of metal, as Fig. 30, but better results will be obtained by making one's own of black paper mounted on glass discs, the diameter of the centre being determined accurately for the conditions under which it will be used. Colour Contrast. — A variation of this method suggested by ILLUMINATION— SPECIAL 27 Eheinberg consists in making the centre disc, the periphery, or both, of coloured gelatine, the results obtained being both beau- tiful and instructive. The above methods suffice for magnifications up to 200 x or rather more, and good results can be obtained with the electric lamp described in the preceding chapter, or even an oil lamp ; but, obviously, the more intense the illumination, within limits, the more brilliant the picture. High Power. For higher powers, owing to the difficulty in centring, focus- sing, etc., a special condenser is invariably employed, either carried in a centring sub-stage, or fitted with centring screws. Fig. 32. Fig. 33. There are two patterns in general use, the paraboloidal and the concentric, diagrams of which we give above, showing the path of the rays through each. Both, owing to the extremely wide angle of the rays employed, must be used with an object slip of a definite thickness, with the object to be examined in fluid, and with fluid, water, or oil between the front lens and the under-surface of the object slip. The aperture of the objective used with either must also be reduced below N.A. 1-0 to cut out any direct rays which might otherwise be transmitted ; this limiting diaphragm, which is dropped on to the back lens of the objective, can be somewhat wider in aperture in the case of the paraboloidal form, although 28 MODEEN MICROSCOPY it should still be under N.A. I'O, as the condenser is fitted with an iris diaphragm, which acts somewhat as a fine adjustment, the closing of which cuts off any rays which would otherwise enter the objective, as shown by the dotted line in the diagram (Fig. 32). The range of magnification for which this method is suitable is 500 to 1,000. The candle-power required for efficient use is 100 to 500. For further particulars of the use of these high-power dark- ground illuminators see Chapter XII., ' The Microscope in Medicine.' CHAPTEB VI STANDS In the preceding chapters we have surveyed in the briefest possible manner the essential parts of a microscope — i.e., the lenses. We now have to consider the best method of mounting these various parts so that there shall be no loss of optical efficiency through mechanical failure. At first sight this may seem a very simple matter, but it must be remembered that few, if any, other pieces of mechanism have to stand such searching tests ; any shake due to looseness of fit is always magnified, sometimes to the extent of 1,000 x , and yet the various movements must be free enough to allow of adjust- ment during observation. By a process of elimination finality has been practically reached as regards the main movements of the present-day instrument, all alike being fitted with a diagonal rack and pinion coarse adjustment, by means of which the object can be brought into view, even with the highest powers and with a lever fine adjustment, which in the smaller stands is usually of a vertical, and in the larger research instruments of a horizontal, type. That this standardization of the essential movements of the microscope has not been attained but by much effort is well shown in a collection of historical instruments such as that in the possession of the Eoyal Microscopical Society (see Ap- pendix III., p. 86). Similarly the various types of mechanical movement for the stage have resolved themselves into two : an attachable pattern for the smaller stands and a built-in pattern for the larger instruments. The stability of the instrument as a whole is assured, in the former by weight rather than spread of base, the adoption of the so-called horse-shoe pattern being now almost universal for 29 30 MODEEN MICEOSCOPY laboratory instruments, which are usually used in the vertical position ; while the larger research instruments are more often fitted with a tripod foot, giving maximum stability at any inclination. All these points will be found illustrated in the next following pages, and in Appendix II., p. 81, particulars of gauges, the universal adoption of which has made possible the interchange- ability of apparatus by various makers. Many attempts have been made to design a stand which shall be universal, but the perusal of these pages, and a careful examination of the illustrations of the types which follow, should prove, even to the novice, that such a consummation is neither possible nor desirable. At the same time, it must be remarked that there has been very little excuse for the numberless patterns which have been produced in the past, apparently merely with the idea of bringing forward something new ; such a method is suicidal in these days of machine construction, and it is to be hoped .that, in the future, makers will confine their efforts to- the production of the types specified by the British Science Guild, particulars of which will be found in Appendix II., p. 82. It should be remembered, however, that these specifications were drawn up by scientists for professional work, and the naturalist would do well to choose, for his first outfit at any rate, something of lower power, preferably binocular. It is true that this type figures scarcely at all in makers' catalogues at the present day, but it is equally true that the old, simple types of binocular microscope, of which there is always a plentiful supply to be obtained second-hand, are more suitable for ele- mentary natural history than those specified by the British Science Guild. But for the fact that, being practically indestructible in the hands of the amateur, they can be obtained second-hand in sufficient quantities to meet the demand, never very great, they would doubtless figure more largely in makers' catalogues, which, on account of their omission, are apt to mislead the beginner into thinking such types obsolete and useless, which is very far from the truth. We will now proceed to illustrate and describe a series of typical stands. PEEPAEATION MICEOSCOPES 31 Holders for Lenses. The former is focussed by sliding it up and down the rod, the object being laid on the opal glass base-plate ; the latter by bending the jointed arm, which is long enough to reach well over a large object. Fig. 35.— Stand with Wrist Rests and Rack and Pinion Adjustment for Focussing Lenses, as Fig. 5, p. 7. The stage aperture, which is large enough to hold a small glass dish, is fitted with removable disc of clear glass, and is illuminated by an adjustable mirror. 32 MODEEN MICEOSCOPY Fig. 36.— Pobeo Eeectok, the Iktehnal Con- stefction of which is shown in fig. 15, p. 12. It can be cariied on a stand similar to Fig. 35, p. 31 ; low-power objectives are screwed into the lower end, the combination giving an erect Image, Fig 37.— The Stephenson Binoculau Microscope with "Wkist-Rests and Rack and Pinion Focussing Adjustment. For diagram of path of rays through this erecting system, see Fig, 14, p. 12. PEEPAEATION MICEOSCOPES 33 Fig. 38. — The Grebnough Binocular Microscope, which consists of Two Complete Mioeosoopes directed to the Same Point on the Object. The Erecting System employed is that of Porro, Fig. 15, p. 12. It is provided with folding arm-rests, rack and pinion focnssing adjustment, inclination-joint, and mirrors, 84 MODERN MICEOSCOPY ^fa^ci^ci( Fie. 39. — Laboeatoet Microscope made to Beitish Science Guild Speci- fication AND EOYAL MlCEOSCOPICAL SOCIETY GAUGES THEOUGHOUT. SeE Appendix II., p. 81. The supplementary figures in liue show the details of the screw focussing and swing-out mount of the Abbe condenser, hidden by the tailpiece in the main illustration, and also the internal mechanism of the vertical lever fine adjustment for focussing the higher powers. OBSEEVATION MICEOSCOPES 35 Fig. 40. — Laboratory Microscope with Attachable Mechanical Stage and Rack and Pinion Focussing Swing-out Mount for Abbe Condenser with Oentrinq Screws. (Gauges and Specification as Appendix II.) In line the mechanism of the two-speed fine adjustment. Fig. 41.— a Typical In- stkumbnt with built- IN Mechanical Stage, Teipod Foot, and Horizontal Levbb Fine Adjustment. Fig. 42. — An Instrument Capable of the Hiohbst Eesbaroh, fitted with Complete Rotation to Stage, Back and Pinion Adjustment for Tube- Length, AND Fine Ad- justment to Sub-Stage. Fig. 43. — A veet Complete Example of the Wenham BlNOCULAK. For diagram of the path of rays through the prism, by means of which both tubes are illu- minated, see Fig. 16, p. 14. Simpler patterns of this type are, of course, obtainable. OBSERVATION MICROSCOPES 39 Fig. 44 — The Hioh- PowEK Binocular Microscope, The above binocular, unlike the Wenham, can be used with the highest powers without loss of resolving power. For diagram of the path of rays through the prism, by means of which both tubes are equally illuminated, see Fig. 17, p. 14. CHAPTER VII AETIPICIAL ILLUMINANTS Lamps, Condensers, Screens. The advantages arieing from the use of an artificial source of illumination have already been referred to in the previous pages. Fig. 45. Fig. 46. Of the various patterns available for use with oil, gas, and electricity, the most generally useful are undoubtedly the electric lamp with frosted bulb (Fig. 45) for routine work (see Chapter VIII.), and the oil lamp (Fig. 46) for general use, including research work with the highest powers (see Chapter IX.) 40 ARTIPrCIA.L ILLXJMINANTS 41 The Oil Lamp possesses most, if not all, the good qualities to be looked for in an artificial illuminant : the source, in this case a flame burns steadily without flickering, can be brought to within a few inches of the table, or raised above the stage of the microscope for the illumination of opaque objects ; when set on edge, not broadside on, as illustrated, it can be sharply focussed in the plane of the object, and the divergent light Fig. 47, emitted can be rendered parallel or convergent by means of a bull's-eye condenser, which can be attached either to the lamp, as Fig. 47, or mounted on a table stand, as Fig. 52a, p. 45. It is sufficiently brilliant to permit the use of a monochromatic screen, such as Gifford's, but for purer monochromatic light, and dark-ground illumination with the higher powers, a more powerful source must be employed. This want of brilliance for certain work, combined with the messiness of oil, has led to the introduction of various patterns of gas and electric lamps. Gas Lamps.— Of these there are three types, all incandescent. 42 MODERN MICEOSCOPY Fig. 48, (1) The inverted gas mantle, as used for domestic lighting, suitably mounted, as Fig. 48, a variation of which adapted for use with spirit is shown in Fig. 49. ARTIFICIAL ILLUMINANTS 43 (2) The thorium disc lamp, in which a small disc of thorium is raised to incandescence by means of a gas flame impinging on it, of which there are two patterns on the market, the Traviss and the Biss. (3) The Barnard lamp, in which a metal tube containing material capable of being made incandescent, rolled in the form of a cigarette, is held at right angles to a bunsen flame, in which the protruding end of the cigarette becomes incandescent (Fig. 50). Fig. 50. These three gas lamps are named in their order of intensity. All are useful for dark-ground illumination, the first and second for low and medium powers, the third for high powers. The second can, in addition, be used for direct illumination, as the disc can be sharply focussed in the plane of the object, in the same manner as the flame of the oil lamp, without showing any sign of structure. Electric Lamps.— Of these other than the one already noticed there are also three types : (1) The Pointolite (Pig. 51), in which a small tungsten ball enclosed in the usual exhausted bulb is rendered incandescent by the current (see pp. 97, 98, and 198). 44 MODEEN MICROSCOPY (2) A miniature form of arc lamp, burning carbons in air, with either hand or clockwork feed. (3) The mercury vapour arc lamp contained in a quartz tube (see pp. 98, 99). Fig. 51. The first and second of these are useful for high power dark- ground illumination, and, on account of their great intensity, can be screened to give monochromatic light of greater .purity than can be obtained with an oil lamp ; but the third is pre- eminently the source for monochromatic light, as the incandescent mercury vapour itself emits light of but few wave-lengths within sharply defined limits, which can more easily be dealt with by means of screens than a source giving a continuous spectrum. Illuminant Condensers. The efficiency of any form of artificial illumination can, for many purposes, be greatly increased by interposing a condenser, by means of which parallel or convergent light can be obtained from the divergent rays emitted by the illuminant. This lens is often attached to the lamp (Figs. 47 and 51), and this is undoubtedly the best plan when used only for transmitted light ; but it is, perhaps, handier for general use on a separate mount, as Pig. 52a, which shows the ordinary bull's-eye condenser consisting of a single lens, such as is usually employed for illuminating opaque objects. AETIFICIAL ILLUMINANTS 45 Fig. 52b shows a two-lens aplanatic condenser with iris diaphragm and centring screws, as mounted for a photomicro- graphic apparatus or optical bench. Fig. 52a. Fig. 52b. An extremely simple but efficient condenser for transmitted light can be made out of the field lens of an eyepiece, as suggested by Nelson. Screens. Monochromatic. — In the preceding pages it has been shown that definition depends largely on the degree of achromatism attained — that is, the correction of the error inherent in a single lens, the outline of the image given by which is surrounded with colour fringes due to the unequal refraction of light rays of 46 MODERN MICEOSCOPY varying wave-length or colour, resulting in unequal magnification and consequent colour fringes. If, therefore, the light rays in the illuminant responsible for the greatest amount of aberration can be cut off before they reach the microscope, a considerable improvement in definition •will be effected ; this can be done by means of light filters, or coloured screens, so chosen that they absorb the rays it is desired to eliminate. Such screens, or filters, can be liquid or solid. One of the best for general use is the Gifford screen, passing blue-green rays only, which can be had in either form. More perfect monochromatic illumination can only be obtained by having screens specially designed for a particular illuminant, such as the mercury vapour lamp (see pp. 44 and 98). Contrast. — A range of Wratten colour screens, the ' M ' set, which can be had in glass or gelatine, will be found almost indispensable in photomicrography, as by their use the contrast between adjacent details or between detail and background can be accentuated. Intensity. — A pale-blue glass disc, inserted in the stop carrier, is usually recommended to reduce glare, but, in cases where the maintenance of colour values is of importance, as in some medical diagnostic work (see p. 97), discs of semi-platinized or half-silvered glass will be found more satisfactory. Such modifiers have the added advantage that the intensity of illumination can be controlled to a nicety by using a set in which the film deposited ranges from light to heavy. CHAPTER VIII METHOD OP USE— ELEMENTARY (FOE ROUTINE WORK) In this chapter we shall deal with the care of and method of using the simpler models for routine work, by which we mean the determination of the presence or absence of some organism or structure already well known. By simpler models we have in mind instruments the com- plexity of which does not exceed that shown in Fig. 39, p. 34, when fitted for routine work with three objectives on a revolving nosepiece, two eyepieces, and an Abbe condenser with iris diaphragm and means of focussing same, the range of magni- fication being approximately 60 x , 240 x , 600 x , with the low eyepiece, and 100 x , 400 x , 1,000 x , with the high eyepiece. Care of the Instrument. The most important point in the care of any instrument of precision is to keep it as free as possible from dust; this can only be done by replacing it in its case when not in use, or by covering it up in some way, by far the most satisfactory cover being a glass shade, a specially thick and correspondingly strong pattern of which can be obtained, called a bell glass, which has a knob at the top, by means of which it can be placed in position and removed. To Clean Mechanical Parts. — But careful as one may be, however, a certain amount of dust is sure to accumulate. This should be periodically removed by means of a soft brush and a duster before it has had time to work into the moving parts and form, with the oil, a clogging, rather than a lubricating mixture. A drop or two of oil may be required at fairly long intervals on all 47 48 MODEEN MICROSCOPY bearing parts, but it is useless to add oil if the movements are gummed up with dried immersion oil and balsam, neither of which can be considered a lubricant ; should any one of the move- ments become stiff from this cause, the fitting should be washed out with xylol sparingly applied, and afterwards relubricated. The stage will also require wiping with xylol from time to time; if bleached thereby, the vulcanite can be restored to its original blackness with a little vaseline well rubbed in. To Clean Optical Parts. — Dust on the lenses is often a source of greater trouble, because it is not so easily located by the inexperienced, but rotation of upper lens of eyepiece, lower lens of eyepiece, objective, or condenser, keeping one eye on the offending particles, or rather the images of same, will soon prove which component requires attention. The three surfaces most likely to need wiping are the outer surface of the eye-lens of the eyepiece, the inner surface of the lower or field lens of the eyepiece, and the upper surface of the condenser ; these can be cleaned with a piece of old handkerchief, moistened if necessary with xylol, not the duster used for the metal work, and not chamois leather nor Selvyt, unless it be kept in small pieces in a covered jar, as both are apt to pick up and retain particles of grit. The front lens of any objective can easily be wiped, and the^ back lens should not get dusty readily if care be taken to stand it front upwards on the table when removed from the microscope for any purp(jse, and to see that, when on the microscope, the nosepiece cover is never left in an intermediate position, but clicked home, and that an eyepiece is always in position. Despite these precautions, should an objective require cleaning, as it will do in the course of years, it is better to return it to the maker than to attempt the job oneself, as, simple as it may seem, it is not an easy job to clean perfectly without scratching a disc of glass a fraction of an inch in diameter at the bottom of a miniature well. To Clean off Immersion Oil.— This should be wiped off immediately after use by rotating the lens mount against a piece of old handkerchief until only the bead of oil on the lens is left, which is so small that one or two strokes of the wiper across the face of the lens are sufficient to clean it perfectly. METHOD OF USE— ELEMENTAEY 49 If, through an oversight, oil is allowed to dry on a lens, more should be added, by which means the dried oil becomes liquefied ; the damping of the wiper with xylol is helpful in such cases, as it renders vigorous rubbing unnecessary, the chief fault to be guarded against when cleaning lenses. Method of Use. Set up the instrument in front of a window, if daylight is to be used, avoiding direct sunlight; or in front of any artificial illuminant, such as the electric lamp described in Chapter IV., p. 17, and figured as No. 45, p. 40. 1. Set the draw tube at the point for which the objectives are adjusted, and, bringing the plane mirror uppermost, tilt it until the light is reflected up through the condenser, low-power objective, and eyepiece to the eye, making sure that the diaphragm controlling the amount of light passed by the con- denser is wide open and that the objective is held central by the clicking spring on the nosepiece. 2. Focus the low-power objective on the object by means of the rack and pinion adjustment, and the condenser to give maximum illumination ; cut down the amount of light by means of the iris diaphragm until the image is free from glare. 3. The medium power may now be rotated into position, which can be done without fear of damaging the specimen, as the low and medium power objectives are made par-focal for the tube- length for which they are adjusted — that is to say, that both focus in approximately the same plane; it is only necessary, therefore, to use the fine adjustment when changing from low to medium power or vice versa. 4. It will be found that, with the medium power in position, the diaphragm of the condenser can be opened somewhat, and the draw tube, which for the low power was set at the nominal tube- length, because such powers are insensitive to fine degrees of difference, can now be adjusted with advantage to compensate for any slight difference in thickness between the cover-glass of the object under examination and that for which the objective was set during the course of manufacture. 4 50 MODERN MICROSCOPY 5. If the object requires the highest magnification, the objective jV inch oil immersion can now be brought into position, first racking up the body tube to avoid the possibility of breaking the specimen, owing to the extremely close working distance of this objective, and also to allow space in which to apply the requisite drop of oil ; a very small quantity is sufficient, about the size of a large pin's head. It should be placed on the specirnen as near as possible to the optic axis of the instrument, which will be, of course, the centre of the front lens of the condenser. Into the drop so placed the objective is lowered by means of the rack and pinion, observation being kept on the operation, not by looking down the tube, but by watching at one side, with the eye on a level with the object, until one finds, sight aiding touch, that the front of the objective is touching the cover-glass of the object. A very slow reverse movement, still using the coarse adjust- ment, but watching this time for the image through the eye- piece, will, more particularly if the object is a well-defined one, bring it into view without difficulty, when recourse may be had to the fine adjustment, and, if necessary, to the higher power eyepiece. 6. The amount of light passed by the condenser can now be looked to and, if possible, increased, but, unless the object is a very translucent one, it will be found that the medium-power objective utilized all the light that the condenser was capable of passing. 7. Finally, the tube-length can be readjusted, as, although the question of cover-glass thickness is in this case practically elimi- nated by the use of oil, these high-power objectives are so sensitive to almost imperceptible differences in the conditions under which they are used, and so difficult to adjust to an exact tube-length during the course of manufacture, that a variation of tube-length of ^ to 1 inch either way seldom fails to improve the image. The procedure would be the same whatever the illuminant used, but if it were an oil lamp, or other illuminant with a small emission area, the whole field would not be lighted when using the low and medium powers ; this is not much disadvantage in the medium power, as the band (image of the fiame edge on, in METHOD OF USE— ELEMENTAEY 51 which position it gives most light) or disc of light is of sufficient area, in comparison with the unlighted portion of the field, as to cause little inconvenience, but with the low power the condenser must be racked down until an area sufficient for observation, or the whole field, is evenly lighted. CHAPTEB IX METHOD OP USE— ADVANCED (FOR EESEAECH WORK) In the preceding chapter we described the easiest method of setting up a microscope for routine work, purposely abstaining from any mention of refinements calling for special knowledge or undue expenditure of time, as, no matter what expert micro- scopists may urge, ninety -nine out of every hundred instruments will always be used thus, and, in our opinion, it is neither desirable nor possible that methods essential for research should be applied to routine work. It is, of course, true that a microscopist conversant with the full possibilities of the instrument can, given a stand fitted with every possible refinement, optical and mechanical, see all that the routine worker sees with half the power or less, and, employ- ing the same power, can see considerably more ; but it is also true that the same apparatus, put into the hands of the routine worker without special knowledge, would show him considerably less than the instruments with adjustments reduced to a minimum, such as are usually used in the laboratories, and would, in many cases, militate against the use of the microscope when it might otherwise be used, on account of the extra time required to set it up. In our opinion, therefore, the routine worker's outfit cannot be too simple, if it shows efficiently what research methods have already established, even though the efficiency is attained by what, in the hands of the skilled microscopist, is unnecessary power (see Optical Index, Chapter I.), and the research worker's outfit cannot be too elaborate, provided the knowledge of how to use it keeps pace with the elaboration of apparatus, and no apparatus be added unless it tends to increased seeing power, even though it be to the extent of but a fraction of 1 per cent. 52 METHOD OF USE— ADVANCED 53 We will, therefore, consider the various refinements that have been found essential to maximum resolution. Essentials. Lenses of High Quality.— It is, perhaps, hardly necessary to state that the primary condition is the use of the finest optical parts procurable, especially objectives, and the finest objectives are, undoubtedly, the so-called apochromatics origin- ated by Abbe, who first succeeded, by the aid of new optical glasses and fluorite, in practically removing the chromatic aberration of the image due to the secondary spectrum, and in correcting the spherical aberration for all colours. Objectives of this type are certainly the finest, when judged by white light — that is, light from a source emitting all wave- lengths — but the improvement in the correction of the achromatic objectives has been so great of late years that, when similar powers of these two series are compared by monochromatic light, it is very difficult indeed to detect any difference in the images, and, as the cost of the apochromatic objectives is something like three times that of the achromats, there is something to be said for the cheaper series even for research work. An incorrigible colour defect, of the nature of under-correction (see Chromatic Under-Correction, Appendix I., p. 78), inherent in all high-power objectives, whether apochromatic or achromatic, led Abbe to introduce the same defect, to the same amount, in all the powers of the apochromatic series, in order that it might be corrected by an equal error of opposite sign in the eyepiece. Such eyepieces are called compensating (Fig. 11, p. 9). This error, although present in the high-power achromats, has not been introduced throughout the whole series. Conse- quently, while the performance of an achromatic objective ^ inch is improved by the use of a compensating eyepiece, the perform- ance of the other powers is deteriorated, which fact led to the introduction of an adjustable eyepiece, in which the amount of over or under correction is variable according to the position of the eye-lens (Fig. 12, p. 9). Elsewhere it has been stated (p. 22) that the Abbe con- denser is limited in efficiency to an objective aperture of N.A. 0"70; 54 MODEEN MICROSCOPY it is evident, therefore, that for research work an aplanatic, or even an oil immersion condenser, should be used ; the former -will suffice up to an objective aperture of N.A. 130, but for the highest obtainable objective aperture, N.A. 1 '40, the latter mounted in sub-stage with jSne adjustment as Fig. 42, p. 37. Adequate Illumination. — A small source which can be focussed in the plane of the object is essential, and, for all work for which an approximation to monochromatic light is sufficient, there is nothing better than an oil lamp, as figured and described in Chapter VII., with a blue-green light filter and the flame set edge on ; but for monochromatic light of greater purity, either the mercury vapour arc or Pointolite lamp, suitably screened, will be found more efficient, owing to the greater intensity of illumination. Full Utilization of Effective Objective Aperture. — There exists an intimate relationship between the power and N.A. of the condenser and of the objective. The power should be such that the image of the fiame forms a broad band set vertically in the field of view, the space on either side being comparatively dark. The N.A. should be sufficient to fill three-quarters of the back lens of the objective with a solid cone of light ; the possibility of doing this can be determined by focussing the objective on an object mounted in balsam, and the condenser, with diaphragm fully open, also on the object, so that the image of the lamp flame is seen superimposed on that of the object, which should then be moved slightly so that the rays of light pass through a clear film of ^balsam only ; on removing the eyepiece, the back lens of the objective will be found filled, or nearly filled, with light, according to the respective apertures of condenser and objective. Owing to the depth from back to front of the lamp flame; there is some little latitude in the position of the condenser ; it can usually be racked up slightly until a point is reached at which the solid cone, or homogeneous illumination of the back lens, begins to break up, as in diagram, where an attempt to' increase the area of homogeneous illumination shown in Fig. 53 as too small to fill the middle ring, representing the diaphragm aperture set for |-cone, results in an appearance as Fig. 54; METHOD OF USE— ADVANCED 55 proving that the N.A. of the condenser is too small, the appear- ance required being as Fig. 55. This relationship of power and N.A. of condenser with the power and N.A. of objective would seem to imply the necessity for a range of condensers, but in practice it will be found that the more sensitive objectives, J to ^\ inch of N.A. 1-30, can be served with an aplanatic condenser of N.A. I'O, as the removal of the front lens reduces its power to that most suitable for an objective i or | inch without reducing the aperture to a point below that required by such objectives. There is not the same need of accuracy with the low powers, and for these the rays of light from the lamp can be parallelized by a bull's-eye, thus illuminating the whole of the field, and the aperture cut down by the iris diaphragm. Fig. 53. Fig. 54. Fio. 55. Perfect Centration. — There must, of course, be perfect cen- tration and alignment of the various component parts of the optical system, hence the necessity of using a stand with rack and pinion focussing and centring sub-stage, as Fig. 41, p. 36, with fine adjustment, if an oil immersion condenser be used. Test of Adjustment. — Seeing that in research work the object is more or less an unknown quantity, it is essential that the adjustment of the instrument should be checked on setting up, and from time to time during use, by reference to test objects, as they are called, the structure of which is known. These should be, preferably, objects showing definite periodic structure, such as the silicious skeletons of certain of the Diatomaceae, of which the most useful in this connection is Pleurosigma angulatum, which shows under the higher powers, after removal of the eyepiece, a characteristic diffraction, or back lens image, consisting of six primary spectra arranged as a hexagonal pattern about the central beam. Mechanical Draw Tube. — Owing to variable thickness of 56 MODEEN MICROSCOPY cover-glass, often unknown and unknowable once used, means should be provided, either on the objective mount itself, by correction collar, which is unusual nowadays, or on the stand, foi: compensating for the variation between the thickness of the cover-glass with which the object under examination is covered and the thickness of cover-glass for which the objective was corrected in the course of manufacture. This same adjustment will serve also for accurately determin- ing the tube-length for which the oil immersion objective has been corrected, which differs somewhat in nearly every case from the nominal tube-length. The sliding draw tube is; of course, sufficient for rough adjustment, as described in Chapter VIII., but to obtain the utmost accuracy it is necessary to observe the image whilst making the adjustment, and this can only be done by having a rack and pinion adjustment fitted to the draw tube, as in Fig. 42, p. 37. For further note on this correction see Chapter XI. Method of Use. It is only necessary to draw together the various points referred to above by detailing the steps necessary to set up a research microscope fitted with three objectives— low, medium, and high — adjustable eyepieces, and aplanatic sub-stage con- denser mounted on a stand, as Fig. 42, p. 37, taking as an object the diatom referred to above, Pleurosigma angulatum, and as illuminant an oil lamp with parallelizer (Fig. 47). 1. Set microscope and lamp in alignment with the flame of the lamp edge on ; if the nature of the object will allow, as in this case, the stand can be inclined to a convenient angle and the mirror dispensed with, otherwise the plane one should be used. 2. With low-power eyepiece and objective in position, tube- length adjusted to that for which the objectives are nominally corrected, and the object, Pleurosigma angulatum, on stage, focus both objective and condenser. 3. Close the diaphragm of the sub-stage condenser to a pio- point, and rack back the objective until the image of the iris diaphragm is seen. 4. Adjust centring screws of sub-atage, if necessary, to bring this aperture central. METHOD OF USE— ADVANCED 57 5. Eack objective down again to focus of object, and, if neces- sary, alter the position of the lamp until the flame bisects the field vertically. It should be approximately 10 inches from mirror. 6. Eemove front lens of condenser, set up parallelizer in front of lamp in order to fully illuminate the field of the low power, focus the sub-stage to give maximum illumination, reduce aper- ture of condenser to J-cone,* and insert coloured screens if the illumination is still too brilliant for the eye; finally, adjust eyepiece and iris diaphragm of condenser. 7. Proceed to examination with the medium power by removing parallelizer, after which refocus condenser, bring lamp flame central by altering the lateral screw of sub-stage if, owing to some slight difference in the centres of the objectives, such adjustment be necessary, remove eyepiece, and adjust the dia- phragm of condenser to |-cone ; replace eyepiece, adjust tube- length for cover-glass thickness ; finally, adjust eyepiece and the iris diaphragm of condenser. 8. To examine with the high-power objective xV iiich oil immer- sion, with the medium-power objective still in position, replace front lens on condenser and refocus, adjusting lateral centring screw if necessary, rack up body tube, and rotate objective xV iiich into position; apply oil to object, and rack down, watching the movement from one side, with the eye level with the object, until the cover-glass is lightly touched ; then, watching for the image, rack up slowly until it appears, adjust lateral screw of sub-stage if necessary, and amount of light passing to the objective by removing eyepiece and shutting down the diaphragm to |-cone, replace eyepiece, adjust tube-length, eyepiece, and iris diaphragm of condenser. * This J-oone illumination, advocated by Nelson as giving maximum resolution, is intended merely as a guide. The final adjustment of the iris diaphragm of the condenser ia dependent on the nature of the specimen and the quality of the objective. CHAPTER X EECOEDING APPAEATUS Position (Finders), Size (Micrometers), Shape (Drawing Apparatus), Detail (Photomicrographic Apparatus). The methods used for recording observations range from the very simplest, calling for nothing more than an engraved mark on the stage of the microscope, to the most complex apparatus such as is used in high-power photomicrography. Position (Finders). It is obvious that, when dealing with an object invisible to the unaided eye, the first step towards a record of any sort is to devise some means by which one may be able to find it again readily whenever required. Single Mark. — ^The simplest means of doing so on a plain stage is to engrave a single dot, which can be filled in with white paint, about the size of a very small pin's head, on the surface of the stage, at such a distance from the optic axis of the instrument, either right or left, that it will not be hidden by the label when any part of the specimen is brought into view. An ink or diamond mark on the glass over this spot renders it possible to replace the specimen on the stage at any time in approximately the same position, if care be taken to see that, before marking, the long edge of the glass slip is parallel to the front edge of the stage, and that parallax be minimized as much as possible by placing the eye immediately over the spot whilst marking. Wright's Finder.— With some microscopes, however, having a large stage opening, the spot would be in such a position that it would be frequently hidden by the ordinary label, as used on a 3 X 1 inch slip, and, as the method adopted must be 58 EECOEDING APPARATUS 59 applicable to any specimen, a series of squares, called a Wright's finder, is frequently engraved on the right-hand front corner of the stage, by means of which the position of one corner of the slip can be noted, the series of lines forming the squares being numbered in both the vertical and horizontal directions for this purpose. Graduated Mechanical Stage.— When the instrument is so fitted similar references will sufQce, but all such marks or references are, of course, only of use for refinding on the same instrument. Marker. — An attachment is sometimes employed which screws on in place of the objective, and draws on the cover- glass with a diamond a minute circle round the desired field ; this is the most direct method, and practically the only satisfactory one, when a particular object or field is to be examined on an instrument other than that on which it was first found. Size (Micrometers). Most microscopic objects can only be measured optically, but low-power objects can be measured mechanically. The Mechanical Method calls for a fixed point or line in the eyepiece, such as a spider's web or hair stretched across the diaphragm or a glass disc ruled with a diamond line placed upon it and a graduated mechanical stage reading by verniers, the readings of the stage being taken when either end of the object it is desired to measure are coincident with the line in the eyepiece, the difference between the two readings being the measurement required ; such a method is speedy and fairly accurate for low powers, but optical methods are to be preferred, and for high powers are essential. The Optical Method. — Measurements can be obtained by : 1. Actual projection. 2. Successive superposition of image of object and scale. 3. Simultaneous superposition of image of object and scale. The last, owing to the ease and speed with which measurements can be made, is usually adopted ; care, however, is required in checking the results, as this method is open to a source of error non-existent in the more cumbersome methods. 60 MODERN MICEOSCOPY All optical methods depend on the use of a stage micrometer, which is merely a finely divided scale ruled with a diamond point to 0-1 and 0"01 millimetre, or 0"01 and 0-001 inch ; on the usual 3x1 inch slip. Actual Projection. — The microscope is set up horizontal as for photomicrography, as described later in this chapter, the image received on a screen placed at any convenient distance, and the two points, the exact measurement of the distance between which is required, taken off by means of a pair of dividers ; the stage micrometer is then substituted for the object and the dividers applied to the image of scale, from which a direct reading is thus obtained. Successive superposition of image and scale. — ^This is a similar method to the above, except that a camera lucida is used (for description of which see below) giving a subjective image instead of the objective image given by actual projection; it is not, perhaps, quite so easy to use until one has mastered the use of a camera lucida, but, once this difficulty is overcome, it is quicker and likely to be more accurate, in that the paper on which the images are seen is in a more convenient position for accurate measurement. Simultaneous superposition of image and scale. — This is the most usual method. The scale referred to is not the stage micrometer, as it is impossible to focus two objects at once in the microscope, but a supplementary scale placed upon the diaphragm of the eyepiece, and known as the eyepiece micrometer, the equidistant spaces of which are calibrated once for all, by means of a stage micrometer, for each objective eyepiece and tube-length used by focussing the stage micrometer upon the stage and adjusting the draw tube until a whole number of divisions of the upper scale exactly superpose a whole number of divisions of the lower scale, from which, the value of the divisions of the lower scale being known, it is easy to find the value of a single division of the upper or eyepiece scale ; but it is essential that the tube-length at which this determination is made should always be used when measuring objects with it, hence the need of careful checking referred to above. Such a scale in the eyepiece, if the microscope be fitted with a mechanical stage, will be found easy to use, but without some EECOEDING APPARATUS 61 mechanical means of placing the edge of the object to be measured against one of the lines of the scale the task, especially under a high power, is extremely difficult ; a film of water between the object and stage will be found useful when measuring on a microscope without a mechanical stage, as it slows down and regulates the movement of the object slip. Various sliding and screw micrometer eyepieces have been devised from time to time, but are now rarely used; it is, however, a convenience to have a special eyepiece for the work with adjustable eye-lens, so that the scale can be accurately focuBsed. Care should be taken, when calibrating the eyepiece scale, to take as many as possible of the central divisions into account, as the subsequent division by the number taken, in order to obtain the value of each division, tends to minimize any possible error ; but the divisions at the edge of the field should not be taken, either when calibrating or actually measuring, as, owing to spherical aberration, they are less accurate than the central ones. Shape (Drawing Apparatus). The general outline of objects can readily be sketched by any- one with sufficient accuracy to constitute a record useful for reference or even illustration, if one or other of the various appliances designed for the purpose be used to aid eye and hand, but it is given to few to be able to produce a drawing which shall be both accurate and finished in the artistic sense. The aids referred to can, as in the case of measurement, be either mechanical or optical. The Mechanical Method. — All that is required for this method is a glass disc ruled in squares, which is placed on the dia- phragm of the eyepiece so that the object is seen ruled, as it were, with a series of guide lines, drawing-paper ruled in squares to correspond, and plenty of patience. The Optical Method depends on the superimposition of the images of object and paper so that a tracing can be made ; this can be done by projection or reflection. Projection of the image on the paper is by far the easier method, Lut a very powerful illnminant is required except for 62 MODEEN MICEOSCOPY low powers ; special inverted microscopes have been made fitted with an electric arc lamp for such work, but the ordinary micro- scope can be used if set up with the body tube horizontal and a reflector be fitted at the eye end to throw the image down on the paper. This reflector, in order to eliminate the double image given by the two surfaces, front and back, of an ordinary mirror, is usually a right-angled prism, as shown below (Fig. 56). The image thus projected is, however, partially inverted — ^^that is to say, it is reversed as regards top and bottom, but not side to side. Reflection. — If instead of projecting downwards we reflect upwards by means of a transparent reflector, view the microscope image by reflection, and look down through the reflector at the Fig. 56. Fia. 57. paper (Fig. 57), making the reflector of neutral tinted glass in order to eliminate the reflection from the under surface, we have — The Beale Camera Lucida, which is independent of a powerful illuminant ; in fact, for any but the highest powers we shall have to reduce the illumination in the microscope until the luminosity of the image is equal to that of the drawing-paper. This is a most important point in all subjective drawing methods, ignor- ance of which accounts for practically all the diificulties experi- enced in using such apparatus. Another point which is sometimes overlooked with this type of reflector is that the distance from eye-point to drawing-paper must be at least 10 inches, the minimum distance for distinct vision, which means that the average microscope must be built up somewhat, and that, if spectacles are worn for heading, they must be used even though they are not usually used for ordinary observation with the microscope. The disadvantage of having to use the microscope in the EECOEDING APPAEATUS 68 horizontal position with the stage vertical, an impossible one with many more or less fluid objects, combined with the partial inversion, a fault which it shares with the projection method described above, led to a modified pattern, viz. — The Ashe Camera Lucida, in which a double reflection corrects both faults. It is not so easy to use, however, as the Beale, and the position of the microscope, although more con- venient, is confined within narrow limits of inclination. If, therefore, for any reason the Beale reflector is fourfd wanting, the pattern usually chosen is that due to Abbe. Fig. 58. The Abbe Camera Lucida. — In this pattern the observer looks directly down the microscope tube, the image of the paper and pencil being transmitted to the eye by means of two re- flectors, the smaller of which is mounted directly over the eyepiece,' a small aperture in the reflecting surface allowing the rays from'.the microscope to pass to the eye (Fig. 58). It is usable with microscope vertical or at any inclination so long as the drawing-board is made to conform to the angle of the stage from^baek^to front; it must also be inclined to the 64 MODEEN MICEOSCOPY stage sideways, as shown in the figure, to prevent lateral distor- tion ; the amount of this inclination can be accurately deter- mined by using a stage micrometer as object, the board being inclined until the spaces between the lines are equalized. This pattern is free from any partial inversion of image, the drawing being an exact copy of the object as seen in the microscope, and if a medium-power eyepiece be used, which should be the case with all the patterns described (unless adjustment is provided for eyepieces with extra long or extra short eye-point), and attention be paid to equalizing the illumina- tion of object and paper, little difficulty will be experienced in using it. That considerable difficulty is often experienced in using such apparatus is proved by the number of different patterns that have been suggested from time to time, many of which are still obtainable ; they all depend, however, on the principles described above, and will present no difficulty to anyone who has grasped them. Detail (Photomicrographic Apparatus). We have seen above that it is possible for anyone to make satisfactory drawings of the shape or outline of an object ; these outlines can be filled in with a varying amount of detail, accord- ing to the ability of the draughtsman to translate what he sees into line, or masses of light and shade. Some objects lend themselves to this method of delineation ; others, again, are almost hopeless, more particularly when the detail is of a controversial nature, and its reproduction by hand must needs be open to the charge from one side or the other of being biassed. It is in such cases that photography will be found most useful, and, the necessary apparatus once installed, it will not be long before the more tedious process of drawing will be ousted, even for those objects which, before its advent, were adequately recorded with the pencil. A number of works have been written on the art of photographing the microscopical image ; we do not intend, therefore, to treat the matter exhaustively, but rather to set down as concisely as possible the EECORDING APPAEATUS 65 necessary apparatus required and the elementary principles which must be understood before attempting such a method of recording observations. The Apparatus Required. 1. The Optical Equipment. — Nothing special is required except it be a projection eyepiece with an adjustable front lens, by means of which the diaphragm, which is smaller than in the ordinary pattern, can be focussed sharply on the screen ; but such an addition is not essential, the low-power eyepieces already described giving good results, and a large number of such photographs being taken without any eyepiece whatever. The sub-stage condenser becomes perhaps of rather more importance than in ordinary visual work, the extra light given by the better corrected condensers considerably reducing the time of exposure, a very important detail when the image is so much magnified, the slightest tremor at such magnifications being fatal to definition. 2. The Stand. — Any monocular instrument mentioned in these pages, or of similar type, is usable ; but a centring sub- stage is almost essential for first-class woirk, and all internal reflections from the body tube must be eliminated either by inserting a diaphragm or a velvet lining. 3. The Camera. — This can be either vertical or horizontal, many varieties of which will be found figured in the makers' catalogues. In either pattern provision should be made for the visual adjustment of the microscope, and the subsequent linking up of the light-tight connection, without the possibility of greater disturbance than can be rectified by the fine adjustment of the microscope. If a long horizontal camera be used, some provision must be made for gearing up the fine adjustment, so that it can be operated while observing the image on the ground glass screen, which can best be done, finally, with a magnifier fixed in a tube so as to focus at the plane occupied by the sensitized surface of the plate, the ground glass screen being exchanged for a clear glass one, which acts merely as a support for the lens. 4. The Illuminant.— This depends entirely upon the magni- 5 66 MODERN MICEOSCOPY fication required; for low-power work an oil lamp with an efficient condenser, such as the Nelson doublet (Fig. 52b, p. 45), will give good results, but the greater the intensity, the shorter the exposure, the importance of which need not again be re- ferred to, and the greater the possibility of obtaining contrast, without unduly lengthening exposure, by the use of suitable coloured screens, a range of which should be in the possession of anyone making use of this method of recording observations. Method of Use. The essential point is to get every piece of apparatus into alignment. Supposing, therefore, that the microscope in use has centring screws to the sub-stage, that a horizontal camera is available, and that the illuminant condenser employed to paral- lelize the rays has an iris diaphragm and centring screws, the following procedure will be found expeditious and reliable : 1. Set each piece of apparatus as nearly in line as possible by measurement, and mark the position of each, adjusting the height of illuminant and illuminant condenser to the optic axis of the microscope. 2. Remove the microscope and set up as for visual observation, replacing it on the base-board when in perfect adjustment, after sliding back the camera front some 12 inches. 3. Test the position of the illuminant condenser in relation to the illuminant by means of a piece of card, to make sure that the illuminant is situated at the principal focus of the illuminant condenser, and that, in consequence, a beam of parallel light is being projected (see bottom diagram. Fig. 18, p. 18). 4. Having determined this distance, adjust the position of the illuminant and illuminant condenser, both vertically and later- ally, until this beam of parallel light is projected centrally upon the aperture of the diaphragm of the sub- stage condenser ; this can best be proved by means of the white card. 5. The card should then be set up against the camera front, and an image of the edge of the iris diaphragm of the illuminant condenser, closed down to a very small aperture for the purpose, should be projected through the microscope upon it ; this can be done by r;acking the sub- stage condenser very slightly in or out of the position at which it was set when placed on the base-board. EECOEDING APPARATUS 67 If not central with the field, adjust centring screws of the illuminant condenser until centration is attained. 6. Reopen iris of illuminant condenser until the field projected on the card is filled with light, and no more ; adjust projection eyepiece if in use. 7. Check centring of illuminant with illuminant condenser by placing card screen against the sub-stage condenser iris, and adjust, if necessary, by moving illuminant. 8. Having thus obtained an evenly lighted field, bring forward camera front, so as to link up the light-tight connection, and proceed to adjust image on screen, slipping into position any contrast screen decided upon before final focussing. 9. No exposing shutter is necessary, as the sensitized plate can be protected until the moment of exposure by interposing a piece of card between illuminant and microscope. By carrying out the above instructions, anyone with a know- ledge of photography would be able to do good work from the beginning, even with improvised apparatus ; but photo- micrography is essentially an art in which, if there is any considerable amount of work to be done, it is cheaper to obtain apparatus the efficiency of which has been proved than to muddle on with makeshifts. CHAPTEE XI TESTS Under the heading of tests we will now describe the apparatus and methods employed for testing the qualities of an instrument, both optical and mechanical, for which the makers are respon- sible, and the working adjustments of the instrument, for which the operator is responsible, recapitulating and amplifying where necessary explanations already given, and adding methods which could not be conveniently dealt with in the preceding chapters without loss of continuity. The tests of quality comprise: Magnification of objectives and eyepieces, Numerical aperture of objectives and condensers, Optical performance of objectives — (a) spherical aberration, (b) achromatism. Mechanical movements, the findings of which are unalterable by the user. The tests of adjustment comprise : Efficiency of illumination, axial and dark-ground, Cover-glass correction, Test objects. The exact determination of the character of the light employed, the findings of which serve as a guide to possible improvement. We will proceed to take these various tests in the order in which they are set down above. Magnification of Objectives and Eyepieces. Objectives. — The initial magnification can be determined by projecting through it an image of a stage micrometer upon a screen 10 inches from the back lens of the objective. TESTS 69 This method, although not strictly accurate (see below), will suffice for all practical purposes. The combined magnification of objective and eyepiece can be determined in the same manner, the distance being measured from the point at which the rays cross on emergence from the eyepiece, the so-called Ramsden disc, which can easily be located by holding a small screen of ground glass or card immediately in front of the eyepiece and moving it away until a point is reached at which the disc of light is brightest and of least diameter. Eyepieces. — From the figure found for the magnification of objective and eyepiece the magnification of the eyepiece itself can be determined, but it must be remembered that the initial magnification of the objective is affected by the tube-length. For instance, if an objective the initial power of which has been found to be 10 x gives, with an unknown eyepiece on an 8-inch tube, a total magnification of 80 x , the power of the eyepiece will be 10 x , not 8 x , as, in order to arrive at the magnification of the eyepiece itself, we must divide the total magnification by the proportionate part of the initial magnifica- tion of the objective developed on an 8-inch tube — viz., /^; then, 80-f- (10x^5) = 10. The power thus found would differ slightly with the higher, and still more with the lower, powers, owing to the fact that we have taken for our basis in the measurement of the initial magnification of the objective a mechanical tube-length of 10 inches rather than an optical tube-length ; but although such points must be taken into account in advanced work, they have no practical bearing on the use of the instrument, the elementary rules for which must be based on approximations. Numerical Aperture (N.A) of Objectives and Condensers. The numerical aperture of an objective can be measured in numerous ways. Objectives — dry. — No particular apparatus is necessary if some plan such as that suggested by Conrady be adopted. The procedure is as follows : On a dark background, such as a table, place two white cards 70 MODERN MIOROSCOPY with their inner edges parallel to each other and a suitable distance apart. If a dry objective is held at a sufficient distance above the table and directed towards a point midway between the two cards, images of the latter will be seen at the back of the objective, and by approaching the objective to the table these images will recede from one another, until finally they can be got to disappear under the margin of the back lens. It is obvious that when this disappearance takes place the inner edges of the two cards are lying in the direction of the most oblique rays which can enter the objective. In other words, they form, with the focal point of the objective, its angle of aperture. In order to get numerical results, the two cards must be placed at a measured distance apart, and the objective made to slide up and down along ,the edge of a divided scale, such as an ordinary foot rule. When the point of disappearance of the images of the inner edges of the cards has been reached, the distance from the front of the objective to the table is read off on the scale. This must be corrected by deducting the working distance of the objective — that is, distance between objective-front and object when used on the microscope in the ordinary way. Then : 1. If a table of trigonometrical functions is available, divide the corrected distance from objective to table by half the distance between the inner edges of the two white cards. The quotient is the cotangent of half the angle of aperture ; find its value in the table, and take from the table .the sine of the same angle. This is the desired numerical aperture. 2. If no trigonometrical table is available, the numerical aper- ture is found as follows : Square the reduced distance from objective to table, also half the distance between the two cards. Add the two squares together and extract the square root of the result. Then the numerical aperture is found as the quotient of half the distance between the pieces of card and the value of the above square root. In order to make the calculation involved as simple as possible, it is manifestly an advantage to make one of the lengths entering into the calculation unity, which is easily done by placing the two cards a distance of two units apart. TESTS 71 For objectives of low numerical aperture 2 inches will be found a suitable distance ; for those of higher numerical aperture a distance of 2 decimetres. This last measurement is suggested so that the experiment may be in its simplest form. Objectives — immersion. — For these a special piece of appa- ratus is required, such as the apertometer devised by Abbe, or the simpler form suggested by Cheshire, as illustrated below, which can be used for dry objectives also. It gives a reading at sight without calculation, and consists of a circular disc of glass with a focussing mark on the upper surface and a series of concentric circles beneath, each circle corresponding to a variation of 0"1 N.A. The reading is taken by removing the eyepiece and counting the number of rings visible in the back lens of the objective. With a high-power objective, however, owing to the small size of the image, this is not an easy matter. To facilitate reading, therefore, a special eye- piece giving an enlarged image of the rings is supplied, which is inserted in place of the usual eyepiece. Condensers. — The total numerical aperture can be measured on this apertometer, but the yig. 59. total aperture is not of so much consequence as the aplanatic aperture (see Appendix I., p. 78), which is best measured by illuminating with the condenser under trial a series of objectives of known aperture. If the image of a lamp flame be focussed in the plane of the object by the condenser, the object, which should be mounted in balsam, being shifted slightly out of the optic axis, after focussing objective and condenser so that the light passes through the film of balsam only, it will be possible to find two objectives in the series, the back lens of one of which is completely filled with light, and the other only partially filled. From which it is possible to say that the aplanatic aperture of the condenser is more than the numerical aperture of the one objective and less than that of the other ; the exact figure can only be arrived at by estimation. The more complete the series of objectives and the greater the skill and experience of the operator, the more accurate will be the determination. Owing to the depth from back to front of the lamp flame, a 72 MODEEN MICROSCOPY somewhat greater reading can sometimes be obtained by racking up the condenser slightly, without destroying the homogeneity of the illuminated area ; this should always be done, therefore, before making the final estimate. Optical Performance of Objectives. (a) Spherical Aberration. — Freedom from this defect must not be confounded with flatness of field ; the proof of good correction is to be found in the possibility of being able to focus every part of the field by use of the fine adjustment, not in an image which shall be equally sharp in every part of the field at one setting of the fine adjustment. Flatness of field, however much it may be appreciated, and greatly as it is to be desired, is, unfortunately, impossible of association with objectives of fine quality. With low powers up to J inch it is generally obtainable for a considerable portion of the field, especially with objectives of small numerical aperture. The compromise which is to be made to secure it is not such in the low powers as to materially affect the general performance. It cannot, however, be given in objectives of medium and high power. A well-corrected objective inevitably has a curved field, and the more perfectly it is corrected, the more apparent does it become. This has become increasingly recognized, and it is now conceded that it is better to get the utmost perfection of definition in the central zone rather than that sharpness should be sacrificed to flatness of field. Flatness of field in any other than low-power objectives cannot, therefore, be expected except at the expense of inferior definition, and this to the critical worker would be intolerable. (6) Achromatism.— Dr. Carpenter's old test for this correction — the examination of the cells in a thin section of deal — will give a very good idea of the colour corrections of objectives. For high powers, the markings on a frustule of the diatom Pleuro- sigma formosum are an excellent test. With the apochromatic objectives these come out quite black and white, while with those of the achromatic series any outstanding colour is at once revealed. Another method is the mercury test adopted by opticians. A small globule of mercury is placed on a slip of TESTS 73 ebonite, and a piece of whalebone or watch-spring is made to snap on it, causing the globule to split up into numerous particles of exceedingly minute size. These globules are then examined with the objective, and can be illuminated by means of a bare gas-jet, lamp, or daylight. Outstanding colour will be revealed by the globules. A cover-glass of proper thickness must be interposed when submitting lenses of considerable numerical aperture to these tests. A more permanent form of this test was embodied in the test plate devised by Abbe, which originally consisted of six cover- glasses of graded thickness, silvered on their under-surfaces, and cemented to an ordinary object slip, across the silvered film of each of which a number of coarse lines were ruled, the test consisting in the observation of the image of the edge of the silvered film. This was later superseded by an improved form in which a scaled wedge was substituted for the separate cover-glasses each of a definite thickness, but such a test-plate requires considerable experience to ensure reliable results, and must be considered more an optician's tool than part of a microscopist's outfit. Mechanical Movements. The necessary tests for the mechanism of a microscope are fairly obvious, more particularly if a high power be focussed. All rack and pinion movements should move freely, but yet not loosely, and should be free from back-lash ; that is to say, that, having brought any such adjustment up to a certain point, there should be no tendency for it to slip back on removing the fingers, and there should be no slack to take up before a move- ment commences. There are, however, one or two points which are not so obvious : The fine adjustment mechanism cannot be tested until the microscope is set up with the illuminant dead central ; for want of this precaution, a misplaced mirror will often condemn an instrument by showing an apparent lateral movement when using the fine adjustment. This adjustment, the most important and most delicate of all the mechanical parts, should be tested, not only when the 74 MODEEN MICEOSCOPY instrument is vertical and inclined, but also in the horizontal position, more particularly if the microscope is likely to be used with a horizontal photomicrographic apparatus, as some types of fine adjustment are very sensitive to position. The centring sub-stage should be tested, not only as to the possibility of centring the condenser to the optic axis of the in- strument, but also to see that the excursion in various directions is approximately equal about that axis. The draw tube should slide easily, and yet be held suffici- ently tight by the collar into which it fits as not to slip under the weight of additional apparatus such as a drawing camera, or the inadvertent pressure of the cheek during observation. All the above tests will serve to give one confidence in one's instrument rather than bring to light any serious discrepancy or falling off from standard if the microscope and accessories have been purchased from a reliable firm. Tests of Adjustment. We will now consider those tests which check the operator's use of the instrument ; such tests are vastly more important to the beginner, as faulty manipulation is responsible for a thousand times more inefficient work than faulty instruments. Ordinary, or Axial, Illumination. — Efficiency can be proved by removing the eyepiece and inspecting the back lens of the objective, the greater portion of which should be filled with a homogeneous or solid cone of light. Very few objectives can be used advantageously with more than three-quarters of the area of the back lens thus illuminated, and even then only on selected objects. With most objectives and objects one must be satisfied with rather less than this, but the |-cone, nevertheless, remains the standard. We need say nothing about position of mirror, centring of sub-stage, focussing of condenser, etc., because, if these are not correct, the even lighting of the back lens of the objective cannot be achieved ; the one test, therefore, of efficient illumination is the observation of the back lens, and if this shows something wrong, one must go through the various steps detailed in Chapter IX. until the fault has been found and corrected. TESTS 75 Dark-ground illumination, as obtained with an ordinary condenser, can be tested in the same way. After moving the object slightly to one side, so as to still allow the rays of light to pass through the medium in which the object is mounted or immersed, the back lens of the objective should appear practically dark, which proves that the stop is large enough. By swinging out the dark-ground stop, closing the iris diaphragm of the condenser so that it is just visible on the edge of the back lens, substituting an objective of higher N.A., and swinging the dark-ground stop back into position, it can be seen, on again opening the iris diaphragm, whether the stop is central and whether its diameter is excessive, having regard to the cor- rection of the condenser with which it is used. Cover-Glass Correction. The importance of making this correction has been referred to in both chapters dealing with ' Method of Use.' We will now detail more fully how this correction can be applied in a systematic manner, either by rotating the collar sometimes fitted to the objective for the purpose, thus altering the relative position of the component lenses, or by alteration of tube-length. One must bear in mind that the aim is to eliminate spherical aberration, which defect may be defined as a difference of focus between the central and marginal zones of an objective. Hence the correct tube-length or the best position of the correction collar has been found when some strongly marked detail or out- line of the object remains in exact focus under any change of illumination, say from a small to a large diaphragm opening beneath the condenser, or, better still, by changing the illumina- tion from central to very oblique, these changes being made with great care, so as not to disturb the other adjustments. The following process will be the safest and quickest: Start with the shortest tube-length, or, when there is a correction collar, with the position corresponding to the thickest cover-glass ; care- fully focus some sharp outline with, say, a \ central cone, then change to a |-cone, or, better still, to very oblique light. Unless the object— owing to an exceptionally thick cover-glass or a 76 MODERN MICROSCOPY very badly adjusted lens— is beyond the range of your adjust- ments, you will find evidence of under-correction — that is, the lens will have to be brought closer to the object with the wide cone, or oblique light, than with central light. Gradually lengthen the tube, or turn the collar, repeating the above observation after each change, until all evidence of spherical aberration has disappeared; the instrument is then in correct adjustment within your own limits of vision. It is advisable to start with the adjustment corresponding to the thickest cover, for the simple reason that this lessens the danger of running through the cover-glass and destroying the object, and possibly the front lens of the objective, when dealing with a lens of a short working distance. The difference between an objective adapted to a 6-inch and that for a 10-inch tube is that in the latter case the back com- binations of the objective are brought closer to the front lenses. This gives a slightly increased aperture. The majority of cover- glasses that are purchased and a large number of those used over commercial objects are more than 0'007 inch thick ; 0"007 inch is a medium thickness of cover-glass, but the tendency is to use thicker ones. It will be found a great advantage to buy only such objectives as are corrected for a medium tube-length,* and having the rackwork before referred to fitted to the micro- scope tube, sufficient latitude would still be allowed if a thinner cover-glass were met with ; but it would often be found necessary to close the draw tubes down to 6 or 7 inches, in order to get the best correction for the thick cover-glasses that are commonly used. Test Objects. It is very difficult to suggest a comprehensive set of test objects, although every microscopist who take^ an interest in this side of the subject usually has some dozens. A mere list of names of objects is meaningless, and a description, more particularly of the detail to be seen, or which ought to be seen, under certain conditions, on the silicious skeletons of diatoms, would be equally so, condensed within the limits of the present * Conrady has advocated that all objectives should be corrected for a tube- length of 8 inches, and with excellent reason, for such an arrangement would be a practical step in the solution of a difficult problem. TESTS 77 work. The following indications cannot but prove useful, however : For correct setting up of the microscope : The diatom Pleurosigma angiilahmi mounted dry on the cover-glass. For experimental verification of the Abbe theory ; The diatom Triceratium favus. The primary structure of this triangular diatom is a coarse hexagonal mesh, giving, with a low power and the narrowest possible illuminating beam, a striking back lens picture of the spectra, which, with the central beam, build up the microscopical image. Although the structure is coarse, these spectra are wide enough apart to be easily cut out singly by stops placed at the back of the objective, one of the most striking experiments thus rendered possible being the exclusion of all but two of the primary spectra, one on either side of the central beam ; diaphragmed in this way, the structure is resolved into zebra-like stripes instead of hexagons, the rotation of the diaphragm making these stripes parallel first to one side and then another of the triangle forming the main outline of the diatom. Owing to its close similarity to the much finer structure of Pleurosigma angulatum, it is of great assistance in understanding the possibilities of this useful test. For colour correction : Low power : Thin section of deal- Medium power : The diatom Navieula lyra. High power : The diatom Pleurosigma formosum. For definition : Low power : Proboscis of blowfly. Medium power and high power : Pleurosigma angidatum mounted dry on the cover-glass. For dark-ground illumination : Low power : Strewn slide of Polycystina or Foraminifera. High power : A minute scraping from the teeth in water showing the living bacteria always present in the mouth. The exact determination of the character of the light employed as regards wave-length can best be determined with a direct vision spectroscope, but this is unnecessary except for advanced work. APPENDIX I GLOSSAEY OF TECHNICAL TEEMS Aberration. — Any deviation of the rays of light when refracted by a lens which prevents the whole of the rays emanating from one point of the object being gathered together in a corresponding point in the image. Achromatic. — Free from chromatic aberration. Aplanatic. — Free from spherical aberration. Apochromatic. — A term coined to designate a type of objective, the image given by which has a greater freedom from chromatic aberration than that given by the achromatic type (see p, 53). Chromatic Aberration. — Want of sharpness in the image due to the unequal refraction of the various wave-lengths — that is, colours — of which the light by which the object is seen is made up. Chromatic Over-Correction. — A term applied to a lens when rays towards the red end of the spectrum are best corrected. Chromatic Under-Correction. — A term applied to a lens when rays towards the blue end of the spectrum are best corrected. Compound Microscope. — An instrument in which the image given by one lens or series of lenses is picked up and still further magni- fied by another lens or series of lenses. Cover-Glass. — The thin glass (average thickness, 0-008 inch) with which the object is covered. Dark-Ground. — A method of illumination by which the object appears self-luminous on a field which receives no direct light, and is therefore dark or black by contrast. Diaphragm. — This is understood in optical instruments to be a circular opening in a plate, the unpierced portion of which serves to cut oif the marginal rays of a beam of light. The adjustable pattern fitted to condensers called an ' iris ' diaphragm is now so universally used that the word ' iris ' is often used by itself as equivalent to diaphragm, although a microseopist still speaks of stopping down a condenser— a survival from the time when separate plates or stops, each with a different sized aperture, were used for this purpose — the word ' stop ' being now reserved for the discs used for cutting out the central beam and thus obtaining dark-ground illumination. Diffraction.— See p. 4. Draw Tube. — The tube, adjustable in length, which carries the eye- piece. Eye-Lens. — The upper lens of an eyepiece. 78. APPENDIX I 79 Eyepiece. — The lens, or combination of lenses, nearest the eye in a com- pound microscope ; sometimes referred to as an ' ocular.' Field. — A contracted form of ' field of view ' — that is, the disc of light visible when looking into the eyepiece, within the bounds of which the object is seen. Field Lens. — The lower lens of an eyepiece. Mechanical Stage. — A stage carrying the object which has movements in two directions at right angles to one another — i.e., lateral and back to front — built into the instrument (see Fig. 41, p. 36) ; or an attachment with similar movements which clips the object-slip and moves it over the surface of the fixed stage (see Fig. 40, p. 35). N.A. = Numerical Aperture. — A measure of the resolving power of a lens (see p. 5). Nosepiece. — The end of the tube into which the objective is screwed. O.I. = Optical Index. — See p. .5. Objective. — The lens, or combination of lenses, nearest the object in a compound microscope. E.I. = Refractive Index. — See p. 2. Ramsden Disc. — The plane at which the rays cross on emergence from the eyepiece. The distance of this plane from the eye lens varies with the power of the eyepiece. Resolution. — The revelation of the ultimate structure of an object. Revolving Nosepiece. — An adapter carrying two or three objectives, any one of which can be rotated into position as required. See Fig. 39, p. 34, showing one in position on the microscope. Secondary Spectrum. — In an achromatic lens the chromatic aberration is corrected for the brightest (yellow or green) rays of the spectrum, and the pronounced colour shown by uncorrected lenses is in consequence removed. A stricter examination, however, shows that rays of a different colour are not brought to the same focus, for owing to the fact that flint-glass, as compared with crown-glass, disperses the more refrangible rays relatively too much, and the least refrangible relatively too little, a peculiar secondary spectrum results from the achromatic combination, the rays corresponding to the brightest apple-green part of the ordinary spectrum being very closely united and focussed nearest the combination, whilst the other colours focus at increasing distances impairs, yellow being united with dark green, orange with blue, red with indigo. The composite effect of these colours is best seen with oblique light, causing dark objects to have apple-green borders on one side and purple ones on the other. Semi-Apochromatic. — In achromatic microscope objectives of the older type, chromatic defects that are worse than the secondary spectrum 80 MODEEN MICROSCOPY are caused by spherical aberration of the coloured rays, the spherical aberration being corrected for the brightest part of the spectrum only. Objectives made entirely of glass, and therefore showing the secondary spectrum, are called semi-apochromatic when the spherical aberration is corrected practically for all colours. Slip. — Practically all objects are mounted, temporarily or permanently, on slips of glass 3 inches long by 1 inch wide, such a mount being usually referred to as ' the slip.' Spectrum. — The band of colours produced by splitting up white light by means of a prism (Fig. 2, p. 2) or a finely ruled grating (see under Diffraction, p. 4). Spherical Aberration. — Eays of light passing through the marginal portion of a lens come to a focus nearer to the lens itself than those rays which pass through the centre of the lens, and the interval between the focal points of rays which pass through the marginal and the central parts of that lens is the spherical aberra- tion. In compound lenses this spherical aberration can be corrected for one or more special rays, and a lens so corrected is called aplanatie. It is only truly aplanatic for the particular rays for which it has been accurately corrected. Spherical Over-Correction. — A condition in which the lens unites the marginal rays at a greater distance than the central rays. Spherical Under-Correction. — The reverse of above. For illustration, see Fig. 28, p. 23. Spherical Zones. — In objectives of considerable aperture the inter- mediate rays may show decided spherical aberration, although the central and marginal rays are united. This defect is meant when spherical zones are spoken of. The degree to which spherical zones are corrected determines chiefly how large a cone of illumination and how deep an eyepiece an objective will bear before 'breaking down.' A high degree of correction for spherical aberration and spherical zones must accompany the reduction of chromatic defects before terms such as ' semi- apochromatic,' and especially ' apochromatic,' can be applied to a lens. Stage. — The table or platform on which the object to be examined is placed. Stop. — A diaphragm with an opaque centre (see Diaphragm). Sub-Stage. — The mechanism fitted beneath the stage to carry the illuminating apparatus. Zone. — A narrow annulus between centre and periphery at any distance from the centre. APPENDIX II OFFICIAL GAUGES AND SPECIFICATIONS The Royal Microscopical Society Standard Gauges. Eyepieces.* Internal diameter of draw tube : Small size ... Large size ... StJB-STAGE. Internal diameter of fitting Objective Screw-Thread. Diameter Core diameter Effective diameter Pitch Number of threads per inch Form Whitworth Screw. 0-917 in. = 23-3 mm. 1-27 in. = 32-25 „ 1-527 in.= 38-786 mm. 0-8000 in. 0-7644 „ 0-7822 „ 0-0277 „ 36. * The standards for eyepieces adopted by the R.M.S. in 1899 were four in number : No. 1.— 0-9175 in. = 23-300 mm. No. 2.— 1-04 „ =26-416 „ No. 3.— 1-27 „ =32-258 „ No. 4.— 1-41 „ =35-814 „ At the present time practically only two eyepiece, standards are in general use, viz., the Nos. 1 and 3, and the Council recommends that these should be known respectively as the small and large size R.M.S. Standard Eyepieces. 81 82 MODEEN MICEOSCOPY The British Science Guild Specifications. Type I. — A cheap instrument for the use of students. 1. Stand. — The modified Continental type, with jug handle; with spiral rack and pinion coarse adjustment. 2. Tube. — Short, with graduated draw tube, allowing for length of nosepiece ; the available tube-length should be from 140 mm. to 180 mm. 3. Fine Adjustment. — Lever type ; lateral milled heads. 4. Stage. — Large, square, fair- sized opening, provided with clips of simplest type, having points of contact level with equator of stage. 5. Mechanical Stage. — Not required, but provision made for its later addition. 6. Sub-Stage Condenser. — Abbe type, accurately centred and fitted with iris diaphragm. 7. Nosepiece. — Dust-proof. Position of objectives marked on nose- piece. 8. Objectives. — Two, of focal length 16 mm. and 4 mm. (or their equivalents), the latter with good working distance, both engraved with focal length, N.A., tube-length, and the magnification at the distance for which the objective is corrected expressed by X ... 9. Oculars. — Two, of focal length 40 mm. and 25 mm. Each ocular should be engraved with the initial magnification, the focal length in mm., and with a magnification number arrived at by dividing 250 by that focal length and expressed by X . . . The microscope should be capable of carrying the additions required for more advanced work. Type IL — A good instrument for advanced pathological work. 1. Stand. — Modified Continental jug-handle type, with spiral rack and pinion coarse adjustment. 2. Tube. — Short, with graduated draw tube ; the scale to allow for length added to the tube by the changing device (viz., nosepiece). The available tube -length should be from 140 mm. to 180 mm. 3. Fine Adjustment. — Lever type. Lateral milled heads of small diameter, but long milled surface. 4. Stage. — Large. Square, with sufficient room to accommodate a large Petri dish. Clips of simple description, having points of contact level with equator of stage. 5. Mechanical Stage. — (a) Detachable type : rack and pinion covered through entire length and actuated by milled heads of small diameter, but with long milled surface. Must work smoothly, and permit of examination of at least 2 inches, of a 3-inch slide. (6) Built-in type. APPENDIX II 83 6. Sub-Stage Condenser. — Abbe type. Rack and pinion, centring, iris diaphragm. 7. Triple Nosepiece. — Dust-proof ; position of the three objectives to be marked on nosepiece. 8. Objectives. — Three, of 16 mm., 4 mm., and 3 or 2 mm. oil im- mersion. Colour of mount of immersion lens to differ from others, and preferably to be black. Each objective should be engraved with the tube-length, the N.A., and the magnification at the distance for which the objective is corrected expressed by X . . . 9. Oculars. — Two, of focal length of 40 mm. and 25 mm. Each ocular should be engraved with the initial magnification, the focal length in mm., and with a magnification number arrived at by dividing 250 by that focal length and expressed by X . . . Typk III. — An instrument designed especially for research work, the character of this to depend on the individual requirements of the different workers. 1. Stand. — With spiral rack and pinion coarse adjustment, and to be equally stable in vertical or horizontal positions. The centre of mass should be kept as low as possible consistent with freedom of access to the sub-stage. 2. Tttbe. — Minimum diameter of body tube to be 50 mm. internally, with double draw tube, so that either long or short tube objectives can be used. Second draw tube to be 100 mm. long, and both draw tubes to be graduated in mm. 3. Fine Adjustment. — Lever type. Lateral milled heads on both sides of stand, of small diameter, but with long milled surface. Fine adjustment must be graduated. 4. Stage. — Circular rotating, with centring screws and provided with a clamp. The mechanical part should be graduated with verniers and be detachable, and so fitted that when removed a plane surface is left. As an alternative, it should be built in with separate and independently operating milled heads. 5. Objective Changing. — Should have sliding objective changers, which should be provided with centring screws. 6. Sub-Stage. — Should have centring screws. Rack and pinion to be exceedingly well fitted and made. Sub-stage iris diaphragm should be rotatable, and should have rack- work lateral motion. 7. Condenser. — An achromatic immersion condenser which can be used dry. 8. Darh-Gi'ound lUuminatm: — Type in which there are spherical reflecting surfaces. Some device in upper surface which, without interfering with the satisfactory working of the accessory, will indi- cate the axial centre. 84 MODEEN MICROSCOPY 9. Objectives. — The following battery of lenses should form the foun- dation, which would, of course, need addition for the particular research work engaged upon: 16 mm., 8 mm., 3 mm., or 2 mm. focal length. Each objective should be engraved with the tube-length, the N.A., and the magnification at the distance for which the objective is corrected, expressed by X . . . Mounting of immersion lenses to be distinctive, preferably black. 10. Oculars.— ^Two to form the basis of equipment, of 40 mm. and 25 mm. focal length. Each ocular should be engraved with the initial magnification, the focal length in mm., and with a magnification number arrived at by dividing 250 by that focal length and expressed by X . . . APPENDIX III MICROSCOPICAL SOCIETIES AND CLUBS The Royal Microscopical Society. (Established 1839. Incorporated by Boyal Charter, 1866.) 20, Hanovbe Square, London, W. 1. President: Professor Frederic J. Cheshire, C.B.E., F.Inst.P. Hon. Secretaries\l°^''^^ .^•.P^'"'^'"'^.-^^'''''-^- LJames a. Murray, M.D. The Society was established in 1839 for the promotion of micro- scopical and biological science by the communication, discussion, and publication of observations and discoveries relating to (1) improve- ments in the construction and mode of application of the microscope, and (2) biological or other subjects of microscopical research. It consists of ordinary, honorary, and ex-olficio Fellows of either sex. Ordinary Fellows are elected on a certificate of recommendation signed by three ordinary Fellows,* setting forth the names, residence, and qualifications of the Candidate, of whom the first proposer should have personal knowledge. The certificate is read at two general meet- ings, the candidate being balloted for at the second meeting. The admission fee is £2 2s., payable at the time of election; and the annual subscription is £2 2s., payable on election and subsequently in advance on January 1 in each year, but the annual subscriptions may be compounded for at any time for £31 10s. Fellows elected at a meeting subsequent to that in February are only called upon for a pro- portionate part of the first year's subscription. The annual subscrip- tion of Fellows permanently residing abroad is £1 lis. 6d., or a reduction of one-fourth. * Forms of proposal for Fellowship, and further information, may be obtained on application to the Secretary, 20, Hanover Square, London, W. 1. 86 86 MODERN MICEOSCOPY Honorary Fellows (limited td 50), consisting of Fellows eminent in microscopical or biological science, are elected on the recommendation of five ordinary Fellows and the approval of the Council. Ex-officio Fellows (limited to 100), consisting of the Presidents for the time being of any societies having objects in whole or in part similar to those of the Society, are elected on the recommendation of ten ordinary Fellows and the approval of the Council. The Council, in whom the management of the property and affairs of the Society is vested, is elected annually, and is composed of the President, four Vice-Presidents, Treasurer, two Secretaries, and twelve other ordinary Fellows. The meetings are held on the third Wednesday in each month from October to June, in the Lecture Hall, at 20, Hanover Square, W. (at 7.30 for 8 p.m.). Visitors are admitted on the introduction of Fellows. The business of the meetings includes the reading of papers, the exhibition of microscopical objects and apparatus (usually on view by 7.30 p.m.), lantern demonstrations, and discussions. Sectional Meetings. During the session additional and less formal meetings are held in the Society's library, and are devoted to exhibits, communications, and discussions. The Biological section meets in the Library on the first Wednesday in each month at 7 for 7.30 p.m. Hon. Secretary : Mr. J. Wilson, 3, West Park Road, Kew Gardens. The Leather Industries section meets occasionally at 7 for 7.30 p.m. Hon. Secretary : Dr. S. H. Browning, 22, Harley Street, W. 1. The Metallurgical section meets occasionally. Hon. Secretary : Mr, F. Ian G. Rawlins, White Waltham Grove, near Maidenhead, Berks. A Paper Industries section is in course of formation, and will deal with researches relating to timber, wood pulp, paper, etc. Those who are interested in the subject and willing to assist are asked to commu- nicate with Mr. James Strachan, F.Inst.P., 74, Blenheim Place, Queen's Cross, Aberdeen. The Library. The Society's rooms, in which are housed the reference and lending library, the extensive and illustrative collection of instruments and apparatus, and also the unique collection of type slides, etc., are open for the use of Fellows on ail week-days (except Saturdays and public holidays), from 10 a.m. to 5 p.m., with the exception of four weeks during August and September. The library and rooms of the Society are also open for the use of Fellows on Wednesday evenings, other APPENDIX III 87 than meeting evenings, from 6 to 9 o'clock, except during the vaca- tions. Situated in the centre of the West End, they are easily reached from Oxford Circus, Bond Street, Piccadilly Circus, or Dover Street Tube Stations. The Fellows residing outside the London area, but within the British Isles, are enabled to obtain by post any of the volumes in the Society's Library (of which there is a printed catalogue) with the exception of a few of the rarest works, and as the Society is also a subscriber to Lewis's Medical and Scientific Library, practically any recent scientific work can be obtained for a Fellow requiring it. The Journal. The Society's Journal is edited by Professor John Eyre, M.D., M.S., F.R.S.E., and Charles Singer, M.A., M.D., F.E.C.P., with the assistance of the Publication Committee, and J. Arthur Thomson, M.A., LL.D., F.R.S.E., Regius Professor of Natural History in the Uni- versity of Aberdeen ; J. E. Barnard, F.Inst.P., A. N. Disney, M.A., B.Sc., J. Bronte Gatenby, B.A, B.Sc, D.Phil., F. Ian G-. Rawlins (Fellows of the Society); and A. B. Reudle, M.A., D.Sc, F.R.S., Keeper, Department of Botany, British Museum. It is published quarterly. All Fellows are entitled to a copy, and it is also sold to non-members, at an annual subscription of 42s. post free. It contains : 1. The transactions and proceedings of the Society. 2. A summary of current researches relating to zoology and botany (principally invertebrata and cryptogamia) and microscopy contained in the leading scientific journals of the world. 88 MODERN MICROSCOPY The Quekett Microscopical Club. {Founded A.D. 1865.) President : D. J. Scourfibld, F.K.M.S. Hon. Secretary : S. B. Wycheeley, F.K.M.S. The Quekett Microscopical Club, founded principally by the late Dr. M. C. Cooke, who presided at its inaugural meeting on July 7, 1865, has, according to the prospectus then issued, 'been established for the purpose of affording to microscopists in and around the Metro- polis opportunities for meeting and exchanging ideas without that diffidence and constraint which an amateur naturally feels when discussing scientific subjects in the presence of professional men.' The ordinary meetings of the Club are held on the second Tuesday in each month from October to June inclusive, when communications relating to microscopic subjects are read and discussed, new instru- ments and apparatus exhibited and described, and interesting objects displayed. Meetings for 'conversations' and exhibition of objects are held on the fourth Tuesday of each month during the year, and on the second Tuesday of the months of July, August, and September. During the summer months excursions are held on Saturday afternoons to various well-known collecting grounds around London, to afford members valuable facilities for becoming acquainted with the haunts and habits of living organisms under experienced collectors and guides. The Club possesses an extensive library of scientific works and a large collection of mounted microscopic objects for lending out to members. The annual subscription is 10s., no entrance fee, and members receive a copy of the Club's Journal of Proceedings as published either once or twice in the year. The Club has over 500 ordinary members and a few honorary members, distinguished for their work in the scientific world. The meetings are held at the Rooms of the Medical Society of London, 11, Chandos Street, Cavendish Square, W. 1, to which all correspondence should be forwarded, addressed ' The Secretary.' APPENDIX III 89 The Photomicrographic Society. President : Joseph E. Baenaed, F.R.M.S., F.Inst.P. 8ecreta/ry : J. G. Bradbury, F.E.M.S., 1, Hogarth Hill, Finchley Eoad, Hendon, N.W. 11. The Photomicrographic Society was founded in 1911 by a small group of microseopists and photographers, the majority of whom were already Fellows or members of the Koyal Microscopical Society and the Royal Photographic Society, with the object of banding together those workers interested in the recording of microscopical observations by photography. Their opinion at the time, amply confirmed in later years, was that the use of photomicrography in those branches of research usually connected with the microscope would be followed by wide extension to commercial and other branches, and at the present day the Society has on its roll of members many who are connected with medical and biological research, engineering, public health, and those trades and professions which use the microscope and have to record the result of their observations. To the efforts of its first President, the Eev. F. C. Lambert, F.R.P.S., the foundation of the Society was mainly due, and others who followed him in this office were the late T. E. Freshwater, F.R.M.S., F.R.P.S., G. Ardaseer, F.R.P.S., J. E. Barnard, F.Inst.P., F.R.M.S. (past President of the Royal Microscopical Society), Dr. G. H. Rodman (now President of the Royal Photographic Society), F. Martin Duncan, F.R.M.S., F.R.P.S., F.Z.S., Commander M. A. Ainslie, F.R.A.S., F.R.M.S., and Dr. Duncan J. Reid, F.R.M.S. Mr. Barnard is now President for the second time, and to his help, especially in its early days, the Society owes a large measure of its success. It is interesting to note that the first member to be actually elected was the late Dr. E. J. Spitta, the well-known author of what may be regarded as the first standard book on photomicrography. From the first the Society was warmly welcomed, and has increased in numbers year by year. It has always recognized that the first essential to success in photomicrography is a thorough understanding of miijroscopic technique, especially as regards correct methods of illumination. Following upon this, its objects have been the study of methods of mounting and staining microscopic objects, the use of light filters, etc., and the improvement of microscopic and photo- graphic apparatus to the same end. A marked feature has been the attention paid by individual members to the designing of new and improved apparatus and improvements to illuminants. The Society's meetings are held at King's College, Strand, at 7 p.m., on the second and fourth Wednesdays in the month from October 90 MODEEN MICEOSCOPY to May inclusive, the first meeting in each month being a lecture or demonstration ; the second meeting, of an informal character, being devoted to discussion, demonstration, and exhibition of members' photomicrographic work. Visitors are heartily welcomed on the lecture evenings. Examples of the Wokk of Members. For examples of the work of the members, see the froatispiece of this volume, the photomicrographs of which were contributed by the following : (A). Diatom, Asteromphalus arachne, X 840. E. A. Pinchin, F.R.M.S. (B). Blood parasites, Trypanosome hrucei, x 1,040. Dr. Duncan J. Eeid, F.R.M.S. (C). Section of Nummulite in limestone, x 9. C. H. CafTyn, F.R.M.S. (D). Surface of plain Portland cement showing blowholes compared with Portland cement mixed with a commercial preparation to render it damp-resisting and homogeneous, x 20. J. G. Bradbury, F.R.M.S. (E). Structure of an electric weld, of the ' V butt ' type, between two |-inch mild steel plates, of the quality used in shipbuilding. Molten steel, in three layers, has been filled into the V-shaped space between the ends of the two plates. The view shows the differences between the characters of the several layers of metal, and also the effect on the plate of the intense heat of the electric arc. It will be seen that the upper part of the first layer and the whole of the second layer have been ' refined ' by the heat, while the bottom of the first layer and the whole of the third layer retain their original course structure. Such low-power views — in this instance only two magnifications — are par- ticularly serviceable for studying welds of this type. Under inter- mediate powers, 100 to 200 magnifications, many of the characteristic structural features are plainly revealed ; but to show those character- istics on which depend the brittleness or toughness of the weld, higher magnifications of 500 to 1,000 diameters are desirable. Professor B. P. Haigh, D.Sc., M.B.E. (F), Eggs of spider, x 10. W. H. Baddeley, F.E.M.S. APPENDIX III 91 The Manchester Microscopical Society. {Affiliated to the Royal Microscopical Society.) President : Professor F. E. Weiss, D.Sc, P.B.S., F.L.S. Hon. Secretary : William Dixon, Broadwater, 43, Pine Boad, Didsbury, Mauohester. This Society was founded in 1880 to associate residents in the city and its suburbs interested in microscopy and natural history. Its members number over two hundred, including honorary, corre- sponding, and ordinary members. Ladies are eligible for election. Subscription. — Entrance fee, 5s, ; annual subscription, 8s. 6d. The meetings are held in the rooms of the Literary and Philosophical Society, 36, George Street, on the first Thursday of the month, except in July and August, when there are no meetings, and September, when the meeting is held on the third Thursday. All meetings commence at 7.0 p.m. A Mounting Section was formed in 1882, with the object of holding demonstrations in the most approved methods of preparing and mount- ing microscopical objects, and in practical biology and microscopy. Membership is confined to members of the Society. Subscription, 5s. per annum. The meetings are held on the third Thursday in the month, except during July, August, and September. An Extension Section was founded in 1898, its object being to bring scientific knowledge, in a popular form, before other societies who are unable to pay large fees to professional lecturers. The work is entirely voluntary and gratuitous on the part of the members. The Society possesses an extensive library, a large collection of microscopical slides, and several microscopes, with the necessary accessories, all of which are accessible to members. Rambles are arranged for most Saturdays in the summer, for the collection of various microscopical objects, ' pond life ' being the most popular. The Society issues a monthly circular to members, and publishes its Annual Report and Transactions in September. The latter is obtain- able from the Secretary at the address given above, the charge to non- members being Is. 6d. (by post Is. 7^d.). " Note. — A number of provincial Natural History Societies have a microscopical section, particulars as to any one of which can be obtained by communicating in the first instance with the Secretary of the Royal Microscopical Society, 20, Hanover Square, Loudon, W. 1. PART II THE MICROSCOPE AND THE SCIENTIST CHAPTER XII THE MICROSCOPE IN MEDICINE— INTEODUCTION By JOSEPH E. BARNA.BD, F.B.M.S., F.Inst.P. Fast President of the Boyal Microscopical Society ; President of the Photomicro graphic Society. In no branch of science is the use of the microscope so widespread as in that department known as medicine, using the term in its widest sense to cover all its allied branches ; no apology, there- fore, is needed for the recapitulation of various details already dealt with in the preceding pages, changing our point of view from the general to the particular. In medicine the instrument is used for educational, diagnostic, and research purposes, the two former being but the offspring of the last, for the research of to-day, the pushing on into the unknown, becomes the ascertained fact of to-morrow, to be utilized educationally to teach a truer conception of the functions of the various organs of the human body and of other organisms, whether parasitic or otherwise, affecting its health, or clinically to aid in the diagnosis of disease. If, therefore, one would understand what is meant by the comprehensive term ' medical research,' as pursued with the aid of the microscope, one cannot do better than peruse the two following chapters, every fact stated thei'ein having once formed the subject of long and patient enquiry, the elucidation of which has opened up still further avenues for attack on problems as yet unsolved, some of which are enumerated at the end of Chapter XIII. It is the province of research not only to discover facts by means of every appliance and refinement of technique possible, but, when found, to suggest the means by which details, so 92 PLATE 11. •%V2 Fig, 60. — Sac. r.\sTrii!i.\xus. St.-vixed. X 675. Fif4. 61. — S,vr. pAsi'iii'.iAxn.'<. D\KK GiioUNii. X 900. 'P^f % #^ t ® ® h ■i,'^: F[f;. 62. — Sac. PASTOr,TAxr. i^j-'^~ k Q- o.i}0 o cs L o °^ yCf. V Hi I (' vfil^^ ' 1 --',' I, Fig. 6G -Starch of AVhkat. Fit;. 67. — Starch of Rve. r O A^ ■ vi r..^\ -- - '(Y. Fio. 68. — Starch of Barley. \ >^ Fig. 69. — Sewage Fungus. ■^^ Fig. 70. — Pesioillium glauoum. ■ h/ ■ V'- ! Fig. 71.— BiciLLus antheaois. \ To face p. 103. CHAPTER XIII THE MICEOSCOPE IN MEDICINE— PUBLIC HEALTH By W. E. COOKE, M.D., F.R.C.P.E., D.P.H., P.B.M.S. The study of state medicine embraces parts of many sciences — geology, bacteriology, entomology, etc. — in all of which microscopy plays an essential part, so that it is imperative for anyone taking up that branch of medicine first of all to become thoroughly acquainted with the uses and abuses of the microscope. The early history of public health is so staged in the dim and distant past that it is difficult to exactly define the period when microscopy first contributed to, or was applied to, the elucidation of its many problems. Perhaps pride of place may be given to the observations of Van Leeuwenhoek on vinegar about 1780. He described Anguil- lula aceti, and proved to his own satisfaction that the sharp taste of vinegar was not due to the pointed tails of the little nematodes, but to some substance which was neutralized by crushed crabs' eyes. He thus showed that A. aceti was a contamination, and not an essential part of vinegar. In 1849 PoUender noticed a rod-shaped organism in blood' of cattle dead of anthrax. The association of the bacillus with the disease recognized by Davaine in 1863 (Plate III., Fig. 71). From these beginnings the microscope has played such an important role that it is difficult to-day to find any part of the Public Health Acts, by-laws, regulations, etc., that is not based upon microscopical observation, or which has not been confirmed by microscopy. Its place in modern state medicine may be imagined from the following summary of its uses : 1. The examination of air, soil, and water. 103 104 MODEEN MICEOSCOPY 2. The detection of adulterations, parasites, and pathogenic organisms in food. • 3. The utilization of micro-organisms for the disposal of sewage. 4. The diagnosis of infectious and parasitic diseases. 5. The investigation and control of the carriers of disease: human, animal, and entomological. 6. Research. A very brief account will enable us to realize what public health in Britain owes to the microscope, and the vast amount of work still remaining to be accomplished. 1. The Examination of Air, Soil, and Water. Air. — Air contains particles of soot ; debris from the bodies of man and animals (hair, epithelial scales, etc.), from street traf&c and industrial operations ; the spores and pollen of plants ; fibres of wool, silk, and cotton ; mineral particles and micro- organisms — moulds, yeasts, the organisms of infectious diseases, the tubercle bacillus, sarcinae, etc. Pure mountain air contains about 2,000 particles per cubic inch. The number increases as the populous districts are reached, until in overcrowded slums more than 3,000,000 may be counted, a large proportion of which are micro-organisms. The air of factories and workshops contains d6bris from the manufacturing processes carried on, such as cotton fibre, woollen fibre, dust, mineral particles, etc., excess of which leads to pul- monary affections, chronic laryngitis, chronic bronchitis, anthra- cosis, silicosis, and pulmonary tuberculosis. In some processes — e.g., wool-sorting and hair-sorting — the air may contain anthrax spores. The microscope has led to better ventilation generally, and the framing of the regulations which ensure freedom from risk, and the much-improved conditions under which industrial workers find themselves to-day. Our knowledge of the aerial transmission of disease is very limited, and much remains to be investigated. Soil. — In addition to the non-pathogenic organisms found in soil, B. tetanus, B. anthracis, the bacillus of malignant oedema, B: typhosus, B. coli, B. enteritidis sporogenes, and B. diphtheria, have all been demonstrated, or cultivated, in the upper layers THE MICEOSCOPE IN PUBLIC HEALTH 105 of the soil. Ankylostovium duodenale and Ascaris lumbricoides may be transmitted by soil. The advantage of the modern practice of thorough drainage of the soil and the rendering impervious of the house site gains confirmation from the above observations. Water. — Chemical evidence as to the character of a water is presumptive only ; the microscope is necessary to furnish the positive evidence as to its purity or otherwise. Suspended matter, such as sand, chalk, clay, cotton and linen fibres, fragments of leaves and other vegetable tissues, fragments of insects, hair, and wool, may be found, also living organisms — Ehizopoda, Infusoria, Hydrozoa, Insecta, Fungi, Algae, Diato- macese, etc. — which, harmless in themselves, indicate the presence of organic matter for their sustenance. The ova of Oxyuris vermicularis (Plate IV., Fig. 74), Ascaris lumbricoides, and Trichuris trichiura are said to be transmitted by water. Bacteeia in Water. — These may be grouped into three classes : the ordinary water bacteria, sewage bacteria, and patho- genic bacteria. The ordinary water bacteria comprise chromogenic and fluor- escent organisms, and organisms of the air and soil. The sewage bacteria include the B. coli groups, B. proteus group, B. enteritidis sporogenes, streptococci, and staphylococci. Found in water, they indicate human faecal contamination, and therefore possibilities of extensive outbreaks of typhoid fever or cholera, and condemn the water-supply at once. The chief pathogenic bacteria found in water are the typhoid bacillus and the spirillum of cholera. The epidemics of typhoid fever that occurred in the last century at Over Darwen, Worthing, Maidstone, and at Lincoln in this century, and the outbreak of cholera at Hamburg, would probably not have occurred if strict microscopical supervision of the water-supplies had been exercised. 2. The Detection of Adulterations, Parasites, and Pathogenic Organisms in Food. Flour (Plate III., Fig. 66) is adulterated with foreign starches — potato and rice — and alum and calcium sulphate sometimes 106 MODERN MICROSCOPY added. Fungi and vibriones and Acarus farince can be detected by the microscope. Coffee is adulterated with chicory, roasted and ground peas, beans, bran, rye, barley, and acorns. Flour, maize, and potato starches and clay are found added to mustard, while occasionally tea contains added mineral matter, gypsum, etc. Barley is associated with actinomyces. The TyroglyphidsB, which infect flour, cheese (Plate IV., Fig. 76), dried fruits, etc., and the Glyciphagi found in sugar, are easily detected by the microscope. Milk. — This is an essential article of diet, especially for infants and young children. Contaminated, it has been respon- sible for many epidemics of scarlet fever, typhoid fever, cholera, diphtheria, sore throat, etc., is a constant distributor of tubercu- losis, and causes an enormous number of deaths annually from infantile diarrhoea. In addition to the foregoing, anthrax bacilli, B. dysenterim, and the paratyphoid organisms, may be spread by milk. The microscope is necessary to check milk- supplies, and gives indications of disease in the cow, the cleanliness or otherwise of the processes of production, storage, and distribution of the milk, and the presence of pathogenic organisms. Suppurative disease of the udder is detected by an increase in the number of milk leucocytes, and lack of cleanliness in the production by the presence of hair, scales, and straw in the sediment, and by the number of bacteria per cubic centimetre. The number varies from 5,000 to 10,000 in Grade A milk to 2,000,000 to 3,000,000 per c.c. in badly produced and stored milk. The pathogenic organisms have already been mentioned. It is obvious that more rigid and constant bacteriological control should be exercised over milk- supplies. Fish. — Epidemics of typhoid fever have been caused by oysters, cockles, and mussels taken from beds contaminated by sewage. B. coli and B. enteritidis sporogenes, certain evidence of sewage pollution, can be constantly demonstrated in shell-fish. The plerocercoid stage of Dibothriocephalus latus is passed in fish, and thus transmitted to man. Meat. — In addition to tuberculosis, glanders, anthrax, and PLATE IV. Fig. 72. — Head of Ct.sticerou.s. Fig. 73.— Trichina spiealis. Fig. 74. — Oxyurls veemiculari.';. Fig. 75. — Ln'EE Fluke of Sheep. .]|^- Fin. 76. —Cheese Mites. Fig. 77.— Segment of Taenia solium. {To face p. 106. THE MICEOSCOPE IN PUBLIC HEALTH 107 actinomycosis, the microscope is necessary to detect moulds, penicillium (Plate III., Fig. 70), mucor, and cladosporium in stored meat, and B. botulinus, B. actrych, B. suipestifer, B. enteritidis, B. paratyphosus, and certain putrefactive bacteria which have been associated with outbreaks of food-poisoning. Meat is also responsible for the transmission of certain para- sites, the larval stages of which are detected by the microscope. Cysticercus cellulosce, Cysticcrcus bovis, and Trichindla spiralis are examples (see Figs. 72 and 73, Plate IV.). 3. The Utilization of Micro-Organisms for the Disposal of Sewage. This method of sewage disposal is practised extensively. Denitrification organisms, many of which are anaerobes, break down the compounds of ammonia and nitrogen in the sewage, yielding free nitrogen, water, ammonia, carbon dioxide, and a small quantity of nitrites. These bodies are oxidized by the nitrifying organisms, first into nitrites by the nitroso-bacteria, and then into nitrates by the nitric organisms — nitro-bacteria. The microscope is necessary in the identification of the organisms used, and to control the processes of purification (Plate III., Fig. 69). 4. The Diagnosis of Infectious and Parasitic Diseases. As the causes of disease become more known, laboratory methods will be increasingly called for. Microscopical diagnosis is utilized in : (a) Bacterial diseases: Typhoid and paratyphoid infections, diphtheria, erysipelas, pneumonia, cerebro- spinal fever, dysen- tery, cholera, plague, tetanus, glanders, anthrax, tuberculosis, and gonorrhcea, to mention a few. (b) Mycotic infections: Actinomycosis, sporotrichosis, nocar- diosis, mycetoma, aspergillosis, etc. (c) Protozoan infections : Amcebiasis, coccidiosis, malaria, relapsing fever, syphilis, and the diseases due to Trichomonas hominis, Lamblia intestinalis, and Balentidinm coli. (d) Diseases due to Trematodes, Cestodes, and Nematodes : Fasciola hepatica (Plate lY., Fig. 75), Tcsnia solium (Fig. 77), 108 MODEEN MICEOSCOPY T. saginata, Dipylidium caninum, Dibothriocephalus latus, the cysticercus stage of T. solium and T. saginata, the larval stage of T. echinococcus, and the nematode infections — Ascaris lumhri- coides, Oxyuris vermicularis (Pig. 74), trichiniasis, uncinariasis, and Trichuris trichiura. (e) Infection by the Arachnida : In England the only common infection is that of Sarcoptes scabiei. (J) Parasitic insects : Pediculi, Cimex lectularius and Pulex irritans, and myiasis, or infection with the larvse of Diptera. The student of public health should remember that he may be called upon to deal with imported tropical diseases, as well as those usually met with in Britain. 5. The Investigation and Control of the Carriers of Disease : Human, Animal, and Entomological. This is one of the most important problems of state medicine: The common diseases in which the human carrier can be diagnosed and controlled are typhoid and the paratyphoid fevers, dysentery (bacillary and amoebic), diphtheria, and cerebro- spinal fever. In the course of time all the communicable diseases will doubtless be controlled and epidemics cut short. The spread of disease through the agency of flies, fleas, bugs, lice, and other insects, is a fruitful field for investigation, and further research may reveal how vast an influence insects have on our public health. The accompanying tables (pp. 109 and 110) will give some idea of the scope of investigation. The spread of infection by direct contagion by the common house-fly, typhoid and paratyphoid fevers, cholera, dysentery, etc. ; the association of Stomoxys calcitrans with anterior polio- myelitis, and Xenopsylla cheopsis with plague, are well known. 6. Research. Lastly, we come to the use of microscopy in state medicine research. About the aerial transmission of disease we know little, and food requires much further investigation. The question of the domestic animals in relation to the spread of infection has scarcely been touched. The causative organism THE MICKOSCOPE IN PUBLIC HEALTH 109 1 £ 6 u a lent in mos- ive. Malaria. ent in louse, contaminated e. Relapsing 0) a jg a g'§ gg ■^^ •as §•3 a a disease. s. ontaminative. IS o S 'a •i-t 1 s 1 .s Si o o c =« CO -43 (D ■4=> a 09 >i o ^1 i§ III . 1i 1 0) " s ra .a c^. . iZ? .E; ^ 3.a ^ 1 g 5b" 1 o u > £: ij "*" o c3 tj § a ^ hi m ^ o ft ft Q O o > <^. hi^ "fe> CD .9 > &0 CO c3 03 N. o 00 "o ll 11 ajj ■§ a a cS (3 n3 « § § ^s i^ s ■s^ § Ph § a ° § S CO CQ I 1 l5 > m 1 CQ 3 o 1 «- 2 J3 1 e3 1 J 1 11 1 ■a-s -2 ° o-a CD •s g ffl gs CD CO 09 CD 03 0) "S "5 "5 s s s ^5-2 >a ^ >s W W 1-^ Ph M ^^ H rt izi o o o 110 M.ODEEN MICEOSCOPY CIQ h-l Ss II 6 t^ 1 CD CO" 03 CD •§ mqa Ai a s u a 03 ^ K a •rl q5 T3 1— ( "o a> s s ■< s^' > "2 ->5 3 .3 a'^.S £8^ 1 0) 60 a M CO CO a 03 0) 'c3 CD a s^ CD -is ■§1 s |.W 1 o •S 1 1" .a f^ rt< "S oi ^ O ^ IB 6 © . n-s '"' >^ . 'o o > CD •^ © 05 ^ 'o rT3 C3 a « o -js o CM Q 53 CO ^ » "S > o ^ S* m f~> a > -, a -« a .^ •l* . OJ m Q> . .^ '^ r4 CO a) -*3 §« BO i: cd 0) -4^ .2 M +3 a to J3 d -+3 o 1^ w 1-1 s O O 5 o 'fev ^ ■ri. 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Now put some of the broken-up material on a slide and examine with a microscope ; pick out the annular vessels on the point of a needle, place them in some clean water, and wash well. Stain in a weak watery solution of acid green, and after washing in water, mount in glycerine jelly. THE MICROSCOPE IN HISTOLOGY 157 Scalariform Vessels. — Treat pieces of the rhizome of Pteris aquilina in exactly the same way as stem of maize. Spiral Vessels. — Treat pieces of the stem of rhubarb in the same manner as annular vessels. Raphides may be isolated, or they can be mounted in situ, in the tissues in which they occur. For the former, take some leaves of cactus, stem of rhubarb, and root of Turkey rhubarb, cut them up into thin slices longitudinally, and place them in a jar of water, covered up to keep out dust, and put away until the tissue has become perfectly disintegrated. This will take several weeks, and the process is more easily carried out by keeping the jar in a warm place. When all the material has broken up, stir well with a glass rod, and strain through a piece of coarse muslin into a shallow vessel, such as a soup plate ; stir up again, and then allow to settle for a minute, so that the raphides may fall to the bottom of the plate ; now pour away as much of the dirty water as possible, add more clean water, and repeat the process until you have got rid of all the disintegrated vegetable fibre. Now pour the raphides into a bottle, and if they are quite clean, pour off the water and add methylated spirit, in which they may be preserved until required for mounting. To mount isolated raphides, clean a cover-glass, fasten it to a slide with the aid of your breath, take up some of the raphides in a dipping-tube, place them on the cover-glass, and spread them evenly over its surface with a needle. Place the slide out of reach of dust until all the spirit has evaporated, and the raphides are quite dry ; add a few drops of Canada balsam, and put the slide away again for twelve hours ; then add a few drops more balsam, take up the cover with a pair of forceps, and mount it on a warmed slip. When the raphides are very large they must be mounted in balsam that is rather thicker than is usually used. Raphides in situ in Tissues. — Harden the stems, roots, or leaves in methylated spirit, and make sections in the ordinary way; dehydrate, clear in clove oil, and mount in Canada balsam. Raphides in Scale-Leaves of Bulhs, such as Onion, Garlic, Lily, Hyacinth. — Strip off a thin portion of the cuticle, place it in methylated spirit for a few hours, and when dehydrated clear in clove oil and mount in Canada balsam. 158 MODEEN MICEOSCOPY Sometimes raphides are rendered too transparent when mounted in balsam. "When this is the case they must be put up in glycerine jelly in the following way : Isolated Specimens. — Pour off the methylated spirit, and add water ; pour off the -water, leaving the raphides at the bottom of the bottle. Clean a cover-glass and a slide. Place a few drops of warmed glycerine jelly on the centre of the slide ; take up a few of the raphides on the point of a penknife, and place them in the glycerine jelly, but do not stir them up. Now apply the cover-glass, and press it down carefully with a needle, giving it at the same time a twisting motion, to spread the raphides evenly between the cover and slide. Put away for an hour or two, sqrape off the excess of jelly with a penknife, wash in water, and then in methylated spirit, dry with a cloth, and apply a coat of black enamel. When raphides in the tissues are prepared in glycerine jelly, wash away all trace of spirit with water, and mount in glycerine jelly as above. Starches {Isolated Specimens). — If the tissue is fresh, scrape the cut surface with a knife, and place the scrapings in a bottle of water ; shake well and then strain through fine muslin into a shallow vessel ; let the starch settle, pour off the water, and wash again with some clean water until the starch is quite clean ; then place it in a bottle, and when it has settled to the bottom, pour off the water, and add methylated spirit. Dried Specimens. — Place in water until the tissue swells up, then, if the material is large enough, it may be scraped and treated as above. If too small — small seeds, for instance — place them in a mortar in some water, and carefully break them up ; strain through muslin, wash with water until quite clean, and preserve in methylated spirit. Starches may be mounted in Canada balsam or glycerine jelly. If the former is chosen, spread a little starch evenly on a cover- glass, let it dry, apply some Canada balsam, and mount it in the ordinary way. For glycerine jelly pour off the spirit and add water, then allow the starch to settle to the bottom of the bottle ; pour away the water. Place a few drops of glycerine jelly on a slide, take up some starch on a penknife, and place it in a little heap in the jelly ; now apply a cover-glass, and press down with a gentle twisting movement until the starch is evenly spread. THE MICROSCOPE IN HISTOLOGY 159 Let the jelly set, scrape away the excess, wash in water, then in spirit, dry, and apply a coat of cement. It is desirable also to prepare specimens of starch in situ in the tissues. Take, for example, a potato, cut it into small pieces of about i inch square, and harden them in methylated spirit. Then embed in carrot and cut the sections, which should not be too thin. Stain in a 1 per cent, solution of methyl aniline violet, wash in water, and mount in glycerine jelly. In mounting starch in glycerine jelly, care should be taken that the jelly is not too hot ; if it be, the form of the starch will be altered. Yeast. — Get some fresh baker's yeast, place a little of it in a bottle of sugar and water, and stand in a warm place for twenty- four hours. Pour off the sugar water, and add camphor water. Make a cell on a slide with black shellac cement, and let it dry ; then apply a second coat of cement, and let this stand for a few minutes. Now take up some of the yeast in a glass tube and place a few drops in the cell ; clean a cover- glass, and bring its edge in contact with the cement on one side of the cell; ease it down carefully, so that no air-bubbles may be enclosed ; now press on the surface of the cover with a needle until it adheres firmly to the cell all round, drain off the excess of fluid, dry the slide with a clean cloth, and apply a coat of cement. Mycetozoa or Myxomycetes. — Most of these fungi can be mounted in glycerine jelly after soaking in equal parts of rectified spirit and glycerine to remove the air, but in those forms which possess lime granules in the capillitium — a character of impor- tance in classification — the calcareous matter disappears when in glycerine in any form. When this is the ease, place the specimen in absolute alcohol until all air is removed, then transfer to clove oil, and mount in Canada balsam. Some specimens may, however, be rendered too transparent by the balsam ; if so, mount them in a shallow cell in some neutral fluid such as camphor water. In their ripe condition they may also be mounted dry as opaque objects. Large fungi, such as Agaricus, should be hardened in methy- lated spirit for a week. Then place the desired portion in water, 160 MODEKN MICEOSCOPY and soak to remove spirit, transfer to gum and syrup, and when penetrated with the gum, freeze and make the sections with a Cathcart microtome, wash away all trace of gum with repeated changes of warm water, and mount unstained in glycerine jelly. Preserving Fluid for Green Algae. — Acetate of copper, 15 grains ; camphor water, 8 ounces ; glacial acetic acid, 20 drops ; glycerine, 8 ounces ; corrosive sublimate, 1 grain. Mix well together, filter, and keep in a stoppered bottle. The above fluid preserves the colour of chlorophyll for a long time ; it may also be used as a mounting fluid. For very delicate specimens leave out the glycerine. The specimens should be well washed in water ; then pour off the water, and add a quantity of the copper solution. To Mount in the Above. — For example, take Spirogyra as a filamentous alga. Make a cell with some black cement, and let it dry ; then apply a second coat of cement, and allow this to nearly dry. Place some Spirogyra in the cell, and with needles separate the filaments ; add a few drops of copper solution, and apply a cover-glass as directed for yeast. Protococcus. — This can be obtained by scraping the bark of trees. Place it in a bottle of water, and let it stand for a few hours ; now add a little copper solution — this will kill the speci- mens, and they will sink to the bottom of the bottle ; pour off the water, and add more copper solution. Now make a cell as for Spirogyra; take up some of the protococci in a dipping- tube, and place them in a cell ; wait a minute for the forms to settle on the bottom of the cell, and then apply a cover-glass ; drain off the excess of fluid, dry the slides with a cloth, and apply a coat of cement. Volvox, gloeocapsa, desmids, etc., may all be preserved and mounted as above. Antheridia and Archegonia of Mosses. — Place some male and female heads of mosses in methylated spirit for a few days, then transfer to equal parts of absolute alcohol and ether, in which they must be soaked for several hours. Pour off the alcohol and ether, and add a thin solution of celloidin, and soak for two or three days ; then remove the stopper of the bottle, and let the celloidin evaporate to about half its original bulk. Now remove a specimen from the celloidin, and hold in a pair THE MICEOSCOPE IN HISTOLOGY 161 of forceps until the celloidin sets, then place it in methylated spirit and soak for an hour or two to complete the hardening. The embedded specimen may now be fastened to a cork with a little celloidin, and longitudinal sections made in a Cathcart microtome, or it can be placed between two pieces of carrot, and the sections made with any ordinary well microtome. The sections must then be dehydrated in methylated spirit, cleared in oil of bergamot, and mounted in Canada balsam ; or, if desired, they may be soaked in water to remove spirit, and be mounted in glycerine jelly. Fertile Branch of Chara. — Chara is usually very dirty ; to clean it, wash well in repeated changes of water, then in very dilute acid for a few minutes only ; again wash in water, and preserve in camphor water. Make a cell with shellac cement as directed above, place a fertile branch of Chara in it ; and examine under a dissecting microscope or lens ; with needles clear away the leaves from the archegonia and antheridia, fill the cell with camphor water, and apply a cover-glass. When a deep cell is required for a specimen to be mounted in acetate of copper, never use one made of any metal. Vulcanite or glass cells must be used. To one side of a cell apply a coat of shellac cement and let it dry ; now take a slide and warm it over a spirit-lamp ; take up the cell in a pair of forceps, and bring the cemented side in contact with the centre of the warm slide, and press it down until it adheres firmly ; then add another coat of cement to the upper side of the cell, and let it nearly dry, put in the specimen, fill the cell with solution, and apply the cover-glass. Prothallus of Fern. — Preserve in acetate of copper and mount in the same fluid in a shallow cell. Sporangia and Spores of Fern. — Place leaves of a fern with sporangia in methylated spirit for a few days to remove the air. Then soak in water for several hours. Warm a slide, and place a few drops of glycerine jelly on its surface, scrape off some sporangia, and place them in the jelly ; now apply the cover-glass very carefully to avoid scattering the sporangia. The object is to keep them in a heap in the centre until the cover is flat ; then press on the surface of the cover with the points of the forceps, 11 162 MODEKN MICEOSCOPY and, if possible, give the cover a little twisted motion. This will spread the specimens ; it will also rupture some of the sporangia and let out the spores. Isolating Antheridia and Oogonia from Fucus.— Take some conceptacles that have been hardened in methylated spirit, and make thick sections by hand only with a sharp knife. Place these in a strong solution of acid aniline green in spirit, and let them stand for two or three hours. Now place in water for a few minutes, and they will at once swell up like a mass of mucus. Place this on a slide and put another slide on top of it, press down the upper slide — this will squeeze out the contents of the conceptacles in little round masses. Separate the glasses, pick up one of the little lumps of antheridia or oogonia, place it in a few drops of glycerine jelly on a slide, then apply the cover-glass, which must be pressed down to spread the specimens. Digestive Glands in Pitcher Plant. — Harden some strips of a pitcher in methylated spirit for a week. Then place in water and soak for a few hours. Then lay the tissue with the glandular surface next to the glass, and with a scalpel scrape away the outer wall. Now bleach the glandular portion in chlorinated soda, then wash well with water, stain in aqueous solution of acid aniline green, wash again in water to remove excess of colour, soak for several hours in dilute glycerine, and mount in glycerine jelly. Aleurone. — Take the endosperm of a castor-oil seed, embed in carrot, place in microtome, and cut sections as thin as possible with a knife wetted with a little olive oil. As the sections are cut, put them on a slide, and place out of reach of dust until you are ready to mount them. Make a shallow cell as directed with black enamel and let it dry, then proceed as directed for acetate of copper mounting, but use castor oil instead of copper solution. When the cover has become fixed, wash away the exuded oil with a soft brush and some turpentine, and, when dry, apply a good finishing coat of black enamel. Water and spirit are apt to injure the aleurone grains, so they should be avoided. Marine Algae. — The best place for collecting specimens is a rocky shore, and the most suitable time is when the tide is at its THE MICROSCOPE IN HISTOLOGY 163 lowest. As a rule, the inshore weeds near high-water mark are green, lower down there is usually a belt of olive forms sheltering red plants beneath them, and where rocks overhang small shallow pools red forms also occur at this level. At extreme low-water mark and beyond it are found brown tangles sheltering red plants again, while at the lowest depths the red weeds occur without shelter. The specimens will be found by searching the rocks and pools, some will be growing on pebbles and on shells, others will be attached to rocks, and varieties may be found stranded on the shore, thrown there by waves, particularly after a storm, the tufts having been torn away and carried inshore from inaccessible regions. For collecting, small tin boxes or an ordinary sponge bag will be found most suitable. A strong chisel mounted on a stout stick will also be required for removing specimens from rocks that are out of reach. Many specimens may be preserved in sea water for a con- siderable time, but, as a rule, the sooner they are mounted the better. Mounting Process. — Eemove the specimen from sea water and wash well in fresh water. Place in a shallow white dish or saucer, select and cut off the portion that is to be mounted, and place it on a slide slightly warmed, drain away as much water as possible, and apply some glycerine jelly ; then, if necessary, lay or spread out the leaves or filaments with a needle and apply the cover-glass, allow the slide to cool, remove the excess of jelly around the edge of the cover, wash the slide in water, dry, and add several coats of enamel or varnish. Corallines, whose tissues are hard and opaque, may be cleaned by soaking for a short time in a weak solution of hydrochloric acid, then wash well in water, and mount in glycerine jelly. 164 MODERN MICROSCOPY ft a OQ «J hJ < rt h- 1 P3 3 'A < % tZ2 Pq 6" o K M CQ ^ H H CQ ft 1 — 1 «1 ft M :zi H -tj 3 m OL! t/f K S5 W < C/J o W « M o Pli Q fi ;zi ;z; ^ij .2S5JS g^S-2 K g ^ s .-s .s .-s 1 j3 ^ C6 Cd m .a .9 .a a^-° S«-S rs ^ S ." -2 -« i^ iJ S:2o3s-^-"' 1 t^r^^. 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', ' .3 .2 a +3 ,fa -*» _t3 Q-+a43-*a-»3-M^-*a Ca .. -ta +3 43 1 -3SfififioS-g apppp|^|p-g|^^ppp 1 n ,£1 :S ffi :3 (§ s s § g s Pi Oi :0 (N § •i • ■ • 5) I 1 I 3 c3 -4A 13 «3 13 ^ t> H PL, O Pq P S !» 60 » §..:;:; :J 'a § 2 2 » S o £ SpmooHooo : : -.3 'it" S 200 .S fe.S 03 P^taOO l-H THE MICEOSCOPE IN HISTOLOGY 167 ft 6 iz; -J) 6" :zi 03 « 1 PP E-l M F-l l-l M « o S 1 r 1 ^ C. Balsam. Ditto. Ditto. Ditto. Ditto. Glycerine jelly. Ditto. Camphor-water. Acetate of copper solution. Glycerine jelly. Acetate of copper solution. Ditto. • Ditto. C. Balsam. Glycerine jelly. Camphor- water. C. Balsam. 1 1 Hsematoxylin Carmine and acid green Hsematoxylin Ditto Borax carmine Methyl aniline Acid aniline green Unstained Ditto Ditto Ditto Ditto Ditto Ditto Ditto Ditto Ditto el rn Iz; 02 t 1 Methylatud spirit Ditto Ditto Ditto Ditto Macerate in water Ditto Camphor-water Acetate of copper solution Dilute methylated spirit Acetate of copper solution Ditto Ditto Macerate in water Methylated spirit Ditto Ditto o "S 1 ......___- o M O H M 1-1 Stems, young Ditto, older Leaves Ovaries Anthers Epidermis for stomata Fibro- vascular tissues Yeast Green algse Bed algse Protococcus Volvox Desmids Baphides Starches Fertile branch of chara .. Antheridiaand archegonia of mosses CHAPTER XVI THE MICEOSCOPE IN GEOLOGY An Introduction to the Use of the Petrological Microscope- By Professor PEEDERIC J. CHESHIRE, C.B.E., P.Inst.P., President of the Boyal Microscopical Society. The petrological microscope is nothing more than an ordinary microscope fitted with certain optical and mechanical adjuncts — as will be seen on reference to Fig. 96, p. 179 — by the proper employment of which a more or less complete quantitative and qualitative determination can be made of the optical properties of transparent crystals, as exhibited in polarized light, and thus the identification of such crystals either effected or facilitated. An elementary knowledge at least of the explanation of polariza- tion phenomena is, therefore, imperative for the intelligent and efficient use of the microscope in question — a fact which is too often overlooked by the microscopist. Polarization of Light. — It is a very curious fact that the understanding of polarization phenomena, when associated with light-waves, should present so many difficulties, when it is remembered that polarized waves are the only forms of wave- motion with which most people are made familiar by their everyday experiences. A pebble dropped into a pond produces a number of small waves, which, starting at the point where the pebble fell into the water, spread outwards in all directions in the form of ever-increasing circles, until they have passed over the entire surface of the pond. But, although these waves travel over the surface, we know that the particles of water con- cerned in their production do not travel, but simply move up and down. A floating cork, for example, is not carried forward by these waves, but simply rises and falls as each wave passes beneath it, 168 PLATE V. INTERFERENCE FIGURES OF ARRAGONITE, (HAUSEWALDT. As seen in convergent sodium light between crossed niools. Fig. 82. Section h mm. thick. Fio. 83. Fig. 84. Section 4 mm. thick. Fig. 86. Pigs. 82 and 84 with extinction directions of crystal parallel to those of nicol. Figs. 83 and 85 with extinction directions of crystal diagonal to those of nicol. [To face p. 168. THE MICEOSCOPE IN GEOLOGY 169 Here, then, we have a case in which waves are propagated from one place to another by the vibratory motion of a large number of particles in one plane only. When waves are transmitted in this way by the motions of particles in a single and constant plane, the wave is said to be polarized. But a simpler and even more instructive example might have been given. Let us suppose that a long rope is stretched rather loosely between two boys, one of whom, by a rhythmic movement of his hand in an up-and-down direction, produces waves in the rope which pass continuously to the other boy. In this case, too, since the vibrations of the particles of which the rope is made up take place in one constant plane only, the resulting waves Fig. 86. — Rope Polariscope: Nicols Parallel. are polarized ones. But now, as in Pig. 86, let the rope be passed through two gratings, A and B — such, for example, as those used for covering street gullies — in both of which the bars are at first arranged vertically. Now let the first boy start waves along the rope — not by confining the motion of his hand to one plane only, but by changing the direction both rapidly and arbitrarily, so that a jumble of waves is sent along the rope in which the vibrations of the particles take place more or less in all directions. It is clear that these waves are no longer polar- ized, since the various particles concerned are moving in different planes. This state of affairs would only exist, however, between the first boy and the grating A, for since the bars of the latter are vertical, it is clear that each wave as it falls upon the grating 170 MODEEN MICEOSCOPY would, in general, be partly stopped and partly transmitted ; and, since in the motion transmitted the vibrations would take place in one constant plane only, it would be polarized. The grating A would thus act as a polarizer, and we should have a succession of polarized waves passing from it to the second grating B, and thence,, since the bars of B are also arranged vertically, to the second boy. Had the bars of the grating B been arranged horizontally — that is, at right angles to those of the first — it is clear that the polarized waves falling upon the second grating would be stopped as in Fig. 87, but in all inter- mediate positions of these bars, between the vertical and Pig. 87. — Eope Polaeiscope : Nicols Crossed. horizontal, the waves from A would be partly stopped and partly transmitted with the plane of vibration changed. Finally, were it required to determine the plane in which the vibrations were taking place in the waves falling upon B, it would only be necessary to rotate this grating into such a position that no waves were transmitted. The bars would then be at right angles to the sough t-f or direction. In this way the grating B could be used to test the polarized condition of the waves falling upon it — i.e., as an analyzer. Polarized Light. — Ordinary light may be looked upon as consisting of waves transmitted through the ether of space by the to-and-fro motions of the particles of that ether in all directions across the line of march of the waves ; but when such a beam of light falls upon a nicol prism, it is polarized — that is. THE MICROSCOPE IN GEOLOGY 171 after passing through the prism, the light-waves are found to have their vibrations in a single and constant plane. The nicol prism, therefore, under these circumstances acts upon light- waves in an analogous way to that in which the grating A acted upon the rope-waves. Similarly, the polarized light produced by the action of one nicol prism being allowed to fall upon a second one similarly arranged, will pass through it, just as the waves from the grating A passed through the grating B, when similarly arranged, as in Fig. 86. The second nicol also being turned through a right angle from the position just described, completely stops the light falling upon it from the first one, just as the second grating B stopped the waves from A in the position shown by Fig. 87. Finally, in all intermediate positions of the second nicol the light will be more or less completely transmitted, but with its plane of vibration changed. To sum up, therefore, we may say that — 1. In ordinary or common light, such as sunlight, the vibra- tions of the ether particles concerned in the transmission of the light-waves take place in every possible direction across the direction in which the light is moving. 2. A nicol prism (or its equivalent) is a kind of optical grating which reduces the vibrations of ordinary incident light to a single direction or plane only, and is thus said to act as a polarizer.* 3. A nicol prism, when polarized light falls upon it, transmits it in general more or less completely with its plane of vibration changed, but in the particular case when it is so arranged that the direction in which it allows ether vibrations to take place is at right angles to the direction in which the vibrations in the incident polarized light are taking place, it stops the incident light altogether, and thus acts as an analyzer. Double Refraction. — The peculiar optical'property possessed by most crystals in virtue of which their examination and dif- ferentiation in a petrological microscope becomes possible is known as double refraction, and is due to what might very appropriately be called ' optical grain.' A piece of wood has a * This action of a nicol prism must not be confounded with that of the grating employed in spectrum analysis, which acts, of course, in an entirely different way. 172 MODERN MICEOSCOPY 5- -3 grain which is usually, it is true, apparent to the eye ; but even in the absence of such evidence the fact could soon be determined experimentally, as by attempting to split the wood with a hatchet in various directions. Eock crystal, the clear and transparent variety of quartz, sometimes takes the form of long prisms of hexagonal cross-section, as shown by Fig. 88. Now, it is found that if a slice be cut lengthwise from such a crystal, as indicated at AA, and smeared with wax on one of its faces, the application of heat to a point on that face will cause the wax to melt. The area over which the melting occurs will not, however — as in the case of glass, for example — take a circular form, but an elliptical one, with the major axis of the ellipse parallel to the geometrical axis of the crystal. This experiment shows that heat is trans- . mitted more rapidly along - ^^ the crystal than across it. In a slice cut across the axis, as at BB, the melted area takes a circular form, showing that across the crystal heat is transmitted equally in all directions. Eock crystal is thus shown to be possessed of a kind of grain running in the direc- tion of its length. In consequence of this grain, which is optical as well as thermal, it is found that if a beam of light be allowed to fall normally upon the face of such a slice of rock crystal, the latter will be found to act as a kind of double grating, sifting and transmitting the incident light as two beams, in one of which the vibrations take place only in the direction of the length of the crystal, and in the other beam across it only. A nicol prism, it may be remarked, is a crystal of Iceland spar in which one of the two beams just referred to is thrown out of the way by reflection on an artificial interface. A beam of light passed along the axis of a quartz crystal is not split up into two beams, but in any other direction it is ; hence rock crystal is what is known as a uniaxial crystal. In other crystals, as mica and selenite, there are two directions in which light passes without being split up ; these are therefore known as biaxial crystals. Fig. 88.— Crystals op Quartz. THE MICROSCOPE IN GEOLOGY 173 Now the two polarized beams into which light is, in general, split up on its passage through a crystal, travel with different speeds, and in most cases in slightly different directions — hence the term ' double refracting.' In the case of rock crystal, for example, the beam in which the vibrations occur along the axis of the crystal travels more slowly than the beam in which the vibrations occur across it.* Let such a slice of rock crystal be placed between crossed nicols, and arranged with its axis inclined to the vibration-directions P and A of the polarizer and analyzer respectively, as shown by Fig. 89. Then polarized light coming upwards from the polarizer towards the observer will be resolved by the crystal into two beams with vibrations in rectangular planes and passing with different speeds. These two beams, falling upon the analyzer, will each again be resolved into two beams — one with horizontal and the other with vertical vibrations. The first of these will pass the analyzer, the second will be stopped. But, further than this, the light which passes , , Fig. 89. — Section of Quartz cut the analyzer and emerges as Parallel to Axis. a single polarized beam is com- pounded of two beams of equal intensities, one of which passed through the crystal with vibrations along aa, and the other with vibrations along hh ; and, since these beams travelled at different speeds, it follows that upon being compounded by an analyzer the waves in one will be more or less out of step with those in the other, with the result that interference will take place, and the waves corresponding to any colour transmitted in opposite phase by the two paths will be destroyed, leaving the trans- mitted beam coloured. Thus, when the crystal is of such a thickness that the waves by the slow path emerge 550 micro- * This statement is made upon the usual assumption that the vibrations of the ether take place in a direction at right angles to the plane of polariza- tion. It should also be remembered that light polarized by reflection at a plane glass surface is defined as being polarized in the plane of reflection. It follows, therefore, from the given assumption and the definition, that the vibrations in tbe polarized reflected light are executed in directions parallel to the surface of the glass. 174 MODEEN MICEOSCOPY millimetres (550 iifi) behind those passing by the fast path, green light with a wave-length equal to this quantity will be cut out, leaving the transmitted beam of the complementary colour red. As shown by Fig. 89, an amplitude oh in the polarizer is, after being resolved along each of the two vibration-directions in the crystal and the vibration-direction (horizontal) of the analyzer, represented by two equal amplitudes oa and oa. Different colours produced in this and analogous ways have been very carefully studied, and are set out in the following table. This succession of colours would be produced by a wedge of selenite with its vibration-directions adjusted diagonally as in Fig. 89, between crossed nicols, the thickness of the wedge increasing from nothing up to about 0"2 mm. The first column gives the retardation — i.e., the distance in micromillimetres which one beam emerges behind the other after passing through the crystal — whilst the second column gives the colour corre- sponding to such retardation. NEWTON'S GOLOUE SCALE ACCORDING TO QUINCKE. Retardaiion in Micro- millimetres. Interference Colour between Crossed Nicols. 1 Retardation in Micro- Millimetres. Interference Colour between Crossed Nicols. 1 40 97 158 218 234 259 267 275 281 306 332 430 505 536 551 Black Iron-grey Lavender- grey Greyish-blue Clearer grey Greenish-white Almost pure white Yellowisb-white Pale straw-yellow Straw-yellow Light yellow Bright yellow Brownish-yellow Beddish-orange Bed Deep red 1 843 866 910 948 998 1,101 Yellowish-green Greenish-yellow Pure yellow Orange Bright orange-red Dark violet-red g 02 1,128 1,151 1,258 1,334 1,376 1,426 1,495 1,534 1,621 Light bluish- violet Indigo Greenish-blue Sea-green Brilliant green Greenish-yellow Flesh colour Carmine-red Dull purple 565 575 589 664 728 747 826 Purple Violet Indigo Blue (sky-blue) Greenish-blue Green Lighter green - Ci ■§ •u 02 1,652 1,682 1,711 1,744 1,811 1,927 2,007 Violet-grey Greyish-blue Dull sea-green Bluish -green Light green Light greenish-grey Whitish-grey i ■ 3 O pq THE MICEOSCOPE IN GEOLOGY 175 Rotary Polarization. — Certain crystals possess in a certain direction the remarkable power of rotating or twisting the plane of vibration of a polarized beam passing through them. Thus, if a slice of rock crystal 1 mm. thick, cut from the crystal normal to the axis as at BB, Fig. 90, be placed between crossed nicols in white light, it is found that the analyzer no longer stops the light, and that no position can be found for it in which it does stop it. In the case of sodium light, however, it is found that a rotation of the analyzer from its crossed position with respect to the polarizer through an angle of 22° again establishes dark- ness. In some instances this necessary rotation has to be made in the direction of the hands of a clock, from the observer's point of view, about the direction in which the light is passing to the eye, whilst in others it has to be made in the opposite direction. In the first case the rotary polarization is said to be right- handed, and in the second case left-handed. If, then, a beam of polarized sodium light, in which the vibrations are vertical, is allowed to pass along the axis of a quartz crystal, the plane of vibra- tion will not remain vertical, but will be gradually rotated or twisted about that axis at the rate of 22° per mm. of length of the crystal. For different colours in the incident polarized white light, the rate of turning is different. Bed light, for example, has its plane of vibration twisted at the rate of 13° per mm., whilst blue, on the other hand, is twisted through as many as 83° in the same length. Thus, if Fig. 90 be taken to represent this action in a slice of right-handed quartz of the specified thickness in incident white light, polarized with its vibrations along the line PP, and passing upwards to the eye, the vibration planes for the red, yellow, and blue will be twisted into the directions BE, YY, and BB respectively, through angles a, yS, and 7, equal to 13°, 22°, and 83° respectively. The analyzer, therefore, set originally parallel Fig. 90. — Section of Qttartz, Normal to Axis, to show Kotary Polarization. 176 MODEEN MICROSCOPY with the polarizer and rotated in the direction of the hands of a clock, would allow in succession the colours red, yellow, and blue to pass in predominance to the eye, so that the crystal would appear to change in colour during the rotation of the analyzer. Optical Adjuncts for the Petrological Microscope. Crystallographic determinations are very much facilitated by the employment of a number of optical adjuncts, the more indispensable of which are set out below. Mica Quarter-Wave Plate. — This consists of a cleavage plate of mica of such a thickness that sodium light, with a wave- length of 589 fjifji, in passing through it, along one of the two vibration-directions possible in a double-refracting crystal, emerges a quarter of a wave-length behind that passing through by the other rectangular vibration-direction. These vibration- directions are often, therefore, referred to as the ' fast ' and ' slow ' directions to differentiate them. If a quarter-wave mica then be placed on a crystal section, so that similar directions in the two sections are parallel to one another, the effect is the same as that which would have been obtained by increasing the thickness of the crystal section, and if the latter should be between crossed nicols with its vibration-direction inclined at 45° to the vibration-directions of the nicols, its colour would rise in Newton's scale — i.e., correspond to a greater retardation. By superposing the mica with its fast direction parallel to the slow direction of the crystal section, the effect would be the same as that which would have been obtained by decreasing the thick- ness of the crystal section, so that in this case the colour would descend in Newton's scale — i.e., correspond to a less retardation. By the use of a quarter-wave mica in this way the fast and slow directions of crystal sections are differentiated. Selenite or Gypsum Plate. — When it is required to differ- entiate the fast and slow directions of a crystal section with small bi-refracting power, a selenite plate of such a thickness as to give a rose colour between crossed nicols is employed. Superposed upon a crystal section with similar directions parallel, the colour changes to blue, whilst the placing of fast on slow changes the colour to red. THE MICEOSCOPE IN GEOLOGY 177 Klein Quartz Plate.— A plate of quartz 3-75 mm. thick, and cut at right angles to the axis of the crystal, gives between crossed nicols, and in virtue of its rotary polarizing power, a purple colour. In this case the orientation of the plate does not affect either the intensity or the colour of the light transmitted. Bertrand Plate.— This plate is made up of four quadrantal sectors of alternately right- and left- hand quartz, cut at right angles to the axis of the crystal, and 2'5 mm. thick. Fedorow Mica-Steps. — This is built up by superposing some sixteen strips of quarter-wave mica, all with similar directions parallel, in such a way that each strip is about 2 mm. shorter than the one immediately below it. Sixteen steps are thus formed, which effect in succession retardations. Fig. 91. Bertrand Plate. Fig. 92.— Mica-Steps. increasing by a quarter-wave at each step, commencing with a quarter-wave, and finishing with four waves. Quartz Wedge. — A thin slice of quartz cut parallel to the crystallographic axis is ground into the form of a thin wedge. Could such a wedge be ground to an infinitely thin edge, it would give at this edge, when oriented with its vibration- directions in diagonal adjustment between crossed nicols, the black of Newton's colour scale, followed by all the colours of the scale in ascending order in passing along the wedge to the thick end. Wedges giving the first six orders of Newton's scale, or some smaller number if required, are thus made. To avoid the necessity for grinding a very thin edge, the quartz plate A from which the wedge is to be made is cemented to a thin plate of selenite B, with the fast direction of one parallel to the slow direction of the other. By this device it is made easy to get the starting black at the thin end of the quartz without reducing that end to a less thickness than the selenite foundation- plate 12 178 MODEEN MICROSCOPY possesses. Sometimes this foundation-plate is made to give the sensitive rose tint of the first or second order, and is made to project for a short distance beyond the thin end of the quartz. The wedge should carry a scale along its length, from which the retardation in micromillimetres at any point of the wedge, and for any given tint, can be determined. It will be found that the optical adjuncts referred to above, as produced by different makers, are unfortunately not uniformly mounted as regards the orientation of their vibration-directions with respect to the length of the plate or wedge. Sometimes the fast direction is coincident with the length of the plate, sometimes it is across it, and sometimes it will be found inclined at 45° to the length. The last disposition has the advantage that by inverting the plate in the cross slot in the tube of the /' 3 A 1 1 1 Fig. 93. — QuAKTZ Wedge. microscope, the optical superposition of fast on fast can be changed for slow on fast without any difficulty. The Construction of the Petrological Microscope. — Pig. 96 shows a first-class modern instrument. The sub-stage polarizer is associated with a condensing system for convergent light, the top lens of which can be turned to one side when plane-polarized light is required. The stage is rotatable, and graduated to read to 5' with the help of a vernier. The collar to which the objective is secured is fitted with centring screws, and is made with a slot into which the usual mica and gypsum compensators can be introduced. The analyzer fitted above the objective can be pushed radially into and out of action. The Bertrand lens slides into position near the middle of the length of the tube, and an auxiliary analyzer with divided circle and a slot for compensators may be fitted over the eyepiece. The fine adjustment head is graduated to read directly to a thousandth of a millimetre. Figs. 94 and 95 show the analyzer and polarizer as mounted for use with an ordinary microscope. THE MICEOSCOPE IN GEOLOGY 179 Fig. 96 180 MODBEN MICEOSCOPY Preliminary Adjustment of a Petrological Microscope. — Before any work is done the following adjustments should be carefully tested, and, if necessary, made : 1. Centring of the objective. 2. Eectangularity of the cross- wires in the eyepiece. 3. Eectangularity of the vibration planes of the two nicol prisms. 4. Parallelism of the cross-wires to the vibration planes of the two nicol prisms when the latter are crossed. Centring of the Objective. — This is done in microscopes of the usual type by the manipulation of two radial set-screws acting against the collar in which the objective is secured. A slide should be placed upon the stage, and a prominent point or feature of it adjusted to the intersection of the cross-wires. Upon a complete rotation of the stage the point selected will describe a small circle in the field of view ; consequently, when the stage has been rotated through 180° only, the point selected will have its maximum displacement from the intersection of the cross-wires. Stop the rotation, therefore, at this point, and turn the adjusting screws so as to move apparently the intersection of the wires half-way towards the selected point. The adjust- ment will now be found to be very nearly correct. Eepeat the operation until it is quite so. Rectangularity of the Cross- Wires in the Eyepiece. — Place a slide with a fine straight line ruled upon it on the stage, and adjust it until the projected image of the line coincides with one of the cross-wires. Note the angular position of the stage, and rotate it carefully through a right angle. The projected image of the line should now be parallel to the second cross- wire. If the centring of the objective has been first effected as above, the projected image in the second case will coincide with the second cross-wire. Rectangularity of the Vibration Planes of the Nicol Prisms. — Darkness of the field is not a sufficiently delicate test for this adjustment, but, if the necessary adjunct — a Bertrand quarter-quartz plate — is not available for a more delicate adjust- ment, darkness should be obtained - a number of times by rotation of the polarizer alternately in opposite directions. The mean of the various readings should be taken as the true one. THE MICEOSCOPE IN GEOLOGY 181 To obtain a better result, place a Bertrand plate upon the stage, Bet the polarizer to zero, and slide the analyzer into position. If the vibration planes of the two nicols are accurately at right angles to one another, the quadrants of the Bertrand plate will appear to have the same tint. Otherwise the colours of adjacent quadrants will not match, in which event the polarizer must be rotated until they do match, when the necessary zero correction should be read off on the polarizer. If the polarizer is not fully graduated so as to allow of this being done, a fine vertical line should be drawn across the junction of the polarizer mount and the sleeve into which it is pushed. Better still, if possible, the polarizing prism should be rotated in its mount, until the latter being at zero, the adjustment is correct. Parallelism of the Cross- Wires to the Vibration Planes of the Crossed Nicols. — A needle-shaped crystal such as anhydrite (anhydrous sulphate of calcium, crystallizing in the orthorhombic system), in which one of the directions of extinction is parallel to the long edges of the crystal, should be placed on the stage between crossed nicols, and rotated until extinction is obtained. Pull the analyzer out, when the crystal should be seen ranged parallel to one of the cross-wires. Turn the crystal over on the stage and repeat. Examination and Identification of the Crystalline Con- stituents of Rock Sections. As this chapter does not profess to be anything more than an introduction to the use of the petrological microscope, no attempt will be made to describe the complete and systematic examination usually made of crystal sections by expert miner- alogists. Some of the simpler determinations only will be indicated. Suppose that an angular crystal which lights up and darkens between crossed nicols upon rotation of the stage is to be examined, we could proceed to determine (1) the angles between the sides of the crystal ; (2) the angular positions of the extinction-directions with respect to the sides ; (3) the differentia- tion of these extinction-directions into fast and slow ; and (4) the retardation of the section. To Measure the Plane Angles of a Crystal Section. — Neither nicol is necessary. Adjust the section on the stage 182 MODEEN MICEOSCOPY until one of the sides of the section is projected along a cross- wire. Take the stage (angular) reading. Eotate the stage until the second side is brought into alignment with the same cross- wire. Take a second stage reading. The difference between these two readings gives the desired angle. To Determine the Angular Positions of the Extinction- Directions. — Cross the nicols and adjust the section until a side of the crystal coincides with a cross- wire. Take the stage reading. Eotate the stage to extinction. Take the stage reading again. The difference is the desired angle. Eepeat, and take the mean value of the results. To make a more accurate determination, advantage is taken of the fact that the four sectors of a Bertrand quarter-quartz plate, placed in the eyepiece between accurately crossed nicols, will appear of one uniform tint whenever a bi-refracting plate on the stage is rotated into such a position that its vibration-directions are parallel to those of the crossed nicols. This method necessitates the employment of an analyzer above the eyepiece. To Differentiate the Extinction-Directions. — This may be done by the use of a mica quarter-wave plate in the way already described. When, however, the bi-refracting power of the section being examined is very small, it is better to employ a sensitive selenite plate (so-called red of the first order). When this is introduced into the cross-slot just over the objective, so that its fast direction is parallel to the fast direction of the crystal section, the rose colour changes to a blue ; whilst when the fast direction is superposed on the slow of the section, the colour becomes a bright red. Retardation. — This quantity is most simply determined by the use of the quartz wedge or the mica-step compensator. Unfortunately, however, these cannot be used satisfactorily in the usual slot over the objective, because in neither case, in the final adjustment, is one thickness only of the compensator operative. In the mica-step, for example, two or more steps must be interposed in the path of the light rays proceeding from the objective to form the image in the eyepiece, whilst in the case of the quartz wedge quite an appreciable fraction of the total length must be interposed. Further, in the latter case, THE MICEOSCOPE IN GEOLOGY 183 the retardation scale cannot be used since it is not in focus. When a very low-power objective is sufficient, the compensators can be superposed on the section on the stage, and the retarda- tion determined directly by the position, in the case of the quartz wedge, of the black band on the retardation scale when the fast direction of the wedge is superposed on the slow direc- tion of the section. In the case of the mica-step the retardation may be equal to that of an integral number of steps, but more generally it falls between two of these, and an estimate of its value has to be made. These compensators should always, if possible, be used in the focal plane of the eyepiece. In that event, of course, the usual analyzer must be thrown out of action and one placed instead over the eyepiece. Fig. 97. — The Action of a Convekgent System. Examination in Convergent Light. — To understand the optical action which is taking place in the microscope when it is being employed for the examination of sections in convergent light, it will be better to consider first the simple case (Fig. 97), in which a plate of bi-refracting crystal A cut at right angles to the axis — a plate of calcite, say — is interposed between two nearly hemispherical lenses, B and C. Further, let plane-polarized light, no matter how produced, start from the point b, on the axis of the system and in the principal focal plane P of the lens B, and passing through the lens B, plate A, and lens C, be brought to a focus by the latter at the point b' in the focal plane F' of the lens C. Light from points a and c will similarly be brought to a focus in points a and c' respectively. Now, it will be observed that in each of these three cases the light that actually passes through the plate A is in the form of parallel rays, but that the inclination which any particular bundle of 184 MODEEN MICEOSCOPY k .Uj 0, Fig. 98.— Eay- DlAGRAM POB A Microscope akkangbdeor Convergent Light. parallel rays makes with the axis of the crystal —the line bh' — depends upon the distance be or ba. In the plane F', therefore, it follows that the light falling in the circular line struck with a radius b'c, around the point b', is light, the whole of which has passed through the crystal plate at ,the same angle of inclination to the axis. In the plane F', therefore, we get the familiar interference figure which, when looked at through a crossed nicol, appears as a number of concentric rainbow- tinted rings, with a black cross marking them off into quadrants. In the actual microscope the condenser B, Fig. 98, functions as the lens B of Fig. 97, and the objective C as the lens C of Fig. 97. The interference image shown as being focussed in the upper focal plane of the objective is pro- jected by the Bertrand lens D and the field-lens E of the eyepiece, into the stop-plane of the latter, and again by the eye-lens F on to the retina of the eye of the observer. The analyzer is shown fitted between the Bertrand lens and the objective. The eyepiece and the Bertrand lens thus act together as a low-power compound microscope to magnify the figure in the upper focal plane of the objective C. In the absence of the Bertrand lens the eyepiece projects the interference figure into the Eamsden circle, where it can be very satisfactorily observed with a powerful pocket magnifier. In the latter case a small stop may be placed in the stop-plane of the eyepiece to cut off all light except that which has passed through the crystal under examination. Plate v., from the atlas of the late Dr. Hause- waldt, shows the interference figures in convergent sodium light and between crossed nicols of sections of arragonite — the first pair due to a specimen ^ mm. thick, the second pair due to one of 4 mm. thickness. Figs. 82 and 84 show the figures when the extinc- tion-directions of the crystal are adjusted parallel THE MICROSCOPE IN GEOLOGY 185 to those of the nicol ; Figs. 88 and 85 when those directions are adjusted diagonally. The attention of the reader desirous of further information is directed to a paper by Dr. John Evans in the Proceedings of the Geologists' Association, vol. xxi., part 2, 1909, on ' The Systematic Examination of a Thin Section of a Crystal with an Ordinary Petrological Microscope.' It is to be regretted that this invaluable brochure has not been published in a more accessible form. The following textbooks may also be referred to — viz., ' Anleitung zum Gebrauch des Polarisationsmikroskops,' by Weinschenk, Freiburg im Breisgau, 1906 ; ' Traits de Tech- nique Min^ralogique et Petrographique,' by Duparc and Pearce, Leipzig, 1909 ; and ' Manual of Petrographic Methods,' by A. Johannsen (Second Edition), London, 1918. The last book is strongly recommended for use by the advanced student. CHAPTEE XVII THE MICROSCOPE IN ENGINEERING By F. IAN G. BAWLINS, F.E.M.8. As long ago as the year 1665 Dr. Hooke published in his ' Micrographia ' an account of an investigation which he had made on the edge of a razor, using for this purpose a primitive form of microscope, in which he placed small globules of glass, and by means of which he intended to increase considerably the low magnifications usually obtainable in his time. The result was that, in spite of the tremendous distortion brought about by the use of such an imperfect optical system, he was able to show that the razor edge, apparently so smooth and sharp, appeared under his microscope — to quote his own words — as 'full of ridges and furrows.' The march of two and a half centuries finds engineers and metallurgists engaged on a quest not essentially different from that to which Hooke had devoted his attention ; but the means at disposal and the problems to be elucidated are now enormously increased, and a description of both of these is the primary purpose of this article. It may be well to point out the close connection between metallurgy and its allied occupations of metal casting and founding on the one hand, and engineering, with its employment of the material in a finished state, on the other. Safety, economy, and efficiency in general depend, to no small extent, upon the thoroughness with which metals and alloys are tested, and whether or not they are really suitable for their purpose. The employment of the microscope in engineering has now become almost a necessity, for the complexity of the materials in use is so great that, without some such aid, accurate control 186 PLATE Yl. Fig. 99.— SriERELEisEx. x 70. Fic. 100.— MiLP Steki,. X 50. ■■^--*'i.. ^.'V- /-H--^ > ,. ,. . .-i^^ Fir,. 101. — AxNEALEP Steel. x 70. Fir,. 102.— Ir,ON-Zivc Alloy, x 60. 4«s;* Fig. 103. — Pn.iELiTE (Dark) and Cementite (Light), x 60. Fig. 104. — Steel Fatigued, x 70. [To face p. 186. THE MICROSCOPE IN ENGINEERING 187 both in manufacture and in use is out of the question. Let it be made clear, before proceeding further, that the great advantage gained by microscopy over other methods of analysis is that the material examined is not destroyed, and that imperfections, weaknesses, or other sources of trouble, are laid bare in situ; and that once a cause of failure has been located, it may not be a very difficult matter to avoid it in future. It is worth while to contrast this with chemical analysis, wherein the content of samples is determinate with almost any degree of accuracy, but the material is destroyed in the process, and mechanical or physical defects can hardly ever be brought to light. ' What, then, is the information which can be obtained by the use of the microscope in engineering ?' The answer, so far as metals are concerned, may be given as follows : (I) Examination for defects, physical and mechanical, arising in manufacture. (2) Control of the heat treatment which the substance is to undergo in manufacture, and assurance of its suitability in the case of the finished product. (3) Investigation of failures in use. In the case of bodies other than metals the information sought is usually (1) Natural structure of the substance ; (2) alteration of that structure in service. Owing to their superior importance in most cases arising in engineering, we will take the metals first, though it is of interest to note that historically it was from the study of rocks that microscopy such as we are considering originally sprang. Metals. (1) Defects arising in Manufacture. — These may be taken to include blowholes, pipes, cavities, and all other forms of unsoundness due to faulty casting of ingots. These troubles usually arise from the retention of gases, particularly hydrogen, which is trapped or occluded with vigour by many metals at temperatures near their melting-point ; and it is evident enough that such shortcomings, if incorporated into the finished article, will assuredly lead to failure sooner or later. It is the business of the microscopist to investigate sections cut, both longitudinally and transversely, from ingots and castings, and to report upon the presence or absence of the above-mentioned blowholes, etc. 188 MODERN MICEOSCOPY For this purpose it is often sufficient for a specimen of the metal or alloy to be roughly ground down to a plane surface, and then examined by means of a low-power objective (say, 1^ inches), the specimen being laid, if necessary, across the foot of the microscope ; the stage in such instruments should be removable, in order that such work may be undertaken in the case of heavy pieces. A vertical illuminator with transparent reflector between objective-front and object is best for this work. We have now to consider another type of defect likely to arise in manufacture — i.e., the inclusion of impurities. As an example we will take the case of steel, though analogous instances occur in most other alloys, and, of course, also in the case of so-called ' pure ' metals. Steel, as is well known, is a complex alloy of iron, carbon, manganese, silicon, and phosphorus. The presence of sulphur and phosphorus is due to metallurgical processes into which it is unnecessary to enter, but if they occur to an undue extent trouble is certain. Manganese in the form of Spiegel (Fig. 99) is added in steel-making to combine chemic- ally with the sulphur, forming manganese sulphide, a compound of less objectionable properties than ferrous sulphide, which results if the amount of manganese is insufficient. The micro- scope, skilfully used, will determine whether the above points have been properly attended to by the steel-maker, for manganese sulphide (MnS) appears under high magnification (say, 1,000 diameters) as dove -grey patches, whereas the ferrous sulphide is blacker, but appears slightly yellow by reflected light. Neither compound should occur in really first-class steel ; but if it is only the manganiferous impurity which is present, the results are not likely to be so serious as if the sulphur-iron compound were incorporated. Fortunately, the affinity of sulphur for manganese is greater than that for iron, and therefore with proper precautions it should be always possible to avoid the formation of ferrous sulphide. In cases such as this the microscopical technique needs to be more refined than in the case of blowholes, etc. The specimen should be comparatively small — about ^ inch square — and must not only be ground, but polished, by being held against a revolving wheel covered with fine cloth. The polishing process is one that requires considerable skill, and if this part of the THE MICEOSCOPE IN ENGINEEEING 189 procedure is unsatisfactorily done very misleading results may be obtained when the specimen comes to be examined under the microscope. Numberless other cases occur where physical defects or impurities can be located with certainty, and the reader will notice that in neither of these two cases taken for illustration would chemical analysis have been sufficient : in the first instance, because such an investigation could obviously give no information as to the existence of cavities, etc. ; and, in the second, for the reason that only the ultimate constitution of the steel would have been determined, and not the actual compounds which existed in the specimen. Naturally, chemical impurities may present themselves in a great variety of ways ; but from the point of view of actual size such inclusions are usually small — the blue crystals of stannic oxide, fairly common in copper, for instance — and therefore a yV-i°cb oil-immersion lens should be available, and a vertical illuminator mounted above the objective when such work is contemplated. (2) The Control of Heat Treatment is one of the most im- portant uses to which the microscope can be put in engineering practice. The properties of metals and alloys depend to an enormous extent upon the temperatures at which they have been cast, forged, annealed, or tempered ; and these temperatures are made manifest by the microstructure of specimens when properly prepared and examined. At this stage it may help to make matters clearer if we consider very briefly the nature of these processes, for without some such knowledge it is impossible to appreciate their effects on the structure of the substance. Casting simply consists of the pouring of the molten metal into appropriate moulds. The temperature at which th'is is done is very important ; if too high, the chemical activity of the substance may be largely increased, and it will take up impurities. If unduly low, pouring will be difficult, and the solid phase will have begun to separate before the mould is completely filled. Forging, or the working of metallic objects, must be performed between rigidly defined temperatures. If outside this range, it may be impossible to fashion the material, or else the internal crystalline structure of 190 MODEEN MICEOSCOPY the metal is injured by the succession of blows. Usually after forging the metal is in a state of strain (this may also happen from rapid cooling without additional work), and therefore this unstable state must be relaxed by annealing, or tempering, or both, such processes involving the reheating of the substance to appropriate temperatures. We are now in a better position to appreciate the information which can be gained by the study of microstructures resulting from different heat treatments. We will begin by the considera- tion of steel, partly because of its importance in engineering, and partly owing to the fact that it responds in an unrivalled degree to heating and cooling effects. Now it is obvious that, if we wish to investigate microscopically the structure of steel near its melting-point (approximately 1,500° Centigrade), we must have some means of retaining at ordinary room temperature the conditions really obtaining at high temperatures. Technical applications and the pure interest of microscopy are together in requiring some such arrangement. Therefore the process of "quenching" has been devised. This consists of taking the metal at any temperature desired, and plunging it into oil, water, or liquid air, the rate of cooling being least in oil and greatest in liquid air. Then (with important exceptions of too difficult a nature to be discussed here) the structure characteristic of the high temperature is retained in the cold : intermediate conditions of the body having been suppressed by the great rapidity with which the metal has been taken through the range of temperature. Incidentally this procedure illustrates a great natural truth, a characteristic of matter in general — namely, that a finite time is required for the transforma- tions of nature to occur, and that if by any means we are able to expedite any process involving such changes so that the required time, however short, is denied to the substance, then the sub- stance will continue to exist in the state characteristic of it when the process began, only in a condition of unstable, or metastable, equilibrium. These different structures, characteristic of different tempera- tures and different rates of cooling, are those which we wish to investigate under the microscope. Let us take a few examples from the iron-carbon system of alloys. Imagine a ' mild-steel ' — THE MICEOSCOPE IN ENGINEEEING 191 i.e., one containing a very small percentage of carbon, which has cooled very slowly from the liquid state. All the intermediate conditions have had time to assert themselves, and the body — always supposing that the cooling has been slow enough — is in a state of stable equilibrium. When examined microscopically, it is seen to consist of different sized grains of pure iron (called by metallographists ' ferrite '), and, at the junctions, small dark inclusions (see Fig. 100). This is the constituent ' pearlite,' and is formed of alternate lamellsB of pure ferrite and tri-ferrous carbide (FejC), the latter being known as ' cementite.' Mechanic- ally, such an iron as this is weak ; the large grains are the very reverse of a compact, close structure necessary to withstand the strains encountered in many branches of engineering. If we increase the carbon content, the areas of pearlite grow larger, until at 0'89 per cent, carbon the whole steel is formed of this constituent. Eaising the carbon percentage still higher, the microscope shows coarse membranes of cementite interpenetrating the grains of pearlite ; but in all cases the grain-size is large, as the result of slow cooling, and the material is ill-adapted for many purposes in engineering ; though, of course, pure iron is very important in some branches of electrical work for magnets, where intensity of magnetism is more important than high retentiveness. We may now consider the microstructure of a steel which has had work done upon it at an appropriate temperature and has been thoroughly annealed to remove strain. The large ferrite grains are conspicuous by their absence (Fig. 101), and, instead, we obtain a close-grained structure (needing a magnification of some 100 diameters or more to resolve it), generally the condition most favourable to strength and good physical qualities. In these cases, by suitable heat treatment, followed by microscopical analysis, we are able to produce and control the manufacture of steel having nearly any desired properties, in the way of wearing qualities, resistance to shock, resilience, toughness, or whatever may be the special characteristic desired. It is hardly necessary to point out how great is the power of the microscope, both in making sure that the structure is appropriate for any given purpose, and also, in the case of manufacture, in enabling metal- lurgists to try experiments with a view to producing some metal 192 MODERN MICEOSCOPY or alloy having some desired property. This is of the greatest importance in the case of the so-called ' light-alloys ' for motor- car and aeroplane work, in which branch of technology there is a great demand for alloys of aluminium combining strength with lightness. In these cases recourse to the microscope has been of the greatest assistance. Amongst the problems often encountered under the general heading of heat-treatment is that of recognizing and studying the properties of intermetallio compounds. These bodies are peculiar, not to say remarkable. In the first place they very seldom conform to any accepted rule of chemical valency (though some reasons have been brought forward to account for this), and secondly, they are only stable within well-defined ranges of temperature, so that, in cooling, an alloy may consist, at different periods of its history, of quite different constituents; these breaking up, as the temperature falls, to give place to the next compound characteristic of the particular temperature range. Since heat is always liberated or absorbed in such changes, a study of the cooling curve together with microscopical examina- tion of specimens quenched at the appropriate moment is very fascinating, for it is then possible to observe the existence of some compound just before an evolution or absorption of heat, and with the next specimen to see a structure entirely different, though these materials may only have been quenched at tempera- tures a few degrees apart. Since the physical properties of these intermetallic compounds are very different, quenching and microscopical analysis can obtain results of technical importance. Fig. 102 shows crystals of the intermetallic compound, iron-zinc. 3. Investigation of Failures in Use. — It is now necessary to enquire into the nature of the information which the microscope can afford us as to the effects of wear and tear made evident by microstructure. Firstly, it is well to point out that much of this work is of a controversial character, and therefore, in an article such as- this, only opinions which have gained general acquiescence will be mentioned. There is a vast and increasing literature of this subject to which the reader is referred for more detailed treatment. Perhaps the most interesting case is that of 'fatigue.' It is a well-known fact that metals and alloys forming the moving parts of reciprocating engines, rotating THE MICROSCOPE IN ENGINEERING 193 shafts and pulleys, etc., suddenly fracture without apparent reason, and when this occurs, a thorough microscopical investi- gation, together with chemical analysis, will often go a long way towards elucidating the cause of the accident. If steel is the material, the pearlite will generally be found to be markedly distorted, being dragged out or compressed, as the case may be, in the direction of motion. The author has himself investigated an instance — by no means uncommon — where a structure of parallel bands of pearlite has resulted from ' fatigue." This is in marked contrast to the granular appearance, to which reference has already been made in the subsection dealing with heat- treatment (compare Figs. 103 and 104). The general theory of distortion of metals under stress is very complicated, but it may be mentioned that the view most usually accepted is that the crystals of the substance are caused to slide bodily over one another, and a system of so-called ' slip-bands ' results, when such a strained specimen is examined with a fairly high-power objective. In order that this slipping should occur, it is necessary to postulate the existence of an ' amorphous cement,' which is supposed to fill the spaces between adjacent crystal grains. The microscope has never yet been able to demonstrate the presence of such a material, but the agreement between actual behaviour of a strained metal and the predictions of theory based on the assumption that an amorphous layer is included at the junctions, is so good that it may only be a ques- tion of time before its existence is placed beyond doubt. Again, in the case of metals exposed to the influence of chemically active gases (i.e., in flues, blast-furnace pipes, and in case- hardening), the microscope has demonstrated a gradual absorption of carbon from the edge of the specimen inwards. The reader will no doubt have come to the conclusion by this time that the usefulness and importance of the microscope as an instrument in the metallurgical side of engineering are very great, but before proceeding to a discussion of the actual tech- nique and manipulation involved, some reference is required concerning the part played by the microscope in problems bearing on the second class of material mentioned early in this article — namely, refractories or non-metallic substances. It is difficult to assign a reason why progress in this direction 13 194 ' MODEEN MICEOSCOPY has been slower than in the case of metals, more especially since — for many purposes — the type of microscope required is simpler. However, a mass of information has been accumu- lated, of which the barest outline can be given here. Non-metallic Substances. Canister, the most important, is a hard silicious rock used in lining furnaces, Bessemer converters, and for various other pur- poses. The best quality contains the greatest percentage of silica (Si02), the usual range being anything between 80 and 98 per cent. In the case of three specimens in the examination of which the author was concerned it was quite easy, with low magnification (about 10 diameters) and the usual transparent illuminating arrangement, to note the differences of silica con- tent ; and, from that, to advise the use of one material more than another, for any specific purpose. Fortunately ganister is transparent when" crushed, and resembles quartzite in micro- structure when the percentage of silica is very high. Dolomite. — A mineral rich in magnesium carbonate, and used as a lining in basic steel manufacture, is also amenable to microscopical inspection in much the same way as ganister, and the information obtained with respect to grain-size, compactness, and other desirable properties is often very useful. Slags. — In recent years, too, the study of the microstructure of slags has been actively taken up. A prodigious amount of slag is necessarily produced in steel-making, and as there is no very great demand for this substance for ballast, ^ large portion of the output is taken out to sea and ' drowned.' It is therefore becoming of increasing interest to enquire into its con- stitution with a view to finding more uses for this by-product. The manufacture of slag-wool is an industry of increasing value, and may well provide more problems for the microscopist. Slags are examined either by transmitted light, as in the case of ordi- nary transparent preparations, or else with the vertical illu- minatdr. The structures observed are of the very greatest beauty, and are not unlike the radiating and spherulitic crystals familiar to microscopists who study chemical salts with the aid of polarized light. In some cases very rare minerals have been THE MICKOSCOPE IN ENGINEERING 195 found in these slags, and it is not uncommon for this artificial silicate to resemble closely igneous rocks formed at considerable depths from a molten magma in the earth's crust. Cements. — These also have come in for their share of atten- tion, and the property of ' setting,' so long a mystery, has been very largely accounted for by microscopical analysis. There is now little doubt that different hydrates, corresponding to different degrees of supersaturation, are among the causes that contribute to the characteristic of ' setting.' By subjecting cements to microscopical investigation a great deal has been learnt about these hydrates, and the conditions under which they are formed and can exist in stable equilibrium (see Pig. D, Frontispiece). The Metallurgical Microscope. It is now proposed to devote some attention to the methods of microscopy which experience has suggested when dealing \^ith metals. In the first place, let us consider — The Stand. — The primary essential is that it should be rigid — not that this is a novel requirement — but it is placed before others because many a good instrument is wellnigh useless, owing to the fact that when heavy pieces of metal are being examined the balance is disturbed, and high-power work especially becomes next to impossible. It is important to re- member that in a great number of cases microscopical investiga- tions are carried on in works laboratories, where the conditions are not ideal, owing to the working of machinery in the im- mediate neighbourhood and consequent liability to vibration. The ;Stage is the next consideration. It should be large, have mechanicnl movements in two directions, and also a raising and lowering rackwork, so that coarse focussing can be done with the stage, instead of by moving the body-tube (see Fig. 105). This arrangement prevents the disturbance of the illuminating system with opaque objects. As previously suggested, it is an additional convenience if the stage is removable, or alternatively provided with a large central opening, so that bulky specimens can be observed by laying them across the foot. It is quite unnecessary for the stage to rotate, though some of the more advanced instruments include this feature. The stage suggests specimens and methods of mounting. 196 MODEEN MICEOSCOPY Fig. 105.— a Typical Metalltthgical Mickoscopb. THE MICEOSCOPE IN ENGINEERING 197 Mounting Metallurgical Sections.— Since the majority of these are usually plane only on one surface and rough on the others, one quite efficient method of levelling i^ to take an ordi- nary 3-inch by 1-inch glass slip, put on it a piece of plasticine, place the specimen face downwards on a flat piece of glass at the bottom of a ring, and press the plasticine down on to the rough face of the object, the glass slip resting on the edges of the ring. Other more complicated arrangements are made for the purpose of levelling specimens, but, taken on the whole, the simple ex- pedient just described is quite efficient. The Vertical Illuminator is referred to in another part of this book, but by way of supplementing the information given there, mention might be made of the condenser pattern (Fig. 106) . Two good points in favour of this arrangement are that, con- FlG. 106. taining as it does its own lighting system, the whole apparatus can be attached to the body-tube of an ordinary microscope (moving with it, and therefore dispensing with the need for a movable stage), and also that mounting of objectives in short barrels is rendered less essential than with other patterns of vertical illuminator. Objectives. — First quality achromatic lenses are quite suffi- cient for most purposes, though for the highest-power photo- micrography apochromats are no doubt superior. An objection to the latter is the curvature of field, particularly annoying when specimens have to be examined over large areas. Restricting our attention to achromatic objectives, the most useful powers will generally be found to be 2 inch, 1 inch, § inch, ^ inch, xV inch (oil-immersion). Owing to the fine structures some- times occurring, lenses with high N.A. are preferable throughout. One more point is very important. In designing objectives for opaque objects, it is a great advantage if the back lens be 198 MODERN MICROSCOPY made as convex as the rest of the design will allow, for this throws reflected light towards the walls of the tube, and thus tends to reduce flare. A black velvet lining to the draw-tube may also assist. Eyepieces. — These should be as low in magnifying power as possible. If greater magnification is needed, it is best attained by a higher-power objective. The author seldom uses an ocular of greater strength than x 8. Compensating or special types of eyepieces are generally unnecessary. If an eyepiece micrometer is to be used for measuring pur- poses, it must be very deeply ruled or etched, for the contrast against a metal specimen is feeble, and reading will be found difficult. Illuminant. — Information as to suitable light-sources may be welcome. The author uses a ' Half -Watt ' lamp (electric) and finds it admirable. The ' Pointolite ' tungsten bulb is generally considered the best of all. Even a Welsbach gas mantle is very efficient if electric light is unobtainable, but in any case the illu- mination must be intense, and a bull's-eye condenser (focussed through a vertical illuminator upon the back lens of the objective) is essential, unless the condenser illuminator is used. Etching. — ^In order to develop the microstructure, polished specimens are usually treated with an etching reagent, and dried by means of an air blast. Various preparations, generally solutions of acids or alkalies, are used. The mechanism of the process is complicated. It most probably depends upon the setting up of ' contact electromotive forces ' at the boundaries of adjoining grains. In conclusion, it is hoped that this brief outline of the part played by the microscope in engineering will be sufficient to show the possibilities and problems that await workers in this field of research. CHAPTEE XVIII THE MICROSCOPE IN AGEICULTURE By D. ward cutler, M.A. (Cantab.) (Rothamsted Experimental Station, Harpenden). Agriculture — the oldest and perhaps the most important industry — has in the past received far less attention from the trained scientist than any of the activities designed to benefit man. But now that the various agricultural problems, and the empirical methods long practised by the farmer, are being more fully investigated, it is becoming evident that some of the most complicated and interesting scientific questions are waiting to be solved. In the early days of research, attention was largely directed towards the chemical aspects of the soil, and Liebig taught that the nitrogenous organic matter was decomposed by a chemical process involving the production of ammonia. During the late sixties and early seventies, however, bacteriology made great advances, and it was demonstrated that all decomposition and purification was largely due to the activities of bacteria. Prom this the deduction was made that the decomposition in the soil was probably brought about by the same agents. Schloesing and Muntz, in 1877, experimentally showed that in sewage beds the conversion of ammonia into nitrate was not a chemical but a biological process ; while Warington, working on Eothamsted soils, proved that it involved two stages associated with two distinct organisms ; but not until 1890 did Winogradsky, by a series of brilliant experiments, isolate these two micro-organisms in artificial culture. This date may be termed the beginning of the biological epoch in agricultural research, and since then this aspect of soil fertility has assumed more and more importance. It is now well established that soil fertility is dependent on the activities of the bacteria ; as already stated, the production 199 200 MODEEN MICROSCOPY of nitrate from ammonia is dependent upon them, as also the decomposition of carbohydrates. Further, recent research has demonstrated that such substance as naphthalein and phenol, ■when added to the soil, are broken up by particular species of bacteria. Unfortunately the short space at my disposal renders it impossible to deal even briefly with agriculture in general, or with the various classes of micro-organisms found in the soil. Since, however, bacteria are dealt with in other sections of this book, and since to-day practically everyone knows something about them, I propose to confine my attention to the less known organisms. We know that every gramme of soil must be regarded as the abode of a vast population. Besides the earthworms and numerous species of insects, there are large numbers of protozoa, algae, and fungi. For a correct solution of many soil problems, it is essential that we know something of the life histories of these micro-organisms as well as of their inter-relationships. Such information can only be obtained by the use of the microscope, coupled with a knowledge of how best to use the various mechanical and optical combinations which have been devised. For these reasons, therefore, it is felt that no apology is needed for the inclusion of a chapter on agriculture in a book of this nature. Protozoa. — The study of soil protozoa is a growth of com- paratively recent origin, and arose out of the investigations as to the cause of increased soil fertility after its treatment by various external agencies. As early as 1888, Frank attempted to explain the increased crop production of soils treated with steam on the assumption that the chemical changes so induced were the operative factors. Numerous other observers, such as Eichter, Konig, Stone, Koch, Peterson and Johnson, adopted this view, but no uniformity of explanation emerged from the work, and serious objections to such a view can be adduced. The treatment of soils by antiseptics, instead of steam, also increases the yield, but contradictory results and opposed explanations were the rule in the early days of investigation. It may, however, be definitely stated that, by heating, the physical, chemical, physiological, and bacteriological properties THE MICEOSOOPE IN AGEICULTUEE 201 of the soil are more or less altered ; coupled with which there 18 an increase in the soluble matter — both organic as well as inorganic — available for plant growth, and, by the use of anti- septics, important changes in the flora, especially respecting nitrification, are produced. Various theories have been put forward to explain these results, though only one need be considered here — viz., that of Eussell and Hutchinson, which was initiated in 1905 by the publication of a paper by Eussell, followed by a second one by Eussell and Darbyshire in 1907. Here it was shown that the absorption of oxygen by the soil is mainly brought about by the action of micro-organisms, and is greatly diminished if the soil has been previously heated to 120° C. After heating, however, to 95° C, the rate of oxidation, instead of being reduced, is considerably increased, in some cases being 50 per cent, greater. Eussell and Hutchinson then began an extended series of experiments on the bacterial numbers of such soils. It was found that treatment, either by heat or antiseptics, effected a ' partial sterilizing ' action in that some groups of bacteria were destroyed — e.g., the nitrification organisms ; but the decay bacteria and those producing ammonia were still present. Moreover, a count of the number present in the soil showed invariably, that though a depression was at first produced, rapid reproduction soon took place, until eventually the numbei's considerably exceeded those present in untreated soil (see Table I.). TABLE I.-BACTERIA IN MILLIONS PER GRAMME OF DRY SOIL. At After After After After Start. 16 Dayi. 30 Days. 74 Days. 200 Days. SoUl: Untreated 27 10 10 45 — ■ Treated with carbon di- 2 17 53 121 — aulphide Soil 2 : Untreated 13 9 4 9 12 Heated to 65° C. 13 21 37 45 60 Soil 3 : Untreated 11 16 9 13 6 Treated with toluene ... 2 43 41 43 18 202 MODERN MICROSCOPY Various experiments demonstrated that in normal soil there is resident a factor detrimental to bacterial development, which factor is eliminated in partially sterilized soil by the treatment employed. Further, this factor possessed the following properties : (a) It is active and not the lack of something. (b) It is not bacterial. (c) It is extinguished by heat or poisons. (d) It can be reintroduced into soils from which it has been removed by the addition of a little untreated soil. (e) It develops more slowly than bacteria, and for some time may show little or no effect ; then it causes a marked reduction in the numbers of bacteria, and its final effect is out of all proportion to the amount introduced. (/) It is favoured by conditions favourable to trophic life in the soil, and finally becomes so active that the bacteria become unduly depressed; this is one of the conditions obtaining in greenhouse ' sick ' soils. It is difficult to see what agent other than a living one could satisfy these conditions. Search was therefore made for soil organisms larger than bacteria, and the protozoa were provisionally regarded as fulfilling the necessary requirements. Thus the impetus given to the study of soil protozoa must be ascribed to the work of Russell and Hutchinson. The general biologist has long been familiar with these lowest forms of animal life ; they have long held an important place in medicine and veterinary science, but they have not as yet been generally regarded as soil organism worthy of serious attention. At present it must be recognized that the significance and effect of the protozoa in the soil is far from understood. Many and varied are the criticisms levelled against the theory of Eussell and Hutchinson, but in no single instance has the adverse evidence adduced been sufficiently strong to justify abandonment of their view. What is required is a solid foundation of pure soil protozoology, quite irrespective of any applied bearing it may ultimately possess, for only by such means will any advance be made. This is undoubtedly a truism, since so-called ' applied ' science, if it is of any worth, is simply the application of ' pure ' science to particular kinds of problems. THE MICEO SCOPE IN AGEICULTUEE 203 Practically all the species of protozoa resident in the soil possess in their life history two distinct phases — the cystic and the active or trophic. In the first the organism assumes a rounded shape, secretes a thick wall, and remains in this quiescent condition for varying periods; but in the second or active stage the animal moves, feeds, and reproduces. Now in the early days of the work it was asserted that bacterial numbers could not be affected by protozoa, because the latter existed in the soil only in the cystic condition. Martin and Lewin, however, by ingenious experimental methods, demon- strated that there are in Eothamsted soils both active flagellates and amoebae. Critics of Eussell and Hutchinson's hypothesis maintained that the soil protozoa must be very few in number, since it was impossible, on examining soil under the microscope, to find any trace of them. The present writer has shown, however, that this is due to a surface energy relationship existing between the soil particles and the protozoa, so that the two are always in intimate contact. In other words, the organisms have the film of water surrounding the particles as their environ- ment, which is probably different in character from that of the free spaces of the soil — a fact that must always be considered when discussing physiological processes. Although the qualitative investigations of Martin and Lewin were at the time valuable, in order to make further advance it was necessary to devise a means whereby the numbers of protozoa in a given soil sample could be ascertained. Such a quantitative method was, however, difficult to provide on account of the relationship between protozoa and soil particles. A direct count of the numbers obviously could not be made. Indirect means have, however, been found depending essentially on dilution methods. In one of these, devised by Cunningham and modified by L. M. Crump, 10 grms. of soil are added to 125 c.c. of sterile tap water and shaken for three minutes. This gives a I in 12*5 dilution. From it further dilutions are made until a sufficiently high one is attained. Petri dishes, containing nutrient agar, are inoculated with 1 c.c. of each of the dilutions and incubated. At intervals of 7, 14, 21, and 28 days the plates are examined, and the presence or absence of protozoa on each recorded. In this way it is possible to calculate approximately the 204 MODEEN MICROSCOPY numbers of protozoa present in each gramme of the soil investi- gated. By means of some such quantitative method the numbers of bacteria and protozoa have been counted, and numerous papers published giving the results obtained. They are, however, inconclusive : thus, on the one hand, Goodey and several American observers concluded that protozoa were probably not agents in depressing bacterial numbers in normal soils, while Miss Crump and Cunningham obtained evidence pointing to the reverse conclusion. Such divergence of opinion was probably mainly due to two causes : firstly, the periods elapsing between the examinations of the soil samples were too great ; and secondly, all the counting methods were unsatisfactory in that they were unable to differentiate between the cystic and active phases of the protozoon life cycle. This is a particularly serious source of error since it is possible in a given sample to have a large number of bacteria, and a large number of protozoa, 90 per cent, of which are as cysts. A count on such a sample would give a result apparently entirely opposed to the theory of action and interaction between protozoa and bacteria, though in reality, as only 10 per cent, of the protozoa are active, and therefore capable of affecting bacterial numbers, the theory would be upheld. The difficulty has, however, been recently overcome by a further modification of the dilution method. Briefly it consists in dividing the samples into two 10-gr. portions. One of these is counted in the usual way, thus giving the total number of protozoa (active + cystic) present. The second portion is treated overnight with 2 per cent, hydrochloric acid (which experiment has shown kills all active forms, leaving the cystic ones unharmed), and counted as before. This gives the number of cysts which subtracted from the total gives the active number. * By employing this method in an extensive experiment striking results have been obtained to be described later. First of all, however, a short account may be given of the types of protozoa found most commonly. * The details of this method and proof of its accuracy will be found in th e following papers : 1. Cutler, D. W. (1920), Journ. Agric. Sci., x. 136-143. 2. Cutler, D. W., and Crump, L. M. (1920), Ann. App. Biol, vii. 11-24. THE MICEOSCOPE IN AGBICULTUEE 205 Martin and Lewin, and Goodey first demonstrated that in Eothamsted soils there exist representatives of the three large groups of Protozoa — viz., the Ehizopoda, the Ciliata, and the Mastigophora. Since then it has been shown that these organisms are very widely distributed, occurring in soils from all parts of the world, and that this obtains, not only for the group, but also for certain species. Of the Ehizopoda two species of amcebse appear to be dominant ; one of them, described by Martin and Lewin as Vahlkampfia soli, is undoubtedly Dimastigamoeba gruberi, originally described by Schardinger in 1899. The second dominant soil amoeba, not yet described, is much smaller and occurs less frequently. Other species, though less in number, are Amoeba gleba (Dobell), Amoeba lawesiana (Goodey), Amoeba agricola (Goodey), Amoeba cucumis (Martin and Lewin), Biomyxa sp., and Nuclearia denticularia. Among the Mastigophora the species most frequently met with are : Oicomonas termo (Ehren), Cercomonas longicauda, Hetero- mita sp. and Manas sp. ,- and, sporadically, Tetramitus spiralis, Tetramitus rostratus, Spiromonas angusta, Copromonas sp., Pro- leptomonas foecicola. The Ciliata are represented by two species of Colpoda : C. cucullus and C. steinii, Gastrostylis affinis, Pleurotricha grandis, which are invariably found, and by less commonly encountered forms such as Vorticella microstoma, Anophrys sp., and Euplotes carinata. Such then are a few of the soil protozoa; the list is by no means complete, but indicates the complexity of the fauna. Let us now consider some of the results obtained in the recent experiment referred to above. It was extensive, in that soil samples were taken daily for 365 days and the numbers of bacteria counted. Further, the species of protozoa were differ- entiated and counts also made of the numbers of active and cystic forms of the dominant ones. In this way a great deal of data was accumulated, consisting of about 16,000 sets of figures. With such a collection it is neither possible nor suitable to deal in an article of this nature except in the briefest manner. I shall, therefore, confine myself to giving some of the salient facts. Firstly, it was found that the bacterial numbers changed 206 MODEEN MICROSCOPY considerably from day to day. Previously it had always been assumed that their numbers remained relatively constant, experiencing only seasonal fluctuations. Thus it was known that in the autumn and spring there was an increase in the bacterial population, but that more frequent variations occurred was not recognized. The recent work demonstrates, however, that the variations from day to day are in many cases as much as or even more than 100 per cent. (Table II.). TABLE II. Sample. Bacteria in MillioTis per Gramme. Active Amceboe. Sample. Bacteria in Millions per Oramme. Adive AmceioB. 223 224 225 226 227 228 229 18-18 21-2 12-6 13-5 8-8 15-7 42-0 386,800 127,000 227,000 221,900 288,000 59,300 2,326 230 231 232 283 234 235 286 15-8 19-75 29-3 40-5 35-2 29-8 54-5 238,000 157,000 Under 50 Ditto 25,000 42,000 2,686 This has an important bearing on many previous investigations where an interval of days or even weeks elapsed between succes- sive counts; because of this some of the conclusions deduced from such experiments will have to be revised. When in relation to the bacterial numbers those of the active amoebss are considered, it is evident that a close connection exists between the two. Throughout the whole of the 365 consecutive counts, with negligible exceptions, when the bacterial numbers were low those of the active amoebae were high, and vice versa. This is illustrated in Table II. and Fig. 107. It may be said, therefore, that the general principle holds that there is in normal soil an inverse relationship between the numbers of bacteria and of the active amoebse. As I view it, in such soil there is always an endeavour to obtain equilibrium between the two sets of organisms ; but, possibly because of varying external conditions, or more probably because of the sequences composing the life cycle of a protozoon, the poise of the balance is continually being upset. Thus, for some reason, not yet entirely understood, the amoebic population increases, whereupon the bacterial numbers are depressed, while at a new THE MICROSCOPE IN AGRICULTURE 207 4 800) Fig. 107.— Numbers of Bacteria and Trophic Am(ebm in Broadbalk, Plot 2, FROM February 9 to March 8, 1920. Daily ooimtings, (Reprinted from the Ann. App. Biol., vol. vii.) 208 MODEEN MICEOSCOPY phase of the life cycle, or because of an inimical environment, the amoebic numbers are lowered to be followed by bacterial increase. It might, perhaps, be said that the amoebae are the cause of the lowering of the bacteria. This would, doubtless, to a large extent be correct, but the word 'cause' is dangerous since it implies a straight and unalterable issue between bacteria and protozoa. There is little warrant for such a view ; nor is so simple a con- ception likely to be a true one ; rather should it be said that the relationship is one factor in a long chain of events leading to soil fertility. As Professor Arthur Thomson said in his Gifford lectures : ' No creature lives or dies to itself, there is no insulation. Long nutritive chains often bind a series of organisms together in the very fundamental relation that one kind eats the other.' In soil such ' nutritive chains ' obtain just as markedly as in other haunts of life. Turning now to the flagellates, an entirely different state of affairs is revealed. It is impossible to establish a direct corre- lation between them and the bacteria, though undoubtedly a small depression is brought about, since some of the species feed on bacteria; the effect is, however, so slight that it is completely masked. All the species investigated, how- ever, exhibit the daily fluctuations in number, but with one exception the variations appear to be quite irregular. Oicomonas termo (Ehren) is a marked exception. As will be seen from Fig. 108, there is a two-day periodicity in respect to the active numbers of this animal, high numbers on one day being succeeded by low ones on the following day, and then by high ones again on the third day. This rhythm has been maintained throughout the year's count, and also obtains in artificial cultures kept under constant laboratory conditions. It is not possible to correlate this rhythm with any of the obvious external conditions, and probably we are dealing with a factor inherent in the organisms and bound up with the life cycle. Oicomonas termo has two methods of reproduction ; asexual, in which by a simple division two daughter animals are produced, each of which may grow into an adult Oicomonas ; and sexual. In this last method two of the organisms fuse into a common mass around which a thick-walled cyst is formed. After an THE MICKOSCOPE IN AGEICULTUEE 209 interval, out of this cyst a single individual is born. As already stated, the rhythm may possibly be explained in terms of such a life cycle, but this cannot be definitely stated owing to our lack of knowledge of it in terms of time. The series of events leading to cyst formation we know, but not the period of time necessary for it. This can only be discovered by work on pure culture, a beginning of which has been made. I would, however, point out that periodicity is a widespread phenomenon in biology, and in particular as regards reproduction. 20.000r 'i 18,000 O I 1 6,000 a S 14.000 P4 1 2,000 .3 1 0.000 I" en B 8,000 6,000 4,000 2.000 Fig. 108. — Active Nttmber of Oicomonas tbrmo (Ehrbn). Daily countings. (Reprinted from Ann. App. Biol., vol. vii.) Before leaving the discussion of soil protozoa there are two other interesting points to be mentioned. As I have already stated, the periodicity of Oicomonas and the obvious external conditions do not seem to be correlated; nor does it appear probable that the daily variations in the numbers of the other species of protozoa can be explained by reference to environmental changes. At present these fluctuations can be accounted for only on the supposition that the operative factors are inherent in the organisms themselves. A great deal of research is necessary to settle this point, but it is of fundamental importance both to 14 210 MODERN MICEOSCOPY biology and for the elucidation of some of the problems connected with agriculture. Superimposed on the daily variations there are seasonal ones. Soil bacteria have long been known to exhibit autumn and spring rises, but only recently has it been shown that the protozoa go through the same changes. As regards the two dominant species of amoebae, the total numbers rise at approxi- mately the same time as do the bacteria ; this might be explained as due to an increased food supply ; but the increase in the total numbers of flagellates does not synchronize with that of the bacteria, each species having its peak at a different time. At present this phenomenon is unexplained. I must now leave the question of the soil protozoa and, in the short space at my disposal, deal with the algae and fungi. Algae. — The algae of the soil have until the last few years been practically entirely neglected. This is possibly due to the assumption that they could in no way be a factor in soil economy ; but recent investigation has shown that in all probability they are, from the agricultural point of view, well worthy of research. They, like the protozoa and bacteria, are found to a depth of about 12 inches, though not uniformly distributed, occurring in greatest numbers in the intermediate depths. They have been obtained from all the soils in which they have been sought, but especial attention has been given to the soils of the German African colonies and Schleswig-Holstein by Esmarch, to those of Colorado by Eobbins, to the Danish soils by Peterson, and to the Rothamsted ones by B. M. Bristol. The flora, largely composed of the blue-green and green algae and diatoms, is especially extensive in cultivated soils; and Esmarch concludes that their not being confined to the surface layer is due to cultivation, in that these operations tend to bring fresh soil constantly to the top with the consequent burying of the surface algse. As other effective factors the burrowing of worms and the percolation of water are cited. The fact that plants containing chlorophyll are capable of continuing vegetative growth in the darkness of the soil is sur- prising, but it has been demonstrated that this can occur, provided that an adequate amount of nitrogenous material is available. Also, Esmarch found that the blue-greens retained THE MICEOSCOPE IN AGRICULTUEE 211 their colour for some weeks, though eventually they became yellowish. There is no doubt but that the algal flora is not confined to the top layers of the soil, but that some species at least are capable of a vegetative existence in the lower layers. It is probable, however, that, under these conditions, they function a little differently ; those on the surface will assimilate COj from the atmosphere and accumulate carbohydrate material and energy, whereas those in the dark will consume such material. Many species of algae have a phase in their life-history called the ' resting condition.' This, in many ways, corresponds to the cystic stage of the protozoa ; for when in this resting condition they have the power of retaining their vitality for long periods under unfavourable conditions. Thus Miss Bristol found that two species, Nostoc muscorum and Nodularia Harveyana, resumed growth after seventy years' desiccation in Eothamsted stored soil, while many species were viable after forty-seven to fifty- nine years' storage. Finally, a consideration of the probable functions of soil algse may be of interest. Frank, Schloesing, and Laurent asserted that they were able to fix atmospheric nitrogen as do species of bacteria. Kossowitsch showed conclusively, however, that this was not the case, but that the presence of algse in certain soils was highly advantageous to nitrogen fixation. He con- cluded that this enhanced fixation in the presence of algse was due to a symbiosis between them and the bacteria, the algse supplying carbohydrate material to the bacteria from the mucous sheaths surrounding certain species, and the bacteria providing their partners with nitrogen, without which they are unable to develop. Such a view is substantiated by other experiments. Thus Boulhac and Gustiani showed that in sand, devoid of organic matter and nitrogen compounds, algse and soil bacteria in combination were able to develop normally, and so to enrich the sand that it was capable of carrying a crop of higher plants. Also, it must be remembered that by their death the algse enrich soil in that they present to the putrefactive bacteria large quantities of organic matter for decomposition. This may have a particular significance in certain parts of the world — for instance, it is well known that the water of the Nile renders the land very fertile, but is itself deficient in organic matter and 212 MODEEN MICEOSCOPY nitrogen. On the other hand, vast numbers of algae are present in it. It may be, therefore, that the fertilizing power is largely due to these forms of plant life. Further, it must be remembered that by photosynthesis — the taking in of carbon dioxide and the giving up of oxygen — and respiration the surface algse have an influence on the soil gases. Thus Harrison and Aiyer, working on swamp rice soils, con- cluded that the film of algse covering the surface induced aeration of the roots of the crop by the oxygen evolved, which was then dissolved in the irrigation water, being carried to the roots. Such, then, are a few of the results achieved by the study of soil algse. The investigations are in their infancy as yet, but there is little doubt that through further research the importance of algse will become more and more evident. Fungi. — As is well known, this group is of enormous import- ance in relation to diseases of plants, but with this aspect of agriculture I do not propose to deal. Of the fungi normally living in the soil we know little, though it has long been recog- nized that there is a large population, numbering more than 200 species. They have, however, been scarcely investigated as regards their function in the soil. This is partly due to the difficulties attendant upon such a study, for suitable methods have not yet been devised. For instance, it is not possible at present to make an accurate count of their numbers in a soil sample ; also, as artificial media is employed for their cultivation, there is no means of ensuring that all the species present will grow. This, of course, applies to research on the protozoa and algse, but with not the same force as in the case of fungi. In spite, however, of the various difficulties, a good deal of work is now being done on these organisms. The genera most commonly found in temperate zones are Mucor, PenicilHum, Fusarium, Aspergillus, Cladosporium, and Trichoderma, though sporadically many others occur. As many of these fungi have the power of withdrawing ammonia from the ammonium salts contained in manure, thus setting free acid, much of the acidity produced in land long dressed with ammonium salts may be ascribed to them. Like all living organisms, fungi require energy, which they obtain from carbohydrates. Unlike the bacteria, however, many species can use cellulose as their -THE MICROSCOPE IN AGEICULTUEE 213 source of supply, and are thus responsible for a certain amount of the decomposition of plant residues. It was once held that fungi were capable of fixing atmospheric nitrogen, but this has been shown not to be the case, with the possible exception of one species, Phoma betce. The source of nitrogen supply is amino-acids, so long as the amount of carbo- hydrate material available is sufficient; when this is not so, Waksman claims that the fungi can use soil protein compounds. Finally, mention may be made of the mycorrhiza, fungal filaments found in association with the roots of higher plants. According to Frank, the fungus attacks the humus and mineral sources of the soil, passing them on as food to the plant. The extreme case of dependence is shown by some species of orchids which possess no leaves, relying practically entirely on the fungi for much of its food. Such a condition is not normal, however ; usually the host plant is able to obtain its nutriment unaided when growing in favourable conditions, only assuming the symbiotic habit when the soil is poor, as, for example, in heaths and moors. Stahl, who has considerably extended our knowledge of these interesting fungi, shows that this co-operative method of living is very widespread, and is especially characteristic of plants growing in dry soils — those poor in mineral salts or rich in humus. Though not of direct agricultural importance, yet this question of mycotrophy has an important practical application. There are many plants, such as the orchids, lilies, etc., which must be grown in soil rich in humus ; when, however, this is supplied, development of such seedlings does not always take place, probably because the appropriate and necessary fungus is lacking. If, however, a little of the soil in which the parent plants are growing is mixed with the soil in which the seeds are sown, germination occurs, and mycorrhiza are found at a very early stage of growth. As a result of this brief survey of some of the micro-organisms it must be realized that the soil is by no means an inert mass of dead material, but rather a laboratory where very complicated chemical reactions are continually going on through the agency of the living population. The soil, even when undisturbed, is never in a state of equilibrium ; and when one also considers the complications brought about by the different agricultural 214 MODERN MICEOSCOPY operations, it becomes evident how difficult a business it is to obtain any clear conceptions of the changes going on, or to formulate definite hypotheses as to the chain of events leading to soil fertility, and ultimately to the crop of wheat covering a field. Many aspects of agriculture I have had perforce to omit, but if I have demonstrated the importance of biology, and especially the importance of the microscope, when in the hands of those trained in its use, the purpose of this chapter will have been served. ADDENDUM For those who wish to extend their reading in the branches of agriculture dealt with above the following references to litera- ture are given. These papers also contain a fairly full biblio- graphy: General Agriculture. Hall, A. D. (1920). ' The Soil,' third edition. John Murray, London. BussELL, E. J. (1921). ' Soil Conditions and Plant Growth.' First volume of the Bothamsted Monographs on Agricultural Science. Longmans, Green and Co., London. Protozoa. Cutler, D. W. (1919). 'Observations on Soil Protozoa,' Journ. Agric. ScL, ix. p. 430. CUTLEE, D. W. (1920). ' Method for Estimating the Number of Active Protozoa in the Soil.' Journ. Agric. Sci., x. 135. Cutler, D. W., and Crump, L. M. (1920). ' DaUy Periodicity in the Number of Active Soil Flagellates, etc' Ann. App. Biol., vii. 11. Crump, L. M. (1920) 'Numbers of Protozoa in certain Eothamsted Soils.' Journ. Agric. Sci., x. 182. GooDEY, T. (1916). ' Further Observations on Protozoa in Relation to Soil Bacteria.' Proc. Boy. Soc. B., 89, p. 297. GooDEY, T. (1916). ' Observations on the Cytology of Flagellates and Amoebae obtained from Old Stored Soils.' Proo. Zool. Soc, p. 309. EussELL, E. J. (1915). ' Soil Protozoa and Soil Bacteria.' Proc. Boy. Soc. B., 89, p. 76. Algse and Fungi. See accounts given in the books by Hall and Russell respectively, and : Bristol, B. M. (1919). ' On the Retention of Vitality by Algse from Old Stored Soils.' New Phytologist, xviii. 29. Bristol, B. M. (1920).' ' On the Algse of Some Desiccated English Soils : An Important Factor in Soil Biology.' Ann. Bot., xxxiv. 85. Brierlby, W. B. (1919). ' Some Concepts in Mycology.' Trans. Brit. Myeo- logical Soc, vi. 204. Rayner (1916). ' Recent Development in the Study of Endotrophic Mycor- rhiza.' New Phytologist, xv. 161. Stahl (1900). Jahr.f. wiss. Botanik., xxxiv. 643. THE MICROSCOPE IN AGRICULTUEE 215 It is essential that anyone desirous of doing research on the micro-organisms of the soil should have a knowledge of modern bacteriological technique and of general physiological methods. As regards apparatus, it is necessary to have a microscope stand made by a well-known firm, and fitted with a sub-stage condenser capable of being centred. A mechanical stage will be found very useful. The objectives mostly employed are nJj-inch oil immersion ; \ inch and f inch, together with a set of eyepieces ranging from 4 to 18 magnification. It is, perhaps, hardly necessary to emphasize the point that to get the best out of the microscope, a general knowledge of its construction and the optical principles involved is essential. PART III THE MICROSCOPE AND THE NATURALIST INTEODUCTION By WILFBED MABK WEBB, F.L.8., Honorary Oeneral Secretary of the Selborne Society. Yeae by year the interest which is taken in the world around us, in the unspoiled works of Nature, continues to increase. It is now also generally recognized that to train the powers of observation is one of the most important necessities in general education, and that it is far better for everyone to teach themselves naturally through the interest aroused by the subjects considered, than to learn nothing but second-hand facts from others. i To the majority of people, whether they are children or not, living things prove most attractive. The general appreciation of them begins most easily and properly out of doors, and may continue as a lifelong pursuit. The detailed investigation of some part of natural history forms an interesting hobby as well as a healthy form of exercise, recreation, and relaxation, par- ticularly for those who are not forced to spend all their spare time upon games, or to whom the more violent forms of athletics and the usual kinds of sport do not appeal. Whatever line of study is taken up, it will be very soon found that if any real progress is to be made — if anything new is to be found out with regard to the structure of the various creatures apart from the obvious — some aid to the vision must be sought, some means of learning details which cannot be seen with the naked eye. Here it is, then, that the microscope comes into play, and it is not too much to claim that, besides being the source of additional interest, the instrument is a great educator — that is to say, it trains, without appreciable effort, the hand to be skilful, the eye to appreciate, and the brain to elucidate. 216 THE MICEOSCOPE AND THE NATUEALIST 217 Moreover, without for one moment suggesting that observations in the open air should not be considered the most essential part of Nature study, we must agree with a recent writer in The Country Home, that 'there will be times when the most en- thusiastic Nature student cannot be out of doors — long, dark winter evenings and wet days even in summer, when indoor work must take the place of outdoor. It is then that work with the microscope will prove such a fascinating hobby, supplement- ing, as it does, the observations made with the naked eye, and leading us into regions where it is impossible to travel with- out it.' The lowest forms of plant-life are unicellular, and often ex- tremely small. The microscope reveals to us that they have powers of locomotion ; it shows us also, for instance, that the green colouring on trees and fences which shows after rain is made up of myriads of minute plants, taking in gases from the air and earth-salts from the surfaces on which they live, and making their food in the same way as the cabbage or the oak- tree. The whole science of bacteriology and the discoveries of the minute fungi which cause disease and putrefaction, which give the taste to butter and the flavour to cheese, entirely depends upon the high powers of the microscope, and though investigations in this direction are not for the young beginner, still, they lie before him when he has mastered the details of his instrument. The story of the interesting, though for a long time hidden, methods of reproduction among the mosses and ferns have been revealed by the microscope. The eggs and motile fertilizing bodies in the so-called flowering heads of the moss have been discovered, and the determination of the species by the syste- matist depends to a great extent upon the microscopic details of the capsules which grow from the egg, and are really another generation, producing spores without fertilization and getting its nourishment from the original moss-plant. In the ferns and their allies, however, we find the sexual organs are borne by a tiny plant like a minute liverwort, which springs from the spores on the fern frond when they fall to the ground, and this little plant nourishes the egg as it develops into a new fern, which, unlike the green moss-plant, is the sexual generation. 218 MODEEN MIGEOSCOPY Fig. 109.— Travelling Microscope Packed in ITS Case, which Measures 9"x3i"x4". Fig. 110— Tbavellino Miorosoope set up for Use. THE MICEOSCOPE AND THE NATUEALIST 219 Before going on to speak of the use of the microscope in the various branches of natural history, we may point out that it can with advantage be used from time to time to lend an additional interest to ordinary Nature study. The youngster who sees the pollen of various kinds of flowers as a powder may well be introduced to the variety of shapes and sculpturing which the grains present. Many of the hairs which clothe and protect the commonest plants are fascinating when their details can be seen. An unfortunate occurrence such as the stinging of a youthful naturalist by wasp or bee may well lead to the examination of the sting of the insect, and possibly the hairs of the nettle, while the delicacy of natural objects compared with those made by man may well be brought home by examining the point of a fine needle with a microscope, and seeing how far more clumsy it is than either of the other two structures to which allusion has been made. We need not deal further with this side of the question, for those who look for suggestions as to microscopic work can glean them from the paragraphs in which more systematic work is discussed. It may be pointed out here, however, that the micro- scope may be used by the young student so soon as the informal stage of nature study is passed. The writer can say from personal experience that the interest which can be aroused is very great, while excellent work with the microscope has been done, for instance, by gardening lads who for years have used no instru- ments of greater precision than spades, and rakes, and hoes. We need not dwell any longer upon the botanical side, except to say that the whole structure of plant bodies lies before the student. There are all the interesting details to be worked out in connection with the formation and storage of starch grains, which vary in different plants; while one may examine the delicate hairs on the roots which take up water and food materials, or the thread-like fungi which sometimes enter into partnership with the roots and do the work of root-hairs, as in the heaths and rhododendrons, not to mention the bacteria-like organisms which produce nodules on the roots of plants belonging to the pea family, and supply their willing or unwilling hosts with nitrogen which they are able to get from the atmosphere. 220 MODERN MICROSCOPY Then there is the structure of the fibres which prevent stems from breaking, and of the tubes which carry water with all the various kinds of thickening which strengthen their walls and prevent them from being crushed in as the plant grows and the pressure within the bark increases. There is the bark itself, made up of many empty brick-shaped cells, and the places known to botanists as lenticels, where the bricks are, as it were, heaped together, instead of making a solid wall, so that air can penetrate to the living' tissues below. With the microscope we see how the annual rings come to be made in a woody stem. We can learn the structure of a bud and the growing tip, and really come to know how a plant is built up. If the living plants should pall, we can cut thin slices of fossils and trace the affinities between plants of bygone times and those of the present day. In fact, there is no end to the beauty and the interest that is revealed by the microscope when it is brought to bear on plant structures. If the material offered by the animal world is not more varied, it is, if possible, even more attractive than that which is to be looked for among plants. To be sure, the botanists have the beautiful flinty shells of the diatoms to study, but among the unicellular animals there is a wealth of forms which are pro- vided with calcareous shells of many shapes, or which build them up with the marvellous discrimination which may exist even in a microscopic speck of protoplasm, from sand grains, or the flinty needles of sponges. It is these shells of foraminifera which, to a large extent, form the ooze which is taken from the very greatest depths of the ocean. Other slightly higher forms have internal silicious skeletons, and may be caught living in fine nets or their skeletons obtained from deposits such as the Barbados earths, which are largely composed of them. There are many of the creatures, such as the bell animalcule, the slipper animalcule, and any number of other Infusoria whose conformation appeals to the eye and whose life-histories are fraught with interest. In the sponges our microscope tells us that amongst the supple horny fibres or the delicate needles and geometrically formed spicules which make up the skeleton there are small chambers in which lie the working cells that THE MICEOSCOPE AND THE NATUEALIST 221 differ in practically no respect from Infusoria. A step, however, takes us to the polyps, and a favourite subject for study is the little fresh-water form, with its waving arms, covered with sting- ing thread-cells that aid it to obtain its prey. Its body, which is all stomach as it were, is lined with cells resembling the proteus animalcules of the ponds, but, unlike them, unendowed with the power of individual locomotion. For charm of shape and delicacy of construction commend us to the skeletons of the polyps, which live in colonies, to the fixed growths on rocks and sea-shells which at first sight look like delicate seaweed, but which on examination are seen to bear innumerable little cups in which the polyps are seated. Almost microscopic, too, are the small jelly-fish which bud off from these colonies, and by an alternation of generations reproduce not them- selves, but colonies like those from which they sprang. Passing over the hedgehog-skinned creatures covered with little nipper- like projections or spines, whose internal structure is of suffi- cient beauty to repay the trouble of grinding sections, and leaving on one side the wheel animalcules, pretty Polyzoa, and the host of creatures known as worms, which offer many problems to the biologist, we come to the molluscs. Their shells are usually large enough to see with the naked eye, but there is a great fascination about the structure of their calcareous coverings. Simple though this may be in the bivalves, it is intricate and puzzling enough in the univalves to satisfy the demands of those who wish to exercise their brains and ingenuity. Then the examination of the tongue-like organs of the slugs, and whelks, and limpets is an aid to the classification of these forms. These structures themselves, covered as they are with minute rasp-like teeth, are so very varied and beautiful that the pleasure of examining them, apart from their scientific investigation, can be well understood. The true snails, again, are often provided with minute calcareous spicules of character- istic shape in the various species, called the ' love-darts,' which are useful for classsification purposes, and form beautiful objects for display. It is when we come to the insects, however, that we meet with even more unlimited material. Some of them, like the tiny fairy-flies — of which, according to the late Mr. Enock, five can 222 MODEEN MICROSCOPY walk abreast through the hole made in a piece of paper with a pin-point — are so small that they have to be examined under the microscope in their entirety, while the parts of other insects show a wealth of detail, and illustrate in a marvellous way the changes which Nature can ring on a single plan. Take, for instance, the mouth-organs of the cockroach. At first sight they little resemble those of the bee, which sucks rather than bites, and appear to have no connection with the proboscis of the butterfly, intended merely for drinking up honey. On careful examination, however, it can easily be seen how the mouth- organs of the two latter have been modified from the first ; and a similar comparison may be made with the stylets of the flea, the piercing organs of the bug^ and the lancets of the gnat. It is only when we examine the wonderful proboscis of the fly that a real difficulty arises. We may mention also the beautiful scales which give the colours to butterflies, and which resemble those found on the more lowly wingless insects known as spring- tails and bristle-tails, which have never known what it is to fly, and have only survived because, when their relatives took to an aerial life, they were thrown out of competition with them. There is, indeed, no end to the work which can be done on insects — their breathing apparatus consisting, as it does, of a series of tubes, from which the air is laid on, as it were, all over their bodies ; their beautiful antennae, and the joints of the legs by which beetles, for instance, are recognized, offer fields for enquiry and objects of interest of which the student will never tire. The dexterous dissector will find full scope for his powers when unravelling the organs of insects, and finding out how these are equally well adapted to the requirements in the smaller forms of life which possess them, as are those of the higher animals, whose general anatomy it needs no microscope to elucidate. In the vertebrates, as in the case of all living things, the minute structure must be learnt from the microscope, and, though the sections are more troublesome to obtain than in the case of vegetable tissues, there are a host of things that can be examined and worked out. Among them we may mention the scales of fishes, the hairs of animals, the feathers of birds. Everyone can see for himself how the feather is built up ; and, THE MICEOSCOPE AND THE NATUEALIST 223 although all the larger feathers are made on the same plan, there are differences in detail. The various birds, indeed, might occupy the attention of a lifetime. It is not needful to dwell any further on the question of the use of the microscope to the student of Nature, or what lies before those who decide to take advantage of it. In conclusion, one may say that it should be part of everyone^s education to learn how to use a microscope, and to have some knowledge of the minute details of the living world. CHAPTEB XIX POND LIFE, WITH SPECIAL EEFEEENCE TO THE ROTIFEEA* By the late C. F. EOUSSELET, F.E.M.S. Methods of Collecting, Preliminary Examination, and Keeping. The fascinating study, under the microscope, of the living microscopic objects found in ponds, canals, and lakes, collectively known as ' pond-life,' requires, first of all, that you should catch your game. The object of this note, therefore, is to discuss those methods of collecting -which, with a good many years' experience, has proved to me the most practical, efficient, and time-saving; it is intended for the young naturalist or beginner who desires to make the personal acquaintance of these minute atoms of life, and thereby gain a better understanding of all living things. A few of the principal localities for collecting pond-life in and near London may be mentioned. The nearest and most con- venient available piece of water is the Grand Junction and Eegent's Canal, which runs from east to west, on the northern side of London, from Victoria Park to Hanwell, and is readily approachable wherever access can be gained to the towing-path. Wimbledon Common and all the great parks have a lake, such as Victoria Park, Eegent's Park, Hyde Park, Eichmond Park, etc., which all afford good collecting-grounds. Smaller ponds are found in abundance in fields and commons in and beyond suburban London, and I need only mention a few such places : Epping Forest, Higham Park, Hadley Wood, Totteridge, * Originally published in Knowledge, and reproduced by permission. 224 PLATE VII. Fio. 111.— ATYPICAL Group of Pond Life Organisms. Bsproduoed by permiasion of the owner of the Copyright, C. Leks CnnTiES, Esq. iTofacep. 224. POND LIFE 225 Hampstead Heath, Ealing Common, Hampton Court, and Putney Common. Apparatus Required.— A few pieces of apparatus are indis- pensable, and these are the following : 1. A Queketter's collecting-stick with ring-net and bottle, and cutting-hook (see p. 88). 2. A flat bottle. 3. A pocket magnifier. 4. A hand-bag with sundry wide-mouthed bottles. The collecting-stick can be obtained from most opticians. It is a hollow walking-stick with an inner rod to increase its length when required, and provided with a screw at the end for the attachment of either ring-net, dipping-bottle, or hook. f\ c Fig. 112. A, C = 9 in. ; angle at C = 140°. The ring is a stout brass hoop, about 6 inches in diameter. The net, which is sewn on to the ring, is made cone-shaped, about 6J inches long, and at its apex is tied a small, rimmed tube of clear glass, about 3 inches long by 1 inch wide (Fig. 113). The material of the net should be either fine muslin, known as ' soft mull,' with meshes fine enough to prevent the Infusoria and Kotifera going through, and yet allowing the water to run out freely, or else a silk material known as ' Swiss bolting silk,' used by millers for sifting the various grades of flour, and obtainable from all mill furnishers ; No. 16 of this silk material has the required fineness. The net is most important, and some care should be taken to have it properly made. Allowing a margin for the seam and for sewing round the ring, the shape and dimensions of the material for a 6-inch ring should be as represented in Fig. 112. This will 15 226 MODERN MICROSCOPY give a net slightly larger than is required, but as the material is sure to shrink a little, it will be of the right size after having been used once or twice. The cutting-hook is a curved knife which can also be screwed on to the collecting-stick, and is intended for cutting roots or water weeds which are otherwise out of reach. The flat bottle can be obtained from opticians, well made, and the parts joined by fusing with fusible cement (T'ig. 113). When invented by the late Mr. T. D. Hardy, it was made by cutting sides and bottom in one piece out of stout india-rubber or similar material, 4 to 5 inches long, by 2 to 2^ inches wide, and f to f inch thick; a square of thin plate-glass of same size, cemented by means of Miller's caoutchouc cement on each side, completed the bottle. A thick piece of india-rubber is, however, so expensive that it is cheaper to buy the finished article. The flat bottle is used for searching over pond-weeds with the pocket lens at the side of the pond, or examining the water which has been collected and condensed with the net. In round bottles it is very difficult to see minute animals clearly, whilst a thin flat bottle allows the whole contents to be readily scrutinized with a pocket lens of considerable power, and one can at once determine whether it is worth while to take home a sample from that particular pond for further examination under the microscope. The pocket magnifier best adapted for field-work is an aplanatic lens, magnifying six diameters, which has a very large flat field, long focus, and perfect definition all over the field. Method of Collecting. — The various groups of plants and animals known as ' pond- life,' found in fresh- water lakes, ponds, and ditches, consist of Algse, Desmids, Rhizopoda, Infusoria, Sponges, Hydras, Rotifera, Polyzoa, Cladocera or Water-fleas, Copepods, Hydrachnida, Worms, and Insect larvae. These can be divided for the purpose of collecting into two groups — the free-swimming, and those that are usually attached to water- plants or submerged objects, and each of these groups must be captured in different ways. All free-swimming or floating forms, which collectively are designated by the word 'plankton,' are best secured with the net. The net is passed through the water two or three or more POND LIFE 227 times, and then held up ; the water will run out in half a minute and quite at the last the condensed animals will be seen entering the little bottle like a cloud, where they can be subjected to a pond-side examination. It is best, however, to empty the contents into the flat bottle, in which the examination with the pocket lens becomes very much easier, and most of the forms one is acquainted with can be recognized at a glance. In this way thousands of Algse, Infusoria, Eotifera, Daphnia, etc., can be captured in a few minutes if the pond be a prolific one. Having thus ascertained that the dip contains some desirable forms, the water is poured into a large, wide-mouthed collecting bottle, of which three to six should be carried in the bag. These bottles should be numbered ; for it is often advantageous to keep the water of different ponds separate, so as to be able to know at home from which pond a particular creature has come. Ponds vary exceedingly as regards their contents in pond-life ; a small pond may be very prolific, whilst another, possibly a larger piece of water only a few yards off, may contain hardly anything worth collecting. By trying all the different ponds, small and large, within reach of an afternoon's walk, one usually succeeds in obtaining a good gathering of free-swimming forms. The net quickly condenses a large volume of water, so that few species, even if present in small numbers only, will escape being captured. Several other methods of condensing pond- water have been devised, but the collecting-net with bottle attached is so simple and effective that we need not trouble about any other apparatus. It may be advisable to try the larger ponds in various places, and both near the surface and also in deep water, as some plankton forms may have collected in one particular corner of the pond and be absent elsewhere ; this is often the case with Volvox globator. The use of a boat on larger lakes is very desirable when available. For rotifers and other active free-swimmers it is not desirable to disturb the mud at the bottom of the pond, but certain species of Cladocera, Hydraehnida, and insect larvae can only be found at or near the bottom. The group of attached forms of pond-life comprise such Infusoria as Carchesium, Epistylis, Zoothamnium, Stentor, etc.; Hydra ; all Polyzoa and Sponges. In searching for these forms. 228 MODEEN MICROSCOPY a quantity of pond-weeds, or rootlets, are brought on shore with the cutting-hook, and selecting some likely-looking, fairly clean branches, but not the newest growth, one twig after another is placed in the flat bottle in clean water, where it can be examined from both sides with great ease, both with the naked eye and the pocket lens. The tree-like Vorticella colonies — Epistylis, Zoothamnium, Carchesium ; the trumpet- shaped Stentors ; the Crown Eotifer Stephanooeros ; the tubes of Melicerta and Limnias; the various Polyzoa ; also Hydra and Sponges, and many others, can at once be seen when present, and in this way good branches can be selected and placed in a separate wide-mouthed collect- ing-bottle containing clean pond-water. A little experience will soon teach one which branches are likely to prove prolific. As a general rule one may say that old-looking, but still sound and green, branches are the best. The Water Milfoil (Myriophyllum) is one of the best water-plants to examine and collect on account of the ease with which its leaves can subsequently be placed under the microscope. Anacharis is more troublesome, but it is occasionally found covered with pond-life, and is an excellent weed for the aeration of aquaria. The rootlets of reeds and of trees growing near the edge of the water should be examined for Sponges and Polyzoa, such as LophopuB, Plumatella, Predericella, etc. In order to obtain some weeds growing near the middle of a pond or lake, a loaded three-pronged hook, attached to a line, may be used ; this is swung round, and may be thrown to a distance of 20 to 25 yards, where it sinks, and the weeds that are caught by the hooks are dragged on shore. By these various means a good collection of pond organisms can readily be made after a little practice. Though the spring and autumn are perhaps the best seasons for collecting, pond-life is never absent, even in the winter under the ice. Having thus filled some bottles with condensed water from various ponds, and placed some promising branches of water- plants in another bottle filled with uncondensed and clean pond- water, the ' bag ' is taken home. It is a great mistake, however, to overstock the bottles with weeds, as the plants in such crowded bottles may begin to decompose, killing most of the animals in a short time. POND LIFE 229 Preliminary Examination. — On reaching home, the first thing to do is to empty the collecting-bottles into small aquaria, so that the captures may be critically examined, isolated, and, if found desirable, placed under the microscope. By far the best and most convenient way of doing this is to transfer the contents of each bottle into a small window aquarium, filling it up with tap-water. The weeds and rootlets that have been brought home are put in another window aquarium in clean pond-water. These small window aquaria, with flat and parallel sides 6 to 8 inches long by 5 to 6 inches high, and only 1^ inches wide inside, are the best nurseries for the microscope. The difficulty of seeing and capturing small objects in a large or ordinary round aquarium is very great, and the use of the pocket lens almost hopeless, whilst in these flat and narrow aquaria no object is out of reach of the lens, and the whole contents can be looked over without difficulty and in a very short time. By placing the tank on a whatnot at a convenient height before a window, or before a lamp at night, most of the free- swimming rotifers will collect against the glass nearest to the light, where they can be examined with the greatest ease and picked up with the pipette if desired. A disc of black cardboard placed some little distance behind produces a very good dark ground, against which the smallest visible specks stand out well. The condensed pond-water is, of course, frequently so dirty with floating particles of d6bris that it is at first hardly possible to see through it; but after standing half an hour it will be found that most non-living particles will have fallen to the bottom, and after several hours the water will be quite clear and every living creature will be readily seen. During the summer months, when Daphnia and Cyclops are abundant, the net frequently collects these in such numbers that they become a nuisance. In order to separate them, when such is the case, I have adopted the plan of passing the water through a small sieve made of material with meshes sufficiently wide to allow the largest Eotifers and Infusoria to go through, whilst keeping back most of the Cyclops and Water-fleas ; the latter are then transferred to a separate tank to be examined by them- selves. It is very desirable to examine the collected objects as soon as 230 MODERN MICROSCOPY convenient, certainly not later than the day after their capture, as many organisms soon die and disappear under the crowded and unnatural conditions in which they are kept in captivity. Keeping. — Rotifers in aquaria can often, particularly in cool or cold weather, be kept for a week or fortnight, and some species, such as Melicerta, occasionally for months if food material in the shape of fresh pond-water can be provided. Failing pond-water, water from hay 'infusions, which mostly con- tain quantities of bacteria and minute Infusoria, may be added. The various species of Polyzoa and Sponges can alsb be kept alive a considerable time by feeding them in a similar way, but Hydras require a fare of Water-fleas if they are to thrive. For keeping microscope life I have found no difference between large and small aquaria, but the small tanks are the more manageable; the great thing to be attended to is the proper aeration with water-plants, of which Anacharis, Fontenalis, and Valisneria are, perhaps, the best, and not to overstock the tank with either animal or vegetable life. The water need not be changed, but a little fresh pond-water should be added from time to time. Larger animals, such as small fish, water-beetles and snails must be excluded altogether from small tanks, and Polyzoa and Sponges must be kept therein in very moderate quantity and small colonies only. In order to ensure success it is essential to maintain a proper balance between the animal and the vegetable life, and also to supply fresh food frequently, for microscopical animals no more than the larger beasts can live long without food. To some extent, no doubt, they feed on each other, but in a small aquarium their hunting-ground is very limited and the game soon becomes scarce. Asplanchna can be seen under the micro- scope to feed on Anursea, Brachionus, Polyarthra, Triarthra, and other rotifers when it can catch them, and their shells and remains are frequently found in Asplanchna's stomach. On the whole, the best plan is to go out and collect a fresh supply from time to time, and as often as may be convenient. I may mention that at the middle of January I had many thousands of rotifers in a tank which I collected two days before in the Grand Junction Canal, near Westbourne Park Station. The canal was covered with blocks of ice, and the POND LIFE 231 time spent near the water did not exceed ten minutes, during which I filled a large bottle with water condensed by means of the ring-net. Aquarium Microscope.— Everyone who has worked at pond- life will have experienced how awkward it is to pick out a par- ticular animal, the size of which requires the aid of a magnifier for unmistakable identification. In order to have both hands free for this operation, and to keep the lens fixed to a particular spot, I devised some years ago a small aquarium microscope (Fig. 113), which is simply a flat metal arm, jointed in such a Fig. 113. way that it allows the lens to be moved all over the surface, but in one plane only, parallel to the side of the window aquarium, whilst the lens is focussed by a small rack and pinion on the left. The whole apparatus is screwed to a small wooden stand, on which the tank is placed. The lens used is an aplanatic combination x 6 diameters, which has working dis- stance enough to focus right through the tank, and sufficient amplification to enable one to recognize most Eotifers, Infusoria, etc., and anything uncommon or new can at once be detected and secured. Moving objects Can readily be followed with a lens so mounted, pond-weeds can be searched for anything that may be growing on them, and the lens fixed firmly in any desired position. I have had this tank microscope in constant 232 MODERN MICROSCOPY use for over twelve years, and can recommend it as thoroughly practical, efficient, and time-saving. APPARATUS FOR MICROSCOPIC EXAMINATION. I propose now to describe those methods which long experience has proved to be the most practical in the examination of living objects under the microscope. Pig. 114 is a photograph of various apparatus used for this purpose, consisting of troughs, pipettes, live-box, and compressor. After capturing a miscellaneous collection of pond-life and transferring it to a window aquarium placed in front of a window, —h Fig. 114. as previously explained, it will be desirable first of all to place some of it under a low power of the microscope — say a 2-inch or 1 J-inch objective — in order to obtain a better general view of the various animals. The free-swimming forms will mostly have collected on the light side of the aquarium, and can there be picked up quite clean and in vast numbers sometimes, with the pipette e, transferred to a square trough as a or b, and placed under the microscope, where the contents can readily be illumin- ated from below, both with transmitted light and under dark ground. I prefer to use dark-ground illumination with low powers when searching over the contents of a trough, and when studying the shape, mode of swimming, ways of feeding and living of Polyzoa, Rotifera, and Infusoria. Moreover, the animals scattered through the trough will soon collect in the POND LIFE 233 spot of light of the condenser, and then the whole field of view will often be a mass of moving, dancing, tumbling, sparkling life. Troughs.— Pattern a, Pig. 114, 3 in. by li in. and ^^ in. thick, is the form I mostly use ; it stands upright on the table, is re- versible, and can be handled without greasing the well part of the glass. The sides are cemented in the fire by means of a fusible glass cement, and thus the trough is, and remains, watertight. The trough h is also a useful type, but it is not reversible, will not stand by itself on the table, and, being cemented with gold size or marine glue, is liable to leak. The troughs usually sold are semicircular in shape, a very bad type, because, in addition to the above defects, the least amount of tilting on the stage will cause the water to run out over the edge. Thicker troughs are objectionable because the sub-stage condenser cannot work through them, and the animal cannot be properly illuminated, though sometimes such troughs may be required by the size and nature of the object. Pipettes. — Efficient pipettes are indispensable for serious work. The old way of using the finger on a straight or curved glass tube to capture pond-life is so unsatisfactory that I have been driven to invent new pipettes for more precise and exact work. Fig. 114 c, d, and e, represent the pipettes in constant use ; e is a glass tube about ,^-^ inch in diameter and 8 inches long, which tapers from the middle to a point more or less fine, according to the size of the animals one wishes to capture. Over the wide end is placed an india-rubber teat, by means of which any single specimen, or scores of animals, can be sucked up with the least quantity of water; d is another type of pipette, having a still finer action ; it is 6 inches long, funnel- shaped at one end, and tapering gradually from the funnel to a fine point ; the funnel is f inch wide, and covered with an india- rubber membrane ; c is a similar, but smaller and finer pipette, 3J to 4 inches long, for picking up small rotifers in a fraction of a drop of water under the dissecting microscope. The slightest touch on the membrane is sufficient to expel or suck up water, so that one has complete control over the amount that is taken up, and there is much less risk of losing the animal one wishes to transfer to the compressor. 234 MODEEN MICKOSCOPY Live-boxes. — The old-fashioned live-box with raised tablet, suitable for insects, etc., is quite useless for pond-life, for the simple reason that the objects cannot be properly illuminated with the sub-stage condenser. This consideration led me long ago to design the live-box /, Fig. 114, in which the glass tablet is flush with the brass plate, and is of small size, thus leaving a wide ring all round. This arrangement allows all objects on the tablet to be perfectly illuminated from below by the sub-stage condenser, both with transmitted light and under dark ground, and at the same time they can be reached and followed from above with both low and high powers, even a ^ inch oil immersion, to the very edge of the tablet, and wherever they may wander. The Compressor. — For more exact work, when it is desired to hold a single animal, and prevent its wandering, I have devised the compressor g, in which the pressure and the thickness of film of water can be accurately regulated by a screw acting against a spiral spring. At the same time, water, or reagents, can be added, if desired, without raising the cover. When properly and well made, this compressor works exceedingly well, and I have had it in constant use for years, but some makers, unfortunately, have introduced variations and so-called ' improvements ' which just take away some of the essential and useful points. The semicircular thin cover-glass must be cemented to the under side of the brass ring with a little gold size, so as to be quite firm and rigid, otherwise its action becomes uncertain, and very small objects cannot be held fast, or else are suddenly crushed. Simple Substitutes for Above. — These are often very useful in an emergency where no live-box or compressor is at hand. An excavated glass slide makes a fair live-cage ; a drop of water con- taining the animals is placed in the cavity so as to just fill it, and no more ; another drop of clean water is placed by the side of the cavity, and a clean thin cover-glass is lowered on to that second drop ; then, by means of a needle, the cover is slowly pushed across the cavity, which can thus be covered without enclosing an air-bubble; the superfluous water is taken up by blotting- paper, the cover-glass being held in position by capillary attrac- tion. This forms a good slide for low and medium powers, but not for high powers. Another good temporary slide can be made by placing three small fragments of No. 1 thin cover-glass near POND LIFE 235 the middle of a glass slip in form of a triangle; the drop of water containing the animals is placed in the centre, and a clean thin coyer-glass is lowered on to the drop so as to rest on the three glass fragments, which prevent the animals getting crushed. If there be too much water it can be removed with blotting-paper. Low and high powers can be used on this slide as far as the movements of the animals will permit, but not oil immersion lenses. Method of Use. — Having detailed the essential apparatus, I will close this section with a few remarks on the practical use of same. The free-swimming organisms, including such forms as Volvox globator, collect on the light side of the window aquarium, and can there be picked up in small or large numbers, and, quite clean, with the large pipette, and placed in the trough ; or any particular species can be selected with the aid of the tank micro- scope, and taken up with the smaller pipette, and transferred to the live-box or compressor in a single drop of clean water, both hands being free for this operation. The fixed forms, such as Polyzoa, Stephanoceros, Melicerta, Floscules, etc., amongst Eotifera, and Stentor, Carchesium, Zoothamnium, etc., amongst Infusoria, require a little manage- ment. If simply placed in a trough, these are often obscured or incapable of being properly illuminated by being too crowded, or by part of the weed over- or under-lying the objects, and also by floating particles in the water. The best result is obtained by trimming — that is, by cutting off a very small piece of weed or leaf on which the animal is attached — in a watch-glass under a dissecting microscope, if necessary — and then transferring it with the pipette to the compressor into a drop of clean water ; it can then be arranged with a needle or bristle 9,s may be desired, and after lowering the cover-glass, fixed and held fast, at the same time giving the animal perfect freedom to expand. In this position the animals can be reached with the sub-stage condenser from below for transmitted light and dark-ground illumination, and also with low and high powers, and even oil immersion objectives from above. The Microscope.— The Wenham binocular (p. 14) is decidedly to be preferred to the monocular microscope. Using both eyes, prolonged work can be undertaken without undue strain, and the 236 MODEKN MICEOSCOPY stereoscopic image gives a very much better idea of the true shape of the animals, though the images are not quite so sharp as with the monocular tube ; but this binocular form can im- mediately be changed into a monocular for high-power work, or whenever desired, by pushing the small prism out of the way. The binocular is to be used only with the low powers up to the |-inch objective ; with higher powers the stereoscopic effect is lost, because the depth of focus, or the plane of distinct vision, is then exceedingly small, and becomes more and more a mere optical section of the object. A mechanical stage is hardly necessary ; for ordinary work a well-made sliding stage or bar is preferable, and should be pro- vided. Stage-clips, of which opticians are so fond, are abomina- tions, and should be consigned to the dust-bin. Of illuminating apparatus, the Abbe form of sub-stage con- denser, achromatic if possible, is the only one that is really useful for all powers, and that need be considered both for transmitted light and for dark-ground illumination. It should be provided with an iris diaphragm and an arm carrying a central stop ; it completely replaces all the older sub-stage apparatus — condenser, spot lens, paraboloid, etc. An illuminant condenser, however, is necessary to render parallel the rays of the lamp-flame, but it should be mounted on the lamp, so as to move with it, once it has been adjusted to project the beam from the edge of the flame on to the flat mirror of the microscope for dark-ground illumination with low powers. All apparatus used in the examination of pond-life — troughs, live-boxes, compressors, and pipettes — should always be carefully cleaned and dried immediately after use, and in no case should the water be allowed to evaporate in them. Much trouble will be saved by the observation of this rule, and the apparatus will always be ready for use. PEESEEVING AND MOUNTING. There are few observers of pond-life who have not felt a keen desire to preserve and keep these small highly organized sparks of life instead of letting them die and disappear in a few days. For a close study of this group, well-preserved type POND LIFE 237 specimens are of the greatest possible assistance and importance, and if such had existed formerly much confusion and inexacti- tude in their description and classification would have been avoided, particularly in the giving of three or four different names to the same species, which causes so much trouble to the student. The total absence of type specimens of rotifers to refer to when required led me to attempt the task of working out a method of permanent preservation, and it is now some twenty years since the first successful experiments at preserving them in a fully extended and natural state were made. The method, although so simple now, took fully three years to work out until the right and most suitable narcotic, fixing agent, and preserving fluid were found. By the use of suitable fixing agents not only the external shape of rotifers can be preserved, but also all the internal structure, to the minutest anatomical details, such as the striated muscle fibres, nerve threads, vibratile tags or flame cells, sense hairs, cilia, etc., and frequently important details can be more readily observed than in the living animal. Narcotizing. — As is well known, no killing agent is sufficiently rapid to prevent the complete retraction of rotifers, and few other animals can contract into such a shapeless mass when we attempt to kill them by ordinary means, such as poisons, alcohol, heat, etc. It is, therefore, necessary to use first a suitable narcotic, which has been discovered in hydrochlorate of cocaine. As a result of many trials, the best solution for most rotifers has been found to be the following mixture : 2 per cent, solution of hydrochlorate of cocaine, 3 parts ; alcohol (or methylated spirit), 1 part; water, 6 parts. Another narcotic which is also very suitable for rotifers is a 1 per cent, watery solution of hydrochloride of eucaine, recom- mended by Mr. G. T. Harris, for Infusoria and other animals. These narcotics, even so dilute, are not to be used pure, as they would cause the rotifers to contract at once and not expand again. The principle to be followed throughout is to use the narcotic so weak that the. animals will not mind it at first, but continue to expand or swim about freely. After a short time its effect will make itself felt on their nervous system, and then some more of the narcotic may be added, until complete 238 MODEEN MICEOSCOPY narcotization is produced, or until the animals can be killed without contractings. But before the operation of narcotizing is begun, it is very necessary to isolate the rotifers in perfectly clean water. The best way is to pick them up under a dissecting microscope by means of a finely-drawn-out pipette, having a funnel-shaped en- largement at the other end, covered with an elastic membrane (Fig. 114 c, p. 232). This pipette forms a most delicate siphon by means of which any selected rotifer can readily be taken up with the least quantity of water, and transferred to another trough or watch-glass full of clean water. This preliminary precaution is necessary, because particles of dirt in the water readily attach themselves to the cilia of dead rotifers, render- ing them unsightly under the microscope. Another advisable precaution is to separate the different species, because most species require a slightly different treatment, and because the small species too readily adhere to the cilia of the large species. Having then isolated a number of free-swimming rotifers in a watch-glass half full of perfectly clean water, one drop of one of the above narcotics is added and well mixed. After five or ten minutes, if the animals continue to swim about freely, another drop is added, and so on until the effect of the narcotic becomes visible, and until the motion of the cilia or the movements of the animals slacken or almost cease, when they are ready for killing. The effect of the narcotic varies very much with dif- ferent species ; some are most sensitive to it, whilst others can stand a considerable quantity for a long time. Killing and Fixing. — Some practice and patience are cer- tainly required to find out the right time to kill the different species ; no general rule can be given, as the time piay vary from fifteen minutes to several hours. It is very essential, however, that the rotifers be still living when the killing fluid is added to prevent post-mortem changes in the tissues, which begin at once on the death of the animals. For killing and fixing several fluids are suitable — namely, I per cent, osmic acid, or Flemming's chromo-aceto-osmic fluid, or Hermann's platino-aceto-osmic mixture. On the whole, I now prefer the last named, which gives a finer fixation of the POND LIFE 239 cellular elements of the tissues and does not stain them so much. It may be explained that the term ' fixing ' implies rapid killing and at the same time hardening of the tissues to such an extent as to render them unalterable by washing and subsequent treatment with preserving fluids. Proper fixation is very essential, as no good preservation can be obtained without it. When the rotifers are narcotized and ready for killing, a single drop of one of the above fixatives is added, and mixed with the water in the watch-glass. A few minutes is sufficient for fixing small creatures like these, and then they must be removed again by means of the pipette to several changes of clean water to get rid of the acid, otherwise they will become more or less blackened. When dealing with marine rotifers, sea-water must be used for washing out, for the difference in density between fresh and sea water is sufficient to cause swell- ing by osmosis, and the consequent spoiling of the specimen. After thoroughly washing, the rotifers are transferred to a preserving fluid, the density of which does not materially differ from that of water. The best preserving fluid found so far is a 2| per cent, solution of formalin, which is made by mixing 2i CO. of the commercial 40 per cent, formaldehyde with 37^ c.c. of water, and then filtering. The above are general directions according to which the great majority of rotifers can be preserved. When under the narcotic, the animals must be watched until it is seen that they can swim but feebly, when, as a rule, they will be ready for killing. If they contract and do not expand again, it is a proof that the narcotic used is too strong, and it must be further diluted. The whole method undoubtedly requires great care, and is a delicate operation, which must be performed under some kind of dissect- ing microscope, but by following the directions here given, and with some perseverance, anyone can learn to prepare a large number of species of rotifers. I would advise that a beginning should be made with some such forms as Brachionus, Anursea, Synchseta, Asplanchna, Hydatina, Triarthra, and Polyarthra, which are easy, and, moreover, occur, and can, as a rule, be collected in large numbers. A few genera, however, are exceptionally difficult. These are Stephanoceros, Floscules, 240 MODERN MICROSCOPY Philodina, Rotifera, and Adineta, and it will be better to leave these until considerable experience in dealing with the others has been acquired. It will have been noticed that the rotifers must always remain submerged in a watery fluid, and be transferred in a drop by means of the pipette. Fluids' of lesser density than water, such as alcohol, as well as fluids of greater density, such as glycerine, are unsuitable because they set up strong diffusion-currents by osmosis, which cause the animals either to swell or to shrivel up completely. Some species of rotifers, such as Triarthra, Polyarthra, Pedalion, Mastigocerca, etc., have an outer surface^ which is strongly water-repellent, and when these come in contact with the surface film of the fluid even for an instant it is most difficult to submerge them again, and, as a rule, they are lost and spoiled. Having then successfully narcotized, killed, and fixed the rotifers fully extended, and finally transferred them into 2| per cent, formalin, the animals may be kept in little bottles, or mounted in the same fluids on micro-slides, either in excavated cells or shallow cement cells. Mounting. — In the cell of an excavated slip, place a drop of the formalin solution, then transfer the prepared rotifers into this drop with the pipette, and examine under the dissecting microscope to see that no particle of foreign matter has been introduced. Then place another drop of the fluid on the slide by the side of the cell, lower the cleaned cover-glass on that drop, and push the cover cautiously and gradually over the cavity. The super- abundant fluid is removed with blotting-paper, and the slide closed by tipping damar gold size cement all round the edge with a fine brush. The permanent closing of these cells has been a matter of very considerable difficulty. As the result of the experience gained, it is recommended that the cells be closed first with a coat of a varnish consisting of two-thirds damar in benzole and one-third gold size, then two coats of pure shellac dissolved in alcohol, and finally four to six coats of pure gold size. Each layer of cement must be allowed to dry thoroughly well ; three days for each layer is not too long. POND LIFE 241 By the method described above, I accumulated within the period of ten years, 1902 to 1912, a collection of over 500 slides con- taining nearly 300 different species of rotifers, probably the only collection of the kind in existence, which is of the greatest use for the identification of species and for the general study of this interesting class. Entomostraca should be narcotized with the same solution as used for Eotifera, then killed with a ^ per cent, solution of osmic acid, and mounted in a 2| per cent, solution of formalin. COLLECTOE'S CALENDAR* January. January being the most severe month of the year, lakes and ponds are often frozen over or difficult to approach. Micro- scopic pond-life, though less abundant than in the spring and autumn, is, nevertheless, nearly always present, even under ice many inches thick. All the following species of rotifers have been taken in January in and near London ; but no doubt a great many more could be found by systematic search : Asplanchna Brightwellii and priodonta; Anurcea aculeata and cochlearis ; Brachionus pala and angularis ; Notholca scapha ; Euchlanis deflexa and hyalina; Rotifer macrurus and vulgaris; Polyarihra platyptera ; Synchceta pectinata, tremula, and oblonga ; Conochilus unicornis ; Coelopus porcellus ; Diaschiza lacinulata and ventripes ; Proales decipiens and petromyzon ; Diglena forci- pata; Dinocharis poeillum ; Monostyla cornuta ; Colurus caudatus ; Mellcerta ringens ; Limnias ceratophylli; CEcistes crystallinus ; Floscularia cornuta; and Stephanoceros eichhornii. Diaptomus and Cyclops and their larvse are abundant, whilst Water-fleas are almost absent. Aquatic vegetation having died down, the fixed forms of rotifers and Infusoria should be looked for on the root- lets of trees growing near the edge of the water. Floscules and Melicerta were once found covering such rootlets very thickly. January seems to be the time when the males of Stephanoceros and other tube-dwellers are found, and their presence is often * For particulars of the Quekett Club excursions for the purpose of collecting, see p. 88. m 242 MODEEN MICROSCOPY betrayed by the thick-shelled, fertilized, resting eggs in some of the tubes, and numerous smaller male eggs in others. February. In the early part of the year, when the weather is still cold and ponds are covered with ice, some Infusoria may be found in abundance, particularly the various species of Vorticella — Carchesiumpolypinum, Zoothamnium arbuscula, Epistylis Jlavicans — attached to submerged rootlets. Eotifera to be looked for in lakes and ponds, particularly duck-ponds : Anuraa aculeata, Anuraa cochlearis, Asplanchna priodonta and Brightwellii, Notholca scapha, Polyarthra platyptera, Euchlanis deflexa, Syncliata tremula. The water-plants having mostly died down, the following fixed forms are found attached on Anacharis, or on submerged rootlets of plants, or on trees growing near the edge of ponds and lakes : Melicerta ringens, Limnias ceratophylli, Stephanoceros eichhornii, Floscularia cornuta, and other species, (Ecistes crystallinus and other species. March. The same species as those mentioned for February are still to be found, but in greater abundance. Some new Infusoria will have made their appearance, such as Stentor polymorphus, which will be found covering the rootlets of Duckweed and other sub- merged plants, Peridinium tabulatum and the free-swimming colonies of Synura uvella, etc. Then the very minute and beautiful colonies of Collared Monads, Codosiga umbellata, and other species of this group may be looked for, attached to the stems of Vorticella trees. All the Eotifera forming the winter fauna will become very abundant in March, and as the food-supply in minute Algse and Infusoria increases, fresh species make their appearance with every rise of temperature. The following additional species may be looked for : Brachionus angularis ; Notholca acuminata, spinifera, and labis ; Euchlanis oropha ; Dinocharis pocillum; Diaschiza lacmulata ; Proales decipiens and petromyzon; Mono- styla cornuta; Diglena forcipata; Rotifer vulgaris. POND LIFE 243 April. All species of Infusoria and Rotifera mentioned as occurring in March are likely to become more abundant in April, which is one of the best months for collecting. The ponds are full of water, whilst they have become approachable, and Daphnias and Cyclops have not yet crowded out the rotifers, as sometimes occurs later on. Volvox globator may be looked for, together with the little parasitic rotifer, Proales parasitica, inside the green spheres. Of larger Infusoria, Bursaria tiuncatella, Choenia teres, Amphi- leptus gigas, and flagellatiis will be found, and, of course, crowds of Eugleiia viridis. Of Rotifera, Syncliata pectinata will be abundant, and Asplanchna priodonta and Brighttvellii will have made their appearance in larger lakes and canals ; also Brachionus pala, quadratus, and Bakeri; Euchlanis triquetra and hyalina; Triarthra longiseta, Diaschiza semiaperta ; Rhinops vitreci, Pterodina patina, Mastigocerca bicornis, and many others. May. All the various pond organisms that die down in winter and in various ways produce protected germs to tide over this, for them, unsuitable season, will now have come to life again and begin to multiply at an increasing rate. Many kinds of Desmids should be found in shallow, mossy pools, or along the edge of rivulets. Among Protophyta and Protozoa the green spheres of Volvox globator will be found in many localities more or less abundantly, and the various kinds of Acineta should be looked for in quiet, undisturbed waters, where many kinds of free- swimming Infusoria will also be found. Of Rotifera there are few species which may not be found in May. At one excursion of the Quekett Club to Totteridge in the middle of May forty different species were obtained. To mention only a few : Notops brachionus, one of the most attractive rotifers, will have made its appearance ; then various kinds of Anuraea, Asplanchna, Brachionus, Coelopus, Cathypna, Diaschiza, Euch- lanis, Furcularia, Mastigocerca, Metopidia, Pterodina, Synchseta, Scaridium, Stephanops ; also Stephanoceros eichhornii, Floscules, Melicerta, and Limnias in abundance. 244 MODEEN MICROSCOPY On rootlets of trees growing near the edge of ponds and lakes will probably be found various kinds of Polyzoa : Fredericella Sultana, Paludicella, and Plumatella repens. June. If the months of April and May are abnormally cold, pond organisms which usually make their appearance in May are likely to be retarded, and will only come on in June. There are, however, summer forms which hardly ever occur earlier than June, and the most interesting of these amongst rotifers is Pedalion minim, with its six arthropodous limbs ; Syncliceta stylata, with its long- spined floating eggs, and Synchceta grandis, the largest species of this genus, may also now be looked for in lakes and water reservoirs, as well as the rare free-swimming Floscularia pelagica. In the same waters will be found two free-swimming colonies of Vorticella : Epistylis rotann and Zoothamnium limneticnm. In June it often happens that certain water- fleas, Daphnia and Bosmina, also Cyclops and their larvae, increase to such an e intent as to render the existence of free-swimming rotifers almost impossible in these waters, and the latter consequently disappear, though they may have been swarming a few weeks earlier. In ponds, however, where this does not occur, rotifers of many genera may be found, and attached to submerged water- plants Lacinularia socialis and Megalotrocha albo-flavicans should be looked for, whilst in reedy ponds the free-swimming spheres of Conochilus volvox may occur. Mossy pools, in addition to their special rotiferous fauna of Philodina, Callidina, Adineta, Cathypna, Distyla, and Monostyla, will also contain water-bears and shelled Ehizopods, such as Diflugia and Arcella and numerous free-swimming Infusoria. Polyzoa, such as Plumatella repens, Fredericella Sultana, Loplwp\ig crystallimts, and Cristatella mucedo, though not common, should be abundant in suitable localities. July. Collecting in July is usually not so profitable as one would expect, because as a rule most of the shallow ponds are dried up by this time, or have been reduced to a muddy swamp, and in the others Crustaceans, Cladocera, and_Cyclops have multiplied POND LIFE 245 to such an extent as to leave little room for the more interesting forms of pond-life. Pedalion mirum should be looked for in large and small lakes, as it will probably have greatly increased in numbers. The some- what rare and very large Asplanchna amphora and ebbesbornii, as well as Asplanchnopus myrmeleo, are summer forms which occur at this season. Other rotifers that appear in warm weather are : Dinoj)s longipes; Triphyllas lacustris; Notops clavulatas; Scaridium eudactilotum anilongicandum; then the free-swimming Lacinularia natans and Conochilus volvox ; also the fixed Lacinularia socialis and Megalotrocha, which are found attached to submerged water- plants. All these are very beautiful objects under the microscope, but by no means common. Volvox globator will certainly be found in abundance now in secluded ponds, and inside the green spheres the little parasitic rotifer, Proales parasitica, should be looked for. The Polyzoa, mentioned last month, will have become more abundant where they occur ; undisturbed ornamental lakes and canals are the best places to find them in. August. For the collector of Cyclops, Diaptomus, Water-fleas, and aquatic insect larvae, August is a very capital month ; not so, however, for the collector of the more interesting Infusoria and Eotifera, which are usually quite crowded out by the more vigorous Crustaceans in the few remaining ponds and pools not wholly dried up. In larger lakes, however, it is possible to find occasionally a number of interesting forms, particularly free- swimming rotifers, such as Asplanchna priodonta a,nd Brightwellii, Synchceta pectinata, and the rarer summer forms, Synchceta stylata and grandin. Where a ' green ' pond can be found full of the flagellate Infusorian Eiiglena viridis, there are usually present also a number of rotifers, such as Hydatina senta, Eosphora aurita, Diglena biraphis, etc., feeding on the Euglena. In shady forest pools, overgrown with Sphagnum, quite a peculiar fauna of moss-haunting rotifers will be found, particu- larly various species of Callidina, Distyla, Metopidia, Cathypna, in addition to numerous interesting Khizopods with shells of various forms. In similar ponds the large but very rare rotifer, 246 MODERN MICROSCOPY Copeus spicatus, should be looked for. Of other rotifers that may be met with in lakes, more or less abundantly, the following can be mentioned : Brachionus pala ; Anuria aculeata, brevis- pina and hypelasma ; Dinocharis pocilhmi ; EucManis triquetra, hyalina, and oropha; Mastigocercabicornis, elongata, and stylata; Polyarthra platyptera; Synchceta tremida and oblong a ; Pedalion mirum, and many others. September. In normal years many of the dried-up ponds begin to fill up again in September, and become then most prolific in intusorian and rotiferous life, because the disturbing Crustaceans, Cyclops, and Cladocera have been to a large extent eliminated. But also in larger ponds and lakes, which do not dry up, the Crustaceans decrease in numbers and give the Rotifera and Infusoria a fresh chance of increase. The following free-swimming forms may often be collected in immense numbers : Asplanchna piiodonta, intermedia, and B7ightwellii ; Triarthra longiseta ; Polyarthra platyptera; Synchceta pectinata, tremula, and oblong a ; Anurcea aculeata and cochlearis ; Brachionus angularis ; Pedalion mirum ; Conschilus unicornis, and the much rarer Floscularia pelagica- Of the fixed forms, Limnias ceratophylli and annulatus, Cephalo- siphon limnias, Lacinidaria socialis, Melicerta ringens and conifera should be looked for on submerged water-plants, such as Ana- charis, Ceratophyllum, Nitella, and on the rootlets of Duckweed. Polyzoa such as Plumatella, Lophopus, Cristatella, should be found in abundance in disused canals and backwaters of rivers and the larger lakes, from which they may be dredged with a loaded hook and line. It may be taken as a general rule that all the more interesting forms of pond-life become more abundant in September, pro- vided only that the weather is not too hot, but tempered by re- peated showers to fill the dried-up ponds with a fresh supply of rain -water. October. October is one of the best months for the pond-hunter ; the weath er is cooler, the ponds have become filled with rain-water again, with plenty of food material in the shape of flagellate Infusoria, and the Crustaceans are on the decline. In this POND LIFE 247 month the greatest variety in species of Eotifera is usually found, particularly of the smaller and rarer kinds, and not in- frequently thirty to forty species may be obtained in one or two small ponds. As a general rule one cannot expect much variety when a few species are present in excessive abundance. The following is a list of forty-four species of rotifers actually col- lected on one occasion in three ponds on October 15, 1898, showing what may be looked for : Floscularia regalis, ornata, cornuta, ambigua, edentata, and annulata; Lhnnias annulatus, var. granulosus; CEcistes crystal- linus ; Philodina megalotrocha ; Rotifer vulgaris ; Syncliceta tremula and oblonga ; Asplanchna priodonta ; Notops hyptopus; Polyarthra platyptera; Eosphora aurita; Furcularia longiseta, sterea, and forficula ; Proales felis ; Diglena biraphis ; Mastigo- cerca rattus and bicor>iis ; Ccelopus porcellus and tenuior ; Rattulus bicomis ; Diaschiza exigua ; Distyla Jiexilis ; Monostyla lunaris ; Ditiocharis pocillum ; Stephanops lamellaris ; Cathypna tuna; Euchlanis oropha ; Metopidia acuminata; Brachionus angtolaris and Bakeri; Povipholyx sulcata; Notholca labis and scapha; Anurcea aculeata, cochharis, tecta, hypelasma, and stipitata. On the other hand, the various kinds of rotifers known as summer forms will now have disappeared. Pedalion mirv/m is such a form, which may occasionally still be seen during a warm October, but is then usually very scarce or absent. November. With the advent of November, pond-life all round becomes less abundant, and fewer species are to be met with. By degrees many of the water-plants die down, and the fauna is reduced to such forms as can subsist through the winter. Those animals which cannot do this, such as Polyzoa, Daphnia, some Eotifera, etc., have by this time produced so-called winter eggs or resting germs. The winter fauna, however, is much more numerous than is usually assumed. Among rotifers, several species of Synchaeta — S, pectinata, tremula, and oblonga — seem to like the winter as well as the summer : Asplanchna pnodonta, Anurma acideata, Polyarthra platyptera, Rotifer vulgaris, Euchlanis deflexa, Triarthra longiseta, Brachionus angularis, Conochilus unicornis, Diglena forcipata, Diaschiza lacinidata 248 MODERN MICEOSCOPY and ramphigera, Dinocharis tetractis, and others. Among the Infusoria, the Vorticella in particular seem to like the cold season, and a number of different species, and often large colonies can be found attached to submerged rootlets of trees growing near the edge of the water. Attached to the fine stems of Carchesium, Zoothamnium, and other stalked colonies of Vorticella, the very much more minute but beautiful colonies of Collared Monads, Codosiga, etc,, are often found, and deserve careful examination with the higher powers. In canals and lakes where Gristatella has been abundant during the summer, their spiny stadoblasts may now be found liberated and often in large masses floating near the edge of the water which lies opposite to the. direction of the prevailing wind. These should be collected and placed in a jar full of water with some Anacharis in a warm room at home, where they will hatch by the end of December or January, and the beautiful young Polyzoa can be seen emerging from their box-shaped prison. December. Severe weather in this country does not, as a rule, set in in December, and the lakes and ponds are not usually frozen over in the early part of the month. The winter fauna has now become more pronounced, but includes quite a number of Inf usorians, Eotifers, and Crustaceans. The following species of rotifers have been collected in December in lakes and canals in and round London, some of them in great abundance : Anurcea aculeata and cochlearis, Asplanchna Bnghtwellii and priodonta, Brachioims angularis, Diaschiza semiaperta, Euchlanis cleflexa, Melicerta ringens, CEcistes crystallinus, Limriias ceratophylli, Floscularia corniita, Synchceta pectinata and treimda, Conochilm wiiicornis, Rotifer vulgaris and macrurtis, Polyarthra platyptera, Notholca scapha, Triarthralongiseta. Of Crustaceans, Diaptomns castor and various Cyclops and their larvae are abundant, whilst Water-fleas- die down. A minute red flagellate Infusorian often seems to form the chief food material of the above lake fauna. PLATE VIII. BRITISH HYDEACARINA. v-^' 'L^t; Fig. 115. — Arkhenurus bruzelii. Fig. 116. — Auhhenurus bruzblii. (KoEN.) Dorsal Sukface (J. (Kobn.) Dorsal Surface $. Drawn from specimens found at Wroxham Broad. -n Fig. 117. — ATURt'.s tntermedius. Fig. 118. — Aturus intermedius. (Protz.) Dorsal Sukeace $. (Protz.) Dorsal Surface ?. Drawn from specimens taten by Mr. Hulbert, Wicklow, Ireland. \ / %^: k i Fig 119.— Thyas vigilans. (Pier.) Fig. 120.— Thyas vigilans. (Pier.) Dorsal Surface $. Ventral Surface ?. Drawn from a specimen taken on Sunningdale Common. CHAPTEB XX THE COLLECTION, EXAMINATION, AND PRESEEVA- TION OP MITES FOUND IN FRESH WATER By 0. D. SOAK, F.L.S., F.B.M.S. Anyone with a love for natural history wishing for a hobby for his spare time would find the study of fresh-water mites (Hydrachnidse) an extremely interesting one. For variety and beauty in colour, and for differences in form and structure, they are not to be surpassed by any other organisms found in fresh water. Wherever there is a pond, ditch, or stream, the collector is nearly sure of being rewarded for. his search by finding one or more species of these interesting creatures. They are easily caught, and can be seen with the naked eye ; they are, however, very seldom recognized without the aid of the microscope. They can be kept alive for a considerable period at home, and are easily preserved when killed. Life-History. — The life-history of these creatures is so imper- fectly known that there is wide scope for an observant naturalist. Although the life-histories of some species have been fairly investigated, the number of such is very limited compared with the species known, and the variety of species which have been recorded in Great Britain are behind the recorded collections of Germany and elsewhere. These creatures are caught in three distinct stages — the larval, the nymph, and the imago. In the larval stage they are very small, and only have six legs. When they first emerge from the egg they are free-swimming, but they soon become attached as parasites to some other form of pond-iife. They will often be found hanging like small red pear-shaped appendages on a great 49 250 MODERN MICROSCOPY number of aquatic insects. In many cases the six legs they started life with disappear after they have become firmly attached by their mouth-organs to their host, and they spend the remainder of this period of their existence without any. This stage is succeeded by the nymph ; the little creatures are then much larger and have eight legs. During this term of their existence they are free-swimming, and can be caught in the net in numbers, but it is impossible to distinguish the sexes. In the, last stage — the adult or imago — all the structure and form are present, but many may be taken that are not fully developed. In the majority of species, the male can be dis- tinguished from the female and the specific difi'erences recog- nized ; but there are some in which the sexes are so much aHke that it is almost impossible to tell one from the other. In others, again, the sexes are so different — as, for instance, in the Arrhenuri — that one would be disinclined to think they could be of the same species. The three figures (Plate IX.) are intended to convey to the be- ginner the three stages. Pig. 122 is the larva of Pio7ia longi- palpis. Fig. 121 the nymph and adult of Hydrachna glohosa (Geerj, showing the ventral surface and the epimeral plates to which the eight legs are attached. Fig. 123 is the larva of an Hydrachnid, parasitic on Dytiscus viarginalis and Nepa cinerea. There is another point in the adult stage to which it will be well to draw attention. When the mite has first made its appearance from the inert period it spends between the nymph and adult stage, the hard and chitinous parts appear to be nearly fully developed, but the soft parts are not so. The body often appears very small, while the palpi, legs, and epimera, etc., are very large in proportion ; it is also very poor in colour. It would be well to ascertain that the mites are quite developed before making drawings and taking measurements. In my ignorance, when I first began the study of water-mites, I had to discard a number of drawings I had made of different speci- mens because they afterwards proved to be only different stages of growth of the same species of mite. Collecting. — For collecting there is no better apparatus than the collecting-stick used by pond-hunters, having a metal ring attachable at its end which carries a cone-shaped net made PLATE IX. m ^^^ r~ ti .J— % u n O o z ■< 1-3 2 S-S" 4lO I I ■~.s so ^ ?!<5 1) S so iiss •rH tH 4J ro fa H O Ph Q 1-3 H "A rt O W a ° d g S P^-^- 60.. ■'^■^ .9 be. t> =: J5 - ^ o s J2 ^T! 5- " H S S-*:" *^ CO