FROM THE LIBRARY OF WILLIAM A. SETCHELL,i864-i943 PROFESSOR OF BOTANY PLATE I. '*:: TEST OBJECTS PLATE II, TEST OBJECTS n A GUIDE FOR THE MICROSCOPICAL INVESTIGATION OF VEGETABLE SUBSTANCES FROM THE GERMAN OF DR. JULIUS WILHELM BEHRENS \\ TRANSLATED AND EDITED BY REV. A. B. HERVEY, A.M. ASSISTED BY R. H. WARD, M.D., F.R.M.S. ILLUSTRATED WITH THIRTEEN PLATES AND ONE HUNDRED AND FIFTY-THREE CUTS Bo0ton S. E. CASSINO AND COMPANY 1885 COPYRIGHT 1885 BY S. E. CASS1NO & CO. BIOLOGY LIBRARY - K B i AUTHOR'S PREFACE. THE preparation of this work has engaged my time for sev- eral years. In the beginning of 1880, I finally concluded to work up for publication the material relating to the microscop- ical investigation of vegetable substances, which I had previously collected for private use. Several friendly botanists, to whom I communicated my designs, counseled me very earnestly to carry them out. As the publishers also were prepared to un- dertake the work at once, the preparation of the manuscript and the printing of it have gone on simultaneously since about Easter, 1880. For a work to be useful in those microscopical inquiries which are most important in the botanical laboratory, it need teach neither optics nor histology. The student will, therefore, find in the work before him a brief description only, of the microscopical apparatus applicable to his uses (Chapters I and II), together with directions for its use. If he wishes to become acquainted with the instru- ment from the standpoint of the optical physicist he must go to the larger manuals of Hartig, of Nageli and Schwendener, or the shortly to be published hand-book of Dippel and Abbe. These are works which, if studied with the necessary care, will furnish a very perfect understanding of the performance of the microscopical apparatus, but which on account of their lengthy theoretical analyses are but poorly adapted for the table of the practical microscopist. The first and second chapters treat of the microscope and its accessory apparatus, while the third contains directions for the preparation of microscopic specimens. Every one knows that the preparation of specimens cannot be learned by the mere iv AUTHOR'S PREFACE. reading of these detailed statements. In this matter manual instruction is the main thing. However, the study of this chap- ter will open in many places new points of view to the youn^ .microscopist, and give him occasion here and there independ- ently to apply new methods. I have to thank my friend, Dr. Conwentz, for kindly under- taking the preparation of the section relating to fossil plants. The most important part of the whole work is in the fourth and fifth chapters. They contain what has heretofore incor- rectly been called micro-chemistry. The fourth chapter treats of microscopical reagents and the fifth of the microscop- ical investigation of vegetable substances. Until the middle of 1880, there was no useful compilation of the matters pertaining to this subject which was at all abreast with the science of the day in existence. But in the meantime .the "Botanical Micro-chemistry" of Poulsen, a brief compilation of the methods of micro-chemical reactions, has appeared. I believe, however, that notwithstanding this little book has justly had a wide circulation, the value of the corresponding chapters of the present work will not be materially lessened. Poulsen's work is designed mainly for beginners and there- fore contains only the most important methods of reaction in the barest outline. On the other hand, the chapter of this work which deals with the microscopical investigation of vege- table substances, furnishes an exhaustive treatment of these matters, and at the same time is so arranged as to make the specialist quite independent of the widely dispersed literature of the subject which is often hidden and not seldom difficult to obtain. But at the same time, a compilation of the literature, as complete as possible, greatly facilitates reference to the orig- inal works. It is evident that the point of view thus briefly outlined must require a handling of the case fundamentally differ- ent from that which Poulsen has given it. I have kept the chemical (i. e., the physio logico-chemical) point of view in the foreground throughout, not only in the arrangement of the whole, but also in the management of each separate section. The arrangement of the subject matter follows closely that of the AUTHOR'S PREFACE. v new edition of Husemann and Hilger's "Vegetable Substances," ("Pflanzenstoflfe") which I regret to say is not vet completed. Thus the use of that work in connection with my compilation is made more convenient. I am firmly convinced that the mi- cro-chemist will have many interesting outlooks opened to him, and many new methods suggested by the study of Husemann and Hilger's work. It seems to me that the separate micro- scopical investigation of vegetable substances is the only way (leading out from their chemical qualities) to attain a true comprehension of the methods of microscopical research. The discerning reader will soon discover that the whole chap- ter is by no means a mere compilation, but that I have critically sifted the existing materials. The useless I have rejected. The useful, however, I have taken not altogether on trust and faith, but as far as possible carefully tested. Indeed, I have tested everything it was possible to in the nature of things, and this experimentation has already consumed more than three years of working time. For the presentation of the whole I have chosen as brevity also seemed to require a purely objective form. Subjective views are kept entirely in the background, and at no time have I entered into argument. Various new discoveries, the results of my experimentations, will be published later in a separate monograph. In the compilation of the literature I have attempted the ut- most possible completeness. For my success in this, I am mainly indebted to the University library, which lacks scarcely a single treatise of all the literature quoted. With hardly a noteworthy exception, I have seen and read it all. But a small portion of the illustrations for this work are cop- ies. Much the greater part are original drawings which I have for the most part made upon wood myself. So far as this re- mark applies to the microscopical apparatus, they have been photographed under my directions with the use of a very small diaphragm ; they appeared almost black, but for engraving, I finished the pictures, which bad been photographically trans- ferred to the wood, in lines with sepia or India ink and white. vi AUTHOR'S PREFACE. Since the printing went on continuously with the elaboration of the manuscript, I could not in some sections refer to the very latest literature, as, for example, in Cellulose, Strasburger's beautiful work on the structure and growth of the cell wall could not be cited, likewise some recent monographs concern- ing the structure of the nucleus. But as far as it was possible I have cited the very latest literature. I shall be much obliged to those gentlemen who may give the fifth chapter critical attention if they will communicate to me any accidental omission or error. W. BEHRENS. GiMingen, Dec. 18, 1882. TRANSLATOR'S PREFACE. IN presenting to the English-speaking public a translation"^ Dr. Behrens' invaluable work, a few explanatory words seem to be needed. It is the first purpose of this work to guide students in all those inquiries relating to the physical products of cell-life in plants, which may be conducted under the microscope, by means of chemical and other reactions. It undertakes so to in- struct him that he can make a thin section of any part or organ of a plant, and, putting it under his lens, answer to himself the questions : What have the life processes thus far produced here, and what are they now producing? It deals with the anatomical constitution of the cell, and of plant tissue, and yet, its inquiries relate far more to physiolog- ical and biological processes and results than to matters purely anatomical and histologies!. The unit of life is the cell. The physical embodiment of that life is the protoplasm, the physio- logical center of which is the nucleus. The formed matter, the finished product of life, is the cell wall and some cell contents, and in the tissue the middle lamella also. The life of different cells and its embodiment are absolutely indistinguishable. The protoplasm and nucleus of, for example, wood-forming cells and cork-forming cells are microscopically and chemically alike. Dif- ferentiation appears only in the finished product of the life pro- cess, viz. : in some cell-contents, and mainly in the cell wall. It is to the nature of this, therefore, largely, in all the various kinds of vegetable tissue that our inquiries relate. But not alone to this : for a full investigation of nearty all the other elements of plant life is carefully marked out, of functional protoplasm and reserve protoplasm or proteid matter, of starch, chlorophyll, sugar, etc., elements so largely concerned in the life processes. The treatise occupies a field almost entirely to itself in the bo- tanical literature both of Germany, and now of the English- (vii) viii TRANSLATOR'S PREFACE. speaking world. It is sincerely hoped that its publication in this form will stimulate in this country investigations into the deeper problems of plant life. It will be seen by reference to the literature cited, that there is an open field for American bot- anists, since the works referred to almost exclusively embody the results of German research, while a few are of French origin, fewer still of English and none whatever of American. I alone am responsible for the translation, and have endeav- ored to take a medium course in it, following the text neither too literally, nor yet translating so freely as to introduce shades of meaning not in the author's mind. 1 am inclined to believe my errors are those more often leaning to the former than to the latter side. In that direction, I suppose, lie the tempta- tions for the conscientious translator, though in the interests of a good style he would be more readily pardoned for sinning on the other side. Early in the enterprise, Dr. K. H. Ward very kindly con- sented to undertake the revision of the two chapters which deal with the microscope and its accessories. He states the plan upon which he has made the somewhat extensive changes in these chapters as follows : " The changes in Chapters I and II consist wholly in the omis- sion of illustrations and descriptions of apparatus in the Conti- nental style, which is comparatively unused and unavailable here, and the substitution of American forms. It was desired, and deemed necessary, that a work intended as a practical hand- book should describe and discuss such instruments as are likely to be most generally preferred and used by the majority of its readers. The author's full and very valuable discussions on the methods of work, and on the construction, testing, care and use of the optical parts, and of the most important accessories, are retained without abridgment or material amendment. "The construction of the Stand is illustrated by several models, in order to exhibit the most common varieties believed to be eligible for such work, and (incidentally) to establish, by com- parison of characteristic stands by prominent American makers, the claim to the existence of a new, serviceable and American type in addition to the two styles, Continental and English, TRANSLATOR'S PREFACE. ix heretofore recognized. It will be noticed that the American type, as illustrated by Plates III to VI, Villa, and IX to XI, is intermediate in size and complexity between the other two, but built upon a radically different model from either ; having much of the simplicity and portability of the Continental with much of the efficiency and versatility of the English style." The changes in the third, fourth and fifth chapters, for which I alone am responsible, consist almost entirely of additions to the text. They relate in the third chapter exclusively to those methods, tools, materials, etc., which experience has taught American investigators to consider important in their work. The few additions to the remaining chapters relate only to those researches in this field, which, up to the beginning of 1884, and subsequent to the close of the author's work, had come under my notice. The matter introduced into the text by the American editors is inclosed in brackets [ ] and usually signed with the respec- tive editors' initials. Foot-notes by either of the editors are likewise mostly signed, and are referred to in the text by sym- bols, while those of the author are all numbered. We are indebted to various firms of opticians, as well as to other parties, for the use of electrotypes, for which we here desire to express our cordial thanks. The work of translating and editing this treatise has been done in the midst of the engagements of a busy professional life, in hours snatched at irregular intervals from the demands of pressing public and domestic cares ; yet I have verified by actual experiment the larger part of all the statements and methods given by the author. A. B. HEKVEY. Taunton, Mass., Feb. 26, 1885. TABLE OF CONTENTS. CHAPTER I. THE MICROSCOPE. I. Introduction, . II. The Compound Microscope, . 14 I. The Microscope Stand, ..... . 15 A. The Student Microscope, .... . 16 B. The Model Microscope, ..... 17 C. The Acme Microscope, ..... 18 D. The New Student Stand, .... . 18 E. The Physicians' Stand, . 19 F. The Continental Student Stand, . 19 G. The Illustrator's Stand, .... . 20 H. The Histological Stand, .... . 20 I. The Biological Stand, . . . 21 J. The Universal Stand, ..... . 21 II. The Objective, . . 22 A. The set of Achromatic Lenses, . 23 B. The Objective System, . 23 The System in Practice, .... . 28 The Immersion-System, .... . 30 The Correction-System, .... . 32 III. The Ocular, 35 IV. 38 V. The Eye Shade, . 42 VI. Magnifying power of the Modern Microscope, . 43 VII. Testing the Optical Powers, .... . 53 A. Testing the Definition, . 54 B. Testing the Resolution, .... . 57 VIII. The Microscope-Tube, ..... . 71 IX. Nose-Pieces, ....... . 74 X. Fine Adjustment, ....... . 77 XL The Stage, . 80 XII. The Illuminating Apparatus, . 82 82 B. Diaphragms, . . . 84 C. Condensers, ....... 87 (xi) xii CONTENTS. D. Illuminating Combinations, ..... 90 E. Opaque Illuminators, ...... 93 F. Observation by Artificial Illumination, ... 94 XIII. The Microscope Foot, 95 XIV. Rules for the Use of the Microscope, . . .96 CHAPTER II. MICROSCOPICAL ACCESSORIES. I. The Preparing Microscope, 100 II. Apparatus for drawing Microscopic Pictures, . . .110 III. The Micrometer and Microscopical Measuring, . .120 1. Objective Micrometers, 121 2. Ocular Micrometer, . . . . . .122 3. Ocular Screw Micrometer, 126 IV. Camera Lucida as a Measuring Apparatus, . . . 127 V. Microscopic Measuring in general, . . . .128 VI. Polarizing Apparatus and Goniometer, . . .133 The Goniometer, . 137 VII. The Micro-Spectroscope, 139 CHAPTER III. PREPARATION OF MICROSCOPIC OBJECTS. I. Introduction, . . . . . . . .156 II. Preparation of Objects without Cutting Instruments, . 160 A. Objects for immediate Observation, . . . 1 00 B. Macerating or Softening, 162 C. Incinerating and Calcining, 164 III. Instruments for preparing Microscopic thin Sections, . 1 65 IV. Cutting Microscopical Sections, . . . . .177 1. Free hand cutting, 179 2. Cutting by means of Elder-pith and Cork, . . 183 3. Cutting in embedding Media, 185 4. Cutting sections with a Microtome, . . . 187 V. Further treatment of the section, . . . .196 A. Removing the Air, . . . . . . .196 B. Handling the Section under the preparing Microscope, 197 C. Clarification of the section, . . . . 198 VI. Preparation of Microscopic specimens of Fossil Plants, . 203 CONTENTS. xiii VII. Preparation of permanent Mounts, .... 213 1. Object-slide and Cover-glass, 214 2. Preserving Media,* 217 VIII. Mounting in Preserving Fluids, ..... 228 IX. Cementing and Finishing the Mount, .... 234 1. Cements, ......... 234 2. Cementing angular Cover-glasses, .... 237 3. Mounting with circular Cover-glasses, . . . 239 Self-centering Turn-tables, ...... 239 X. Labelling and Cataloguing Preparations, . . . 245 XI. Storing permanent Preparations, 247 XII. Examination of living Organisms, .... 249 XIII. Drawing Microscopic Objects, .... 254 1. Aids to Microscopical Drawing, .... 254 2. Conducting Microscopical Drawing, . . . 259 3. Drawing Materials, ....... 265 CHAPTER IV. MICROSCOPICAL REAGENTS. I. Introduction, 267 II. Apparatus for the preparation of Reagents, . . .272 III. The Volumetric Method, 278 IV. Enumeration and preparation of Microscopical Reagents 283 A. Inorganic Combinations, ..... 283 1. Water, 283 2. Nitric Acid, 284 3. Sulphuric Acid, 284 4. Hydrochloric Acid, 284 5. Phosphoric Acid, '. 285 6. Solutions of Iodine, 285 7. Potassium Hydroxide, 288 8. Potassium Chlorate, 291 9. Potassium Nitrate, 291 10. Potassium Bichromate, 291 11. Sodium Chloride, 291 12. Ammonia, . . . . . . . .291 13. Ferric Chloride, 292 14. Chromic Acid, 292 15. Copper Sulphate, 293 16. Cuprammonia, 293 xiv CONTENTS. 17. Mercuric Chloride, . 295 18. Millon's Reagent, . 296 296 B. Organic Combinations, . . 297 20. Alcohol, . 297 21. Ether, . . . 297 22. Acetic Acid, . . . . . . 297 23. Cupric Acetate, . 298 24. Sodium Nitro-Prussiate, ..... . 298 25. Potassium Ferrocyanide, .... . 298 26. Oxalic Acid, . 298 27. Asparagin, ....... 298 28. Cane Sugar, ....... . 299 29. Aniline Coloring Matter, .... . 299 30. Aniline Sulphate, . 301 81. Phenol, . . . 302 32. Phloroglucin, . 302 33. Indol, . 303 34. Eosin, . . 304 35. Hsematoxylin, ..... 304 36. Cochineal Extract, . 305 37. Carmine Solutions, . 306 38. Picro-carminate of Ammonia, . 309 39. Alcanna Tincture, . 310 CHAPTER V. MICROSCOPICAL INVESTIGATION OF VEGETABLE SUBSTANCES. A. Substances of Universal Distribution, . . . 315 I. Cellulose and its Modifications, 315 1. Cellulose in the narrow sense, 318 2. Mucilaginous Cellulose, 327 3. Wood Substance, 330 4. Middle Lamella, 343 5. Corky Cellulose, 347 6. Fungus Cellulose, 353 II. Starch, 357 III. Dextrine, 365 IV. Vegetable Mucilage, 367 V. Gum, .... 371 CONTENTS. xv VI. Inulin, 375 VII. Grape Sugar, . . . 377 VIII. Cane Sugar, 378 IX. Albuminous Matter, 379 1. Reserve Proteid Matter, ...... 380 2. Functional Proteid Matter, 392 X. Chlorophyll, . . 403 XI. Coloring Matter of Flowers, 421 XII. Asparagin, 425 XIII. Inorganic Vegetable Elements, .... 428 B. Plant Substances of Limited Distribution, . .431 XIV. Glyeoside, 432 XV. Tannin, . , 435 XVI. Alkaloids, 438 XVII. Fats, 439 XVIII. Essential Oils, 441 XIX. Stearoptene, 441 XX. Resin, Balsam, Turpentine, ..... 442 XXI. Coloring Matter of Flowering Plants, . . .447 XXII. Coloring Matter of Cryptogamic plants, . . 448 CHAPTER I. THE MICKOSCOPE I. INTRODUCTION. THE microscope, as an instrument of observation, holds at the present time the most prominent position in the department of scientific botany, especially in vegetable anatomy and to some extent also in physiology. Vegetable anatomy treats entirely of the investigation of the constituent elements of vegetable organisms, and the minuteness of these parts makes the use of the instrument in question almost always neces- sary The cultivation of vegetable anatomy has been so far dependent upon the development of the microscope that those periods in which most improvements have been made in it correspond in many cases to those in* which the principal de- velopment of this science has occurred. On the other hand also, the wider development of phytotomy has not been without its influence upon the improvement of certain parts of the mi- croscope. The name of the apparatus as well as its invention belongs to the later middle ages, somewhere in the fifteenth or six- teenth century. The instrument was called the microscope (;j.>.7.o<>s, small, and ffxo-sw, to look upon, to observe by means of the sight) because it allowed very small objects, grains of sand, insects and the like to be seen magnified. One must not think, however, that those first very timid attempts to construct magnifying glasses can in any way be compared with the instruments fabricated in recent times. The first of these productions were the "flea-glasses," Vitra pulicaria and THE MICROSCOPE IN BOTANY. muscaria, which served the learned men of that time more as a "highly curious and frightful microscopical amusement for eyes and mind " than for scientific observations. They consisted of the kind which we see to-day in our toy shops, and were made of a single glass lens which was the segment of a sphere of small diameter. This lens was fastened into a wooden tube which bore at its lower end in the focus of the lens a small glass plate on which a crushed flea, a gnat, a fly's leg, or a like object was pasted. The "flea-glass'' belonged to the indispensable requisites of a learned man of that time. It magnified about six to ten times, equivalent to an average magnifying glass of to- day. It makes the most comical impression on one to-day to read the descriptions which these Faustian savants give of their observations with the "flea-glasses." Many, amazed, de- scribed real monsters which they thus observed, while the less expert believed they saw the Devil himself in the innocent instrument. Leeuwenhoek (1632-1723) one of the first who really set about scientific observations with the microscope, a man who, by his discovery of the infusoria, is known in the widest circles, used a simple microscope exclusively in his observations, but which, measured by the ideas of his time, possessed very strong magnifying powers. He understood how to grind very perfect little lenses with short focal distance, which he fastened between two right-angled plates of silver or brass (from 4 to 5cm. long, and about 3cm. broad) screwed upon each other in such a way that one could look down through two corresponding holes in the plates and through the lens lying between. Behind this mounted lens was a small pointed instrument capable of move- ment in all directions, on the end of which the object to be observed was impaled. The whole instrument w r as then held towards the light and the lens brought as near to the eye as possible. Leeuwenhoek's microscopes magnified from 40 to 100 times, a few 150, and one even 270 times. But already before the time of Leeuwenhoek, that is, still be- fore the close of the sixteenth century, or perhaps in the be- ginning of the seventeenth century, the compound microscope had been invented. It is very essentially distinguished from INTRODUCTION. 3 the one already mentioned by this : that a picture is produced by the lens nearest the object, the so-called "objective," and that this image is viewed through a lens placed near the eye, the " ocular," relatively magnified. This principle is fundamental in all microscopes, in those also of the present time, though they may be constructed never so differently from those of the first inventor. Who should be regarded as the inventor of the first com- pound microscope was for a long time a doubtful and frequently disputed question. According to the literary studies of Hart- ing, 1 it appears pretty nearly beyond doubt that this merit should be ascribed to two spectacle makers in Middelburg in Holland, Hans & Zacharias Janssen, father and son. Pre- viously Fontana, Galileo Galilei, and the Netherlander Drebbel also had been regarded as the first inventors of our instrument. In a work by Borel, a Frenchman, is a letter from a countryman and friend of the younger Janssen who among other things describes the first microscope in the following way. 2 "It pos- sessed not, as such an instrument would now, a short tube, but one almost a foot and a half long. The tube itself was of gilded brass and was fixed in the middle at a height of two fingers on three bronze-crested dolphins. The foot consisted of a disk of ebony that carried various small instruments and minute objects which we viewed from above in almost miraculously magnified form." Although with the invention of the compound microscope in general, there had been worked out the proper arrangement which should be given to the apparatus, still the construction of magnifying glasses during the century succeeding the in- vention was so faulty that it was little or not at all fitted for making those observations which have been attained in recent O times. During that period the object under investigation was viewed with reflected light only, it being concentrated upon the object from the lamp which was the source of light, by means of a 1 Harting. Das Microskope Braunschweig, 1859, pp. 586-596. 2 The Latin text is printed in Halting, 1. c., p. 589. The work of Borel bears the title " De vero telescopii inventore, cum brevi omnium conspiciliorum historia. Accedit etiam centuria observationum microscopicai-uin. Hag. Comitum, 1655. 4 THE MICROSCOPE IN BOTANF. globe filled with water, or by a condensing lens. Such an in- strument adapted only for superficial illumination is illustrated, for example, by Robert Hooke, 3 who also states that a thin section through a flask cork, on a dark background, looks like a honey comb, in which could be distinguished open spaces (pores) and separating walls. 4 The illuminating reflector (mir- ror) which is indispensable to us, and which we place under the object, in order to view it with transmitted light was first intro- duced for the use of the compound microscope about the year 1735, by Culpeper and Scarlet, 5 and a few years later (1740) by Wilson, 6 for the simple microscope. Thus an important step forward was made. But at that time, the construction and combination of lenses for the compound microscope were very faulty. One did not then as now employ an ocular, made of two glasses, and an objective of several lenses, but each consisted at that time of but one glass. The consequence was that the microscopic image often appeared very much bent, because only its middle por- tions were clearly shown, while the edges were distorted be- yond recognition. This explains what Wolf 7 expressly declared, in the year 1723, that at that time the simple microscope was much more in use than the compound (see above, Leeuwen- hoek) and that one would much rather use the former, es- pecially in high magnifications, than the latter. From the following, it becomes clear how these microscopes gradually gave place to those with flat fields of view. The in- struments of Drebbel, Galilei and probably also the one of Janssen, above described, possessed, as remarked, two convex lenses, the one serving as ocular, and the other as objective. Fontana inserted in his microscope an intermediate concave glass between the other two lenses. Hook did the same, but princi- pally with a view to enlarge the field of vision, and he let it remain when he saw the image clearly and distinctly. A fur- ther step was made towards this improvement by Divini, about 1670, who first united two plano-convex lenses in one ocular, 3 R. Hooke, Micrographia, or some physiological descriptions of minute bodies made by magnifying glasses. London. 1667. 4 J. Sachs, Geschichte der Botanik. p. 247. 5 Harting, 1. c., p. 672. e Hurting, I. c., p. 615. 1 Sachs, 1. c., p. 267. See also, Hurting, I. c., p. 688. INTRODUCTION. 5 as it is done to-day. Soon after that, doublets also, which had already been in use for some time as simple microscopes, began to be employed as objectives. At last, toward the end of the seventeenth century, there came in combinations of lenses as objectives. They were either biconvex or plano-convex 8 lenses of unlike foci, permitting therefore by different combina- tions the production of lower or higher magnifications, while in the oldest compound microscopes the different magnifications had been brought about in this way, the tube was constructed like that of a telescope of three or four parts which slid into each other, and the magnifying power was increased by drawing these out. But the power of the microscope was so small that a scholar of that time praised a microscope which magnified eighty diam- eters, as being "certainly quite gigantic in its magnification" (quod certe insigne augmentum est). AVhile we have seen that in the fabrication of the microscope it has gradually come about that the spherical aberration has in great part been overcome by means of suitable forms and combinations of lenses, it was not until the concluding third of the last century that another great defect of the instrument was removed, namely, the chromatic aberration. Xewton had indeed already, towards the end of the seventeenth century, expressed the opinion that glasses might be constructed which should pro- duce a colorless image by a combination of two lenses, made of material having the greatest possible likeness of refractive power, and the greatest possible difference in color-dispersing power, although it was not possible for him to bring the matter to ex- perimental proof. 9 Dollond had indeed, in 1758, constructed the first achromatic telescope, and so had reduced that theoretical calculation to practice. Euler also, in 1771, had established a coherent theory of Achromatics, and Fuss in 1774, in accord- ance with the analyses of the latter, gave directions for con- 8 While it is naturally impossible to correct the spherical aberration by a combination of biconvex glasses, it is quite easily done by the use of plano-convex lenses, whose plane side is turned under and brought next to the object. Although toward the end of the last cen- tury plano-convex object-lenses were generally used, they were constantly so placed that the convex side was turned toward the object. Chevalier and Amici first used them re- versed, and so reduced the spherical aberration to a minimum. 9 Through inexact observation he was led into theerror of supposingthat materials, with nearly the same refractive index and very different color-dispersing power, did not exist. THE MICROSCOPE IN BOTANY. structing an achromatic microscope. But it was held to be impossible to construct achromatic glasses of the small size required in microscopic objective lenses, and it was believed therefore that a microscope could never be made which would give at the same time in some measure high magnification and a clear sharp image. According to Hailing, 10 it was again a Hollander, van Deyl, (1807), who first constructed a really achromatic microscope, forming his objective out of two biconvex crown-glass lenses, and a biconcave flint-glass lens lying between. His instrument, however, for reasons stated in note 8, p. 5, possessed a very considerable spherical aberration. A short time after that, Brewster made the attempt to substi- tute a fluid for the flint-glass middle lens (Newton had attempted to do the same thing already). It is known under the name of Brewster's achromatic globe. It is a glass globe filled with water in which two biconvex lenses are made to lie pole to pole in the optical axis. The contrivance has however never come into use. After achromatic objective lenses for microscopes had been prepared by Fraunhofer, the most gifted optician of all time, there appeared in France, Chevalier (about 1824), and in Italy, Arnici (1827 and later), who constructed objectives such as to-day are generally in use, under the designation of "apla- natic." ] In them the spherical, as well as the chromatic aberration, was so far suppressed that they no longer essentially hindered microscopical observation. We shall become more intimately acquainted with the aplanatic lens in the subsequent portions of this chapter. The names which in more recent times have had most signif- icance in microscope-making, and with which, together with their contributions we shall become more intimately acquainted, in the proper place hereafter, are principally, Hugo von Mohl, Ober- hauser, Hartnack, Nachet, Merz, Plossl, Beneche, Wasserlein, Pritchard, Ross, Zeiss, Seibert, Krafft, Winkel, Schieck, Leitz, Powell and others.* 10 Hartins, L c., p 691. 11 From d privative and n-Aacaw, to deceive, to lead astray. * Certainly the names of our two greatest American opticians, Spencer and Tolles, should not be omitted from this list. A. B. H. INTRODUCTION. 7 In our historical review, we have thus far drawn attention only to the optical part of the microscope, while the bearer of these parts, the stand, has been left out of sight. In the period directly after the invention of the instrument, very little attention was devoted to the microscope stand. It commonly consisted of an elaborately turned piece of wood, while the cylindrical draw-tube was often made from pasteboard. The first important improvement which the mounted compound microscope received was the introduction of the illuminating mirror by Culpeper and Scarlet, as already noticed above. The inicroscopist now began to work with transmitted light. The ob- ject for observation was placed upon a perforated plate, fche stage, which occupied a position between the mirror and the objective. In order to focus the object, that is to say, to bring it ex- actly in the focal point of the object-glass, we may proceed in either of two ways, viz., the object table or stage may be made fast, and the adjustment produced by moving the tube which carries the optical apparatus towards it, or on the other hand the tube may be fixed and a vertical movement may be given to the stage. In the first compound microscopes with illuminating mirrors, from the factory of Culpeper and Scarlet, the stage was fixed, and the focussing was done by shoving the tube by hand. But already, about the middle of the eighteenth century, Cuff had employed the setting screw to accomplish a more exact focussing. In his microscopes the tube was fastened to a metal hinge which could move up and down on a perpendicular metal rod. By this manipulation, the approximate, or so-called , coarse adjustment was accomplished, while by a clamping screw, the hinge was made fast, and a very small shortening or lengthening of the hinge itself was accomplished by a second screw (the so- called micrometer screw) thereby making the fine adjustment. In later microscopes (of Martin, Jones, van Deyl, etc.) the tube was moved by a simple rack and pinion. In the instruments of Chevalier the opposite plan was adopted, the stage was moved by means of a hinge, -*vith a clamp screw on a vertical prismatic metal rod. The fine adjustment was produced, as in the instruments of Jones, by means of a care- fully cut, small thread, fine-adjustment screw. THE MICROSCOPE IN BOTANY. Afterwards these last contrivances were generally abandoned ; only the microscopes of Amici now have them, and in the oldest stands of Plossl both the tube and the stage are movable on the same vertical rod. In the instruments of the present time (or at least on the larger and medium stands), without exception, the adjust- ment is produced by moving the tube vertically in a direction perpendicular to the stage, which is made fast to the rod. The coarse adjustment is produced directly by free hand or by means of a rack and pinion. The fine adjustment is made by a fine screw. While .the stands of the first microscopes, which were made throughout of polished or unpolished wood, could satisfy but very modest demands as to their outward appearance, stands made of brass came into very general use at the end of the last century. There came a time when the outside appearance of the stand received by far the most attention, while the optical part was very little improved. There was brought into use with the stand also, at that time, every possible useless acces- sory (the so-called microscopical accessory apparatus) so that the observer was often more hindered than helped by it in his work. 12 It was the famous phytotomist Hugo von Mohl who first ex- pressly demanded the simplest constructed microscopes, and rebuked with sharp words the coquettish toying with stands which was becoming the fashion. He speaks about it, 13 for ex- ample, as follows. "The simpler the construction of the micro- scope is, the more easily and more quickly will one accomplish all the necessary movements. The more complicated the con- struction the more will they cost in time and reflection, and the more will the attention be distracted thereby during the observa- tion. Whoever has not the manual dexterity to work with a sim- ply constructed microscope, and finds it necessary to use a screw 12 The author has recently had :m opportunity to take a look at snch an English instru- ment in the. physical collection of the technical high school in Brunswick, of which one literally cannot get at the stage on account of screws, magnif3 - ing glasses and other things, and which one can recognize as a microscope geneially only after the most exact consideration. 13 H. v. Mohl, Mikrographie, Tubingen, 1846, p. 89. INTRODUCTION. instead of his fingers for every movement, is on that account disqualified for a microscopical observer, for he will labor in vain to prepare a usable specimen." After this short historical survey of the invention of the mi- croscope, we will now turn to a consideration of the instrument itself. Since we shall assume that those general laws of optics concerning the refraction of rays of light, which may be found stated in every text-book are already known, it will be our aim in the following treatise to give a representation of the microscope without admitting special theoretical deductions. Afterwards the reader will be made acquainted with the methods of prepar- ing botanical microscopic specimens, and at last there will be shown to him how their methodical investigation should be conducted. But it will not perhaps be useless to insert here first of all the following general remarks. From the start it must be clearly understood that the micro- scope is not an instrument to which one only needs to turn and look in, in order to behold some great discovery. The micro- scope is, on the contrary, an instrument whose use and manage- ment must be learned, but which then, if it be used with understanding and with regard for the most careful precautions, permits things to be seen which would be forever shut out from the unaided eye. Under the microscope, however, we see always only a very small part of a natural body, and what is more important, we see that only in two dimensions, namely, in length and breadth. We can never at the same time perceive its thickness. We must therefore, in order to come to a clear conception of the microscopical structure of organs having a corporeal appearance, to the naked eye, contemplate different sections through it, made in the direction of the three dimensions of space, and then combine these by means of our mental eye. Thus there is re- quired for seeing and understanding the microscopic image, not only activity of sense but activity of mind, also. I quote here, ill reference to this, the declaration of one of the most highly 10 THE MICROSCOPE IN BOTANY. accomplished of living observers, Julius Sachs, as he writes in the History of Botany : M " Seeing is an art which must be learned and cultivated, a definite purpose must stimulate the will of the observer, to will to see exactly and rightly, to distin- guish and combine what is seen." .... "By the invention of the microscope the eye became capable not merely of seeing small things larger, and in general of seeing the invisibly small, but much more ; there was combined with the use of magnifying glasses the one other advantage, viz. : that then first we learned in general to see exactly and scientifically. In that we armed the eye with a magnifying glass, the attention was concentrated on a single point of the object, the seeing was in part indistinct and always of but a small part of the whole object. The per- ception of the visual nerve must be accompanied with a purpose- ful and intense reflection, in order to make the object observed in fragments by the magnifying glass, clear, in its inner connec- tions, to the mental eye. So the eye, by being armed with the microscope, became itself a scientific instrument, which no longer ran over the objects with thoughtless movements, but received strict discipline from the understanding of the observer, and was kept to methodical work." "As in every other science, so in the investigation of the structure of plants, the sense perception must be worked over by the understanding, to distinguish the important from the unimportant, and to bring the single perceptions into logical coherence, to follow a purpose in the investigation, but this purpose can be none other in the last instance for the plant anatomist, than that the whole inner structure of the plant in its collective coherency shall be so clearly comprehended that it may at any time, in all its details, be perfectly reproduced in perfectly sensible definiteness from the imagination. To attain this is not easy, because the more powerfully the microscope magnifies the smaller the part of the whole which it shows. Skilful and superior preparations, care- ful combinations of different images, and long practice are necessary to attain that object. The history of vegetable anat- omy shows how difficult it has been for observers to gradually form a clear, coherent conception from these fragmentary views J' " Sachs, 1. c., p. 236, ff. INTRODUCTION. 11 But there is also, for those who have already attained some facility in the use of the microscope, one important aid which is indispensable to methodical observation. Every one knows, and has often been compelled to make the remark to himself, that our memory can be said to be trustworthy only to a limited extent, and that the subjective impression which the brain re- ceives is but imperfectly and often but temporarily fixed. It is especially necessary to resort to the help of the graphic art if one would preserve in the memory, with any exactness, a series of microscopical observations. This may be done in either of two ways. In the first place by keeping an exact record of the observations, and in the second place by endeavor- ing to fix the microscopic image on paper by means of pencil or brush, in other words by drawing it. The latter presupposes a real manual dexterity, a certain technique. But it will not be difficult for microscopists who are but little practised in draw- ing to acquire the necessary skill for this purpose. Besides this there is, as we shall show farther on, abundant and man- ifold apparatus, which allows the microscopic image to be thrown down by means of a reflecting prism upon a sheet of paper tying near the microscope, where it may be traced with the pencil without further trouble. But these contrivances are to be used, especially by the beginner, only with the greatest care. For the microscopical drawing should not be merely the spiritless crude copy of the image seen, but it should receive into itself all the experiments, all the studies which the observer has made upon the object ; in a word it should be idealized. In connection with the opinion just now expressed I will quote a restrictive remark of Sachs: "A microscopical drawing, like illustrations of objects of natural science generally, cannot quite lay claim to replace the object itself; all the more then should it repeat with all distinctness what the observer has perceived and in so far support the description in the text. The drawing will be all the more perfect, the more the eye is trained in ob- serving and the understanding in interpreting the forms. The illustrations should show to the reader nothing other than what has been traversed throughout by the mind of the observer, for only so can it serve to bring the two to a mutual understand- 12 THE MICROSCOPE IN BOTANY. ing. But the case has still another significance : even during the drawing of a microscopic object it is necessary for the eye to dwell on single lines and points, in order to comprehend their true dependence in respect to all the dimensions of space ; it often happens thence that relations are perceived, which, pre- viously, even in the most careful observation, were not noticed ; for, however the question being investigated is determined, new questions are opened. Thus as the eye is first, by the use of the microscope, trained to scientific seeing, so first by careful drawing of the object will the educated eye become a growing councillor of the investigating mind." 15 One already skilled in drawing, when he begins to make mi- croscopical observations and drawings, will at first produce quite imperfect pictorial representations ; but by the constant use and practice of the eye in microscopical seeing, the drawings will tend to ever greater perfection and instructiveness. Much has been written as to the personal characteristics which the microscopist should possess. We limit ourselves, therefore, under the supposition of making a theoretical presentation, to the four following factors required by him : a skilful hand, good eyes, a tranquil mind, and self knowledge. For the preparation of microscopical specimens, which pre- supposes the careful management of the most various tools, a certain skilfulness of hands is one of the first conditions. In relation to the eyes, the short-sightedness so common to scien- tifically educated people, is in no way a hindrance to microscop- ical observation. On the contrary, it is often very useful in the preparation of specimens, as a brilliant example may be quoted to show, in the case of one of the most accomplished micros- copists and vegetable anatomists, Wilhelm Hofmeister, who, in preparing objects, brings them very close before his imspecta- cled, Very shortsighted eyes, and in this way sees relatively very large, thus using the eyes in place of a mounting micros- cope. 16 Observe with the right eye, but one should avoid 16 Sachs, 1. c., p. 280. 16 Spectacle wearers do best when they remove the glasses during exact microscopical observation, and they should be used in preparing microscopical drawings, only by ex- tremely shortsighted people, when it cannot be done without them. In this manipulation there frequently results (at least to the shortsighted author) this discomfort : since the right INTRODUCTION. 13 pinching up the left eye meanwhile, because the muscles used in shutting it will soon thereby become sensibly affected. One should look at the same time with the left eye upon the table (the darkest possible, for example, a dark green colored one). The inclination to squinting, arising from this use of the eyes, may be easily prevented by giving the eyes timely rest after working with the microscope.* Respecting the mental condition of the observer it may be mentioned, that as it lies in the nature of the case only that temperament of mind can be serviceable to investiga- tion in which we find ourselves absolutely passionless. Says Harting, 17 " But for the exercise of the critical judgment, in microscopical observations, there is need not merely of the sim- ple purpose, but we must find ourselves in that condition of mind which makes it possible for us to see with unclouded vision, and to judge with unprejudiced understanding. As the principal requirement thereto I name mental tranquillity during the investigation. As easy as it might seem to satisfy this requirement, experience teaches that the opposite most often prevails. In microscopical investigations this is of great im- portance ; for these often occasion lively mental impressions which are incompatible with the desired mental rest during the observations." Of the four above named indispensable characteristics of the microscopist, self knowledge plays the chief role. The micros- copist must be a sceptic through and through ; he must take nothing for granted ; he must approach every object to be inves- tigated with a certain mistrust. He will much more easily be trained to be a good observer if he continually seek to detect himself in a false observation, than if he goes at his work with the self-satisfied consciousness that nothing could make him see spectacle glass so constantly strikes against the ocular, the part which lies upon the nose will be pressed upon the skin covering the bridge of the nose, in such a way as finally to produce a very troublesome pain, which may bring on headache. This discomfort may be avoided by soldering upon the bearing place of the spectacles a thin plate of gold some three millimeters wide, and 35 to 40 mm. long, which has previously been bent into the ex- act form of a nose saddle. 17 Harting, I. c., page 327. * The evils here mentioned are greatly mitigated by the use of the binocular microscope, or. with monocular instruments, by employing an eye shade such aa that shown in Fig. 17. B.H.W. 14 THE MICROSCOPE IN BOTANY. anything falsely or imperfectly. The microscopist must exer- cise self-criticism. As easy as it is to criticise others, it is very difficult for many men perhaps for all to lay upon themselves the strict, critical measuring rod, with which they are so ready to measure others. Egoism, plainly, rules the world. Yea, still more, not only is self-criticism necessary to the microscopist, but also love of truth for its own sake. He should not delude himself, by thinking that he has already seen this or that with exactness, or perfect correctness, when it has first darkly dawned upon his consciousness. He must con- stantly protect himself from accepting for positive fact, what is but probability or only possibility. How this last-named qual- ity of the microscopist, and of all who would become such, is to be attained is not easily put down in words on paper, in respect to that every one must go through his own self-discipli- nary school, but the inscription upon the Delphian temple, "Know thyself," should in spirit constantly hover before him. II. THE COMPOUND MICROSCOPE. The compound diopteric microscope 18 is an optical apparatus which, by means of a convex lens, produces a magnified image of an object, which again is viewed through a glass that still further enlarges it. The compound is distinguished from the simple microscope essentially by this fact, that by means of the latter we view the magnified object itself, while by the former we look only upon the enlarged image of the object. From this statement it must follow that the compound microscope must consist of at least two glasses, viz., of one which is brought near to the object to be magnified, and which produces the image (image producer, objective), and of a second which stands near to the observing eye, and through which the picture already produced is viewed (image viewer, ocular). The two glasses must naturally have such a mutual position in respect to each other that the image will Ml exactly in the focus of the ocular. In microscopes of the present day, however, neither "Compound catopteric microscope there is none the catadiopteric is temporarily ex- cluded from our consideration. PL. III. Zentmayer's American Student Stand. THE MICROSCOPE STAND. 15 the objective nor the ocular consists of only one lens, but the former is [most commonly] constructed of three plano-convex achromatic lenses, and the latter of two glasses of which the one ( the collecting lens ) is placed beneath the image and [sometimes] has a biconvex form, while the true ocular is above the image and is plano-convex with the plane side turned toward the eye. For the production of a good microscopic image it is required : #. That these lenses shall by skilful grinding be given that curvature which corresponds to the magnification as it has been previously determined by calculation. b. That the five lenses shall be exactly centered ; that is, that their foci shall lie in one straight line in the longer axis of the microscope. The above described optical apparatus must be mounted in a stand so constructed as to permit : a. Objective and ocular to be placed at a definite distance from each other which remains unchanged during the observation. b. The bringing of the object to be observed exactly in the focus of the objective. c. The furnishing a sufficiently large quantity of light for the object, to give its image the desired degree of brightness. I. THE MICROSCOPE STAND. [For the purpose of introducing the forms, modifications and nomenclature of the various parts of the compound microscope as now made in this country, a few stands will be figured and described ; the selection being made with a view to obtain (with the exception of plates VII and VIII) typical American forms, of moderate size and free from ostentatious display of unnec- essary mechanism, and especially those which have been instru- mental in bringing recent improvements into use.] [Stands of the class represented by plates III to VI, varying in style according to the skill, ingenuity, or caprice of their makers, can be obtained from all dealers at a cost of from $25 to 830, or with a minimum outfit of objectives and accessories at $40 to $50. Stands of the class represented by plates IX, 16 THE MICROSCOPE IN BOTANY. X and XI, should cost with a minimum outfit about $50 to $75 ; though they are capable, by a judicious increase of ex- penditure, according to the needs or means of the purchaser, of being developed into instruments of a far higher and costlier grade.] [A. THE STUDENT MICROSCOPE.] [As a sample of the smallest, simplest, and least expensive instruments really worthy of being commended as available for scientific work, may be mentioned the ?f Student " microscope of Joseph Zentmayer of Philadelphia, which is represented at one- third actual size in plate III ; the plate representing the parts usually known in the aggregate as the stand.] [The foot is a support widely spread at the bottom, having three points of rest upon the table, and prolonged upward at the center into a conical pillar, which bears at its summit, by a trunnion joint cnpable of rest in any position from vertical to horizontal, the parts required for holding, illuminating and viewing the object.] [The stage, occupying a central position, is a firm plate of blackened brass, nearly square in form, perforated in the optical axis of the instrument with a circular opening for the transmis- sion of light, and designed to support the object. A glass slip, or other contrivance carrying the object, is held in position, while lying upon the stage, by two spring clips under which it is placed. The size of the central opening of the stage and the amount of light passing through it, are regulated by means of a circular revolving plate or diaphragm, let into 'its upper surface, and supplied with a series of apertures of various sizes, any one of which may easily be brought into use.] [The illuminating portion is a mirror, plane on one side and concave on the other, placed below the stage, and so mounted that it can be readily turned toward any source of light. It is supported by a tail-piece or mirror-bar, a radial arm having a swinging motion around a center corresponding with the po- sition of the object on the stage, by which motion any desired obliquity of light can be obtained with great facility, or the PL. IV. The Model Microscope THE MODEL MICROSCOPE. 17 mirror can be carried above the plane of the stage for the purpose of reflecting light upon the top of opaque objects.] [The main tube of the instrument is called the compound body. It contains an ocular, or eye-piece, slipped into its upper end and has a screw at its lower end for the reception of any desired objective. Its normal length for use in an inclined position, as shown in the plate, is partly secured by means of an inside sliding tube, known as the draw-tube, which can be pushed in for the sake of greater compactness when the stand is to be used, as is frequently necessary in laboratory work, in a vertical position. The whole compound body, carrying the essential optical parts of the microscope, slides smoothly through a fixed outside tube, so that the required distance between the magnifiers and the object can be approximately secured by a push witli the hand. By using the thumb and fingers adroitly, and giving a screwing motion to the sliding tube, this adjust- ment can be made safely, and with sufficient precision for even moderately high powers. When greater accuracy is required it is attained by means of the fine adjustment, a sliding motion upon planed surfaces of brass just back of the compound body, this motion being controlled, with great delicacy, by means of a screw with finely cut threads acting upon an intervening lever. The milled head attached to the top of this screw appears on the extreme left of the instrument, at the top of the curved limb connecting the stage with the compound body.] [33. THE MODEL MICROSCOPE.] [This microscope, made by the Bausch and Lonib Optical Co., of Kochester, N. Y., is represented in plate IV, one-third natural size. It is a rather larger instrument, of convenient form and good workmanship, having two pillars instead of one. The coarse adjustment is made by a rack and pinion movement, the large milled heads of the pinion appearing in the plate just behind the compound body ; this adjustment being more con- venient though not more precise, in skillful hands, than the adjustment by sliding tube. The fine adjustment screw is in the same position as before, though acting upon a clock-spring 2 18 THE MICROSCOPE IN BOTANY. system, to be described hereafter, instead of upon a lever. The stage is round, and concentric to the optical axis of the instrument, as are all other round stages worth mentioning ; and to it may be added an extra revolving plate with a movable object-carrier, by which means the adjustment of the object beneath the objective is much facilitated. The diaphragm, seen just beneath the stage, or other substage apparatus, is slipped into a substage ring provided for that purpose.] [C. THE ACME MICROSCOPE]. [Somewhat similar to the last in size and general efficiency is the new model Acme No. 4, represented in Plate Y, made and sold by James W. Queen & Co., of Philadelphia. In this in- strument the fine adjustment screw is removed to an exceptional location below the limb, and the pinion of the coarse adjustment is placed very high, close to the top of the limb, in order to secure the long range of adjustment required for low-power objectives. The diaphragm, being attached to a movable arm, can be swung out of position, as seen in the cut, when not in use, and a substage ring, also shown in the cut, attached in its place for the reception of an illuminating lens or other ap- paratus. A material advantage of this stand is the possession of a body sufficiently large for oculars of ample size ; thus admitting adequate oculars of its own and permitting the fre- quently convenient interchange of any oculars, not exceeding that reasonable size. The diameter of the ocular is about 1 J inch (32 mm.), which is the size recently recommended as a standard by the committee on oculars of the American Society of Microscopists, but not yet acted upon by the society. A larger and more elaborate Acme stand, No. 3, by the same manufacturers, has a body of the same size, but possesses a rotating stage and a substage, somewhat like those in Plate X ; the substage being attached, however, to the same bar as the mirror, as in Plate IX.] [D. THE NEW STUDENT STAND.] [Another excellent instrument of the same grade, of the latest American type, is the New Student Stand, made by PL. V. The Acme Microscope. PL. VI. Bulloch's New Student Stand. THE PHYSICIANS' STAND. 19 Walter II. Bui loch of Chicago, and represented f natural size in Plate VI. The peculiarity of this instrument, as compared with the preceding, is the possession of Mr. Bulloch's form of fine adjustment described hereafter. A sliding object-carrier which can be adapted to the stage is shown lying near the foot of the stand.] [E. THE PHYSICIANS' STAND.] A very solid and serviceable instrument of this type is the Physicians' Microscope of L. Schrauer of New York, shown in Plate VIII A. The body is large, admitting an ocular of 32 mm. in diameter, and is adjustable by means of its draw-tube to any length from 16 to 25 cm. or more. The diaphragm is inserted in the stage ; and a glass sliding stage is provided, in the Zentmayer style, held in position by a spring with ivory tip. Such a stage has a smooth motion and wide range, is available for use Avith the Maltwood finder (a photographed scale of great use for recording the exact location of mounted objects on a slide and enabling them to be promptly found when wanted again), and is unaffected by those reagents which might, in certain cases, mar a brass stage. The joint by which this stand is inclined has a set-screw for securing it in any position. The disk of the swinging mirror-bar is graduated as in all the higher class stands of this type, for the purpose of determining the obliquity of illumination or the angular aperture of objectives.] [P. THE (CONTINENTAL) STUDENT STAND.] [\Vhile the stands heretofore and hereafter described may be considered as representing the characteristic American type, there have always been some observers who preferred the small, compact stands of the French and German model, known as the " continental " style. Some makers have accordingly adopted a model of this type. Mr. J. Grunow, of Xew York, one of the earliest American makers, has always been distinguished for this class of stands, and his excellent workmanship has gone far toward making them popular for medical and histological work. His Student Stand No. 2, represented by Plate VII, is a very 20 THE MICROSCOPE IN BOTANY. efficient instrument of this class, -a solid little stand, with short body and limb, a draw-tube, a low square stage with included rotating diaphragm, and a heavy horse-shoe base.] [G. THE ILLUSTRATOR'S STAND.] [Of somewhat erratic model is the microscope called the Illus- trator's. It is made by T. H. McAllister of New York, and shown in Plate VIII B. It is one of the simplest and most prac- tical of those designed to hold several objects at once. It consists of a broad circular base from the center of which rises a pillar that carries a mirror, a circular stage 25 cm. in diame- ter rotating concentrically about the pillar, and at the top a horizontal transverse bar with its vertical compound body. The body is focussed by sliding through a tube in the transverse bar, the motion being controlled by a pin working in a spiral slot. The stage is capable of carrying twelve slides, radially, at an equal distance from its center, which can be successively brought under the lenses by rotating the stage. The microscope is best adapted to large objects under low powers. It is adapted to certain uses in teaching, where a number of forms are to be shown in comparison with each other to a class. In research, its use is mainly limited to the rapid comparison of objects, as in the classification of unfamiliar objects, the study of adulter- ations, or the comparison of samples of merchandise.] [H. THE HISTOLOQICAL STAND.] [This stand, made by Mr. Zentmayer, and represented, with the addition of the Wenham Binocular arrangement, Fig. 16, in Plate IX, is of the same size as his Student Stand, most of the castings being identical, but is a far more efficient instrument. This superiority is due mainly to the possession of asubstage, a horizontal ring or short tube, designed to support the diaphragm or other apparatus that may be required between the stage and the mirror. This substage is carefully centered around the axis of illumination between the mirror, in whatever position it may be placed, and the object on the stage ; and it has a smoothly sliding vertical movement by which it may be readily located at PL. VII. Grunow's (Continental) Student Stand. 00 co PL. IX. Zentmaysr's Histological Stand. PL. X. Bul loch's Biological Stand. PL. XI. The Bausch and Lomb Universal Stand. THE UNIVERSAL STAND. 21 any point of that axis. This stand is made with a glass sliding st;ige, or with a round rotating stage, if desired. In the monoc- ular form, the cost may be reduced by substituting for the rack and pinion coarse adjustment a sliding tube like that in Plate III. It is most commonly made monocular, as in Plate III, but it can be made binocular as figured, at an extra cost; as can also the Acme No. 3, and the Biological and Universal. It is one of the earliest instruments to which were applied several of the expedients just now termed the modern improvements of the microscope ; and it presents, in combination with them, the low square stage, and the small body, of the continental style.] [I. THE BIOLOGICAL STAND.] [Of larger instruments, capable of utilizing all necessary accessories, and believed by the writer to be large enough for any histological work, Mr. Bulloch's Biological Stand, repre- sented in Plate X, was one of the first to assume substantially its present form. In this stand the tail-piece is made double, one portion carrying the substage and the other the mirror ; an arrangement which is essential to the efficiency of this modern device, since the substnge frequently requires to be in a position axial to the compound body, for the purpose of holding illumi- nating lenses or prisms, for instance, at the same time that the mirror is being used in an oblique position. The usefulness of the whole arrangement is impaired in such cases unless the different parts can be moved independently of each other.] [J. THE UNIVERSAL STAND.] [This stand, made by the Bausch and Lomb Co., is repre- sented natural size in Plate XI. It comprises the same general features as the one last named, but by a slight increase of distance between the stjige and the table sufficient space is secured to admit the use of the largest illuminating or polarizing apparatus, etc., that is usually employed on the largest stands. In fact there is scarcely any of the accessory apparatus of the highest-priced microscopes that cannot, with a few slight modi- 22 THE MICROSCOPE IN BOTANY. fications ill non-essential particulars, be easily and efficiently combined with this. This stand can be obtained as shown in the cut, in a very simple and inexpensive style ; but it is capa- ble of a much higher development. It has been constructed, for the use of the writer, with the addition of lengthening mirror bar, graduated draw-tube for use in micrometry and in drawing to scale at any desired amplification, centering adjust- ment to stage, and graduated rotation of the same, centering substage moved vertically with rack and pinion, and graduated fine adjustment screw with index point, for use in measuring .approximately the thickness of objects or cover-glasses. It is named by the makers the " Universal," from the belief that it is possessed of the working capacity of the most elaborate stands. The stage is well adapted to the use of a glass sliding stage ; and a mechanical stage moved in all directions by special mech- anism can be added if desired. R. H. W.] We proceed now to the more particular consideration of the separate parts of the microscope and begin with the most im- portant part of the optical apparatus, viz., the objective. II. THE OBJECTIVE. The objective consists, as we have seen on p. 15, of several achromatic double lenses joined together in a system. The ob- jective lenses as a rule are plano-convex and are formed of a. biconvex converging lens of crown glass, Fig. 1, I a, and a plano-concave dispersing lens of flint-glass, Fig. 1,16. The under, convex side of the former corresponds exactly to the upper, concave side of the latter. The two are cemented FIG. i.i, ir. together to form a whole by means of perfectly transparent, colorless Canada balsam. Very rarely and only in glasses made for the greatest magnification, the underside of the flint glass lens, is given a very slight con- cavity, Fig. 1, II, 6, so that these in the same manner as the others become an achromatic, concavo-convex lens. Formerly THE ACHROMATIC SET. 23 each achromatic lens was mounted by itself, and before being used several were combined according to need. This was known as a "set" of achromatic lenses. Now, at least for the larger instruments, these achromatic lenses are permanently combined in the optical manufactory and constitute the "ob- jective system." A. THE SET OP ACHROMATIC LENSES. Each achromatic double lens is mounted in a short brass tube, as is shown in Fig. 2, I, II. In II, d, is the achromatic lens with the plane side of the flint-glass lens turned downward. It is placed in the middle of the short tube in the upper part of which is cut the matrix b, into which the whole of the patrix of a similar short tube, I c, may be screwed. Each tube is designated by a successive number, and the lens of least power bears the lowest figure and that of the greatest the highest figure. In III is represented, for example, a set of lenses belonging to an old instrument of Schieck. [Such lenses may be used singly or combined into sets of two or three, the smallest lenses, when the size varies, having the highest power and being placed at the bottom of the combination. R. H. TV.] These sets of lenses were given up a long time ago, and are applied now only to the very cheapest instru- ments. In scientific botanical investigations we shall very seldom be in a situation where we must use them. T\ e consider now : B. THE OBJECTIVE-SYSTEM. Aside from the advantages already mentioned which follow the permanent combination of several double lenses into a sys- tem, certain essential improvements in respect to both aberra- tions (see pp. 5-6) may be aimed at in the proper arrangement of the lenses; for, as Lister^ first pointed out, the special 19 Lister in Philosophical Transactions, 1S30, p. 19S. ff. 24 THE "MICROSCOPE IN BOTANY. aberration of double lenses may be made to compensate each other by placing them at proper distances apart. It is not possible to correct both aberrations perfectly, for the difficulties attending the construction of very small achromatic lenses with short foci is commonly very great, the grinding of the most powerful lenses being done with the help of the microscope itself. Thus the best systems of lenses are not altogether free from faults, but these faults are reduced to the lowest possible limits. We shall just here briefly mention some general qualities upon which the value of microscopical glasses depends and which w T e already in the foregoing have many times designated by name. There are two principal requirements of a good objective- system. It must give, first, a field of view of the greatest possible size and brightness, and second, an image of the greatest possi- ble distinctness. The quantity of light which may pass through a lens depends upon its superficial area, and the amount of light which may pass through two lenses of different sizes is proportioned to the square of their diameters. That is, a lens of n mm. in diameter will afford a field of view four times as bright as one | mm. in diameter. The degree of brightness of the lens is measured < by its "angle of aperture." What this is we understand by drawing as in Fig. 3, I, straight lines from two diametrically opposite points on the edge ., p n W of the lens g h to the focus B. The angle EBF in this case is the " angle of aperture." It is divided in halves by the principal axis of the lens AB. In a combination of lenses the angle of aperture will not be determined by the ex- treme peripheral rays which will enter the front lens L from a luminous point, lying in its focus B, II, but by those which will pass through the whole combination L and L'. The angle of 20 The principal axis of a lens is that straight line which joins the middle point of the spherical surfaces of the lens. It also passes exactly through the middle point (o) of the lens. THE OBJECTIVE-SYSTEM. 25 aperture in the combination illustrated by Fig. 3, II, is not eBf but EBF.- 1 From this it follows that an objective- system which shall give a large and bright field of vision 22 must be so constructed as to have the widest possible angle of aperture. But it has been found in practice, that there is a certain maximum, which must not be overstepped, or, in the highest magnifications, the image will be materially damaged in other respects. The angle of aperture, as appears in the foregoing, is great- est in such lenses as have the shortest focus, and the most highly curved surfaces. But here again conies in the defect, at least if it have a spherical surface, which we have several times des- ignated as spherical aberration. Let us suppose that in front of a lens, Fig. 4, in its principal axis AB is found a luminous FIG. 4. point A. This emits rays Aa Ab Ac Ad on the upper half, and Ab' Ac? Ad' on the lower half of the lens. The ray Aa falls perpendicularly upon the surface of the lens in the direction of its principal axis, and will pass through unbent, while Ab and Ab 1 will be bent in a definite angle, and will have their meeting point behind the lens in B. Likewise Ac and Ac' after being bent will be united again in (7, and Ad and Ad' in D. That is to say, the farther removed the point is from the center of the lens through which a ray passes, the more it will be bent, and the shorter 21 A very simple contrivance for measuring the angle of aperture of a microscopical objective-system is given by Dippel. (Das Mikroskop. Braunschweig 1872, Bd I, p. 86 f.). According to Dippel (L c. p. S3 ff.) the resolving power of an objective-system depends upon the size of the angle of aperture. While Hurting (I. c. p. 219 ff.) traces this back to other causes. 26 THE MICROSCOPE IN BOTANY. the distance behind the lens where they will be reunited. We get three points behind the lens where the rays which have been emitted from A come together again ; viz., B, C, and D. Nay, many more, since A sends out not only the rays we have supposed but an infinite number of others, so we shall have an infinite number of focal points all lying between B and D. We have assumed that the rays Ab and AU are the nearest possi- ble to the principal axis, and Ad and Ad 1 the nearest possible to the edge of the lens. This variation in the focal points we call the spherical aberration, and the distance B to Z), the length of it. If now we hold a translucent screen or a piece of ground glass at the point B, we shall see thereon a picture of A, but it will be made more or less indistinct by the rays which come from the outer portions of the lens, and have crossed and been dispersed at C D, etc. So we must get rid of these so-called "marginal rays" Ac Ac f Ad Ad', etc., by shutting them off. This can very easily be done by means of a diaphragm. To accomplish this, all that is necessary is to place an opaque disk with a circular hole in the middle of it between the lens and Q A, or the lens and B, so as to permit only the middle rays Aa Ab Ab', etc., to pass through. This is indeed a very simple method of overcoming the spherical aberration, but it is only to a certain extent satisfactory, since naturally what such a dia- phragm shuts off, reduces the angle of aperture by so much, and in consequence thereof, the field of vision will be made smaller and its brightness much diminished. It is on this account, of the greatest importance, that the spherical aberration should be reduced in some other way, viz. ; by the peculiar construction of the lens itself. If a lens be not alike convex on both sides, but if one side have less curvature than the other, or the lens be plano-convex or plano-concave, its spherical aberration will be diminished, as experience teaches, if the side of least curvature, the plane or concave side of the lens be turned towards the object (at least with microscopical objectives). 23 It has been shown that those biconvex lenses, which have surfaces of unequal curvature and 23 With telescopic lenses, whose object is very far removed, we must reverse this rule and present that side of the lens which has the greatest curvature to the object. THE OBJECTIVE-SYSTEM. 27 the least spherical aberration are those whose curvature on one side has a radius six times as long as that of the other. 24 The spherical aberration is diminished also, by combining a bi- convex lens of crown glass, and a plano-concave lens of flint glass. In a biconvex converging lens the spherical aberration is of that nature that the marginal rays come to a focus in front of the true focus of the lens. A diverging plano-concave* lens has an aberration of like nature only with this important difference, that the marginal rays come to a focus behind the focus of the central rays, or the true focus of the lens. If now the two lenses be made of glass, of different refractive powers, and then combined to make a whole, and rays of light be passed through them, the relative direction of the central and marginal rays will be reversed by the action of the diverging lens. It comes about, thence, that by the united effect of the two lenses the focal point of all the rays will be brought very near together ; only it must be understood that the lenses should be made with a definite curva- ture on their contiguous sides, which indeed we need not here more particularly specify. Finally, there is another very effective means for materially lessening the spherical aberration, which consists in uniting three plano-convex doublets in one system. In this we follow Lister, 25 who discovered the one peculiar characteristic of the achromatic doublets, and gave formulas for their combination based on this discovery. The distance apart of the three lensete must be a very definite one. It is the art of the optician by exact and most careful experimentation to give the lenses their right posi- tion in his objective-system. The smaller the achromatics, the shorter their focal distance and the more difficult is it to accom- plish this combination. It is for this reason that good object- ive-systems are so expensive. The great advantage which is attained by this combination of three lenses is that, notwithstand- ing the great magnifying power of the lenses singly, and in spite of correcting their spherical aberration, the angle of aperture is still large and the field of vision still shows great brightness. 24 This proportion answers, however, only for those kinds of glass which have an index of refraction of 1.5. 25 See more particularly Lister, at the place before mentioned and in llarting, /. c., p. 46 f, and 138 ff. 28 THE MICROSCOPE IN BOTANY. It is a generally known fact that by uniting a biconvex crown glass lens with a plano-concave flint glass lens the chromatic aberration of the rays is almost entirely overcome. We have already stated the reasons for this on page 5. We believe that in this cursory review of the physics of the matter we shall not be expected to go into it more particularly. 26 We will therefore only add in brief, that in refraction through a single lens, the violet rays have their focus nearest the lens, are the most converged, while the focus of the red rays is farthest removed from it. The focal points of all the other colored rays lie between, naturally, in the order in which they appear in the spectrum. An achromatic lens-combination is now so corrected that with it the focal distance for red and violet light is the same. In this way these two colors of the spectrum fall upon the same point, but not so with those which lie between. These, as is easily seen, give secondary dispersion images which appear as colored borders of different tints, commonly yellow or green. To remove these it is only necessary to make a combination of several doublets, and in fact this requirement is met in a combi- nation such as is the present objective- system. As in these systems the spherical aberration is reduced to its lowest terms, so also the chromatic aberration is, for the most part, overcome. At the present time, if the flint-glass lens is given a very slight preponderance the result is that the system shows a very soft, and to the eye a very pleasant blue tinge, in the micros- copic image. We call such a lens as that over-corrected. If the lens on the other hand shows a red border (by reason of a preponderance of crown glass) we say it is under-corrected. Since the objective-system is capable of all the wished-for corrections, of which we have spoken, we have in it what we call an "aplanatic," that is, a lens with the least possible error, the smallest amount of both spherical and chromatic aberration. The Systems in Practice. Now that we have become ac- quainted with the optical principles of the objective-systems, 26 See thereon for example Wullner's Lelirbuch der Experimentalpliysik, Leipzig, 1871, Bd. II, p. 21(5-2-20 v. Quiiitus-Icilitis. Experimentalphysik, Hanover, 1SUG, p. 250ff. Hurting. I, c., p. 37-40. THE OBJECTIVE-SYSTEM. 29 we shall proceed to consider some of the different sorts of objectives. The common system consists of three achromatic doublets of the form shown in Fig. 1, I. They are arranged as is shown in Fig. 5, a, >, c. The smallest, which is also the strongest magnifier, is nearest the object ; the largest and weakest is removed farthest from it. By this combination there is attained on the one side a greater focal distance of the object, and on the other an objective so constructed gives a wide angle of aperture and a very bright image. Differing from this, a very perfect system is sometimes made by combining an under lens of three parts, a plano-concave of flint glass, and two plano-convex lenses of crown glass, with a middle lens of the form of Fig. 1, I, and an upper lens constructed of a middle bi-concave of flint glass and two bi-convex lenses of crown glass. The mounting of an objective-system is clearly represented in Fig. 6, which is a medium system of natural size from the manufactory of Seibert. The three plano-convex doublets are contained in the lower half cZa, which part is screwed into the upper db, the latter having no lenses. Both parts are made permanently fast to each other. At b the system is provided with a screw thread by which it is made fast to the microscope. In this manipulation one takes it with the thumb, index and middle fingers of the right hand, on the two edges cc, their milling enabling one to hold it fast. On the smooth surface e between the two edges the number [and maker] of the system is engraved. [Somewhat similar ob- jective-svstems are shown in situ at the bottom of the mi- croscope tube, or compound body, in Plates IV and VI to XI. R, H. AV.] The use of such a system (the so-called dry lens) is very easily understood. But the use of two other systems of con- struction, mainly applied to the production of much higher magnifications ; viz., the "immersion-system" and the "correction- system," is very much more elaborate. 30 THE MICROSCOPE IN BOTANY. The Immersion- System. In order to understand this system, it is necessary to assume that the botanical object to be viewed by the microscope lies under a thin glass plate the cover- glass and is surrounded by a layer of water or other fluid, glycerine, essential oils, etc. The light, which enters the optical apparatus of the microscope from the object has to pass, on its way, through one after another the several media, water, glass, air. Since the glass and the air are very different in their re- fractive power, and the light enters from a thicker medium, glass, into a thinner, air, a number of the rays will be so far dispersed that they will not be able to' enter the objective-system, and the consequence will be that the microscopic image will be correspondingly darker ; and further, a considerable reflection of the rays of light will take place from the under plane surface of the lens. In order to remove this defect we have for a long time adopted this contrivance, with high magnifying powers, of substituting a thin film - of water for the layer of air which intervenes between the cover-glass and the front lens of the objective. We owe this especially to Hartnack. Since the refractive power of the water is much nearer that of glass than is that of air, it is obvious that the interposition of the film of water will very considerably diminish the before-mentioned dispersion of the rays of light, and will cause therefore, many more to enter the objective and will give to the microscopic image a consider- ably greater brilliancy. The result of this arrangement is essentially the same as if the angle of aperture of the objective were considerably increased. By this means the reflection of the rays from the under surface of the lens and also from the upper surface of the cover-glass is altogether obviated. Such a system, whose lowest lens is immersed in water is called a "water-system" or "immersion-system." This objective is prepared for use in the following way. The objective is turned bottom side up, and a drop of distilled water from a flask, by means of a glass rod or hair pencil, is placed upon the lens. Here it will round itself up into a little hemisphere. Now reverse the objective and the drop will remain in place by the power of adhesion. The objective should now be screwed into THE IMMERSION-SYSTEM. 31 the microscope tube, and the tube pushed down by hand or by the rack and pinion till it comes near the cover-glass upon the object which lies upon the stage. Now if one breathes a little upon the cover-glass and then carefully brings the drop down till it touches it, it will easily unite with the surface of the glass and form the desired film of water between the glass and lens. Then, by means of the fine adjustment screw, the object can be exactly focussed. Particular care should be taken that the drop of water used does not contain even the smallest bubble of air, else the microscopic image will be ruined. After use, the drop of water which adheres to the objective should be carefully wiped off with a piece of soft old b'nen cloth which has been washed in distilled water. [It is sometimes, moreover, convenient to plunge the objec- tive directly into the medium in which objects are situated, for the purpose of examining them without preparation or selection, and under strictly natural conditions. When the medium is not corrosive, ordinary "immersion" objectives may be used in this manner, provided they have sufficient screw-collar movement to make the necessary corrections ; and objectives specially corrected for this use have been constructed by Tolles and others. Such lenses thus used, though not inapplicable to certain botanical researches, have been heretofore mostly em- ployed in zoology and pathology. By a modification of this plan, however, dry lenses of lower powers may acquire a new value to the botanist. By surrounding the objective with a brass cylinder open above and closed tightly with a thin cover-glass below, it may be plunged into water or various solutions, with impunity, and a clear and satisfactory view may be obtained of objects in the fluid, or lying at its bottom, in saucers, dissecting troughs, or other suitable vessels. Of course the vessels, or their bottoms, must be of glass if transmitted light be required. By this method, not only the unavoidable tremor of the upper surface of the liquid, which renders study of objects below by usual methods difficult and quite unsatisfactory, is ren- dered harmless ; but the object may be freely manipulated with needles, scissors, or dipping tubes, under objectives of from two inches (51 mm.) to a low-angled J or , without interrupting the 32 THE MICROSCOPE IN BOTANY. FIG. 7. view. Small objects, in small quantities, may be thus examined under the higher powers in watch-glasses. For larger quanti- ties and lower powers, nothing is more convenient than the little glass dishes occasionally sold as individual butter plates, or the glass jars sold as seed and drink cups for bird cages. W T hen the quantity of material is unlimited, and especially when manipulation is required, nothing is more convenient than plain glass preserve dishes one inch (25 mm.) deep, and four inches (10 cm.) wide. For dissecting purposes with reflected light ex- clusively, china dishes, or the hard rubber dissecting troughs sold by microscope dealers, may be used ; the bottoms being lined with thin sheets of cork if it be desired to fasten down the objects with pins. A most ^_J \_ convenient apparatus for this quasi-immersion use of dry lenses is the " objective-protector," Fig. 7 ; shown in section and in situ upon the objective in Fig. 8. It was proposed by Mr. R. E. Dudgeon of London, and is made by J. H. McAllister of New York. R. H. W.] The Correction- System. We will now FlG - 8 - suppose that an object Fig. 9, p, lies on the microscope, under the cover-glass DD (a highly magnified section in the illus- tration) through which we send rays of light pv, pa, p(',pe, . . . pa', pc r , pe', from the illuminating mirror, to the objective. The rays impinge upon the under sur- face of the cover-glass, and those which fall upon it ob- liquely do not proceed in the same direction as here- tofore, but, at the surface between the water and the glass, are refracted and within the glass take the direction a A, cC, eE, . . . .a'A^c'C^e'E'. Further, in passing through the upper surface of the cover-glass into the air or into the water, as the case may THE CORRECTION-SYSTEM. 33 be, in dry or immersion objectives, they are again refracted in the direction AF, CG, EH, . . . A'F', C'G', EH. The rays of light are more widely dispersed on emerging in propor- tion to the acuteness of the angle at which they enter the cover- glass from p. If we now construct the uniting points of the twice refracted rays we shall have a series s along the line^v, one point above another, each of which represents the image of the preparation p, as a luminous point. The distance a swill be less or greater in proportion to the thickness of the cover- glass DD. There results from this exactly the same phenome- non that we have learned to know as spherical aberration. The use of the cover-glass, therefore, will cause a certain indistinctness of the microscopic image similar to that caused by the use of an objective which has not been corrected for spherical aberration. 27 We have alreddy learned that the defect of spherical aberra- tion can be corrected by-placing the three objective' lenses in a certain right relative position to each other. It is also evident, that the optician can likewise eliminate the damaging influence of % the cover-glass, if the same thickness of cover-glass were always used, and he had taken into account this particular thick- ness in correcting the objective for spherical aberration. This is now almost always done and the objectives for medium mag- nifications are commonly so corrected that they give the clearest possible image with the use of a cover-glass .1 to .2 mm. thick. For high power objectives, chiefly for strong immersion lenses, with which the influence of the cover-glass is most dam- aging to" the clearness of the image, we have followed Ross 28 in hitting upon a contrivance which almost entirely eliminates this bad influence, viz., a device for changing at will the relative distance of the lenses, and thus obtain an objective which is aplanatic for every thickness of cover-glass. We name this the " adjustable lens," the " correction system," the " system with correction for thickness of cover-glass." 27 More particularly H. v. Mohl, I. c.', p. 157/. Harting, I. c., p. 146-149. 28 Ross discovered this influence of the cover-glass in 1837 and sought at once to obviate it by the screw correction in the system (Harting, 1. c., p. 747). Already before this (1829) Amici had made the same discovery, but constructed however, no correction, system, but added to his microscopes several equivalent systems which were intended for different thicknesses of cover-glass (Harting, I. c., pp. 148, 720). 34 THE MICROSCOPE IN BOTANY. Ross so constructed his objectives that the correction was made, by changing at will, by means of a screw contrivance, the distance between the upper lenses which Avere fastened together as a whole, and the lower lens. Afterwards Hartnack modified this so that the two lower lenses which were made fast together were made movable towards the upper which was fixed in the microscope tube. Finally, several, especially German makers, have modified the system of Hartnack so that the two under lenses are made fast together and to the mi- croscope tube, and the upper lens is mounted in an inner movable sheath so as to be adjusted to the other two. The last two methods of con- struction do not ditler at all in regard to the results produced, and the difference between Ross' and Hartnack's system of correction is of a subordinate nature. In Fig. 10 is represented a longitudinal section through a Hartnack objective which shows the characteristics of the ad- justment. By means of the screw thread b the inner cylinder i which bears the upper lens 3 is made fast to the microscope tube. The two under lenses 1 and 2 are screwed fast to the outer cylinder a into which the inner shell i exactly fits. At c is a ring which by means of a screw thread can be moved up and down on i. This ring bears in an inner groove v the cylinder a. Now if the ring be screwed up, the tube a with the lenses 1 and 2 will be carried up and not at the same time turned around the axis of the objective with the ro- tary motion of the ring. In reference to manipulating the correction in microscopical work the following may be said. Fig. 11 represents the immersion No. VII of Seibert, natural size. The objective has a screw collar c corresponding to c in Fig. 10, which on its upper smooth part is garduated into ten divisions (1, 2, 3, etc.) which graduations play by a mark at e. By a full turn of c the upper lens is removed from or brought nearer to the lower lenses, as the case may be, by the distance of a screw thread. FIG. 11. THE OCULAR. 35 The interval between the graduations corresponds to .1 of this change of distance. Now to enable one to control this move- ment of the upper lens up and down from a certain medium position the device a i has been contrived. A small slit is cut through the outer cylinder a which allows the inner cylinder i to be seen through it. Both a and i are provided with a mark. If they stand at the same height (as they do not in the illustra- tion) then the upper lens is in a normal position to the under. If the middle line i is above the other a the upper lens is moved farther away from the lower, and the reverse. After the lenses have been put in their normal position by bringing the of the ring graduations to the mark e, the objective should be put on to the microscope, focus the object and then turn the ring experimentally back and forth till the image becomes very sharp and distinct. When this is done a note should be made on the slide, of the direction and distance which the ring has been moved. HI. THE OCULAR. We have already seen on p. 15 that the ocular consists of two glasses, the upper one being placed near the eye and is the essential image-viewer, and in the narrow sense the ocular-glass, while the under one called a " collecting o lens," is only in a limited sense a part of the ocular and might with much greater propriety be considered a part of the ob- jective. But since they are always united to make this part of the microscope it has always been customary to designate the two glasses together as the ocular. Fig. 12, which represents a somewhat conventional longitudinal section of a Hartnack ocular, will enable us to under- stand the arrangement of oculars. In a FlG 1 cyb'ndrical brass shell, which exactly fits into the tube of the microscope, there are screwed two end pieces, one c above, and the other d below, of which the one 36 THE MICROSCOPE IN BOTANY. carries the ocular lens at a and the other the collecting lens at b. Inside of e at about equal distance from a and b is placed the diaphragm /, intended to cut off the marginal rays coming through 6, which are so deleterious to the beauty of the image. Both lenses have a plano-convex form, and the convex side of both is downward. This arrangement of the lenses with their curved sides down, essentially influences the size of the field of view and the sharpness of the image. A reversal of them would materially diminish the field as well as the sharp- ness and flatness of the image. The ocular is set in the top of the microscope-tube. The oc- ular lenses should be exactly centered with the objective lenses, that is, so made that a straight line drawn through the middle FIG. 13. FIG. 14. point of the objective lenses shall also, on being prolonged, pass through the middle point of the ocular lenses. This presup- poses exact workmanship. Oculars have the form of a simple cylinder Fig. 13, or in later times that represented in Fig. 14. The first is found in the microscopes of Hartnack, Merz and Nachet, and the second in those of Gundlach and Seibert [and ? n those of most English and American makers. R. H. W.] That part of the ocular which slips into the microscope tube should exactly and easily fit into it. In putting the ocular into the microscope-tube it should always be allowed to sink down to cc Figs. 13, 14, in the tube. The distance of the ocular from THE OCULAR. 37 the objective is a definite one and should not be changed at will, save in those cases to be described farther on in treating w microscope-tubes." For the ocular is placed at such a distance from the objective that the image produced by it would appear above the collect- ing lens. In other words the collecting lens is below the point where the cone of refracted rays from the objective would meet. If we suppose a cone of refracted rays of a magnified object, from the objective, to be represented by a A, bB, cC, dD, eE, b'B\ cfC', d'Z>', 4E 1 , Fig. 15, it would present itself to us in e d c ba b'c'd'e . Flo. 1.3. the extended form represented by EE 1 . Now we shove down beneath this image the collecting or "field lens," LL, corres- ponding to b, Fig. 12, and the rays which fall upon it at fc, r, fl, Jf, fo', jp', (T, t't will be refracted inward toward the axis aA, and take the direction fcg, dfc, 4g, *tf, fc'', *'$', A'g 7 , t'd 7 , the diverging bundle of rays changed to a converging, and the image thus modified by the field lens will fall at $ (&'. The collecting lens has really diminished the image. But this loss of magnification is in various ways an advantage, for now more of the image can be seen than when the rays are diverging. It is also evident that the interposition of the field lens increases 38 THE MICROSCOPE IN BOTANY. the brightness of the image by concentrating the given number of rays upon a smaller surface. In like manner the distortion of the image by the unequal magnification of the central and mar- ginal portion is obviated. To this end also the diaphragm mentioned above affords no small help by cutting off most of the distorting marginal rays. The effect of the ocular glass a Fig. 12, is that of a simple magnifying glass, by which the picture at $ $' is enlarged. It is not necessary to mention that the ocular lens should be so placed that the image $ $>' will be exactly in its focus, or that combining the ocular and field glass with the objective will materially assist in correcting its spherical and chromatic aber- ration. They can be adjusted by somewhat over correcting the aberrations of the objective and somewhat under correcting those of the oculars. The oculars of most microscopes have the arrangement just now described. It is called the Campanian or Huygenian, ocular (negative ocular). This is at the present time sometimes so altered that the field lens is made of a con- cavo-convex flint-glass and a biconvex crown-glass cemented together with Canada balsam, thus making a biconvex achro- matic doublet. This modification of the Huygenian ocular was first made by Kellner and was called the orthoscopic ocular. The aplanatic ocular of Plossl is much the same thing, made of two achromatic plano-convex lenses, and are combined for the most part as shown in Fig. 12. A very useful variation is found in the Kamsclen or positive ocular. The lenses have the same form as in Fig. 12, but the field lens is permanently reversed, turned with its plane side toward the objective, while its distance from the ocular lens is much less than in the Huygenian oculars. For common obser- vations the positive ocular is seldom used. It is mainly useful in fine microscopical measurements, but is not absolutely neces- sary even for these. [IV THE BINOCULAR OCULAR.] [For the sake of the advantages of stereoscopic vision, and of the comfort secured by using both eyes instead of one, the THE BINOCULAR OCULAR. 39 pencil of rays above the objective, or above an erector inserted within the draw-tube, is sometimes divided into two portions, one of which is transmitted to each eye. When the apparatus is permanently attached to a modification of the compound body, it is termed a binocular microscope, and when mounted separately and capable of removal like simple oculars, it is called a binocular ocular, or eye-piece. Among those who, when the stereoscope was an interesting novelty, undertook to apply its principles to the microscope, the first to succeed was Prof. J. L. Riddell of Now Orleans, La., who is therefore justly credited with the invention of the binocular microscope. He bisected the pencil of rays above the objective by a pair of rectangular prisms which turned the parted halves of the pencil, by internal reflection, horizontally across each other to a dis- tance apart equal to the distance from each other of the pupils of the eyes, at which points they were again reflected directly upwards by a second pair of prisms to the two oculars. Shortly afterwards, Nachet of Paris slightly modified aud improved this plan, dividing and crossing the rays by internal reflection from the opposite sides of a single equilateral triangular prism, the rays passing thence to a pair of prisms below the oculars in a direction not horizontal but inclined upward. Not long after- wards, the late R. B. Tolles of Boston, still further improved this apparatus by transferring the whole system of prisms to a position just below the oculars and above an erector attached to the lower end of the -apparatus, the whole being removable together, and leaving the tube ready for the reception of any simple ocular. Notwithstanding its excellent workmanship, good definition, fine stereoscopic effect, ease of removal, and applicability to all powers however high, this ocular never came into use, less perhaps on account of its considerable cost, than because of the small, high power oculars and parallel tubes required in its construction. Oculars of high power are tire- some to the eyes at best, and especially when the light has also passed through the numerous refracting and reflecting media composing an erector and set of reflecting prisms ; and to pre- serve the parallelism of the axis of the eyes as required for dis- tant vision, when looking intently at an object known to be 40 THE MICROSCOPE IN BOTANY. near, is a continual strain, since the eyes under such circum- stances tend instinctively to turn their axes in a converging di- rection towards the object of vision. At least, these seem to be the causes of failure to the writer, who has endeavored perse veringly at various times since the introduction of this binocular to overcome the diffi- culties of its use, but always with such loss of comfort as 'to lead to an abandonment of the attempt.] ["Lately, Mr. Stephenson of London has devised a binocular in which the pencil of light is bisected by a pair of small prisms inserted very close to the objective, the image being erected and reflected obliquely forward by a mirror or prism placed in the tube above. This arrangement requires the stage to be permanently fixed in a horizontal position, the di- verging tubes thus taking an inclined position favorable for every use. It is not converti- ble to a monocular. Being an erecting binocular it is especial- ly adapted to use as a dissecting or preparing microscope.] [Meanwhile, Mr. F. H. TTenham also of London had abandoned the idea of a sym- metrical division of the rays, and introduced a little prism into the pencil just above the ob- jective, which should reflect the right half of the pencil obliquely THE BINOCULAR MICROSCOPE. 41 across the left half to the left eye, while the left half passed without interruption to the right eye, as shown in section, Fig. 16, where A is the "Wenham" prism which reflects half the light through the oblique body B, and D, E are two draw-tubes by slightly raising or lowering which, the distance apart of the two eye lenses may be adapted to the eyes of various observers. Such a binocular is shown, complete, in Plate VII. This ar- rangement gives one field of vision with definition absolutely unimpaired, an advantage as yet gained by no other binocular. It is also interchangeable while in use from the binocular to the monocular effect, by simply slipping the prism into or out of the pencil of light. These advantages proved greatly to outweigh its somewhat cumbersome mounting which is not removable from the stand, the unequal light and definition of the two fields, and its practical limitation to medium powers and apertures ; and it immediately became and has thus far remained the binocular of England and this country. It is best adapted to powers of from one to two inches (25 to 51 mm.) up to one-half or one- fourth inch (13 to 6 mm.). With low powers of ample aper- ture the capacity of the objective is sensibly reduced by the brass mounting of the prism, which serves as a diaphragm to cut down this aperture, and with powers of from one-half inch (13 mm.) upward, the angle of aperture of the objective used should be moderate, and the illumination should be arranged to light both fields freely without flooding the object with two much light.] [Nachet soon changed his binocular so as to embody some of the advantages of the Wenham form, likewise incorporated into the stand itself, allowing one portion of the pencil to pass with- out intentional alteration through glass with parallel surfaces directly to one ocular, while the other portion was diverted twice by internal reflection in two prisms, and then directed ob- liquely to the other ocular. This form was well adapted to the small stands of continental model, and is still in use.] [Just now another binocular, in the form of a removable ocu- lar, is coming into use, devised by Prof. Abbe and manu- fractured by Zeiss of Jena. In arrangement of the parts and direction of the rays it resembles the last mentioned form, but 42 THE MICROSCOPE IN BOTANY. instead of a division of the light into lateral halves a part of the light from the whole pencil is transmitted to one eye, and the other part reflected to the other. By stops over the eye lenses, cutting off the opposite lateral portions of each, the fields can be so differentiated as to produce a stereoscopic effect when de- sired.] [Of the numerous ingenious and plausible binoculars invented, the above mentioned are all which have come into actual use sufficiently to demand notice. It is evident that the binocular has been received with more favor here and in England than on the continent, for the reason that it is better adapted to the work of the one than to that of the other. It is most applicable to medium powers and low angles, and is most valued by those who use such powers for general natural history work, where stereoscopic effect is available and serviceable. On the other hand, it is least valued or used, if at all, by histologists, medi- cal microscopistSjdiatomists, and other classes who use mostly the higher powers and larger angles, that render its use less satis- factory if not inexpedient. R. H. W.] [Y. THE EYE SHADE.] [When the monocular instrument is used, the fatigue of the observer's eyes is greatly lessened by habitually keeping the unemployed eye open, and protecting it by a black eye-shade, placed before it. The greatest comfort is attained when light is arrested by a central stop of limited size, which does not wholly darken the eye, but only prevents the formation in it of an image of the objects on the table. This prevents the confusing effect of an image in the unused eye, and the fatiguing effort required to keep the observer's attention confined exclusively to the microscopic image in the other; but still avoids tiresome contrast between the two eyes by allowing the entrance of much diffuse light, and permits, without abrupt transfer from dark- ness to light, the frequent changes required from rest over the shade to inspection of books or of other objects upon the well- lighted table. For this reason, probably, and for their own clumsiness, the large shades or hoods first used were soon aban- THE EYE SHADE. 43 donecl. Lately, some very neat and useful shades have been used ; but being attached to the top of the ocular, they could be used only on oculars of exactly similar size and form, and were invariably removed and required to be replaced with every change of ocular. For these reasons the writer caused to be constructed a new form represented in Fig. 17, which springs upon the compound body just below the ocular, and without re- gard to the style of ocular or fiuish of tube, is securely held at a convenient distance from the face, is not affected by change of oculars, and can be instantly transferred to any microscope body of suitable size, or reversed to shade the other eye. Being made of hard rubber it is of proper color and light weight, is little inclined to scratch the brass work with which it comes in contact, and is sufficiently elastic to be placed without altera- tion upon tubes varying 6 mm. in diameter. It is made by the Bausch and Lomb Optical Co., of Rochester, of any size desired, and is cheap as well as efficient. The writer's expe- rience leads him to believe that some such contrivance should always remain fixed upon any minocular microscope while it stands upon the table ready for use, even occasional glances into the tube being more tiresome without this accessory than with it. R. H. W.] VI. THE MAGNIFYING POWER OF THE MODERN MICROSCOPE. The optical powers of a microscope depend on several differ- ent factors. A microscope which will satisfy modern require- ments must give a large, flat and bright field of vision, in which the magnification of the margin is essentially the same as that of the middle. The image produced must be perfectly distinct on the edges. The fine structural relations of the object must 44 THE MICROSCOPE IX BOTANY. be sharply brought out, and the magnifying power of the in- strument should not be too small. The size and brightness of the field of vision depend chiefly upon the angle of aperture of the objective, and on the proper position of the field lens of the ocular, while the sharpness and colorlessness of the image are dependent upon the correction of the spherical and chromatic aberration. Under the designation "resolving power," we understand the capacity of the objective to bring to view the fine structural rela- tions of the object ; the more and finer these relations which an objective will discover, the greater is its resolving power. Independent of this quality is the magnifying power of the microscope, that is, the capacity to produce an image of an ob- ject which exceeds in extension many-fold the object itself. In microscopical magnification we speak only of linear enlarge- ment. It is expressed in terms of one dimension of space. There are various contrivances and methods to determine the magnifications of a microscope; they are all, however, grounded upon the use of a very fine measuring scale ( e. #., a millimeter divided into 100 parts), which is made on a glass slip and viewed urrder the microscope to determine the distances apart of two or more division marks of the millimeter, so as to find thence by simple division the amount of the magnification. Latterly the opticians very exactly determine the magnification of the various combinations of their lenses and furnish a table of the same with their microscopes, so the microscopist is now seldom required to determine for himself the magnification of an object ; for this reason he is referred to the more compre- hensive works of other authors. 29 The magnification is produced in great part by the objective and in a less degree by the ocular. Each objective will show different magnifications by the use of oculars of different powers, since the image produced by the object will be more powerfully magnified by oculars of greater curvature than by those of less. 29 See Harting, 1. c., p. 131, p. 244jf.; Harting, Recherches Micrometriques II. v. Mohl, 1. c., p. 215jT. Jacquin in Zeitsch. /. Physik & Mathcmatik. 18-28 [IV], p. 1. Ettingliausen ibid 1829 (V), p. 316jf. Pohl in Berichte d. k. k. Acad. d. Wiss. Wien, XI, p. 504J. Dip- pel, 1. c., p. 92-100, etc. THE MAGNIFYING POWER. 45 In order to show in what relation these different glasses some- o times stand, we will take an example from the magnifications which the Hartnack oculars 1-6 give in combination with his objectives No. 2, 4, 5, 7, 10. OCULARS. OBJECTIVES. 1 2 3 4 5 6 2 25 30 45 4 60 70 90 140 5 100 125 160 240 7 200 240 300 450 600 750 10 520 600 750 1100 1500 1800 It is not a matter of indifference in what manner the oculars are combined with the objectives. The principal burden should always be laid upon the objective. For the production of a given magnification, there should be combined the strongest possible objective with the weakest possible ocular. Resolving power, the delineation of the details of the image, is alone an attribute of the objective. The ocular does indeed more or less enlarge the image produced by the objective, and indeed, nat- urally at the cost of its brightness, but it will have only the least possible influence upon its improvement, on the resolution of the finer structural relations of the image. Supposing we de- sired to produce a magnification of, say, 240 diameters, in ac- cordance with the above quoted table, we should not combine ocular 4 with objective 5, but objective 7 with ocular 2. Or if we desired a magnification of 600 diameters, we should not take objective 7 and ocular 5, but objective 10 and ocular 2. Or if we wanted 750 diameters, we should not put objective 7 with ocular 6, but objective 10 with ocular 3. The amount of linear magnification attainable to-day with a good microscope is a very considerable one, especially is that of the immersion- systems abreast of what formerly could be produced with dry lenses, and was called a very prodigious one. In order to give an idea of what this really is, we will instance the objective mngnifications of instruments from some of our best 46 THE MICROSCOPE IN BOTANY. optical manufactories. In each case, only the linear magnifi- cation will be given which would be produced by the use of the weakest ocular which the firm furnishes. An asterisk (*) indi- cates that the magnification is produced by an immersion-system, the rest are by dry lenses. TABLE OF MAGNIFICATIONS. MAKERS. OCULARS. OBJECTIVES AND MAGNIFICATIONS. No. 1 2 34567 8 9 10 11 Nachet. No. 1. 30, 89, 180, 2GO, 300, 350, 460,* 580,* 775,* 900,* 1150,* 1320, 12 1700.* No. 1 2 3 45678 9 10 11 12 Hartnack. No. 1. 15, 25, 50, 60, 100, 150, 200, 250, 350, 410,* 520 ,* 600,* 710,* 13 14 15 16 17 18 820,* 930,* 1040,* 1200,* 1400,* 1560.* No. 9 is both dry and immersion. No. 00 I II III IV V VI VII VIII IX X Seibert. No.O. 10, 18, 30, 45, 66, 100, 200, 305, 460,* 650,* 950,* 1450.* No. 123 4567 8 9 10 11 12 Schieck. No. 0. 20, 40, 70 90, 150, 200, 275, 400, 450, 500,* 600,* 750,* 13 14 15 16 850,* 930,* 1100,* 1400.* No. a aa A AA B BB C CC U DD E Zeiss. No. 1. 5, 18, 45, 70, 110, 180, 220,* 240, 380, 400,* 680.* No. 1 2 3 4 5 6 7 8 9 10 11 Winkel. No. 1. 25, 54, 74 102, 184, 222, 275, 366, 458, 500, 584. In the following table is presented the strongest ocular mag- nification of the above named six firms. MAKER. OBJECTIVE SYSTEM. OCULAR. MAGNIFICATION. Schieck. System 15. No. 5. 6500* Diameters. Hartnack. " 18. No. 6. 5400* " Nachet. " 12. No. 4. 4500* '< Seibert. " X. No. III. 4400* " Zeiss. Imm. " 3. No. 5. 2300* ' " Winkel. " 11. No. 6. 1600 THE MAGNIFYING POWER. 47 [In America and England, objectives are universally named from their equivalent focal lengths in inches ; a one-inch object- ive, for instance, being a lens system whose amplifying power is -the same as that of the simple lens of the specified focal dis- tance. This nominal focal distance is usually much greater than that called the working focal distance, between the object and the nearest portion of the combination of lenses. Oculars are similarly named by several makers. It follows from this most convenient system of nomenclature, that the amplification is approximately made known by the name. Thus assuming, ac- cording to usage, the round number 10 inches (25 cm.) to be the distance of most distinct unaided vision, a one inch (25 mm.) lens would magnify ten times, a two inch (51 mm.) five times, a one-half inch (13 mm.) twenty times, a one-fifth inch, (5 mm.) fifty times, etc. Furthermore, the combined amplifi- cation of objective and ocular, as used in the compound micros- cope at a distance of ten inches (25 cm.) from each other, will be represented by the multiple of their individual powers ; a one-fourth objective, for instance ( X 40) with a one-inch ocular ( X 10) giving a resultant power of 400. The following table shows the theoretical powers, thus calculated, of a few combi- nations, other powers being in the same proportion : OCULARS. OBJECTIVES. 1 1 1 1 1 1 1 ^ 5 3 1 2 5 10 15 20 30 50 *c ^ 2 o S 127 76 25 13 5 2.5 1.7 1.3 0.8 0.5 * 3 POWER. 2 4 10 20 50 100 150 200 300 500 In. mm. a 2 51 5 10 20 50 100 250 500 750 1000 1500 2500 1 25 10 *2 H 20 40 100 200 500 1000 1500 2000 3000 5000 | 13 20 I 1 40 80 200 400 1000 2000 3000 4000 6000 10000 * 6 40 z 80 160 400 800 2000 4000 6000 8000 12000 20000 [Such tables have been used for an indefinite period as a means of assisting the memory in respect to the powers em- ployed. They have not been used with the understanding that 48 THE MICROSCOPE IN BOTANY. they were precisely accurate as applied in practice, but with the distinct explanation that they were only approximately correct, since it would be required, to make them literally correct, to locate the ocular and objective by means of certain optical planes which obviously do not coincide with any recognizable part of the mounting, and which cannot be determined, by any known method, with sufficient facility for popular use. It has, therefore, been somewhat customary to approximate, by taking an eight and one-half inch (21 cm.) length of tube, assuming that the rest of the ten inches (25 cm.) will be more^ or less evenly supplied by the mounting of the objective and ocular. The position of the planes certainly vary much in different lens systems of equal power, but they are stated to be usually, near the diaphragm of the ocular, but more or less behind the back lens of the objective. This explains the over-estimate of am- plifications, especially in the lower powers, obtained by com- putation. It also shows that exact accuracy in such estimates is scarcely to be expected under any plan that may be adopted, and that to attain even average accuracy, it would be necessary either to lengthen the tube to an inconvenient extent, or else to habitually underrate, by general agreement, the powers of either the oculars or the objectives.] [The following table represents the magnifiying powers of the Bausch and Lomb objectives and oculars in their various combinations, determined by actual measurement with a mi- croscope tube-length of eight and one-half inches (21 cm.) in addition to the objective and ocular.] S" OBJECTIVES. J 3 1 4 1 j 1 1 1 4 3 2 1 1 To 4 5~ IT 8 10 12 16 A a c 12 18 25 4B 50 92 130 210 275 325 400 550 650 800 B o 15 23 30 54 70 110 160 250 325 390 490 650 775 9SO C ft 23 30 45 80 90 165 240 375 485 580 750 970 1160 1500 D 30 45 60 108 140 220 320 500 650 780 980 1300 1550 1960 ^ 1 TABLE OF OBJECTIVES. 49 [By including in this table, objectives up to one-fiftieth, and oculars up to one-fourth or one-eighth, all of which are made, the resultant powers would extend well up into the tens of thousands ; such powers, however, are but little used or es- teemed. These oculars do not correspond to even fractions of an inch, and are here designated by letters, after the early Eng- lish style which is not yet wholly obsolete ; but the makers in- tend hereafter to conform to the numerical plan.] [As the nominal focal lengths of objectives, while liable to moderate errors and discrepancies, indicate the amplifying powers sufficiently well for most purposes, it is only necessary in stating the powers made by the American manufacturers, to tabulate the equivalent focal lengths of their objectives. As to oculars, the lowest is usually a one and one-half or two inch (38 to 51 mm.) ; the one inch (25 mm.) is much used and is said to be the power employed by prominent makers in correct- ing their objectives, and those of from one-half inch (13 mm.) upwards are used for such special purposes as inicrornetry, or counting fine lines.] [The amplifications with two inch (5cm.) ocular, given in the fourth column of the table on pages 50 and 51, are calculated for the ten inch (25 cm.) distance, and are therefore somewhat overrated practically. It should be added that the highest powers named are seldom if ever made or used ; and that, to say the least, the work of most microscopists can be done more easily, if not better, with a 1 or JL than with a . JL or A.] [In the table the latest published price (1884) of each lens is annexed, to show the present value of such work, and to in- dicate the significant relation, in the various lenses of each maker, between angular aperture and price. In a few instances other considerations overrule this law, and give an unexpectedly high or low figure. It is claimed by the makers, though often earnestly disputed by others, that the excessive prices of the high-angled lenses are not fictitious values based upon a monop- oly of the supply, but they only adequately represent the amount of labor actually expended in overcoming the practical difficulties of construction. On the other hand, it is certain that the day has fortunately passed away when lowness of price 4 50 THE MICROSCOPE IN BOTANY. TABLE OF AMERICAN OBJECTIVES. roc 8 1 US. c POW B <1 ER. ^ J5 P BAUS LOJ 9 5 f o Pi <; CH & IB. 1 3 $ Aperture o ft cs ow. oo $ Aperture X .ACH. to 6 $ Aperture g V. JER. 1 3 Is Aperture ^ > r,ES. 1o O 3 $ ZEN MAYI - < T- :R. "a $ 5 127 2 10 /5-3\ UnJ 18 /5-3\ \in.) 20 (**} \ln./ 15 4 102 24 13 6 10 12 6 13 18 12 18 8 /4-2\ UnJ 8 20 1 /4-2\ VtaJ 8 20 9 12 3 76 8* 17 9 12 16 6 13 18 12 18 11 13 8 20 8 13 7 20 12 17 2 4 1 51 5 61 25 33 12 15 22 6 13 18 15 20 22 15 J8 20 15 18 6 20 10 16 20 7 18 30 38 16 24 32 6 15 20 15 22 30 12 18 25 18 24 6 18 23 17 23 15 25 10 50 20 36 45 6 15 25 25 30 50 15 20 35 15 24 26 36 85 5 12 25 20 80 25 33 40 12 22 40 25 17 8 10 3 4 20 19 12 62 26 ?2 10 18 13 67 27 40 8 15 2 3 17 15 20 75 100 25 35 65 15 22 30 25 36 40 43 6 10 15 25 32 86 47 12 22 30 30 17 32 18 1 2 13 42 65 98 9 18 30 65 18 27 40 50 72 110 7 10 20 40 50 50 n 70 100 15 25 50 4 10 1 3 10 25 30 125 150 70 110 13 34 50 75 100 18 25 35 60 80 110 20 20 to 75 95 115 30 35 40 60 80 22 30 8 120 35 1 4 1 5 6 5 40 50 200 250 n 75 100 125 na.h 1.40 14 17 24 100 n 75 20 n 75 n 80 na.hi 1.20 14 25 60 n 100 115 13 na.di 1.28 16 30 40 70 100 135 i 170 25 90 120 18 35 110 130 18 28 90 110 135 20 35 40 135 na.hi 1.40 22 90 35 40 THE OBJECTIVE-SYSTEM. 51 TABLE OF AMERICAN OBJECTIVES. Continued. Inches 3 1 us. i s PO^ \Viih2in. w ocular F fS a.mi.iodv CH& MB. 3 o i Aperture O * d ow. 6 $ GUXD ! o LACH. * Aperture " w V, rt ER. 5 1 Aperture ^ LES. 1 $ ZEN MAY 1 s. T- KR. ,0 $ 1 6 4 60 300 140 i 165 na.hi 1.18 na.hi 1.40 40 23 45 70 n 100 110 165 20 25 40 n 80 na.gi 1.12 na.hi 1.20 16 30 45 di 175 na.i 1.21 na.lii 1.35 40 40 70 1 b 3 80 400 115 135 i 170 na.hi 1.18 na.hi 1.40 21 30 25 50 75 150 45 135 na.hi 1.40 24 80 120 i 135 di 135 150 di 175 25 25 32 45 40 1 10 1 12 2.5 2.1 100 120 500 170 na.hi 1.18 na.hi 1.40 28 50 80 130 i 175 30 50 na.gi 1.12 na.hi 1.20 25 35 na.i 1.25 na.hi 1.27 nahi 1.35 50 60 80 135 170 35 45 600 130 i 175 na.hi 1.18 na.hi 1.40 27 30 55 90 135 na.hi 1.40 40 90 1 15 1.7 150 750 i 160 75 i 150 150 na.i 1.17 40 60 66 i 170 65 1 16 1 18 1.6 160 180 .800 i 175 na.hi 1.18 na.hi 1.40 35 65 125 na.gi 1.12 na.hi 1.20 na.hi 1.40 40 50 120 135 na.hi 1.35 40 125 1.4 900 di 175 70 1 20 1 25 1.3 200 1000 i 170 100 na.hi 1.20 na.hi 1.40 75 160 160 1 250 1250 na.hi 1.40 200 na.hi 1.20 nahi 1.40 120 220 na.hi 1.35 150 100 1 32 1 50 0.8 320 1600 na.hi 1.20 150 0.5 500 2500 na.hi 1.20 200 na.hi 1.17 270 1 75 0.3 0.2 750 3750 9901 na.hi 1.20 260 na.lii 1.17 ??? 1 100 1000 na.hi 1.20 300 [References in table: i = immersion; di = dry or immersion ; gi = glycerine immersion ; hi = homogeneous immersion; na=numerical aperture; n = non-adjustable, all lenses of over 65, if not so marked, having screw collar adjustment for thickness of cover.] 52 THE MICKOSCOPE IN BOTANY. indicated poorly finished or imperfectly corrected lenses ; the cheapest objectives, by the best makers, being carefully corrected and neatly mounted, and owing their cheapness to the ease with which low-angled lenses can be corrected and the simplicity with which they may be mounted. They are there- fore adequate for any work, however important, that requires the use of such angles ; and they are most commonly supplied in connection with the various grades of students' and physi- cians' microscopes.] [The focal distances and angular apertures in the table on opposite page are given on the authority of the several makers. There is reason to believe that these data, on account of ex- tensive agitation of the subject, are more carefully determined and more accurately stated than formerly. The apertures given are the air-angles, unless otherwise stated. In lenses of the highest grade the numerical aperture is necessarily given, there being no corresponding air-angle. The Aperture Table of the Royal Microscopical Society, founded upon the elaborate re- searches of Prof. Abbe, is reproduced here as a most convenient means of comparing dry, water and homogeneous immersion lenses, and of applying the doctrine of Numerical Aperture as a measure of their theoretical illuminating, resolving and penetrat- ing powers. For instance, 180, the theoretical maximum of air angle, corresponds to unity of numerical aperture, and of illu- minating and penetrating power ; this being practically equiva- lent to only 97 31' of water angle or 82 17' in homogeneous media corresponding to crown glass, and having a theoretical resolving capacity for 96,400 lines to the English inch. Higher apertures, as readily shown by the table, are practicable only in immersion lenses, and give increased illuminating and resolv- ing but lessened penetrating powers ; lower apertures having exactly the reverse characteristics. R. H. W.] There is a widespread opinion that the more a microscope magnifies the better it is. This is really the case, however, only under certain conditions. A microscope which magni- fies very powerfully but at the same time gives imperfect, indistinct, and poorly illuminated images is far less to be preferred than one which gives much smaller magnification but produces APEKTITKE TABLE. The " APERTURE" of an optical instrument indicates its greater or less capacity for re- ceiving rays from thu object and transmitting them to the image, and the aperture of a Microscope objective is therefore determined by the ratio between its local length and the diameter of the emergent pencil at the plane of its emergence that is, the utilized diameter of a single-lens objective or of the back lens of a compound objective. This ratio is expressed for all media ami in all cases by n sin u, n being the refractive index of the medium and u the semi-angle of aperture. The value of n sin u for any partic- ular case is the "numerical aperture" of the objective. Diameters of the back lenses of various Dry and Immersion objectives of the same power (4 in.) from 0-50 to 1 52 N. A Numerical Aperture. (7t bin u = a. Angle of Aperture (= 2 u.} Illuminating power. ( 2 -) Theoretical resolving power, in lines 10 an Inch. (A = 0-S2C9 M = line K.I Penetrating power. (^) if! "o Water- iwmerKion objectives. (?t = 1-33) Ilovwf/eneons- immcrxion olljcctives. ( = l-52.) 1-52 .. 180 0' 2-310 140.528 658 1-50 .. .. 161 23' 2-250 144,000 607 1-48 .. 153 39' 2-190 142,072 076 1-46 >t .. 147 42' 2-132 140,744 685 1-44 M .. 142 40' 2-074 138,816 094 1-42 .. 138 12' 2-016 136,888 704 1-40 134 10' 1-900 134,900 714 1.38 >> tm 130 20' 1-904 133,032 725 1-36 rt 126 57' 1 850 131,104 735 1-34 .. 123 40' 1-790 129,170 746 1-33 tt 180 122 6' 1-770 128.212 752 1-32 105 50 120 33' 1-742 127,248 758 1-30 M 155 38 117 34' 1-0'JO 125,320 709 1-28 148 28 114 44' 1-038 123,392 781 1-26 142 39 111 59' 1-588 121,464 794 1-24 137 30 109 20' 1-538 119,536 806 1-22 133 4 106 45' 1-488 117,608 820 X^"~~^^^ 1-20 .. 128 55 104 15' 1-440 115,080 833 ff ^\\ 1-18 125 3 101 50' 1-392 113,752 847 M 1*16 j] 1-16 121 20 99 29' 1-340 111,824 802 v JJ 114 1 118 00 97 11' 1-300 109,896 877 ^^^^s 1-12 lt 114 44' 94 50' 1 254 107.908 893 1-10 111 30' 92 43' 1-210 100,040 909 Q 1-08 1-06 1-04 1-02 100 0-98 180' 0' 157 2' 108 30 105 42' 102 53' 100 10' 97 31' 94'50' 90 33' 88 20' 80 21' 84 18' 8217'i 80 17' 1-100 1-124 1-082 1-040 1-000 900 104,112 102,184 100,250 98,328 90.400 94,472 920 943 902 980 1-000 1-020 0-96 147 29' 92 24' 78 20'! -922 92,544 1-042 0-94 O-92 0-90 0-88 140 6' 133 51' 128 19' 123 17' 89 50' 87 32' 85 10' 82 51' 76 24' -884 74 30' -840 72 30' -810 70 44' -774 90,616 88,088 80,700 84.832 1-004 1-087 1-111 1-136 086 118 38' 80 34' 68 54' -740 82.904 1-163 0-84 114 17' 78 20' 67 0' -700 80,976 1-190 /* s \ 0-82 110 10' 70 8' 05 18'; -072 79.048 1-220 ( *80 / 0-80 106 10' 73 58' 03 31' -040 77.120 1-250 V J 0-78 10231' 71 49' 01 45' -008 75,192 1-282 ^+~~*S 0-76 98 50' 09 42' 60 0' -578 73.264 1-316 0-74 95 28' 67 30' 58 10' -548 71^336 1-351 0-72 92 (>' 05 32' 56 32' -518 09.408 1-389 0-70 88 51' 63 31' 54 50' -490 07,480 1-429 0-68 85 41' 61 30' 53 !)' -462 05,552 1-471 0-66 82 30' 59 30' 51 28'! -430 03,024 1-515 0-64 79 35' 57 31' 49 48' -410 01,096 1-562 0-64 0'60 0-58 70 38' 73 44' 70 54' 55 34' 53 38' 51 42' 48 9' -384 40 30' -360 44 51' -330 59,708 57,840 55,912 1-613 1-667 1-724 0-56 68 6' 49 48' 43 14' -314 53,984 1-786 54 65 22' 47 54' 41 37' -292 52,050 1.852 0-52 62 40' 46 2' 40 0' -270 50,128 1.923 1 0-50 00 0' 44 10' 38 24' -250 48,200 2-COO EXAMPLE. The apertures of four objectives, two of Avhicli are dry. one water-immersion, and one oil-immersion, would be compared on the angular aperture view as follows: 10(> (air), 157 (air), 142 (water), 130 (oil). Their actual apertures are, however, as -80 (air), -98 (air), 1'26 (water), 1-38 (oil), or their numerical apertures. COMPARISON OF ENGLISH AND METRIC MEASURES. Scale show- ing the rel;itioii of Millimeters, etc., to Inches, mm. and cm. in. Jfierc M ins. 1 -000039 2 -000079 3 -000118 4 -000157 5 -000197 6 -000236 7 -000276 8 -000315 9 -000354 10 -000394 11 -000433 12 -OOU472 13 -000512 14 -00055 1 15 -000591 16 -000030 17 -000669 18 -00071,9 19 -000748 2O -000787 21 -000827 22 -000866 23 x -000906 24 -000945 25 -000984 26 -001024 27 -001063 28 -001102 29 -001142 30 -001181 31 -001220 32 -U01260 33 -001299 34 -001339 35 -001378 36 -001417 37 -00 14.-) 7 38 -001496 39 -001535 4O -001575 41 -001614 42 -001654 43 -001(593 44 -001732 45 -001772 46 -001811 47 -001850 48 -001890 49 -001929 50 -001969 60 -002302 70 -002756 80 -003 1 50 90 -003543 100 -003937 200 -007874 300 -011811 millimeters, etc., int mm. ins. 1 -039370 2 -078741 3 -118111 4 -157482 5 -190852 6 -236.'23 7 -275593 8 -314963 9 -354334 10 (1 cm.) -393704 11 -433075 12 -472445 13 -511816 14 -551186 15 -590550 16 -629927 17 -669297 18 -70*668 19 -748038 2O (2 cm.) -787409 21 -826779 22 -866150 23 -905520 24 -944890 25 -984261 Z6 1-023631 27 1-063002 28 1-102372 29 1-141743 3O (3 cm.) 1-181113 31 1-220483 32 1-259854 33 1-299224 34 1.338595 35 1-377965 36 1-417336 37 1-456706 38 1-496076 39 1-535447 4O (4cm.) 1-574817 41 1-614188 42 1-653558 43 1 -692929 44 1-732299 45 1-771669 46 1-811040 47 1-850410 48 1-889781 49 1-929151 5O (5cm.) 1-968522 decim. 1 2 3 4 5 6 7 8 9 10 (1 mete j Inches, etc. mm. ins. 51 2-007892 52 2047262 53 2-086633 54 2-126003 55 2-165374 56 2-204744 57 2-244115 58 2-283485 59 2322855 60 (6 cin.) 2-362226 61 2-401596 62 2-440907 63 2-480337 64 2-519708 65 2-559078 66 2-598449 67 2-637819 68 2-677189 69 2-716560 7O (7 cm.) 2-755930 71 2-795301 72 2-834671 73 2-874042 74 2-913412 75 2-952762 76 2-992153 77 3-031523 78 3-07094 79 3-110264 80 (8 cm.) 3-149635 81 3-189005 82 3228375 83 3-267746 84 3-307116 85 3-340487 86 3-385857 87 3-425228 88 3-464598 89 3-503968 9O (9 cm.) 3-543339 91 3-582709 92 3622080 93 3-661450 94 3-70082U 95 3-740191 96 3-779561 97 3-818932 98 3-858302 99 3-897673 100 (10 cm. =1 dcm.) ins. 3937043 7-874086 11-811130 15-748173 19-685216 23-622259 27-559302 31-490346 35-433389 ) 39 370432 = 3 280869 ft. = 1-093623 yds. Inches, etc., into micro- millimeters, etc. ins. ft T^TTO 1-015991 Tufoo 1-269989 -ndnnr 1-693318 To-tanr 2-539977 snAnr 2-822197 Tifoir 3-174972 ToW 3-628539 FoW 4-233295 TuW 5-079954 4iiW 6-349943 snAnr 8-466591 at-tar 12-699886 -roW 25-399772 mm. aio -028222 - 8 ta -031750 7^0- -036285 d-u -042333 050300 056444 063499 ;rb -072571 ib -084666 ^b -101599 -dro -126999 T5o- -169332 TO -253998 5*6 -507995 1-015991 - 2 V 1-269989 iV 1-587486 -iV 1-693318 i^ 2-116648 iV 2-539977 i 3-174972 4-233295 & 4-762457 | 5-079954 (5-349943 -^ 7.937429 | 9-524915 cm. fr 1-111240 ^ 1-269989 iV 1-428737 * 1-587486 U 1-740234 1-904983 fl|- 2-063732 I 2-222480 || 2 381229 1 2-539977 2 5-079954 3 7-619932 decim. 4 1-015991 _5 1-269989 6 1-523986 7 1-777984 8 2.031982 9 2-285979 10 2 539977 11 2-793975 1 ft. 3-047973 meters. 1 yd. = -914392 si ifl *; - ; : - 3 E a : - f " = E n I _ to * I L E to - 10 - * C : I p oi r I 400 -015748 500 -019685 600 -023622 700 -027559 800 -U314!)(5 900 -035433 1000 (=1 mm.) 1000 /u. = 1 mm. 10 mm. = cm. 10 cm. = 1 (in., 10 dm. = 1 m. TESTING THE OPTICAL POWERS. 53 clear and sharp images. It may be said, on the contrary, that those instruments are comparatively the best which, with rela- tively low magnifications, still show the details which appear in poor instruments only with the use of high powers. In judging of a microscope the first things to be thought of are these : Does it show a sharp and clear image, all details, all fine structural relations? What are its defining and resolv- ing powers? Then, one may inquire how many times it mag- nifies. We shall now proceed to show by a simple method how one may satisfactorily judge of the defining and resolving powers of a microscope. VII. TESTING THE OPTICAL POWERS. The defining and resolving powers of a microscope may be best tested by means of so-called "proof objects" or "test objects." These consist of small parts of animals or plants, and also of very small whole organisms which are prepared in a certain way. They should be examined with a known magnifica- tion, which should be produced mainly by the objective. It should then be ascertained if the image appears the same in this as in some notoriously good microscope, or comparison should be made with some distinct and clear illustration or exact description which may be accessible. If, then, one recognizes all those details which the illustration or description, with a like magnification, furnishes, it is a sign of the good quality of the microscope. But if the outlines of the form and the fine structural relations are more indistinct than in the illus- tration, or if they are in general not visible, it would thence follow that the instrument Would not fully satisfy modern re- quirements. Since the testing of the optical performance of a newly ac- quired instrument is the first thing to be taken in hand, the maker usually takes care to provide some test-object by which this may be done. Testing the instrument, particularly with objects of difficult resolution, should take place on a not too dark day, when the heavens are uniformly covered with a veil of transparent clouds ; 54 THE MICROSCOPE IN BOTANY. at all events, not when 'the sky is filled with numerous gray, rap- idly moving clouds which produce constant changes in the light. The microscope should be placed close before an open window facing the north or east. The illumination of the object should be by central light from the mirror, oblique light being in almost all cases unsteady. The size of the aperture in the diaphragm under the stage to be employed will depend upon the power of the objective in hand. It is also recommended that people who use spectacles should lay them aside when observing test objects, since the least particle of greasy matter, such as might easily be left on the glass by the motion of the eyelashes, would render the microscopic image less sharp and clear. It is self- evident that the objectives, oculars and the glasses holding the test-object should be perfectly, clean. We will first discuss those objects by which we test the de- fining power and afterwards those by which we test the resolv- ing power of the microscope. A. TESTING THE DEFINING POWER. All the objects used for this purpose in the magnifications employed must show an altogether distinct, clear, delicate and colorless outline. The test-objects used exclusively for defini- tion should be employed only with low or medium powers, while those for resolution should be used with the highest powers, they likewise giving at the same time tests of the definition. The most important tests for definition are the fol- lowing : 1. The Calcareous Plates of the Synapta. That part of the echinoderm group known as Holothurians or "sea-rolls" con- tains animals of a mostly cylindrical form whose bodies are cov- ered with a leathery skin. Imbedded in this skin are a great number of microscopically minute, perfectly colorless (rarely somewhat colored) calcareous bodies. The genus Synapta, of which representatives are found in the Mediterranean, but whose more numerous species occur in the warm seas of Polynesia and southern Asia, shows an extraordinarily delicate example of these small, calcareous bodies. The integument of these long TESTING THE DEFINING POWER. 55 worm-like animals is pretty thin, and growing in it are rectan- gular or roundish, perforated, calcareous plates (Plate I, Fig. 1, b) in which anchor-like hooks are fastened (Plate I, Fig. 1, a). 30 The fully formed calcareous anchors have a length of 0.97-0.98 mm., are as clear as glass and perfectly transparent. The end of the handle is slightly bifurcated and a little warty- rough. The anchor hooks are curved bow-like and bluntly rounded at the end. The rectangular little plates are about 0.8 mm. long, and are perforated with from 34 to 40 holes great and small, the larger being in the middle and the smaller towards the ends. 31 The anchors of the Synapta are suitable for testing the re- solving power of very low objectives only (10 to 50 magnifica- tions). It should show a contour in this magnification sharply bounded by a thick black line. In the perforated plates this contour, corresponding to its greater thinness, is more delicate than in the anchors. Poor glasses show an imperfectly defined outline with a soft haze around the edges. Lenses imperfectly achromatic produce a numercusly colored border near the edges. The illustration in Plate I, 1, is prepared from a magnification of about thirty diameters. The specimen should be mounted in balsam. 2. Transverse section of Coniferous wood. While the ob- jects already described are suitable only for testing the lower powers, we have, in a transverse section of the needle-bearing trees, a very good object for testing the definition of medium, and even stronger powers. It is a matter of indifference which one of the coniferous species one chooses from which to make the prep- aration. The one illustrated here, Plate I, Fig. 2, is a piece of the cross section of the young stem of the common fir tree (Pinus sijlvestris) which may be found anywhere. The prep- aration was made in this way. The most delicate possible section was made through the young stem. Xo dull places in the knife should be permitted to leave their marks upon the 30 The calcareous bodies from the following and other species of the Synaptae are suitable for test-objects. Synapta inhaerens, glabra, Godefroyi, recta, similis, molesta, Kefersteinii, Besselii, diyitata. 31 There are also species (for example, S. inhaerens') in which the calcareous plates have a few large holes with toothed edges. 56 THE MICROSCOPE IN BOTANY. section. How this is to be avoided will be shown more exactly hereafter. The section should then be put for some minutes in absolute alcohol to remove the resin from the resin tubes, 3 ' 2 then it should be washed in distilled water and mounted in glycerine- jolly or glycerine. For our purpose we shall consider the wood layer which forms one concentric ring. With high magnification the wood cell 33 presents the three following layers in its walls. The one layer which is common to the two cells, and which we shall with Sachs name the "middle layer," a, is thin and highly refractive. Then follows toward the inside a second stronger layer, which is compounded from several concentric lamellae, the intermediate thickening layer 6, and upon this lies a soft layer which lines the inside of the cell, and is called the inner thickening layer, c. All these layers should appear clearly and sharply differentiated from each other with a linear magnifi- cation of from 450 to 800 diameters. The middle layer is com- monly somewhat easier to see than the inner thickening layer. Hence the former will become visible earlier when the prepara- tion is put under the lens, and the latter as it is progressively subjected to higher and higher powers. The inner layer is best adapted to give account of the definition of high power lenses. With good objectives the inner layer should show itself sepa- rated from the cell cavity in sharp outline, a very sharp delicate line bounding it. Poor objectives, on the other hand, show a broad gray line about the cell cavity which becomes clearer in- wards, with no sharp limits but gradually loses itself in a delicate haze. It should be noticed that thick and imperfect sections produce the same kind of an image. On this account, only the thinnest possible sections, as already mentioned, should be used for this investigation. o Longitudinal sections of the woody parts of this plant furnish good test-objects for these magnifications and for somewhat lower powers. Here we find the great rounded " bordered pits" 34 which are very suitable for testing the definition of au 32 Sach's Lehrb. clev Bot., IV, Aufl. p. 95, Fig. 78. 33 Sachs, 1. c., p. 75, Fig. 57. s * Sachs, L c., p. 25, Fig. 23. TESTING THE RESOLVING POWER. 57 objective. Their entire outline should appear sharp and clear, as simple lines. 35 3. Scale-dots of Lycaena. We may for the same purpose, with good results, employ the scales of the "Bluelings." I in- deed recommend that the three species be used in the investi- gation : Lycaena Alexis F. (= Icarus Hbst.), L. argiolus L. or L. argus L. As with all butterflies their wings are closely covered with numerous small stalked scales, to which they are indebted for their lively colors. On the upper as well as on the under side of the wing are two kinds of scales, one of a longer form which bears on its surface delicate longitudinal markings, and another which consists of a longer style joined to an ellipti- cal plate. The latter are provided on the upper surface with a few (6 to 10) dotted longitudinal markings, Plate I, Fig. 3. These scales sKould be mounted in Canada balsam for test-objects. Good medium objectives magnifying 35(Xto 450 diameters should show in the former kind the longitudinal markings clearly as double lines. And in the latter the dots should appear as small circles which have a minute dark dot in the middle. The markings and the dots should not mingle or blend with each other. The specimen is somewhat more difficult to resolve when mounted dry, than in Canada balsam, and appears colored. B. TESTING THE RESOLVING POWER. In order to be assured of the superior quality of the higher and highest power objective-systems we must employ the best test-objects, which in the first instance permit us to judge of the resolving power, and at the same time are a test of the definition. Both qualities of the objective are essentially bound up to- gether. An objective-system with unsatisfactory definition will never resolve difficult images. But it sometimes happens that two systems of very nearly the same resolving power, showing the resolved details in sharper or fainter outlines, do not possess 83 Preparations of potato starch grains maybe employed as tests of definitions for medium powers. They should be examined in water or glycerine, and their separate layers, which are grouped about an excentric formation-point, should be bounded by a sharp, strong and delicate outline. (See Sachs, 1. c. Fig. 51. on p. 62.) 58 THE MICROSCOPE IN BOTANY. the same power of definition. The test-objects most often used for resolution are butterflies' scales and the siliceous frustules of different species of diatoms. 1. Scales of Hipparchia janira and Lycaena argiolus. Our common white butterfly, Hipparchia janira, has on its wings several kinds of scales, short, medium and long. A scale of medium length is illustrated in Plate I, Fig. 4. It has a breadth of 0.059 mm., and a length of 0.156 mm. It is rec- tangular, has three broad points at top, and is heart-shaped at bottom, ending in a short style. Its surfaces are covered with 22-24 longitudinal ridges, which have an average distance apart of 0.00266 mm., so that about four of these go to 0.01 mm. (10/Jt =10 micromillimetres). Magnified as in the illustration (305 diameters) we see very many delicate cross lines between the longitudinal elevations. The higher the magnification, the more the finer details are brought out, and the butterfly's scale therefore furnishes a very excellent preparation for testing the resolving power of the strongest objective-system. In recent times, Dipper 36 has most accurately investigated the butterflies' scales employed as test-objects, and we shall give here in his own words the results to which that naturalist has arrived in his very exact studies of these important tests. "The longitudinal flutings are made by the elevation of the upper surface, between which run furrow-like depressions so that the scale seen in section has a wavy aspect. When viewed with lower powers, and oblique light falling upon them in a direction perpendicular to their length, they appear to be bounded by two sharp lines. With higher magnification and central light, and with an objective of good definition, they assume a toothed appearance, and because of the cross-lines which lie in the same plane, and come into the focus at the same time, they take the appearance of being thickened at these points. With the lower powers, this structural relationship, on ac- count of its delicacy, shows with scarcely half the sharpness of the boundary lines. With oblique illumination, even with the medium higher powers, it is- overlooked, because the shadows cast by the longitudinal ridges apparently obscure 36 Dippel, /. c., p. 118 /. TESTIXG THE RESOLVING POWER. 59 these fine lines. Herein we see the foundation for the widely divergent views which microscopists have expressed about the real nature of these longitudinal markings. Thus, for example, Brewster (Treatise on the Microscope) declared that the cross markings did not exist at all, but that the longitudinal ridges c5 O O were beset with small teeth. Chevalier (Les Microscopes, etc.,) described the scales of Pieris brassicae as beset with longitudinal ridges which were formed by minute balls placed near each other, and held that the true test of an objective- system consisted in making these globules visible. Some English microgra pliers agreed with this. Others, for example, Goring, then under the German H. v. Mohl, controverted it, and asserted the existence of sharply defined longitudinal and transverse markings, and held Chevalier's description of this test-object to be a plain witness that the latter had misinter- preted his microscope. Brewster is only partly in the right with his statement, since he quite overlooked the cross-lines, or rather failed to see them altogether ; but Chevalier decided correctly. I have examined the same object anew, with several of the best objective-systems of recent times and find them formed as Chevalier asserted. , The well-known heart- shaped scales of Pieris brassicae 37 are, especially over their upper surfaces, both on the longitudinal markings and interven- ing spaces, beset with small, irregularly angled, or roundish bodies, whereby, under certain conditions of illumination the appearance of cross-lines is produce.d, which run between and near the longitudinal lines, but which by direct illumination and good defining objectives are seen to appear in the way pointed out by Chevalier. In most scales, the cross markings run in a direction perpen- dicular to the axis of the longitudinal ridges ; in others, in an oblique direction, as well over the summits of them, as across the intervening spaces, without interruption. In those adjust- ments of the microscope, however, with which one commonly chooses to see the cross marking between the others with dis- tinctness, these facts easily escape observation. Only by a certain medium adjustment do they come forth clearly. The 37 See Dippel, 1. c., Figs. 61 and 62, on p. 119. 60 THE MICROSCOPE IN BOTANY. cross markings are not like the longitudinal, elevations, but rather depressions between the more or less regularly rectangu- lar to roundish bodies, which as a rule stand in series of four between each two of the longitudinal markings. Thence be- tween each two of the stronger longitudinal markings, there are three others very much more delicate, and which are far more difficult to see than the transverse markings, and afford good tests of the strongest lenses. The best objective-system in which is united the greatest resolving power with the best definition as well as the most perfect chromatic correction, can alone make us acquainted with this structure. 38 Bruno Hasert had already, in 1847, traced out this structure and since then more fully examined it. (Official report of the 34th meeting of German Naturalists and Physicians, at Karlsruhe, p. 212.) At his suggestion, I have likewise most carefully examined this object with my most powerful objectives, and have convinced myself definitely of the correctness of his representation. Tims we have established a solid footing as to how these cross markings ought to look through good objectives. In the same way we explain the diagonal markings on these scales, which one perceives with certain illuminations and with strong objec- tives. If we examine the transverse lines with direct light and a magnification of 300 to 500 diameters they will appear as if serrated, but they must be sharply defined for that. With higher magnifications the separate little bodies will appear with clear delicate outlines, as soon as the spherical aberration of the objective is perfectly corrected. Oblique illumination, on the contrary, is the cause of obscuring the true structure with low magnifications, and affords sharply marked linear cross stripes, as they have been heretofore represented by those mi- croscopists who have in such testing worked with their mirrors excentrically placed. Objectives also which are not strictly first-class with respect to definition, or have imperfect correc- tion of chromatic aberration will give that kind of an imnge, with high magnifications. On this we may ground objections to a system which is known to be otherwise excellent, that it shows the cross markings of the Hipparchia scale as serrated. On the 38 See Plate I, Fig. 7, which represents a copy of Dippers, Fig. G9. TESTING THE RESOLVING POWER. 61 contrary, we must accept it as evidence of the good quality of an objective, that it shows these markings with central illumi- nation quite distinct and sharp, while one which does not thus show them betrays a lack of defining power. The diagonal lines come out when the oblique light falls upon the scale at an angle of 30 to 60 to the axis of the longitudinal markings, while the delicate longitudinal lines come out most clearly when the oblique light falls perpendicularly upon the longitudi- nal axis. In respect to the visibility of the two last named systems of lines, the butterflies' scales furnish test-objects of a difficulty almost equal to the diatoms, without, however, afford- ing a sufficiently perfect series of comparisons." It should be added that Plate I, Figs. 5, 6, and 7, shows us single pieces of the scale of Hipparchia janira under different magnifications, Fig. 5 X 500, Fig. 6 X 1450, Fig. 7 X 1920 times, the last two after Dippel. 39 Lycaena argiolus has scales which are somewhat more difficult to resolve than those of Hipparchia janira in respect to their corresponding fine markings. Fig. 8, Plate I, represents a whole scale x 305 times. Fig. 9, a piece X 500, and Fig. 10, a small piece X 1450 times (after Dippel). 40 Butterfly scales are commonly mounted in Canada balsam for test-objects. Mounted dry they are somewhat more difficult. Glycerine mounting seems to me very serviceable also. 2. The Siliceous frastides of the diatoms. There occurs in the mire and on plants in stagnant waters, and also in the sea, a group of single-celled algse in an almost endless variety of forms and species, known in general as diatoms. They are throughout of microscopical minuteness, and are distinguished from all other algae by having their cell walls covered with a framework of pure silex, which consist of frustules that fit upon each other and represent a perforated lattice- work of the most delicate structure. This siliceous shield consists of two separable halves. If the diatoms are boiled in a mixture of potassium chlorate and nitric acid the entire organic substance will be destroyed and only the siliceous frames will be left over so Dippel, 1. c., Figs. 63 and 69, on p. 119 and 122. *>Dii pel, 1. c., Fig. 73, 0.1 p. 1-23. 62 THE MICROSCOPE IN BOTANY. with the two halves parted. These separated halves of the frustules are what furnish the most excellent test-objects. They are prepared in two ways, either mounted dry or in bal- sam, and the method of examination as test-objects is determined by the particular kind of mounting. On the whole, those mounted in balsam are more difficult to resolve than those mounted dry. Dry mounts are most suitably made as follows. After the diatoms have been separated by the mixture of nitric acid and concentrated solution of potassium chlorate, the fluid containing the diatoms is put into a high and narrow test tube, and the diatoms are allowed to settle to the bottom. They are then repeatedly washed in distilled water till litmus paper no longer shows any trace of the acid. A sample of the diatomiferous fluid is taken out with a pipette, and placed on a clean slide, and the water allowed to evaporate in some place free from dust, (for example in a drying chamber) and a cover glass cemented on over it, as will be described further along. The balsam mount is made in the following way. A small portion of the cleaned diatomiferous fluid is put in a watch glass and evapo- rated. The residue is mixed with pure oil of turpentine, and then a drop of the turpentine oil with the suspended diatoms is mingled with a drop of Canada balsam on a slide ; lay on a cover glass and fasten with gentle warming. 41 Several species of the genus Pinularia make tests for very low powers. The cross markings on the long sausage-shaped frustules of Pinularia nobilis Eh. can be easily and clearly seen with a magnifying power of thirty diameters, but the like form- ations on the elongated elliptical* body of P. viridis^Rh. must be magnified 209 diameters to be distinctly seen. Since we have shown how the butterfly scales may most suitably test the 41 Except in rare cases, we shall not ourselves undertake the preparation of the diatom test-objects. They can be had at the best microscopical institutes (Dr. Kaiser in Berlin, Mollerin Wedel, Holstein, Rohdig in Hamburg, etc.) [In America all of the principal dealers in microscopical goods keep diatom tests on sale, and I believe Mr. C. L. Petico- las of Richmond, Va., has made a specialty of the preparation of these diatom test-objects, A. B. H.| at the price of one to two Marks each (25 to 50 cts.) Moller furnishes a diatom test plate which contains under the same cover-glass, a number of diatom frustules for test-objects so arranged that tiiose at one end are easiest, and they become progressively harder of resolution towards the other end, so that it is possible to test the power of resolu- tion of all systems by means of this one preparation. TESTING THE RESOLVING POWER. 63 lower powers, we need not further consider the diatoms as tests for objectives of that kind. For objective-systems of medium magnifications, the various species of the genus Pleurosigma make most extraordinarily beautiful test-objects. They are almost all good tests also for the higher powers. The species of the Pleurosigma are easily recog- nized by the peculiar sigma-like (?) curvature of their bodies. A doubly curved central line is drawn along their whole length which in the middle is expanded into a lengthened nodule. On each side of the central line, the frustule is covered throughout with a framework of delicate lines, which now lay claim to our exclusive attention. The species used as test-objects, may be separated into two groups according to the construction of the above mentioned framework or skeleton. The first includes the species Pleurosigma balticum and PL attenualum; to the second belong PL angulatum and PL formosum. (a) We shall first consider Pleurosigma balticum Sm. This little plant is from 0.29 to 0.33 mm. long, and should be mounted in Canada balsam when used as a test object. Plate I, Fig. 11-14. By a magnification of 100 diameters the object appears as a delicate hyaline form, of the shape of Fig. 11. We distinguish the lateral boundary edges, and the beautifully curved central line with a knot in the middle. With this mag- nification we may perceive either by central or oblique light, minute carvings on the surface of the frustules. If the magnifi- cation be now raised to 200 diameters, by means of a stronger objective and the weakest possible ocular, the surface will appear to be covered with a very delicate lattice-work. We see at once that this consists of longitudinal and transverse striae. By different focussing, now the former, and now the lat- ter, will be made distinctly visible, particularly towards the outer edges. Now, if we choose a higher ocular, taking the same objective, and make the magnification 300 diameters, the image will become a little clearer since the single lines will be farther apart. Two transverse lines stand at a distance of 0.0007 mm., so that about fifteen of them go to make 0.01 mm. An objective magnification of about 460 clearly resolves, the lattice-work into two systems of lines which stand perpendicu- 64 THE MICROSCOPE IN BOTANY. lar to each other, one running lengthwise and the other across the diatom. We now without difficulty see that at the inter- secting points of the atrice of the two systems are knot-like thickenings, Plate I, Figs. 12, 13, the form of which is appar- ently rectangular. A little stronger ocular magnification (550 to 590) Fig. 12, 13, makes the image still somewhat more distinct. Finally, the markings appear to be perfectly resolved with a magnifica- tion of 950, Plate I, Fig. 14. The knots have now clearly a four- sided form, and by a still stronger magnification (1400 to 1450) we recognize the fact that they are in reality six-sided, but the six angles are not regular, two opposite sides being shorter than the other four. 42 (b) Pleurosigma angulatum Sm. forms the diatom test which is properly most in use for medium and higher powers. They should be mounted dry. If mounted in Canada balsam, they are considerably more difficult of resolution, and the fol- lowing description would not apply to a balsam mount. PL an- gulatum, which may easily be distinguished from all other species of the genus by its form, attains a mean length of 0.24 to 0.32 mm. Both sides of the middle portion of the frustule are drawn out somewhat sharply angular, Plate II, Fig. 1, whence it gets its name. The middle rib differs but little from PL balticum only being a little slenderer and lacking the slight curvature each side of the central nodule. With low magnification PL angulatum has a bright yellow-brown color. We shall now examine the structure of the frustule of this diatom with low, medium, higher and highest magnification. 1. Low powers (50-150 diameters). The surface shows a perfectly homogeneous aspect. The color is uniformly clear 42 To those who are disposed to undertake the testing of microscopes for themselves by means ol'these objects, we especially commend the work of Fritschnnd MUller. -'The carv- ings and finer structural relations of the Diatomacece, with reference to the use of the spec- ies as test-objects. Part I, 12 plates, photo-micrographic illustrations, Berlin, 1370, 4to. Price 16 Marks, each plate singly 1.6 Marks." The plates represent beautiful photographs direct from the microscope : I. Diatom type plate, No. II of J. D. Moller in Wedel, magnified 90 diameters. -II. Arachnodiscus ornatus Ehrbg. X 530. III. Triceratium favus Ear. X 545.- IV. Pinularia nobilis Kg. X 545. V. Navicula Lyra Ehr. and var. X 530. VI. Stauroneis P/teemce??*mw Ehr.X545. VII. Pleurosigma balticum Sm.X545. VIII and IX. PI. angulatum Sm. X 515, 1200. X. Grammatophora marina Sm X 545. Gram, oceanica Ehr. = G. subtitissima; X "00. XI., XII. Surarella gemma Ehr. ; X ^ co , 1200. TESTING THE RESOLVING POWER. 65 brown. Oblique illumination reveals no further details on the surface. The diatom cannot be used as 11 test for lower powers. 2. Medium powers (200-400 diameters). With a mag- nification of 200 to 250 diameters, produced in conjunction with the weakest ocular, we first recognize traces of markings, Plate II, Fig. 1, which come forth in a dark brownish shade. Concerning the nature of these markings, this magnification permits us to say nothing further . But if one raises the mag- nification by a somewhat stronger ocular, to about 300 diarn*- eters, and uses oblique illumination, the markings will be distinct, and recognized as consisting of three systems of stria-,. which are inclined to each other at an angle of about 120. With this magnification, however, with the use of oblique ilr lumination, we recognize these systems only when the light falls upon them severally at an angle of 90. Concerning this quality which it is well to regard, Dippel 43 first remarked : "If the oblique light be applied to one side, there will appear one or the other of the systems of lines according to the situation of the diatom. When the oblique light falls parallel with its long- est axis the somewhat more widely separated cross-lines will appear. If now we turn the light about 90, the more closely drawn diagonal systems of lines will appear, with somewhat the same sharpness. On the contrary, only one of these systems of lines will become sharply visible when the longitudinal axis of the frustule forms an an^le of about 45 with the direction of C the rays." Condenser illumination, without stopping off the central rays, gives on the whole a much clearer picture than the above described process. 3. Higher magnifications (450 to 900 diameters). When the objective magnification reaches 450 to 480 diameters, the three systems of lines on the surface of the frustule become distinctly visible, mutually crossing each other at an angle of 120, Plate II, Fig. 2. But we next observe that what at first appeared to be striae are not straight lines, but that they are hexagons lying side by side in rows in the same direction, which cover the whole surface of the diatom with the most extraordi- narily delicate lattice-work, Plate II, Fig. 4. With a magiiiti- Dippel, Das Mikroskop. Bd. I, p. 12S. 5 66 THE MICROSCOPE IN BOTANY. cation of 450 to 480 diameters, the hexagons can be seen distinctly only when the light is central and the focussing upon a given place is extremely sharp. If the illumination is excentric some one system will prevail, according to the direction in which the lattice-work is illuminated, as is indicated above. 44 Especially, with central condenser illumination, the image becomes very clear. By right focussing, the little hexagonal surfaces will appear quite colorless, and their sharply bounded contour some- what chocolate colored. 45 When the image is produced with a good objective, it ought to bear considerable ocular magnification without impairing its distinctness. Produced with a good Gundlach's immersion- system, VIII, the image bore an ocular magnification of 1000 to 1400 diameters, still showing the corners of the hexagon per- .fectly angled and sharply defined. 4. The highest magnifications (900-2000 diameters). With magnifications of over 900 the resolution of the hexagonal .network will become still more distinct. Since the true diam- eter of the hexagon is about 0.005 mm., it is evident that it may be very distinctly perceived when magnified, we will say, to 1000 times. We have repeatedly studied PL angulatum with the Seibert immersion-system, No. IX (x 950, 1430, 2170, 2880), and have seen its minutest structure with central and oblique illumination and with the use of all oculars ; and most beauti- fully by the use of a condenser with the middle rays shut out. Plate II, Fig. 4, shows the image with a magnification of 2880 with the above mentioned system ; Fig. 3, with a linear enlargement of 1200, the latter from the photo-micrograph of Fritsch, Plate IX. The species of the genera Grammatophora and Nitzschia are connected with those of Pleurosigma in affording the very best test objects for the higher and highest powers of the microscope, as is also Surirella gemma whose delicate siliceous frustule can be resolved only by the highest and best magnification. 4* This unequal prevalence shows very well in Plate VIII of the above cited work of Fritsch and Muller which represents a Pleurosigma angulatum with excentric illumination. The photograph should be examined in different places with a good magnifying glass. 45 By wrong focussing, exactly the contrary effect is produced ; the surfaces are dark and the contour bright. TESTING THE RESOLVING POWER. 67 (c) Grammatophora marina Sm. The body of the Grammat- ophora species can be compared, as to its form, with nothing better than a cigar case. The exterior aspect of this genus of diatoms presents the form of a more or less elongated rectangle with the angles blunt or rounded, Plate II, Figs. 5, 7. Next to the middle, run two zigzag-like bent, coarse lines, whose form and position vary according to the species. Outside of these longitudinal lines, on the whole extent of the outer border, runs a zone which is filled with the most delicate transverse striae. These exclusively claim our attention here. If we examine the Gr. marina (in Canada balsam) with a magnification of 200 diameters, the cross lines spoken of are scarcely visible. We shall see them only after looking a long time. The markings beceme somewhat more distinct, with the use of excentric illumination, or better still with condenser il- lumination. The weaker ocular magnifications change the im- age but very little, but if it be raised to about 600 diameters, and excentric illumination be used, every cross line will appear to consist of a clear bright and a dark or shadowed line. If now w r e raise the objective magnification to 450480 diam- eters, the markings become very distinct, both by central and central-condenser illumination. Light which falls upon them obliquely, and especially in a direction perpendicular to the markings, brings out the image much more distinctly. With central illumination and very high focussing, the striated part of the diatom appears to be covered with minute points ; deeper, the cross lines seem to be granulated. The image is most distinct when the focus is made to touch the middle between the upper and lower surface of the diatom. 46 Fig. 6 represents Gr. marina magnified 600 diameters. Stronger magnifications (900 to 1200) change the image but 46 After a very exact study of the Grammatophora species, I here express the view that the transverse strife are formed of a series of granular elevations standing one behind an- other. In this 1 come into controversy with Dip|el who supposes that besides the smooth transverse striation, there are other systems of lines which lie over these. "In all other species, there occurs, as in PI. angulatum, along with the transverse strice, another diagonal system of lines which cross these, and which in Gr. subtilissima are extremely difficult to see, and for this resolution it is necessary to have the most favorable illumination by the use of well regulated oblique light." (Dippel. /. c.. Bd. I, p. T29.) I hold on these grounds that Dippel's illustrations, Figs. 87-90, are not altogether true to nature. Compare them with Fritsch and Moller, I. c.. Plate X. 68 THE MICROSCOPE IN BOTANY. little. Gr. marina is principally recommended as an object for the weaker immersion systems. (d) Grammalophora oceanica Ehrbg.:=6rr. subtilissima . This species is distinguished from the one described above by its differing form (it is slenderer and longer than Gr. marina) and by the considerably greater fineness and difficulty of resolution of the transverse fitrice. While the transverse lines of Gr. ma- rina stand about 0.00041 mm. apart, those of Gr. oceanica are removed but about 0.0003 1 mm. from each other. An objective magnification (dry lens) of 200 diameters does not resolve the markings in the least, neither by the use of central, oblique, or condenser illumination, nor even if the magnification be raised by means of oculars to 400 or 500 diameters. The same thing happens with the lowest immersion system whose lowest ocular magnification does not exceed 400 or 500. It is only when the magnification reaches 700 with a low ocular that the cross bars appear in the form of very delicate lines, Plate II, Fig. 7. With still higher immersion systems, 1200 to 1500 di- ameters, they appear still more distinct and differ in no import- ant way from those of the species described above. Fig. 8 shows this in a balsam preparation. (e) We may briefly make mention here of a very good test- object from another genus of diatoms, Nitzschia linearis, Plate II, Figs. 9, 10. It has a peculiar wand-like form, deeply chan- nelled on the outer edge. Proceeding from this there are drawn over the whole surface transverse lines, which in respect to del- icacy, and distance from each other, stand between the two species of Grammatophora (0.00036 mm.). A clear resolution of these can be reached only by the strong immersion-systems. Fig. 10, Plate II, represents likewise a balsam preparation. (f) An altogether superior diatom test-object, which can be perfectly resolved only by means of the most powerful sys- tems, is the Surirella gemma Ehrbg. It should be mounted dry, and even in that condition it is extraordinarily difficult to resolve. Plate II, Figs. 11-13, represents a view of it magnified by object- ive-systems 500, 700, and 1200 times. The form of the j$u,ri- rella is oval and the ends are but a little pointed. The oval surface is marked over with a framework of strong siliceous TESTING THE RESOLVING POWER. 69 bars which run across, at one side are quite irregularly joined to the thickened border, and on the other to the longitudinal middle line. In the fields between these bars not the least trace of markings is discernible, even with a magnification of 500 diameters. See Fig. 11. With an objective magnification of nearly 700, lines become visible in the fields which run parallel with the cross bars. See Fig. 12. Indistinctly with this mag- nification, but clearly with one of 1200 to 1500 diameters, these lines appear to consist of small dots. The whole field makes the impression as if it were filled with a basket-like tissue. See Fig. 13. This, according to Dippel, 47 gives ground for the suppo- sition that over the continuous cross lines which are relatively strong, run very delicately drawn longitudinal lines, the latter being seen only, for the most part, with oblique illumination. "These diatoms give a right beautiful image when the latter method of illumination is used, and when the rays touch the- longitudinal strice at about an angle of 25 to 30." We herewith conclude the series of objects which serve us as the best tests for the optical powers of the microscope. The most important of these are, however, the scales of the Hipparchia Janira and the frustules of Pleurosigma angulatum. To these therefore we have devoted the most particular description. Still we might here add a remark as to test-objects in general. Many dealers furnish test objects with cover-glasses 0.15 to 0.20 mm. thick. We esteem these of no practical use. For if we undertake to use one of them with very high powers, we shall find the cover-glass so thick as to prevent our focussing down to the object itself, and so in spite of the correction screw the image would be ruined. We should follow the lead of Moller, and mount test objects under a cover-glass not more than 0.05 to 0.08 mm. thick. 48 To Leopold Dippel belongs the credit of having exactly de- termined the direct distance between these transverse or longitudinal markings upon the scales of the butterflies' wings, the diatoms, etc. In furtherance of our present aim we give in the annexed 7 Dippel, I.e., p. 131. Moller in Wedel, Holstein, offers such at the price of 1.50 Marks. 70 THE MICROSCOPE- IN BOTANY. table the results arrived at by this well-known naturalist. 49 (Cross markings are always meant except in Surirella where longitudinal lines are intended. In Ijycaena, a signifies a bright colored and b a dark colored scale.) NAME OF TEST OBJECT. Manner of Mounting. Number of strife to the 0.01 mm. Distance apart of the strice in Millimeters. Balsam 46 0.00208 78 0.00153 Drv 10 12 00099 10 11 00096 6 H 14 15 00074 1 l.'l ]-.'i III 14 15 00074 Drv 22 23 0.00046 25 00041 <( 28 29 0.00036 Dry 30 32 0.00032 32 34 0.00031 Applying the tests in the series in accordance with the ar- rangement in this table, one can come to a clear understanding of the excellency and of the working qualities of his instrument. In conclusion it may be mentioned that Nobert for a long time has been making and furnishing an apparatus for testing the microscope without the use of test objects. It is called "Nobert's test plate" (Probeplatte). It consists of a number of groups of very delicate lines which are cut in glass by means of a diamond, or eaten into it by means of hydrofluoric acid. The lines of the several groups are at different distances apart, as for example, those of the first are 0.002256 mm. and of the last group, 0.000282 mm. asunder. The distance apart of the lines of the groups lying between these, gradually pass in value from that of the former to that of the latter. It is evident that with the help of this plate one may very easily determine the resolving power of a system, if he will begin with the first group and work progressively through toward the more difficult till he has reached a point where his lens will no longer resolve the lines. The Nobert test plate would certainly supersede all Dippel, 1. c., p. 134/. THE MICROSCOPE TUBE. 71 other test objects, did not its very high price, made necessary by the almost incredible fineness of the work, interpose the greatest barrier to its general distribution.* Since we have now finished the consideration of the optical parts of the microscope, we will undertake to describe the other parts, only however in respect to their principal features, the stand and the illuminating apparatus. We will first describe the 'microscope-tube. VIII. THE MICROSCOPE-TUBE. The microscope-tube is a solid tube of brass, whose length is adjusted to the construction of the optical apparatus and rel- atively to the cooperation of the objective and ocular. In the medium and larger microscopes, its length varies between 18 and 28 cm. Beneath, it carries the ["Society"] screw for re- ceiving the objective-system. Within its upper opening which is made cylindrical to receive it, the ocular is set. Within the tube are placed several diaphragms for cutting off certain rays which would injure the microscopic image. The tube is blackened on the inside, at least in the lower part and up to the topmost diaphragm. [In some instruments, lining the inner tube 'with lusterless black cloth will improve the definition by remov- ing a scarcely noticed glare of light reflected from the imper- fectly deadened surface of the brass tube. In other stands, diffused light enters the short oculars from the adjacent surface of the tube that has been brightened by contact with the longer oculars : this is prevented, by some makers, by so shaping the oculars or so guarding the inside of the tube that all oculars, whatever their length, will come into contact with the tube for exactly the same distance. R. H. W.] The larger microscopes commonly have draw-tubes also. The tube then consists of two parts, one shoving into the other like a telescope, so that it may be drawn out to different lengths, according to need. On the advantage of this arrangement, Harting has remarked as follows i 50 50 Harting. Mikr., page 157/, *Ou account of the recent death of Xobert. his rulings will become increasingly scarce; but plates approaching them in quality, and serving the same purpose as tests, are now ruled at a far less cost by C. Fasoldt of Albany, N.Y., and by other makers. R. H. W. 72 THE MICROSCOPE IN BOTANY. "In a microscope to which belong several oculars and objec- tives, it would be unreasonable to expect that one and the same length of tube would be best for all combinations. An exam- ination previously made will serve to show at what length of tube the various optical parts will do their best work, and this can be noted and followed in the future." "A second advantage of this contrivance consists in this, that by the inward and outward movement of the inner tube, the magnification can be brought to any definite number. For mak- ing micrometric measurements this is very important. It is simpler for instance to have the diameter of the image divided by 500 than by 487 or 513, or by 100 than by 93 or 107. Also in many observations, it is important to have the field of view of a certain definite size, 1, 2, 3, etc., mm. But this can be brought about only by increasing or diminishing the distance between the ocular and the objective, and this may be done with- out damage to the image if certain limits be not overstepped." "For this purpose the inner tube should be graduated. The optician, or the owner of the microscope, can then, by careful investigation, construct a table which shall indicate the point on the graduation which will be of service in actual work with various combinations and magnifications." [A third use of the draw-tube is, by being pushed in, to re- duce the length of the body to much less than its usual standard, for the purpose of adapting it to the vertical position often re- quired in laboratory work. To this end, such stands as are most suitable for histological work are made with a very short body, about 12 cm. long, which may be increased to the ordinary length when using the instrument in an inclined position, by extending the draw-tube as represented in Plate III. In Plate X the draw- tube is wholly closed. The stand shown in Plate XI has two draw-tubes, one within the other, to give greater range of length. By this extreme shortening of the body, the ocular is brought as near as possible to the table and one is enabled, with a min- imum of discomfort and fatigue, to lean over the stand and look down the vertical tube. While the optical corrections are visibly disturbed by great shortening of the tube, there are many ob- jectives of moderate capacity whose performance is not materially THE DRAW-TUBE. 73 injured, and others whose screw collar adjustment is capable of fairly correcting the evil produced.] [The draw-tube usually is, and should in all cases be, provided at its lower end with an adapter having the society screw for the reception, when required, of an analyzing prism, a spectro- scopic arrangement or an objective to serve as an erector. An objective, having too great focal length to be employed in the usual manner, may likewise be inserted here and, by sliding the draw-tube, it may be focussed through the empty nose-piece upon the object below it. Should the screw become bright from use and reflect false light, it must be guarded by a ring of hard rubber or blackened brass screwed into it. This will not only render it harmless but will become an efficient diaphragm to stop stray light from other sources. R. H. W.] The microscope-tube is moved by a propelling mechanism, rack and pinion, or by free hand. In the latter case it is nec- essary that the tube should exactly fit into the inclosing sheath. This is secured by careful polishing of both surfaces or by lining the inside of the sheath with cloth [or by the use of springs]. The tube should never be oiled, but it and the sheath kept always absolutely clean. When we are examining one object after another with different magnifying powers, it is necessary in each case to screw another system into the tube. [The screw by which the objectives are attached to the tube is of standard size, devised and prepared, in 1857, by the Lou- don (now Royal) Microscopical Society, and therefore known as the " Society" screw. Being, practically, in universal use in both this country and England, and even applied to conti- nental objectives intended for sale here, this standard screw renders objectives of the different makers interchangeable, so that a student may use his set of objectives upon a variety of stands, or may purchase any desired lens without doubt as to its harmonizing with his former apparatus.] [The introduction of the society screw, with all its attendant advantages, was a loss in respect to ease of manipulation, as it put a stop, temporarily, to the use of bayonet catches and other devices designed to lessen the labor and delay occasioned by frequent changes of objectives while the instrument was in use.] 74 THE MICROSCOPE IN BOTANY. [IX. NOSE-PIECES.] [Subsequently double, and even triple and quadruple nose- pieces were used, the upper portion of the apparatus being attached by a society screw to the compound body, while the lower portion or revolving plate carried the specified number of objectives permanently screwed into it, so that any one of them could be rotated into use, in the axis of the instrument. Such a triple nose-piece is shown in Fig. 18. When well made, very light, and of the angular form, this device is very satisfactory in use, and greatly relieves the labor of inves- tigations requiring frequent changes of power. It is however somewhat costly and cumbersome, and its weight, including that of the attached objectives, im- pairs the delicacy of the fine adjustment, especially in some of its older forms.] [During the past two or three years there has come into ex- istence a new series of contrivances, by which objectives though attached one at a time can be changed with ease. In them the nose-piece is a single chuck, permanently screwed into the mi- croscope body, some mechanism being included by which the objective can be instantly seized or released. No change is made in the screw of the microscope-tube or of the objective, and the collar screwed upon the objective should be of such size as to be left in position when the objective is packed in its box. These nose-pieces require excellent workmanship, in order to secure accurate centering and adequate stability, and in their use care should be exercised, especially by inexpert hands dur- ing the manipulations required for the sere w-collar adjustment of the objective, not to unintentionally release the objective from the grasp of the chuck. Should experience give weight to this drawback, slight modifications of the apparatus would doubtless remove the difficulty.*] , * Constant use of the "Facility" nose-piece for more than a year does not reveal the existence of this possible objection. A. B. H. NOSE-PIECES. 75 FIG. J9. [Such a contrivance is the "Facility " nose-piece, Fig. 19, which is simply a self-centering chuck, which seizes the objec- tive by a small ring or collar permanently screwed upon its so- ciety screw. It was devised and is made by James L. Pease of Chicopee, Mass. Of the same general character is the " Congress" nose-piece contrived by Prof. Albert McCalla and made by W. H. Bulloch of Chicago, Fig. 20, in which a chuck, with three slots, grasps, by three pro- jections upon it, a ring perma- nently screwed upon the objective. The contrivance still more recently produced by Mr. Zentmayer, Fig. 21, is one in which a nose-piece is screwed into the micros- cope-tube and a collar permanently screwed upon the objective, the collar and nose-piece being connected by a screw, the opposite quarters of whose threads are cut away, so that insertion can be accomplished without screwing and the inserted collar locked fast by a single quarter- turn which will bring the screw-threads of the two pieces into relation with each other. An index-mark upon the nose and a corre- sponding mark upon each collar indicate the position in which the collar can be inserted ; and a jam-nut enables the nose to be set in such position as to bring its index in front of the microscope or in any location pre- ferred by the observer. This contrivance has been successfully used in the arts, as in the construction of breech-loading cannon and in the coupling of hose for use FIG. 20. FIG. 21. 76 THE MICROSCOPE IN BOTANY. with " fire-engines." It was proposed by Mr. E. M. Nelson at the Qtieckett Club, Sept., 1882, to similarly cut away portions of the society-screw from objectives and stands, as a means of instantaneous attachment. No general effort has been made, however, to introduce it for this purpose, and it is doubtful if the modification could be effected by the various makers with such uniformity that their work would be really interchangeable.] [The latest contrivance of this sort is by Mr. Charles Fasoldt of Albany, N. Y., who makes a nose-piece the alternate sixths of which have the thread cut away, while one of the remaining sixths is movable and capable of being withdrawn from contact by a lever reacting against a strong spring, as shown in Fig. 22. The objective requires no preparation, and can, after starting by pushing back the movable section of the screw, be screwed in in the usual manner. If, how- ever, the lever be pressed down with the thumb, the movable section of the screw is with- drawn so far that the objec- FIG 2> tive can be placed at once in position against the shoulder above. Releasing the lever allows the movable portion of the screw to slide firmly back into place and grasp the threads of the objective, which is held exactly as if it had been screwed in, and which can be tightened up against the shoulder, if neces- sary, by a single screwing movement. A jam-nut is provided, as in the Zentmayer form, by which the lever and index can be set in a position most convenient to the observer. A correspond- ing mark should be made upon the brass mounting of every objective to be used, the mark upon the objective corresponding to the index of the nose-piece, not when the objective is snugly screwed up to the proper tension, but after it has been un- screwed about one-eighth of a revolution. When then in- serted, in this slightly unscrewed position, the threads are grasped with more certainty and effect, and a slight twist, one- THE FINE-ADJUSTMENT. 77 eighth turn to the left, sets it with uniform and unerring firin- iiess against the shoulder. After experience renders this twist habitual, it becomes almost automatic, and is scarcely distin- guished as a part of the apparently single action of putting the objective where it is wanted. When in position, the objective cannot fall out or be pulled out, without being either unscrewed or else instantly released by pressing down the lever that opens the screw. R. H. W.] X. THE FINE-ADJUSTMENT. The fine adjustment screw, as we have pointed out, is a con- trivance by which, when the coarse adjustment has brought the optical apparatus into the immediate neighborhood of the ob- ject, it can be adjusted with almost mathematical exactness, so as to bring the object exactly into the focal point of the lens. How this adjustment was brought about in the older micro- scopes we have already mentioned, p. 7. With smaller micro- scopes, to this day, the fine adjustment is occasionally found on the stage. This is objectionable on instruments used for sci- , entific observation. In microscopes designed for scientific investigation the fine-adjustment screw is placed in the perpen- dicular pillar [or variously shaped limb] which bears the tube. [Until a very few years ago, the fine adjustment of the best English and American stands consisted of a light tube or nose- piece, sliding easily within the main body of the microscope and projecting slightly below its lower end ; this tube being pressed firmly downward by a spiral spring, and being raised by a lever, actuated by a screw which was turned by the thumb and fingers of the observer's hand. The position of the lever and screw varied with the caprice of the maker, but it was most frequently attached to the body in front and near the lower end. The objective screwed into this sliding tube could be moved up and down until its distance from the object was ad- justed with great precision. The arrangement, however, lacked firmness, especially in handling the screw-collar adjust- 78 THE MICROSCOPE IN BOTANY. ment, and it often became much the worse for wear. Some persons, also, feared that the slight change it effected in the dis- tance between the objective and the ocular might be practically as well as theoretically objectionable.] [Meanwhile Continental microscopes were made with a fine adjustment moving the whole body of the instrument. In most of those which found their way to this country or were imitated here, a hollow pillar was provided, within which was contained a solid cylinder firmly attached below to the foot, or to the movable portion of the trunnion-joint. The outer por- tion (a hollow tube sliding over the inner) carried with it by means of a transverse bar the whole body of the microscope, the vertical movement being accomplished by a screw working against a spring ; just as if, in Plate XI, the milled screw-head, high up at the right of the plate, were made (which it is not) to carry up and down the outer and visible portion of the column below it over an inner and concealed column. This form of adjustment lacked smoothness and uniformity of movement, was particularly subject to side motion, and had no good means of taking up the loss from wear. Its use in this country was very limited and mostly confined to stands of low grade.] [In 1876, among the novelties prepared for display at the Centennial exhibition, Mr. Zentmayer transferred the fine ad- justment of his stand from the nose-piece to the limb, making the bar just behind the body slide upon plane surfaces, as in the coarse adjustment, a sufficiently delicate movement being imparted to it by a lever acted upon by a screw at the left. Great steadiness and indefinite capacity for wear are attained in this way. This adjustment is shown in Plate III, the black line just back of the body and parallel with it representing the edge of the sliding surfaces. In Plate IX this is shown com- bined with a rack and pinion coarse adjustment, a separate slid- ing movement being provided for each.] [In Mr. Bulloch's microscopes, Plate X, a similarly situated screw and lever give a very delicate vertical motion to a sliding box, in which is set the pinion of the coarse adjustment itself as a portion of the fine adjustment.] [In the Bausch and Lomb instruments may be found the so- THE FINE-ADJUSTMENT. 79 called "clock-spring" fine adjustment, shown in Plates IV and XI, and in section in Fig. 23. The transverse bar of the stand, extending from the pillar to the body, consists of a hollow box whose top is represented by d, whose front, towards the right, is open, and whose back, to- wards the left, is either at- tached to the pillar c by screws, or cast in one piece with it, as in Plate IV. From the back of this box, and firmly attached to it, project forward two stiff horizontal steel springs aa, which bear at the right the plate e, con- taining the pinion f of the coarse adjustment fg. An arm of e projects backwards and is pressed down by the carefully cut fine-adjustment screw 6, reaction being fur- nished by the springs aa. The horizontal and vertical portions of e being inflexible and continuous, and the pinion-rack and body, f to A, having no other support than the springs aa, it is evident that any vertical movement of the screw b must impart a like movement to the body h and to the optical parts contained therein. Since a and a are parallel and of equal length, the motion of the body h must always be in a direction absolutely parallel to the pillar c and vertical to the plane of the stage. The theoretical move- ment of h to and from c is so slight as to be unnoticed. This form of adjustment is free from lateral motion or lost motion, has no friction except that of the screw itself and does not de- teriorate by age or wear. A somewhat similar system, though not exempt from friction and wear, is employed in the stands of Seibert and Krafft described by Dr. Behrens, in which a par- FlG. 23. 80 THE MICROSCOPE IN BOTANY. allel motion is secured by a system of levers instead of springs. R. H. W.] XL THE STAGE. The object-table, or, for short, the stage, is a solidly wrought metal plate which, in respect to the optical apparatus, assumes an unalterable position. The optical axis of the microscope must be exactly perpendicular to the plane of the stage. The form of the stage as in Plate X is rectangular, or, as in Plate XI, round. Both forms are alike practical, assuming that they are roomy enough to be convenient to handle. Small stages are objectionable. They should at least be large enough so that the largest form of slide would not reach from side to side. In order to obviate the reflection of rays from the stage it is com- monly blackened [at least on its lower side]. Large microscopes are usually furnished with rotating stages, and this arrangement is very convenient, as it enables one to turn the object quite about upon its axis without being obliged to disturb the slide. And besides, when the rotating stage is graduated and made to work by an index, it can be used with good results in measuring the angles of crystals. In the latter case, the stage should be provided with a screw arrangement by which it may be exactly centered, so as to bring that part of the object to be examined into the exact optical axis of the microscope. In the illustrations, Plates X and XI, such ro- tating stages are represented [centering adjustment being ap- plicable if specially ordered]. The circular plate can be set in rotary motion upon the stationary tinder-piece. It has a milled edge so as to be moved more easily by hand. [Many of the small and cheap microscopes are now made with a plain round stage, a form unobjectionable in itself and capable of a higher development than the square. The stage, for instance, in Plates IV, V or VI, is of extreme simplicity, but is capable, by reason of its circular form and location (con- centric with the optical axis), of receiving, either originally or subsequently, such an upper plate as those shown in Plates X and XI, thus making a simple but serviceable revolving stage. THE STAGE. 81 Such ail arrangement, besides its other good qualities, is com- patible with that extreme thinness of stage which is now con- sidered essential in order that freedom of illumination be not interfered with. R. H. W.] It is sometimes necessary to make the object fast to the stage in order to devote a considerable time to the examination of a single point, or in order to be able to turn the microscope down to an oblique position. For this purpose a simple clamp ar- rangement is sufficient. [The commonest form, and one answering, in skilful hands, nearly all useful purposes, is a pair of spring clips of steel or brass attached to the stage by screws or pins, as shown in Plates III and XI. Beneath these the object-slide is placed and can be successfully manipulated, even under moderately high powers. A bar of glass or brass, sliding under short clips, forms a suffi- cient ledge for the support of a trough or receptacle too large to be placed under the clips. A some what more delicate adjust- ment can be obtained by using a brass object-carrier sliding over a glass stage as in Fig. 24, which is designed as an addition to the stand shown in Plate IV ; or a glass object-carrier, or sliding stage, which is especially adapted to chemical work and is easily applied to any stage of the model shown in Plate IX.] [While elaborate " mechanical stages," on which the object can be moved in every direction by racks and screws, are not required for general use and are much less esteemed and used than formerly, still there are some procedures, such as microm^ etry and the use of extremely high powers, where a stage having some sort of mechanical movement is a material advantage. o Such stages are now made in simplified forms, thin enough to conform to the present demand for thin stages, and small enough to be applicable to the smallest stands described in this book. R. H. W.] An arrangement seldom employed by the botanist, but more frequently used by the zoologist, is a warming stage. By means of it, the preparation may be kept at a higher temperature than 6 82 THE MICROSCOPE IN BOTANY. that of the surrounding atmosphere. Max Schulze 51 has invented such a stage capable of being heated. It consists of a metal plate which is fastened to the stage by clamps. Corresponding to the opening in the stage there is a place bored out in it for illumination. It carries in front, in the middle a diagonally placed thermometer and two long arms extend out far beyond the stage ; under these are placed two spirit lamps as heaters. The lower end of the thermometer is wound about the opening on the plate and so gives the exact temperature of the object. XII. THE ILLUMINATING APPARATUS. The illuminating apparatus consists essentially of three in- struments, viz. : the mirror, the diaphragm and the condensing lens. The mirror is placed beneath the stage, but the diaphragm .is placed near, if not upon the under side of the stage itself. A. THE MIRROR. The mirror is formed of a circular metal frame of 30 to 50 mm. [or more] broad, in which is mounted, on one side, commonly, a plane glass mirror, and on the other, a concave mirror of spherical form. [The mirror is fastened to the arm, as seen in Plates III and XL] The mirror frame is mounted upon an arm so as to revolve upon it and the arm is so constructed as to enable the mirror to be moved right and left, and placed in any position with reference to the opening in the stage. It is by no means a matter of indifference, for the illumination of the object, which of the two mirrors, the plane or the concave, is used, as will be clear from what follows. We will suppose that there is a source of light I, Fig. 25, which sends rays upon the mirror s, as shown in the illustration, Za, Ib, Ic, Id, le. It is known that the concave mirror converges the rays that it reflects. It will be seen without further elucidation that, by giving the mirror its proper position and distance from the 6i Frey, Das Mikroskop, page 65. THE MIRROR. 83 object, all the rays which fall upon it will be concentrated upon the object that lies at p, upon the stage between the slide and the cover-glass. In this case very many rays are concentrated upon a very small space, the object, which must consequently be very brightly illuminated. The parallel rays of daylight, which are commonly used in microscopical investigations, behave, when they fall upon the concave mirror quite like the diverging rays we have just been considering. The plane mirror works very differently. The parallel rays falling upon that are reflected at the same angle, consequently run parallel on their way to the object. If we now suppose FIG. 25. that there falls upon the surface of the mirror n rays of light and the stage diaphragm has but L the extent of the mirror surface, it follows that the plane mirror will afford but JB. of the light, to the object, that it receives, while under the same con- ditions, the concave mirror would contribute to. the illumination of the object, all of the rays that it receives. Still less of the illumination will be given to the object when it is received from a near source of light as in Fig. 25, for then the rays are di- verging, and on falling upon the plane surface, will be still more diverged, and thus spread more widely, and, so to say, thinly, over a given space. For obvious reasons, therefore, the plane mirror should be used with low powers, and the concave with higher and the 84 THE MICROSCOPE IN BOTANY. highest powers. From the above considerations also, it is easily seen why the plane mirror affords, on the whole, stronger lines of definition than the concave. Very delicate structures, which are to be observed with low powers, appear with much sharper outlines when illuminated by the plane than by the concave mirror. For many objects, with medium or high magnification, illumi- nation must be had from the concave mirror with oblique light, in order to be able to recognize certain fine details. This oblique illumination is produced by moving the mirror to a certain angle right or left, and adjusting the diaphragm so that the cone of rays may still reach the object on the stage. Now the outlines will give broader shadows than before and so will be much more easily recognized. The different effects of central and oblique illumination become clear by using first one and then the other illumination upon the diatoms, as, for example, Pleurosigma angulatum, PL formosum or PL balticum. B. DIAPHRAGMS. Only in rare cases will one be able to obtain the desired illumination by means of the mirror alone. In many cases it is important to shut off from the microscopic image the border rays and even the central rays that are reflected from the mirror. Both can be accomplished by means of the diaphragm attached to the stage. 1 . The Revolving Diaphragm. In the older instruments and in the smaller ones of the present time the marginal rays are shut off by means of the revolving diaphragm, Fig. 26. It is a FIG - 26 - plate of metal, not too thick, blackened on both sides and provided with several round holes of different diameters, but whose middle points are at the same distance from the center of the disk. The diaphragm is fastened to the under side of the stage [or to some supporting apparatus DIAPHRAGMS. 85 beneath the stage] by a screw passing through its center, and about which it is made to turn by the fingers, its openings being brought successively under the opening in the center of the stage. But it is difficult faultlessly to center this diaphragm, and what is a worse evil it is not on the same plane with the top of the stage but with the under surface, and the consequence is that between the diaphragm and the object a cone of dispersing rays is formed which very considerably injures the clearness of the microscopic image. To avoid this fault the .diaphragm is sometimes made in the form of a concave segment of a globe, and the under side of the stage is hollowed out to correspond. But this contrivance allows no very near approach to the object, and the difficulty of correct centering is still greater, and the objec- tion to this kind of diaphragm increases with the power of the objective employed in the investigation. [In a few American stands, as in some of those made by Mr. Grunow of New York, and in one of Mr. Zentmayer's, the re- volving diaphragm plate is, for this reason, let into the upper sur- face of the stage, so that when in use it lies almost in contact with the object-slide. In most American microscopes, however, it is attached to the under surface of the stage or fixed at a moder- ate distance below it, in order to secure that control of the an- gular breadth of the illuminating pencil, which is obtained by locating the diaphragm at a sensible distance below the point of convergence, p, in Fig. 25, of the cone of rays condensed up- on the object by the concave mirror. By far the best arrange- ment is to have the diaphragn^-plate so mounted, whether in a sliding-tube or upon a sub-stage, that it can be set at any level from the plane of the top of the stage to a plane five or ten mm. below it, as in Plates IX to XI. R. H. W.] 2. The Cylindrical Diaphragm is a contrivance which quite obviates the evils mentioned, as belonging to the disk dia- phragm, and it is so simple and so well adapted to its purpose that it has quite driven the others out at the present time. It consists of a hollow cylinder exactly turned without and blackened within. The upper end is drawn suddenly in to form a raised ledge of somewhat less diameter than the opening in 86 THE MICROSCOPE IN BOTANY. the stage. Over this upper edge of the cylinder are placed small metal caps which are provided with central openings of various sizes. [This excellent piece of apparatus, which has not yet come into very general use in this country, is a survival of the "dark well" or "dark chamber" of the early days of the modern microscope. It may be slipped from below into a ring or short tube like that projecting from the lower surface of the stage in Plate V, or supported by the sub-stage, as in Plate X, or in microscopes of very simple construction inserted into a special carrier to be presently described.] 3. [ The Iris Diaphragm is, by far, the most perfect means of limiting the cone of illuminating rays, and is now produced in various forms by numerous makers. That of the Bausch and Lomb Optical Co. is represented in Fig. 27. It consists of a dark well, closed at the top by a series of thin movable plates, which by a very simple mechan- ism may be made to close the opening altogether, or to open gradually and form a practically circular aperture of any desired size up to the maximum capacity of the well, the size of the opening being controlled by the milled head at FIG. 27. the bottom. uch ease and precision are secured by this contrivance, in adjusting the amount of light admitted, while the object is under observation and all the adjustments of the stand and light remain undisturbed, that a person once accustomed to its use is little likely to be satisfied without it. How it may be combined with tlje condensing lens will appear further on. R. H. W.] 4. [Special Diaphragm-stops.'] All the contrivances thus far considered are adapted to shut off the marginal rays which the mirror reflects upon the object. Latterly, attention has been given to correct the central rays which proceed from the mirror. It has been found that they frequently injure the mi- croscopic image ; particularly so is this true in the use of a condenser, where one must often provide for their elimination. This can be done very simply by the use of a "central-stop." A central-stop is a small circular metal plate which is supported CONDENSEKS. 87 on the slenderest possible metallic arm [or on a thin glass disk], and which by some contrivance may be brought into the middle of the condensing lens. [A horizontal slit, consisting of a central opening much longer than wide, in the diaphragm plate, is used to great ad- vantage in connection with the condensing-lens, in the illumi- nation of binocular microscopes, sufficient angular breadth of illumination being thus secured to light both fields of the instru- ment freely without such an excess of light as would impair the view of the object. A corresponding effect is attained by the use of a pair of horizontally arranged circular apertures sepa- rated at such an angular distance apart that each one will admit the pencil of light required by one tube of the instrument.* The pair of apertures requires to be adjusted with more skill than the horizontal slit, and ap- parently without compensating FIG. 28. advantages. With these stops, shown in Fig. 28, the use of the binocular may be extended to higher powers and angles than without them. Special stops for oblique light or other effects may be used at the option of the student. Any of the special stops may be cut in certain portions of the revolving diaphragm-plate or in some of the caps of the cylindrical diaphragm which must be substituted for the iris diaphragm during the time of their use. R. H. TT.] C. CONDENSERS. For certain purposes it is recommended to interpose a lens between the mirror and the object which will concentrate the rays of light from the mirror exactly on one point. This can be done best with a condensing lens of very short focus. We commonly use a plano-convex with the convex side strongly curved. [Such a condensing lens is often required, not only to in- *See a figure and description of this method of binocular illumination by the writer in the American Naturalist of December, 1870, p. 636. 88 THE MICROSCOPE IN BOTANY. crease the amount of light reaching the object, but also to secure effects dependent upon the obliquity with which the light passes through it. The simplest arrangement and one of the best is a nearly hemispherical lens, 10 to 12 mm. in diameter, stuck to the bottom of the object-slide, directly beneath the object, by a minute quantity of glycerine or oil of cloves. The lens should be less than a hemisphere by about the average thick- ness of an object-slide, so that when the two are united the object will be at the center of curvature. Light, either par- allel from the plane mirror or condensed by the concave mirror, may then be passed with a peculiarly brilliant effect, directly to the object from the mirror in whatever position, from axial to the level of the bottom of the stage, in which the mirror may be placed. If the obliquity chosen be in excess of the semi- aperture of the objective* light will pass through the object but not directly into the objective ; the object, if neither too opaque nor too translucent, then appearing brilliantly illuminated upon a dark field, the same effect being produced with less intensity by the prism mentioned below, or with very limited brightness and only for very low powers, by the concave mirror alone in a very oblique position. The condensing power of the hemisphere is small, on account of its large curvature and the position of the object far within its focus. If greater refraction be desired, the " Wenham button" may be substituted, whose sharp curvature and more precise focus give a more intense illumination, but one adequate only for minute objects and applicable chiefly to the higher powers. For illumination with parallel instead of converging rays a small triangular prism may be similarly at- tached to the slide, the effects being the same except that the light is not condensed and that its obliquity is limited to one angle, or if the prism be revolving and not equilateral, to two or three angles. Such a prism upon a convenient mounting is shown in Fig. 29. It is of far less general applicability than the hemispherical lens. Both lens and prism are somewhat difficult to locate exactly in the required position, and are liable to slip out of place if the stand be inclined, especially if too much of the connecting liquid be employed. For these rea- sons they should be mounted, for stands having a sub-stage, at CONDENSERS. 89 the summit of a vertical wire rising from the center of the sub- stage. When there is no sub-stage, this wire may be supported by an arm attached to the stage itself, as in the ingenious device of Jas. W. Queen & Co., which appears, with prism at- tached, in Fig. 29.] [If the lens be mounted at the top of a dark well, its angular capacity will of course be limited by the posi- tion of the lower edge of the tube which should, for this reason, be short and broad. It can then, however, be slipped away from the object, down- FIG 29 wards, its glycerine contact being omitted, and focussed upon the object from below. In this case its capacity as a condenser is greatly increased by placing be- neath it a still larger lens called a collecting lens. Condensers of two large non-achromatic lenses have been extensively used with great success for many years, largely through the influence of Dr. Beale in advocating the use of an ocular for that purpose; and, notwithstanding recent improvements in this direction, one may still with much satisfaction transfer his highest power ocular to a ring beneath the stage, as a condenser. In the or- tlioscopic ocular, similarly used, and the " Webster" condenser, an achromatic upper lens is employed ; while in the latest and now most approved form, introduced by Professor Abbe and hence called the Abbe condenser, both of the lenses are non- achromatic and of such great thickness that the top of the lens will nearly touch the object-slide when focussed upon the object. By this arrangement not only is great aperture (n. a. 1.42 and upwards) readily obtained for use with the highest-angled ob- jectives, but water or glycerine contact with the object-slide becomes practicable, giving an "immersion" illuminator with increased working capacity. This simple and inexpensive combination seems to be superseding, with good reason, all the elaborate and carefully corrected achromatic condensers for- merly used.] [By combining a black center-stop with a condensing lens or system, a central cone of light below the object and a corre- 90 THE MICROSCOPE IN BOTANY. spending inverted cone of light above the object will be sup- pressed, each having the same angular aperture as the obstructed portion of the condenser. If an objective of less than this aperture be used, it will of course receive no direct light, but will view the object illuminated by the oblique rays from the unobstructed marginal position of the condenser, the field mean- while remaining dark. For low powers exclusively, a single thick lens with a central black stop attached to its upper sur- face, known as the spot lens, is sufficient. An achromatic con- denser with center-stops, or the Wenham paraboloid, a truncated glass paraboloid, with a center-stop, to give by internal reflec- tion a hollow cone of rays condensed at a large angle, has been heretofore employed with the higher powers ; but the large lens-systems of the different varieties of Abbe condensers, with their large apertures and immense amount of light, if pro- vided with suitable center-stops, leave little to be desired, with either high powers or low. Objectives of too large aperture for this method of illumination are frequently brought within its scope by inserting a diaphragm behind them to temporarily reduce their aperture to a practicable limit. This so-called black ground or dark field illumination is very effective with many delicate vegetable hairs, fibres, etc., which should usually be viewed dry or in water, as balsam renders them too trans- parent to arrest and disperse sufficient light. R. H. W.] [ D. ILLUMINATING COMBINATIONS. ] [1. The Universal Accessory. For the sake of convenience, some of the opticians combine into one piece of apparatus, to be fitted below the stage, several of the sub-stage appliances, such as diaphragms, condensing lenses, polarizing prism, etc. A simple arrangement for this purpose, being inexpensive and applicable to smaller stands, is the "Universal Accessory" of Bausch and Lomb, shown in Fig. 30. It consists of a rather thick stage-plate intended to lie upon the stage in place of the object slide, and to carry the slide under a pair of spring clips upon its upper surface. Set into the centre of this plate and projecting below it through the central opening of the stage, is a short ILLUMINATING COMBINATIONS. 91 revolving tube to receive a cylindrical diaphragm, polarizing prism, or condensing lens ; the latter becoming, with a black centre stop, an efficient spot lens. This apparatus is especially suited to stands having no sub-stage conveniences.] FIG. [2. Ward's Iris Illuminator. A more elaborate and effective arrangement, constituting an illuminator suitable for work of a higher grade, is a combination of the condensing lens with a decentering iris diaphragm, devised by the writer and made by Bausch andLomb. It is shown in Fig. 31, and consists of any FIG. 31. or "immersion," under and desired lens system, either " dry close to which is mounted an iris diaphragm set in a sliding plate so that it can be moved into any position from the center to the periphery of the system, without altering the position of the latter. Thus not only the obliquity of the light, but the 92 THE MICROSCOPE IN BOTANY. exact amount desired or found advantageous at any chosen ob- liquity can be regulated with perfect precision by a touch of the hand to the decentering screw and to the adjusting collar of the diaphragm. This contrivance can be applied, without out- growing the limits of the customary 1J inch (38 mm.) sub-stage tube (which size of sub-stage is being very generally adopted, and it is hoped will soon be made " standard") , to any condensing system whose posterior diameter does notexceed 21 mm. (|j in.) . It is well adapted to a simple hemispherical lens, a large-lens achromatic condenser, or the doublet of thick non-achromatic lenses adopted by Prof. E. Abbe of Jena. In using the first or the last of these three, which have nearly superseded the late "achromatic condensers," it should not be forgotten that the best performance is nearly always obtained by connecting the illuminating lens with the object-slide by a drop of water. A blue glass disk, for correcting the glare and color of artificial light, is fitted to a tube that can be inserted into the bottom of the dark well of the diaphragm. A special adapter is also pro- vided for the use, in place of the iris diaphragm, of central stops for securing dark field illumination ; and a revolving tube, slipping inside of this, carries a horizontal slit, or pair of horizontally arranged apertures, for the better illumination of binocular mi- croscopes (see page 40), or special stops for the production of any effect desired by the user. In similar fittings, may be mounted a polarizing prism and selenite plate, a small Nicol's prism being sufficient, in connection with the condenser, to give adequate illumination for moderately high powers. The whole apparatus rotates about its own optical axis, which remains coincident with that of the microscope itself. By removing the lenses from the top of the apparatus, the iris diaphragm, with or without its blue glass disk, or the polarizer, will be found in position for use by itself. Except for very low powers, however, the illuminator may be considered as a part of the stand and kept habitually in place, the changes of light required for a great variety of work being readily accomplished by its aid. It can be applied to almost any microscope, whether with or without a sub-stage.] OPAQUE ILLUMINATORS. 93 E. OPAQUE ILLUMINATORS. [The foregoing methods of illumination pertain to objects sufficiently transparent or translucent to be viewed by light passed through them from below. Opaque objects, viewed by light reflected from their upper surface, as frequently becomes necessary in botanical study, can seldom be adequately illumi- nated by the general light of the room. They usually require for satisfactory exhibition the condensation of light upon them by means of a lens or mirror. A small condensing lens may be FIG. 32 a FIG. 32 b attached to the microscope itself, or mounted upon a stand of its own. Large plano-convex condensing lenses called "bull's eyes," having great thickness and short focal distance, are usually mounted on a separate stand as shown in Fig. 32. They 94 THE MICROSCOPE IN BOTANY. are used near the microscope, the object being in the principal focus, to condense light upon the object, or near a lamp, the flame being in the principal focus, to give a beam of intense and nearly parallel rays for use at a convenient distance from the flame. In stands having the modern style of swinging tail- piece, as in Plates III to XI, the concave mirror can be readily brought above the stage for the concentration of light upon the object. For higher powers a. small concave silvered mirror, or side reflector, is attached to the objective or to some neighboring part of the stand. Opaque objects under the highest powers can be sufficiently lighted by mak- ing the objective its own condenser, as in the case of the Beck illuminator, shown in Fig. 33, where light entering an aperture in the side of the nose-piece is reflected downward by a thin cover-glass in the center of the tube, and is condensed by the objective upon the object in its focus. In the use of such contrivances, of which there are many in use, much care and tact are necessary to avoid the glare of false light. R. H. W.] F. OBSERVATION BY ARTIFICIAL ILLUMINATION. The microscope is an apparatus of the day. The best source of light for it is diffused daylight, when the heavens are evenly covered with a transparent white veil of clouds. The blue of the cloudless heavens, the changing light of hurrying clouds, and the direct sunlight are alike objectionable for careful micro- scopical investigations. But if one has necessary microscopical work to do on a sunny cloudless day, his best course is to take a white wall or a large white paper screen which reflects sunlight as the source of his illumination. But it will now and then happen to the microscopist that he must work by the light of the lamp, it may be to make the evening hours help out the short dark winter days, or it may be to study the reproductive processes in the lower cryptogams, which take place only during the night hours. In these cases one OBSERVATION BY ARTIFICIAL ILLUMINATION. 95 has to contrive some way to improve the artificial light which in and of itself is in the very highest degree unserviceable for mi- croscopical observations. The yellow light of gas or petroleum consists, for the most part, of rays which belong to the first half of the spectrum and are, more than any other, hurtful to the eyes of the observer. Besides, if one turns the mirror di- rectly to the flame, the field of view is commonly too bright, and if toward the lamp shade, it is too dark. In order conven- iently to modify the brightness and color of the light, the following means is suggested and recommended. Direct the mirror to the brightest part of the flame and then interpose, close to the mirror, an upright screen of thin translu- cent paper, which one can easily make in a few moments. This subdues the brightness of the field so much as to make it painless to the eyes. In order to change the yellow color to a blue tint it is only necessary to bring between the mirror and the diaphragm, a little plate of blue cobalt glass. I use a little round glass disk, about 13 mm. in diameter and 2 mm. thick. Its color is a faint blue and the underside is slightly and uniformly ground. This little plate is placed directly under the diaphragm. In this way the illumination of the field is made uniform and soft bluish. The following arrangement is still better and more convenient. Fill an ordinary cobbler's globe* with a pretty dark blue solution of cupric oxide of ammonia, and place it between the source of light and the mirror in such a way that the mirror can be directed towaid the brightest spot of white light produced. In this way one gets an illumination for evening which leaves very little to be desired. The right concentration of the cupric oxide of ammonia is easily made out after a few trials. XIII. THE MICROSCOPE FOOT. Of the microscope foot two things are required. The mi- croscope must rest on it firmly and securely. The foot should *I am told that, in Germany, the cobblers, when at work in the evening, suspend a glass globe filled with water between themselves and the lamp, which seta upon the table before them, in such a position that it concentrates the light directly upon their work. A. B. H. 96 THE MICROSCOPE IN BOTANY. be neither too light nor too small. Formerly the microscope foot was made in a disk or plate-like form, of a thin plate of brass loaded with lead. But to this form of foot there was this objec- tion, that, unless the surface upon which it rested was exactly flat and even, the microscope would totter or wabble with every motion. So at the present time the microscope is made with the horse-shoe foot [or with a tripod giving three widely sepa- rated points of support. The latter effect is most frequently gained by means of three solid projections on the bottom of the brass plates or arms as shown in all the Plates but especially in Nos. V, X and XL It is now customary to give more satisfactory means of contact with the table by supplying the feet with disks of soft India rubber projecting slightly from their lower surface. R. H. W.] The folding microscope foot, which consists of three legs, is altogether objectionable. ' They never give a firm support and are the most impracticable things ever devised for the microscope. At best this form is applicable only to travelling microscopes where its compendiousness is its only recommendation. XIY. RULES FOR THE USE OF THE MICROSCOPE. A good instrument is a very precious article, but a microscope ruined through neglect is the most hateful thing one can see. By proper treatment a microscope, though much and often used, will remain unchanged for many long years, assuming that it be not too much exposed to its mortal foe, the dust, and that it be cleaned each time after using. It may not be superfluous here to add some hints about keeping the microscope clean. It is an easy matter to keep the metallic part clean. It is only necessary after each using to rub it carefully with chamois skin or linen. Commonly it will be sufficient to rub it dry, to restore the brightness, but sometimes water may be used espe- cially in cleaning the stage. Alcohol and the like should under no circumstances be used on the polished parts of the micro- scope, because it will be sure to dissolve the lacquer with which they are covered. But its use is not necessary, since no oil or RULES FOR THE USE OF THE MICROSCOPE. 97 glvcerine or the like should bs put on the screws or any part of the stand. The dull parts of the oculars so far as they sink into the microscope tube, the tube as far as it runs in the outer sheath, and finally the diaphragm cylinder, should be carefully cleaned. Keeping the glasses cleaned demands still greater care. Many people believe the optical parts of the microscope are clean when no dust can be seen in the field of vision. But this is proof only that the under ocular glass, the collecting or field, lens is clean. Particles of dust on the objective cannot be seen: in the field of vision as a little reflection will show, but they exercise a damaging influence on the microscopic image, since they cause a cloudiness and impaired definition. It is therefore necessary to clean, occasionally, both oculars and objectives. For this purpose, old, very soft linen washed repeatedly in dis- tilled water or a soft hair pencil with distilled water should be used. The ocular should be cleaned in the following way. Suppos- ing it to be quite soiled, both glasses should be unscrewed and first of .all wiped with dry linen. Then dampen a clean piece of linen with distilled water and rub each glass holding it by both edges. This may be repeated as often as necessary, and when it is rubbed perfectly dry it should be swept with a fine hair pencil to remove any fibres from the linen which may be adhering to it. In this way we get perfectly clean glasses. To test the purity of a cleaned glass it should be breathed upon a little. If it is perfectly clean the dampness will all disappear at the same time. But if there is a particle of dust it will gather in a little zone about that and evaporate there a little later than it does on the clean surface. The objective is cleaned in the same manner, but since its three lenses are joined in an air-tight mounting it is scarcely possible for dust to collect between them. So we shall have to clean only the upper and under surface of the lens-system. Usually it may be done as with the oculars. But since the upper object glass is difficult to come at we should prepare i wooden stick with some soft linen bound over the end of it, and with this reach down and clean off the glass. Should the 7 98 THE MICROSCOPE IN BOTANY. lower lens get besmeared by the careless use of the reagents it should be at once cleaned by repeated washings with distilled water. Water will suffice in most cases. But if it be necessary to use alcohol it should be done with the greatest caution and celerity. For by employing alcohol we are in the greatest .danger of ruining the whole system, by the alcohol penetrating within the mounting of the glasses and partly dissolving the Canada balsam by which the crown and flint glass lenses are cemented together. The cleanliness of the objective may be tested in the same way as the ocular, as indicated above, or by examining the reflected image of a window in the glass by means of a magnifying lens. For the preservation of the microscope, besides cleanliness, the handling of the screws is of importance. This particularly concerns the matrix of the tube and the screw of the objective- system which fits into it. Since by almost every change in the magnification this must be screwed on and off, and since by putting the objective on a little obliquely, the whole screw arrangement may easily be injured, the following method of screwing on the pails is commended as one certain to work no injury to the apparatus. The objective should be set upon the tube with its thread close up against the end ; then it should be turned backwards, as in the act of unscrewing, till one hears the short click which shows that the thread has fallen into its proper place, when the system may be reversed and screwed up. Further, the greatest care should be used in focussing. With high magnifications the objective is brought very close to the object and must be carefully guarded in the act of focussing, or both objective and object will be ruined. Many microscopists do the focussing in this way. The tube is pushed down by hand, or by the rack and pinion, till it comes very near to the objects and then exactly focussed with the fine-adjustment screw. With the use of low powers this is satisfactory, but with higher magnifications, and in the use of the lower by beginners, the following, reverse, method is recommended. By looking across the stage from the side one can bring the objective down very close upon the preparation, still without touching it, bring it RULES FOR THE USE OF THE MICROSCOPE. 99 within the focal distance. Then by hand or by the rack and pinion run the tube upward till the coarse adjustment is reached and finish with the fine-adjustment screw. This method secures perfect safety for both lens and specimen. Respecting the preparation to be examined, it is clear that both slide and cover-glass should be perfectly clean before it is put under the lens. Permanent preparations should be wiped each time before using, with a piece of linen, and if they are used with an immersion fluid it should be carefully removed after each using. If a preparation is to be studied for a considerable time with- out removal from the stage, a glass bell should be placed over the microscope and preparation, which, in case it rests upon the same leather or cloth-covered wooden plate, with the microscope, will effectually exclude the dust. If one brings the microscope in winter from a cold to a warm room and undertakes to use it at once, the ocular glass becomes dimmed by the condensation upon it of vapor from the body or the atmosphere. It is better, therefore, to bring in the mi- croscope some little time before the work is to begin, and set it near the stove to warm up. If the microscope is to be for a long time out of use, it should be inclosed in its mahogany case, and put away in some closely shutting cupboard in which is placed a little dish of chlorate of lime. This insures the safety of the steel parts from rust, and prevents the formation of verdigris. Never should the micro- scope, and under no circumstances should the objectives, be stored in a closet in which the reagents ar^ kept, for out of the closest-stoppered reagent bottle, there will come some vapor of acid, which will at length cause the greatest injury to every kind of optical apparatus. CHAPTER II. MICROSCOPICAL ACCESSORIES. UNDER this term we include a series of implements which finds frequent use in microscopical investigations. The greater part of them are connected with the optical apparatus of the microscope itself. The most important microscopical acces- sories of which we shall here speak are the preparing micro- scope, the apparatus for drawing or photographing microscopic pictures, the micrometer, the polarizing apparatus, the goni- ometer and the micro-spectroscope. I. THE PREPARING MICROSCOPE. (DISSECTING OR MOUNTING MICROSCOPE.) The preparing microscope is of use, in order to prepare those objects which have previously been made ready for examination by means of the section-cutting instrument, and need to be examined first with a low power, in order to lay them rightly on the object-slide, or, by means of small needles and knives, to further treat and manipulate. 1. The Simplex. The preparing microscope consists essen- tially of a mounted magnifying glass in the focus of which the slide bearing the object may be brought ; beneath this is placed the mirror to furnish the necessary light for the object. A simple magnifying glass gives comparatively but small magnification. To get strong magnifying power it would be necessary to make the lens with a high superficial curvature, and this would involve a short focal distance and too great prox- imity to the object to allow a convenient use of the dissecting needles. For this reason we have for a long time ceased to use (100) THE PREPARING MICRO SG.OPE/ simple magnifying glasses and instead use a combination of two or three. By this means we secure a shortening of the focus and greater magnification without diminishing the working dis- tance between the object and the under lens. Combinations of two or three of these glasses are called respectively doublets and triplets. [For the lowest powers of the preparing microscope, single lenses, either double-convex or plano-convex, of from two inch to one-half inch (51 to 13 mm.) focus are commonly employed. By some of the makers they are so constructed that two may be used together.] [2. The Coddington Lens. For powers as high as one-fourth inch (6 mm.) and as low even as one inch (25 mm.) the Cod- dington lens, Fig. 34, is available. This is a solid glass cylinder whose ends are ground to spherical surfaces both of which are portions of the same sphere, the center of the curvature of both being identical and situated in the center of the glass. A groove FlG 34 is ground around the circumference of the cylinder and blackened to serve the purposes of a diaphragm. This construction gives an excellent definition which is "less dependent than in any other magnifier on the exact adjustment of the lens ; since, having an optical axis in any available direc- tion, oblique rays pass through it under exactly the same condi- tions as axial ones, and the performance is therefore not marred by imperfect centering. The working focus is rather short. To partially remedy this fault and to render the instrument less clumsy, the cylinder is often shortened, at a slight optical disadvantage, by bringing the convex surfaces nearer together than when in their theoretical position.] [3. The Achromatic Triplet. When a magnifier having the external form of a shortened Coddington is made achromatic, it consists of two double convex or meniscus lenses of large curvature, whose aberrations are corrected by a thick lens of glass of a different refractive index cemented between them * with Canada balsam. The " Globe lens " of Gimdlach is literally an achromatic Coddington, as it is a sphere of flint glass, MICROSCOPE IN BOTANY. ground hollow and filled and corrected by a much smaller sphere of crown glass, the whole being reduced to a cy- lindrical form by cutting away the unused peripheral portion. Being achromatic it does not require the diaphragm groove. A more common combination is a pair of double convex crown glasses corrected by a thick, double concave flint glass ; though some makers, notwithstanding the disadvantage of exposing the softer glass to outside wear, prefer to place the flint, in the form of a meniscus, at each end of the crown. This combination, more or less modified and variously named by different makers, constitutes the achromatic triplet which is now taking the place of all other magnifiers, especially for the higher powers, in the simple microscope. It gives a long working focus and a broad, clearly defined and beautifully lighted field of view, which is a luxury for all purposes and may be considered indispensable for very fine or difficult work. It is usually mounted like the Coddington, Fig* 34. The achromatic objective of the compound microscope is not equally suitable for use in the simple microscope, though the low powers, if mounted short, are sometimes so employed. For this purpose the separating objectives in short tubes, Fig. 2, are available.] [4. The Engraver's Glass, consisting of a pair of plano- convex lenses, about 45 mm. in diameter, mounted in a deep, hard rubber cell, as repre- sented in Fig. 35 and forming a doublet of great size, gives a large field of view, is used with little strain or fatigue to the eye, and is a very serviceable preparing microscope when only the lowest powers are required. The writer has employed these glasses for twenty years with great FIG. 35. J J satisfaction in the exami- nation of pressed plants mounted upon paper as herbarium specimens, in the preliminary examination of hand- writ ing, and THE PREPARING MICROSCOPE. 103 in the selection from among large masses of material, as of fabrics or mixed fibers or other substances supposed to contain inequalities or adulterations, the portions requiring further in- vestigation ; also in performing under the lenses such manipula- tions or dissections as require only a low amplification. Such lenses may be best supported upon the large lens holder shown in Fig. 37.] [5. The Handy Dissecting Microscope. A preparing mi- croscope of extreme simplicity, made by Bausch and Lomb, is shown at its natural size in Fig. 36. It consists of a glass plate into which is screwed an upright brass rod B, which sup- ports the magnifiers at A. These are three simple magnifying glasses, capable of being used singly or together, and mounted FIG. 36. in a form available for ordinary pocket use. The addition of a Coddington lens, Fig. 34, suitably mounted to be attached to the same stem, gives a somewhat higher power, of good quality. [6. The Lens Holder. Finding the lens holders in use to be of too limited applicability, being too light, for instance, to carry the large engravers' lenses, and too short-armed for the convenient study 'of handwriting upon large sheets of paper or of mounted herbarium specimens, or else too unstable for use with higher powers, the writer has devised and employed a 104 THE MICROSCOPE IN BOTANY. form, .arranged somewhat like the stands used by engravers, which is (unlike them) sufficiently firm and manageable for either large or small magnifiers, of low or high powers, and is available for an arm-length of 20-25 cm. It consists, as shown in Fig. 37, of a rectangular frame which slips over the pillar of a bull's eye stand, both it and the bull's eye being often mounted upon the same stand, for the sake of simplifying the apparatus, and because they are often advantageously used in combination. The frame slides smoothly up and down the pillar, being held in any position by an included spring. To an FIG. 37. extension of the bottom of the frame is attached a horizontal arm, having first a horizontal pivot joint, and secondly a ball and socket joint, the tension of these being readily adjustable by means of a screw with a large milled head. By bending the joints, the lens maybe brought near the pillar for use in connec- tion with the bull's eye ; or by attaching the jaws or ring to a longer wire, the total arm length may be increased at will.] THE PREPARING MICROSCOPE. 105 [At the end of the arm rises a vertical pivot upon which can be slipped almost any kind of pocket magnifier, such as a Cod- dington, or achromatic triplet Fig. 34, or a three-lens system like A, in Fig. 36, or a double bellows-shaped arrangement like that shown in situ on the arm. Or, the lenses being removed, a split wire may be inserted into the hollow end of the arm, bearing a pair of hinged semicircular jaws, shown in the figure, for carrying an engraver's glass, or any variety of large lenses not requiring delicate adjustment. For magnifiers of higher power, requiring more precise adjustment, a ring is substituted for the jaws.] FIG. [There is a fine adjustment at the top of the rectangular frame, where a screw with milled head, pressing the pillar against the spring, promptly but steadily depresses the lenses to the extent of about four times its own motion.] [This apparatus is now made for the trade by theBausch and Lomb Optical Co. When supplied with fine achromatic lenses, and kept standing always ready upon the table, it becomes con- 106 THE MICROSCOPE IN BOTANY. stantly useful even to persons well supplied with elaborate ap- paratus. It is worked, if transmitted light be required, over the stage of any dissecting microscope that may be within reach. By turning the jaws or ring into a vertical position, it is well adapted to the examination of living aquatic plants in a glass jar or aquarium ; for which purpose powers of 50 to 100 diameters may become available by using the Briicke magnifier FIG. 39. (p. 107) or the Bausch and Lomb compound dissecting magni- fier (p. 108), which for this use should be screwed, not slipped, into the ring.] [7. The Compact Dissecting and Mounting Microscope is made by Bausch and Lomb, and is a preparing microscope of medium size and cost, and of sufficiently portable form for pocket use. It is represented in Fig. 38. A japanned iron base, 9 cm. square, carries a mirror in a central location for axial PL. XII. Compact Compound Dissecting Microscope. Bausch & Lomb. THE PREPARING MICROSCOPE. 107 illumination, and also supports a pillar which carries, at a height of 9 cm., a stage of blackened brass, slightly smaller than the base. This stage has a central opening of about 32 mm., sup- plied with a removable glass plate. Inside the pillar is a trian- gular rack-bar moved by a pinion with a milled head, which carries a transverse bar with a ring at the farther end, into which, in the optical axis of the instrument, the amplifying lenses are inserted. This ring is also furnished with the society screw, by means of which objectives may be substituted for the simple lenses. The mirror can be instantly transferred to the bottom of the stage for oblique, or to the top of the stage for opaque illumination. Hand rests after the German style can be attached to the sides of the stage, as figured in Plate XII. Both base and stage can be folded flat against the pillar, in order to be packed in a very small case.] [8. The Botanical Dissecting Microscope, Fig. 39, made by Mr. Zentmayer, is a somewhat larger and heavier instru- ment than the one just described ; but it has essentially the same parts similarly arranged. The circular base is 12 cm. in diameter, and the stage is 9 X 12 cm., with a central opening 4cm. With the stage increased to 11 X 15cm., and a larger mirror substituted, compactness would be still further sacri- ficed, but the Avorking capacity would be, in the writer's opinion, correspondingly increased.] [9. The Briicke Compound Dissecting Microscope. A sepa- rating achromatic objective, whose lenses can be used either sing-ly or together, with an ocular in the form of a concave eye lens (as originally proposed by Prof. Briicke of Vienna) in- serted into a small tube sometimes less than 5 cm. high, which acts as a little compound body, constitutes a microscope, upon the principle of the Galilean telescope, which has long been used in France and Germany, and to a more limited extent but not Avith less satisfaction, in this country. The objective lenses commonly used, by themselves give powers of from 12 to 30 diameters, which poAvers are increased by the eye lens to from 40 to 100 and upwards. With the highest powers the working focus is exceedingly long (8 mm.), affording ample room for the use of needles or dissecting instruments. The field of view is rather small.] 108 THE MICROSCOPE IN BOTANY. [10. The Bausch and Lomb Compound Dissecting Microscope. This form, just introduced as a substitute for the Briicke instru- ment, consists of a little compound body (combined with their compact microscope in Plate XII) only 75 cm. long and 19 mm. in diameter, which contains a diminutive objective, ocular and erector. By the use of the draw-tube the erector can be car- ried from near the objective to near the top of the body, giving a range of powers of from 12 to 150 diameters, the working focus meanwhile varying inversely from 38 to 6 mm. The light trans- mitted is less than with the -Briicke apparatus, the field of view averaging about the same for the same powers. The advan- tage of the new arrangement is the ready command of the whole range of intermediate powers by simply sliding the draw- tube. For the higher powers, it may be used without the erector if preferred. The objective is a dividing one, whose lenses, the compound body being removed, can be used sepa- rately or in combination as simple magnifiers.] [11. The Histological Dissecting Microscope, a combined simple and compound microscope made by R. and J. Beck of London* and Philadelphia, figured in Plate XIII, is adapted to a considerable range of family, school and amateur use. The simple or preparing microscope figured at the right, is small, compact and substantial ; and is somewhat suggestive in form of the Zeiss preparing microscope. It is 'promptly converted to a compound microscope as shown at the left, for examinations re- quiring higher powers, by removing the lens from the transverse arm, and by replacing it with a small compound body. As a simple microscope this instrument lacks breadth of stage, and as a compound, it lacks a fine adjustment; still it is used with much satisfaction by many whose wants do not demand a higher grade of apparatus.] [12. Hand Rests. In using any form of preparing micros- cope, not even excepting those with the largest stages, much in- crease both of comfort and of steadiness in manipulation can be secured by using hand rests at the sides of the stage. Thin * This instrument, as well as some others mentioned hereafter, though English, is ad- mitte'd among American apparatus, for the reason that the American business relations of the London house have been such for several years past, that the Beck firm has come to be regarded as partly an American enterprise, and that their wares have become as familiar and accessible as if actually made in this country. R. H. W. , ,., 8. 8 bfl c TJ C ct 15 o O u o CD THE PREPARING MICROSCOPE. 109 metal wings, as in PL XII, can be attached to the stage for this purpose. For frequent and prolonged use, however, a broader and firmer support made of wood and resting upon the table instead of the stage, is more restful and is coming into use. The writer has been accustomed to use a rest, made of mahogany strips about 1cm. thick, and 10 to 12 wide, con- structed as shown in front view, somewhat diagraphically, at about one-fourth size in Fig. 40 ; where there is a base lying FIG. 40. upon the table, the rests attached at one end by hinges and held down firmly with brass hooks, hinged strips supporting the rests at the desired height and in an inclined position, and wooden buttons held by large screws (which for better stability should be fastened with brass nuts below) for holding the base of the microscope firmly in position. The hinges are all so arranged that the strips can be folded together solidly, for portability, as shown in Fig. 41, and held snugly in F Z ::! / \ ^p _^ \ L E J 1 n r 1 !! Pn !! i L ) 1 3iUj 1 JJ K 1 ii ]JJV_ \ r 33E "^ 4^" .ii^ip FIG. 41. that position by the same hooks as when open. The hooks are on the farther side of the wooden strips. Such an arrangement can be purchased from the microscope dealers, or made for one's own use by any person fond of such experiments. By a slight change in size it is applicable to any preparing microscope. It should be made of such size that the upper ends of the rests 110 THE MICROSCOPE IN BOTANY. will be nearly continuous with, or slightly below, the stage of the microscope. Exact approximation is not necessary. When properly adjusted the rest is perfectly firm and steady. When portability is not required, the hinges and hooks may be dispensed with, and the wooden strips fastened together with glue and brads. R. H. W.] II. APPARATUS FOR DRAWING MICROSCOPIC PICTURES. If we suppose that m, Fig. 42, be the tube of a microscope, d the objective and c the ocular, then the preparation o lying on jn FIG. 42. the stage sends out a bundle of rays which, as the mathematical line or, passes out of the ocular in the vertical direct 1311, or, in the way already described. Now place a small glass mirror s directly over the ocular, inclined at an angle of 45 to the emitted rays, and the rays will be reflected at a like angle from the mir- ror, changed now from a vertical to a horizontal direction. If I put my eye at the point a, I shall naturally perceive the mi- croscopic image reflected in the mirror. If now the mirror be transparent and a sheet of paper p be fixed up perpendicularly APPARATUS FOR DRAWING MICROSCOPIC PICTURES. Ill behind it, I shall see through the mirror and look upon the paper beyond and the microscopic image will appear to be ly- ing on the paper, or, in other words, it will be projected through the mirror upon the paper. If we choose, the distance [ab = 10 inches (25.4 cm.), the magnifying power, which is really a question of angular extent, will be always converted to linear measure at a fixed distance, as it should and must be to render a variety of records by different observers comparable with each other. The magnifying power represented by com- paring the size of the drawing made at this standard distance with the actual size of the object itself will also represent more accurately than at any other distance the resolving power of the instrument ; since the power of the microscope to render small objects or fine points of structure distinguishable depends on the angular size of the object as seen in the microscope com- pared with that of the object, not as unseen by the naked eye upon the microscope stage, but as it could be seen by the naked eye at the best distance that could be chosen for that purpose. By common usage this distance is established at the standard limit of 10 inches (25.4cm.) which is assumed to be an average representation of the distance, varying for different eyes, of most distinct vision for small objects. The impropriety of the advice, which does not lack high authority, to project or draw the magnified images, for measurement or comparison, at the exact distance of the object on the stage becomes evident in such extreme cases as using a simple microscope, with the object very near the eye, or a compound microscope having a tube two or three inches or as many feet in length. E. H. "W.] One may observe the working of this simple contrivance, very easily in the following way. Fix a clean cover-glass of the utmost possible thinness to the top of an ocular, by means of a drop of wax, in such a position that it will meet the rays of light from the objective at an angle of 45, Fig. 42, s. Powerfully illuminate the field of view, and put on a lo\v magni- fying power ; then the image of the object beneath will be seen projected, and somewhat darkened, on the paper b. With a pointed lead pencil, one may then easily trace the coarser outlines on the paper, since one can see at the same time both 112 THE MICROSCOPE IN BOTANY. the image and the pencil point. This simple contrivance would be perfectly satisfactory for tracing microscopic images, were it not for two faults. First, the paper surface is in a very unfavorable position, the hand having no support, hence the tracing will be in coarse, rough outlines, and secondly, the image is very poorly lighted. Also, according to the investiga- tions of Fresnoi, in the reflection of a transparent mirror, the 0.944 part of all the rays that fall upon the glass pass through it and only the 0.056 part are reflected and come to be of value in the reflection-image, so that this will have but the 1*8 part of the brightness of the original image. These prevail- ing faults can be overcome in great part in the following way. In order to project the image on a horizontal surface it is nec- essary [to place the microscope body horizontally while draw- ing, or else] to have a contrivance for double reflection. This is represented in Fig. 43, where m again is the microscope-tube bent at a right angle at r, where there is a small thin- glass mirror s placed at an angle of 45 to the ray or. The ray on striking this is re- flected in the direction of w 1 through the ocular c. Here again a transparent mirror is set at an angle of 45 which reflects the rays in the direction FIG ' 43< r'a, and the image will appear to the eye to be projected on the horizontal surface beneath at 5, where, on the already prepared paper, the tracing may be con- veniently done. One can easily construct this contrivance himself, by using a paper tube bent at r, at a right angle and made to fit on over the microscope-tube, and into which an ocular can be put. The mirror s, which should be made of the thinnest glass, may be blackened with India ink on the back side. By making the magnification low, and the light strong, this simple apparatus is very well adapted to the purposes of microscopical drawing. In APPARATUS FOR DRAWING MICROSCOPIC PICTURES. 113 order to save the great quantity of light which passes through the transparent mirror, s', not reflected, a small metallic mirror is substituted, which not being transparent reflects all the light that falls upon it. It should be made of silver, and supported on a very slender arm. It must also be smaller than the pupil of the eye, in order that the eye may look by it and perceive at the same time the underlying paper, pencil, etc., and the image have the appearance of being projected upon the paper. Som- mering 1 invented the small metallic mirror as an aid to micros- copical drawing. The use of the reflecting glass mirror has always had this dis- advantage that it did not give a perfectly sharp image, and for this reason. We will suppose ABC D, Fig. 44, to represent a magnified section of a glass mirror blackened upon the back. On this mirror the light ray on falls at an angle of 45. Here a . part of the ray undergoes reflection, in consequence of which this part of the beam of light takes the direc- tion n m (angle A no = B n m). Another part of the beam, how- ever, passes through the glass being refracted by it in the direction n n 1 to the back side of the glass. Here it is reflected so that angle C n'n = D o o n'n". Here it again suffers refraction at the front surface A B, and in consequence goes as a ray in the direction 71' m' parallel with the ray reflected from the front surface n m. By the use of the glass mirror, therefore, the ray will be divided into two rays which run near and parallel to each other, and whose dis- tance apart will be greater in proportion to the thickness of the glass used. So the image reflected from a glass mirror will be divided into two images which are not exactly superimposed, and the consequence is, the image is less distinct after reflection than before. This evil is obviated by the use of a reflecting glass prism, represented in section as a right-angled isosceles triangle, 1 See H. v. Mohl, Mikrogvaphie, p. 321. Ilarting, Das Mikroskop, pp. 176, 901. 8 114 THE MICROSCOPE IN BOTANF. which makes the reflection from the hypothenuse. The latter must be ground absolutely flat, which is by no means an easy thing to do. Fig. 45 represents two reflecting glass prisms which are so arranged as to correspond to the two glass mirrors in Fig. 43, r and r' in both figures corresponding to each other. We will suppose that the ray o passing in the direction of the arrowpoint falls upon the side of the prism p perpendicular to its surface. It enters the glass unre- fracted and passes to r, striking the hypothenuse surface at an angle of 45. Here it suffers a total reflection and takes the direction rr'. In like manner it enters the second prism p' and is likewise reflected at r f in the direction r'a. It is therefore clear that the two mirrors s and s', Fig. 43, can .be replaced by the prisms pp'. TIG. 45. FIG. 45. [A prism like p', but about half the size figured, is sometimes cemented to the end of a small bar and mounted in front of the ocular, the large prism being dispensed with. It is very con- venient for drawing either in the horizontal or inclined position of the tube ; but as it inverts the image, as do other singly- APPARATUS FOR DRAWING MICROSCOPIC PICTURES. 115 reflecting arrangements, difficulty is experienced in retouching the sketch made by its use. It also requires great steadiness of position on the part of the user. By substituting for the little prism a small steel mirror, also smaller than the pupil of the eye, the Sommering mirror is produced, which acts precisely like the small prism and has much the same advantages and defects. Neither of the above has come into very general use.] [1. The Neutral Tint Reflector. The singly-reflecting cam- era, called the neutral tint reflector, was brought into use at the suggestion of Dr. Beale of London and is shown in situ in Fig. 46. This reflector was originally a thin plate of neutral tint glass, but a common white cover-glass is now employed as a satisfactory substitute. It is supported in front of the ocular at an angle of 45, so that the observer looks ob- liquely through it at the paper and pencil, seeing at the same time and apparently in the same place the microscopic image reflected from the glass. The glass being thin, but little indistinctness results from the confusion of the separate reflec- tions from its two surfaces; while the inconvenience of its inverting the image seems to have been quite overbalanced by its simplicity and cheapness, and the fa- cility with which it can be used even by inexpert persons. Though practically limited, for the reason stated above, to those uses which allow a nearly horizontal position of the microscope body, this con- trivance is probably more used in this country than any other form of camera lucida. R. H.W.] 2. The Wollaston Camera lucida. The little mirror s in Fig. 42, s' in Fig. 43, and the prism j/ in Fig. 45 may be replaced hy an apparatus invented by Wollaston and represented in Fig. 47. 2 It consists of a four sided glass 2 See Wollaston in Phil. Transactions, 1809, No. 38, p. 741. W. H. Wollaston's descrip- tion of the camera lucida, an instrument designed for sketching objects in the neighbor- hood, and for making magnified or minified tracings (Gilbert's Annalen der Pliysik. Bd. XXXIV. N. F. Bd. IV, 1S10, pp. 353-361, I Tafel) Gehler's Physikalisches Worterbuch, Leipzig, 1825, Bd. II, p. 30^. 116 THE MICKOSCOPE IN BOTANY. prism AFGH, in which FGH is a right angle while HAF equals 135. The ray strikes the surface GF in the vicinity of F perpendicularly, so that it passes unreflected through the prism to r and then to r' in each undergoing total reflec- tion, and passing out of the prism in the direction r'a. The eye placed at the point a looks downward through and just beyond the edge of the prism at H, and perceives the sheet of paper lying at p, on which at b the image of the rays seems to be. [The Wollaston camera lucida is usually attached to the mi- croscope by means of a spring-ring slipping over the top of the ocular. Connected with the front end of this ring is a light brass box containing the prism and wholly covering it except that the side FG in Fig. 47, is left unprotected and that the edge His exposed (more clearly seen at F, Fig. 48), by a notch through which the observer looks down upon and through the prism, at the same time that he views the paper and pencil with that portion of the pupil of the eye that is not over the prism. This camera lucida, originally devised for general drawing, is still used more than almost any other for microscopical work. Care is re- quired as in the use of other cameras, to so regulate the intensity of the illumination of the field of view and of the draw- ing paper that neither shall be obscured by the relative bright- ness of the other.* For greater ease and distinctness in viewing the paper, a lens of long focus, like a spectacle glass, is often placed below the prism, just below ?', Fig. 47, and in the line of the ray ab. This camera is generally used with the body of the microscope in a horizontal position, and with the paper lying horizontally beneath the ocular, since a vertical * As the light in the field of view and on the object may be easily and exactly regulated by means of the diaphragm and mirror, the principal difficulty will be found in the man- agement of the light upon the paper. Hence a small movable screen of thick paper, which one may easily contrive lor himself, about 30 cm. long and 20 cm. high, placed more or less directly between the source of light and the paper, thus darkening the latter at will with its shadow, will be found very useful in conducting this kind of microscopical drawing. A. B. H. APPARATUS FOR DRAWING MICROSCOPIC PICTURES. 117 position of the tube would require the paper also to be vertical. R. H. W.] 3. NoberCs Camera lacida. Another camera lucida origi- nated with Nobert and is diagrammatically represented in Fig. 49. It permits the picture to be drawn on a horizontal surface. The rhombic prism, ABCD, bears on its oblique surface AB a small right angle prism EFG, made fast to the surface AB at EF by means of Canada balsam. It is then so placed over the ocular of the microscope that a ray of light proceeding from it strikes the surface FG perpendicu- larly. It goes directly through the mass of glass to a in the direction oa, vmrefracted and losing little light by reflection at r r . Again ray b from a horizontal drawing surface strikes the oblique side BC of the rhombic prism and passes through to the side total reflection in the direction r T) 1 A C 1. FIG. 49. CD at r and suffers a At ^ it is again totally reflected in the direction ra, and reaches the observer's eye at the same time and in the same direction with the image-forming rays from the microscope oa. The observer sees in the micro- scope, by means of this apparatus, not only the object but at the same time also an image of the drawing surface and pencil. The Nobert draw- ing prism as com- pleted by Nachet [and known in this country as Nachet's] is represented natu- ral size in Fig. 50. This apparatus for horizontal d r a w i n g consists of a metal ring r which is put upon the microscope-tube and the ocular is replaced. The metal ring bears a projection h, into which the brass rod f exactly fits, and not only turns upon its axis but likewise may be moved up and down by hand. On f FIG. 50. 118 THE MICROSCOPE IN BOTANY. is fastened a blackened metal plate cZ, which widens into a circle over the ring and has its center bored out with a large opening. The metal box AB is fastened to this plate by means of the screws e. The box contains, within, the two glass prisms more exactly illustrated in Fig. 49. At C and in the corresponding place of the under surface is a circular opening which falls in with that of the plate d just now mentioned. The box must be so large that B shall extend laterally beyond the foot of the mi- croscope. Directly under B is the surface of the paper on which the drawing is to be done. The eye looks down through G and sees in the field of the microscope the drawing paper and the pencil. The box AB may be turned aside at will and leave the ocular free. [4. Grunow's Camera lucid a. Mr. J. Grunow has contrived a camera lucida in which an effect nearly identical with that of Nobert and Nachet is secured, but in a slightly different manner. This device is shown in section in Fig. 51 where P is a rectangu- lar reflecting prism like those in Fig. 45, while P' is a cube formed of two such prisms cemented together with Canada balsam. One of the surfaces of contact, fg, is silvered, except a circular spot in the center about half the diameter of the pupil of the eye, thus bisecting the cube obliquely with a perforated mirror. It is evident that the eye at PN can look directly down the microscope-tube in the direction NM through the cube P' by reason of the central aperture in the silvered surface fg, while the paper on the table at P can be seen with ease at the same time by re- flection from the glass surface P and from the silvered portion of the interior surface fg. This device is mounted and slipped over the ocular. It is used with the same comfort to the eyes as the Nobert form, though not with the same position FIG. si. APPARATUS FOR DRAWING MICROSCOPIC PICTURES. 119 of the microscope, since the Grunow camera is best adapted to an inclined position of the microscope-tube, making the line PP vertical and the drawing upon the table free from distortion when the tube is inclined at about an angle of 30, while Nobert's gives the same results in a vertical position of the mi- croscope, and requires an inclined drawing board when the tube is inclined.] [5. Photo-micrography. The substitution of the camera ob- scura for the camera lucida as a means of preserving or repro- ducing microscopic views, while not without disadvantage in respect of showing the relation of parts, and of selecting typical points in different portions of the field and combining them all in one picture, is often desirable on account of the impartiality and completeness of detail secured by the automatic action of the light itself. Hitherto its use has been mostly confined to the few who happened to possess an exceptional access to, and familiarity with, the mysteries behind the scenes of some photo- graph gallery, and to the still smaller number who were pre- pared to incur the expense of employing the assistance of a professional photographer. Recently, however, the develop- ment of amateur photography as a popular pastime has placed within reach of the microscopist the means of doing this work for himself, without previous experience, unusual mechanical skill, or considerable expense.] [The Bausch and Lomb Optical Co. offer for sale a very compact and beautiful amateur photographic camera, suitable for general work, with a simple attachment by means of which it can be brought into relation with the possessor's microscope, and the magnified image of an object focussed upon its sensi- tized plate. Mr. W. H. Walmsley of Philadelphia, late Am- erican Manager for the Messrs. Beck, has arranged, and has introduced to the trade, a complete outfit, containing not only a camera, and platform for connecting with the microscope, but also the requisite chemicals, and a complete, carefully selected assortment of the various supplies required for the work. This will be a convenience to those who do not wish to incur the trouble and delay of learning their own wants by means of their 120 THE MICROSCOPE IN BOTANY. own experience. Mr. Walmsley's arrangement of his apparatus is shown in Fig. 52, which illustrates clearly the theory and practice of such devices generally. R. H. W.] FIG. 52. III. THE MICROMETER AND MICROSCOPICAL MEASURING. In microscopical investigations it is often a matter of great importance to be able to determine the true size of an object under the microscope. This can be done in various ways. But as different as the various apparatus is which has been designed for this purpose, there are but two principles involved in it, viz., either to measure the object itself, or, to measure the mag- nified image of it which the objective produces. The appara- tus which aims to do the former, we call "objective micrometers :" that which aims to accomplish the latter we name "ocular mi- crometers." Objective as well as ocular micrometers may be either glass- or screw-micrometers, that is their essential part may be fine diamond-rulings on glass, or a carefully cut screw \vhose thread has a definite height. OBJECTIVE MICROMETERS. 121 I. OBJECTIVE MICKOMETEKS. A. The Objective Glass- Micrometer. (Stage micrometer.) This consists of a small glass plate on which, by means of a diamond, fine rulings are cut, a millimeter being divided into one hundred equal pails. [Its appearance under the microscope is as represented in Fig. 53. Subdivisions of the inch, or inch and millimeter compared, are also used.] If such a ruled plate be put under the microscope in place of the object slide and the object to be measured be laid upon it, the length of the latter can be determined. This kind of microscopical measuring is doubt- less the simplest in the world, but, alas ! there come in so many disturbing influences that its use is limited to a few cases only. [It is chiefly used to determine the working value of the divi- sions of the ocular micrometer.] B. The Objective Screw-Micrometer. [This apparatus is a sliding plate attached to the stage, or constituting a part of the stage itself, by which the object while in view in the micros- cope is carried steadily past a fine line fixed in the ocular. The movement is accomplished by the push of a fine horizontal screw acting upon 'the plate, and the distance traversed in carrying the whole object across the line, the same being the diameter of the object itself, is determined by the number of the turns and fractions of a turn of the screw, the width of whose thread is very accurately known. Though manufactured with great skill, and theoretically capable of reading off results with almost unlimited minuteness, directly and without the trouble of computations pertaining to ocular micrometers, this apparatus requires great skill in manipulation and is particularly liable to get out of order. At best it is believed to be inferior to the ocular micrometer in accuracy, since it measures the object directly, and not its magnified image ; any errors in its perform- ance being, therefore, multiplied by the whole magnifying power of the microscope, instead of by the power of the eye lens only. For these reasons it is, as conceded by the author, giving place more and more to the ocular micrometer.] 122 THE MICROSCOPE IN BOTANY. [Some microscopes with a mechanical stage have the adja- cent surfaces graduated, with or without a vernier, so that the position of the stage or the distance it has moved can be read off and recorded. This serves as a finder, by which the position of an object mounted on a specified slide may be registered, and supplies a means of roughly measuring the size of large ob- jects, as for instance, the width of a leaf or the length of an anther. R. H. W.] 2. THE OCULAR MICROMETER. Ocular micrometers by which we undertake to measure the real image of the object are either glass- or screw-micrometers. They have, however, this advantage over the objective microm- eter, that they do not require by far, the same precision of work as that, since a casual error in the rulings, etc., has a mag- nitude in the results of the measurement, equal only to the given . error divided by the whole number of the objective magnification. A. Ocular Glass-Micrometer. This apparatus has a wider distribution in Germany than all other micrometers. It con- sists, as the objective glass-micrometer does, of a glass plate upon which are diamond-rulings. A thin cover-glass is put over the rulings for their better protection. The scale extends for four or five mm., and each millimeter is divided into ten or twenty equal parts, the fifth and tenth line being extended in the ordinary way. for greater convenience in counting. [Whether it be divided into metric or English units is unim- portant, except that it may sometimes be required for use as a stage micrometer, since the working value of the divisions de- pends upon the optical conditions under which they are used, and must be carefully determined after the apparatus is arranged and in order for the proposed work.] [The simplest, cheapest and commonest form of ocular microm- eter is a round cover-glass, properly ruled, which is laid when about to be used upon the diaphragm of the ocular in the focus of the eye lens. The rulings should be turned downwards to THE OCULAR MICROMETER. 123 bring them close to the diaphragm, and if not distinctly visible, they should be made so by slightly unscrewing the lens, and thus bringing them into its focus. When properly adjusted, the ruled scale will appear clearly defined in the field of view somewhat as represented by Fig. 53. Sometimes the glass is cut away at one end of the lines, leaving them at the edge of a half-cir- cular disk ; and thus, without impairing the micrometer, half the field is presented with its definition undisturbed by the additional glass plate. Some makers mount the glass scale in a plate of brass or hard rubber to be slipped into the ocular just above the diaphragm through FIG - 53 ' slits left for that purpose ; the slits being closed by a short, sliding tube, when the micrometer is not in use. Unless a screw be attached, as is sometimes done, giving a delicate lateral movement, the use of any ocular micrometer is much -facilitated by the employment of the mechanical stage, by means of -which a coincidence of certain lines Avith any part of the object to be measured can be the better secured. K. H. W.J Measuring by means of the ocular micrometer is conducted in the following way. The micrometer is put in place, its scale and the object brought in focus, and then by turning the ocular and moving the object, the whole length of the object to be measured is covered with the scale, in such a way that its rulings are perpendicular to the longer axis of the object. Then adjust the object as sharply as possible and count up how many of the rulings are covered by it (whole and fractions) . This number is to be noted down and if one desires very great exactness the measurement may be repeated on different parts of the scale and the results averaged. It is clear enough that one does not get the exact and real size of the object by this process. To get this one must either exactly determine the number of magnifications of the objective- 124 THE MICROSCOPE IN BOTANY. system together with that of the field glass and the eye lens, so as to ascertain the true value of the ruled spaces of the microm- eter, or he must determine their true value by the use of an exact objective glass-micrometer. If he has for instance found that in tbe first case, the objective and the field lens together magnify seventy diameters and the rulings of the micrometer are 0.05 mm. apart, so by the use of that objective-system the space between two rulings will amount to a magnitude in the object of 0.714 of a micromillimeter. The determination of the relative value of the ocular micrometer by means of the objective glass-micrometer is very simple. The latter is used as an object, and then it is determined how many of its divisions answer to a certain number of the divisions of the ocular micro- meter. Divide the first by the last and the result will be the true value of the ocular-micrometer divisions in units of the ob- jective micrometer. If, for instance, 20 divisions of the ocular micrometer cover an extension of 87 micromillimeters of the objective micrometer, the value of an interval in the ocular micrometer will be f & = 4.35^ = 0.00435 mm. [In such work, round numbers are always preferred, for the sake of simplify- ing the labor -of computation and comparison. In the above example, for instance, the 20 divisions of the ocular micrometer could, by lengthening the optical axis of the microscope by slightly raising the draw-tube, be made to cover 100 micro- millimeters instead of 87, the value of one interval becoming 5/j. instead of 4.35,u, and the labor of computation being reduced to almost nothing. So in the author's following illustration, the values 8, 3.63, 1.27, 0.55 would by a judicious use of the draw-tube be changed to 8, 4, 1.5 or even 2, 0.6 or even 1 respectively. Having once found a convenient position of the draw-tube for a certain purpose, much trouble will be saved by recording that position so that it can be promptly restored when next wanted. The draw-tube should be graduated for this pur- pose. Having been set in position, approximately, by means of the graduation, a slight readjustment will instantly secure exact apposition of the desired lines (say 20 to 100) of the ocular and objective scales. R. H. W.] If the manufacturer would take pains to determine the micro- THE OCULAR MICROMETER. 125 metric value for every system furnished it might be designated [approximately, but not with sufficient accuracy for fine work] on the tables of magnification which accompany the microscope. If this were given, as we may suppose for illustration, for the systems I, II, III, IV, of an instrument 8.00, 3.63, 1.27, 0.55, respectively, then I have these numbers by which to multiply the number of units read off from my ocular micrometer in order to determine the true size of the object measured in micromilli- naeters. Thus, the object being measured is found to cover 17 divisions of the ocular micrometer, the magnification being with the sys- tem I. It has consequently a true length of 17 X 8.= 136,^ ; or, the magnification is made by system IV, the object covering 53.5 divisions of the micrometer. Its true size will then be 53.5x0.55= 29.4,* = 0.0294 mm. Some firms have lately furnished adjustable ocular glass-mi- crometers, which are provided with a fine screw by means of which the rulings may be moved in a horizontal direction, and a further adjustment by which the rulings may be brought very exactly iiito the focus of the ocular lens. For fine measurements this is decidedly preferable to the one previously described. [Objective glass-micrometers, here called stage micrometers, if based upon the English units are commonly ruled to 1, A and -JL inch. The metric (decimal) system, however, is com- ing into somewhat general use here, and it must be admitted that 1 mm. (about JL in.) _L and mm. form a more convenient series than any round fractions of the inch, i mm. is, more- over, equal to 10;-*, the micron or micromillimeter, /-*, being the only recogized unit in the world for a minimum unit in meas- urements. At present, the metric values being not yet familiar to all students, stage micrometers are often ruled showing the metric and English scales in comparison with each other. The stage micrometer should be ruled with the utmost possible precision, as its errors are multiplied by the whole magnifying power of the microscope employed. It has- been known for years that the best micrometers in use contained perceptible errors. The standard micrometer of the National Committee, adopted in 1883 by the American Society of Microscopists, 126 THE MICROSCOPE IN BOTANY. represents 1 cm. subdivided to 1, and ^L mm. ; and the value of its spaces, as related to larger standards of length, have been determined with a certainty and precision not known to have been attained before in any micrometric standard. Copies of this standard can now be obtained from the dealers in microscopes ; and the loan of officially certified copies, for com- parison, can be obtained, under certain restrictions, from the officers of the society. R. H. W.] B. THE OCULAR SCREW-MICROMETER. This micrometer is seldom used in Germany but frequently in England, where, however, on the whole, very little and very indifferent microscopical research is made, except indeed, to look at diatom frustules, and to take delight in their markings ; but for that, one has there the most expensive and showy ap- paratus.* The measuring apparatus in question differs very little from the objective screw-micrometer. By means of a micrometer screw whose drumhead is divided into one hundred parts, a movable thread is carried through the field of a Ramsden ocu- lar toward another thread parallel to it. The revolutions are read off from a metal scale in the field of the ocular. [This apparatus, borrowed from the telescope, and familiarly known as the " Ramsden" or " cobweb " micrometer, requires to be well made, and to be attached to a sufficiently firm stand. It is also rather expensive. It is believed, however, to be cap- able of performing a series of measurements with a rapidity and precision not easily attained by any other means. Like other ocular micrometers, it is most readily used upon a micros- cope having a mechanical movement to the stage.] [Fig. 54 shows a cobweb micrometer as now constructed by * The preceding remark is retained out of respect to the author's liberty of opinion, and as a very vivid caution (which might be useful also in this country) against a real and con- ceded evil; but with a very decided belief on the part of the writer, that the distinguished author's information must have come from such partial or prejudiced sources as to lead him to somewhat overstate the faults of his neighbors, and to greatly underestimate the really scientific work which is being done in England. R. H. W. THE CAMERA LUCIDA AS A MEASURING APPARATUS. 127 Mr. Zentmayer. It is of convenient model and excellent work- manship, and is combined, very advantageously, with a gonio- meter attachment represented in the cut by the large graduated circle with vernier. R. H. W.] FIG. 54. III. THE CAMERA LUCIDA AS A MEASURING APPARATUS. Almost all the contrivances described on pp. 114-118, may be directly applied to the measuring of microscopic ob- jects. If one has traced the exact outline of the object, and knows exactly how many times the image has been magnified, it is only necessary to measure the drawing with the metric rule and divide the same by the number of the magnifications, to get the true s^:e of the object in fractions of a millimeter. 128 THE MICROSCOPE IN BOTANY. It is first of all necessary, in this case, to determine with ex- actness the magnification which the camera lucida gives with the objective employed. This may be done in the following way. Set the camera lucida on the microscope and use an ob- jective glass-micrometer for an object on the stage, 3 illuminate it with excentric light so as to bring its markings out clearly, and draw a number of the markings on a piece of paper [at a distance of 10 inches, 25.4 cm., from the eye, and in a plane at right angles, to the line of vision]. This may be done in this way. Draw a straight line with India ink across the paper. Place this line so that it will lie exactly perpendicular to the rul- ings of the micrometer. Then with a sharp pencil mark the points of intersection of the micrometer rulings with the line, and as the lines by magnification have a considerable size, mark the same edge of each, right or left. The markings in the middle of the field should be selected for this purpose. Then ascertain the distance between any two points on the line by the milli- meter scale ; repeat this for a considerable number of the mark- ings, and getting the arithmetical average, divide this by the micrometer unit. The quotient gives the number of the magni- fications. If one draws an object on paper intended for subse- quent measurement, it should naturally be placed at the [stand- ard distance of distinct vision, viz. 10 inches = 25.4 cm. See p. 111].* IV. CONCERNING MICROMETRIC MEASUREMENTS IN GENERAL. In the descriptions of measuring apparatus we have here and there dropped hints as to their management. Some remarks of a general nature may not be out of place here. Measurements, made by the very best micrometers, only approximately express the absolute size of the measured object. The reason for this 3 If one has no stage micrometer he may resort to a good eye-piece micrometer, 1mm. divided into twenty parts and boldly make use of that. It gives, especially when several measurements are combined, perfectly satisfactory results. * If one has a microscope with a draw-tube, he can very easily, with his objective and camera, produce a magnification of any desired round number, which is very handy in simplilying the calculations. MICROMETRIC MEASUREMENTS IX GENERAL. 129 is first of all in the fact that the exact focussing of the object is attended with the very greatest difficulty. Each worker here fol- lows his own subjective judgment, andean follow no other. He works with his eyes and his hands. When two trained micros- copists focus the same object under the same microscope, it may be assumed as a matter of course that the two adjustments will turn out to be different. Indeed, should this not be the case, we must consider it to be purely accidental. That measure- ments made by each of the two adjustments must differ goes without saying, and therefore it comes about that measurements, made by two different persons, can be compared as to their ab- solute value only with the greatest difficulty. For this reason all those measurements which are thought to be so extraordi- narily accurate, and whose possibility was once so long and so widely discussed, become quite or entirely valueless. But most objects which are to be microscopically measured organic f ol - ms appear under very unlike dimensions and it must be a matter of great indifference to us whether the long diameter of a grain of starch from a potato, be given ^ too large or too small. But micrometric measurements are of particular worth when the results of the same observer are compared for the purpose of obtaining their relative value. But as to getting the absolute results of two different observers, "one may confidently maintain that if the measurements of different observers in any one in- vestigation are comparable, this comparability still continues if the single measure selected were 5 or 10 per cent greater or smaller.'* 5 \Ve have still some words to add concerning the designation of the value which we obtain by the micrometer. In former times the line was the unit of micrometric measurement; in France the Paris line ; in England the English duodecimal line ; in Germany the Paris, Bheinish, or the Vienna line. Since the middle of the present century all these units of measure have been displaced by the millimeter ; only the English are so con- servative that they still [partially] maintain the line unit, just as in measuring heat they will not exchange the irrational Fahrenheit thermometer for that of Celsius. On the continent the decimal metric system alone is used. 6 Nageli xind Scbwendener, Das Mikroskop, 1877, p. 285, 130 THE MICROSCOPE IN BOTANY. The fractional part of a millimeter may be expressed in two ways, by a vulgar, and by a decimal fraction. Hugo v. Mohl 6 earnestly pleads for the use of the common fractions. The des- ignation of micrometric values by the decimal fraction, he holds to be a real nuisance. He thinks that one must be a mnemonic expert to be able to form an intuitive conception of magnitude expressed by a decimal fraction. With Nageli and Schwendener 7 we are of another opinion. We believe the decimal fractions to be the only logical terms in which to express micrometric values. [Both the advantage and the difficulty of the decimal system are doubtless equally real; but the perplexity which prevents persons not possessed with a mathematical turn of .mmd from intuitively apprehending the value of fractions ex- ..tending to several places, especially in decimals, does not apply jneasurably to a single place of decimals, tenths. Hence the necessity of a minute micrometric unit, especially, but not .exclusively, in a decimal scheme. The micron (infra) being frequently sufficient without fractions, and never requiring any- thing beyond tenths except for procedures involving expert- ness in mathematics, wholly relieves this difficult}', as soon as it becomes familiar to the mind as a unit having a definite value of its own. R. H. W.] Harting 8 proposed in his time to adopt the one-thousandth part of a millimeter (0.001 mm.) as a unit for giving micrometric values. He would designate this magnitude by 1 mmm., or .shorter by It* ; he named this unit a micromillimeter. This proposition of Harting is doubtless the best and the microinilli- meter has maintained itself to this day. 9 If one reads that an object is 23,u long and knows that I/* corresponds to the unit in the 3d decimal place he can easily construct in his mind the decimal fraction of the millimeter which shall express it (0.023 mm.). But since we usually obtain only the relative size of microscopic objects, so even this latter mental translation is not necessary, for we learn to think of the micromillimeter as a unit, exactly as in common life we do of the centimeter or the mark. Hugo v. Mohl, 1. c., p. 31S/ 7 Nageli tmd Schwendener, I. c., p. 288. Harting, Mikr. p. 506. 9 Listing has also recently pronounced in favor of the micromillimeter as the unit for micrographic measurements. He desires to introduce the name Micron or Micrum for it. (Carl's Repertorium fiir Experimentalphysik, Bd. x, 18G9, p. 5.) MICROMETRIC TABLES. 131 In some exceptional cases, in micrometric measurements, we still have to deal with the old values, the fractional parts of a line. There are now, for instance, scarcely any objective screw- micrometers made, and those who use this instrument will in most cases have to use an old one, which measures in the frac- tions of a line. Likewise those statements of micrometer values which occur in works of the first half of the present century are based on the Paris line throughout, so that in order to compare one's own measurements with those, it will be necessary first of all to reduce the latter to the equivalent fractions of a milli- meter. I have found, by long use, the tables which follow to be of great practical convenience in making these reductions. % TABLE I. COMPARISON OF THE UNITS OF MEASURE. 1 MM. PARIS LINE = i ENGLISH LINE = 1 RHEIN LINE = 1 VIENNA LINE = Millimeter. 1.0000 2.2558 2.1166 2.1802 2.1952 Paris Line. 0.4433,. 1.0000 0.9384 0.9964 0.9732 English " 0.4724 1.0659 1.0000 1.0299 1.0371 Rhein " 0.4587 1.0347 0.9710 1.0000 1.0070 Vienna " 0.4555 1.0275 0.9642 0.9930 1.0000 TABLE II. REDUCTION OF THE UNITS OF MEASURE TO MTCROMILLTMETERS. MICROMILLIMETER PARIS LINE ENGLISH LINE RHEIN LINE VIENNA LINE 1 M (0.001 mm.) = 0.000443 0.000472 0.000459 0.000455 2 " = 0.002 mm. = 0.000887 0.000945 0.000917 0.000911 3 "=0.003 " = 0.001330 0.001417 0.001376 0.001366 4"=0.004 " = 0.001773 0.001890 0.001835 0.001822 5 " = 0.005 " = 0.002216 0.002362 0.002293 0.00-2277 6 " = 0.006 " = 0.002660 0.002834 0.002752 0.002733 7 " = 0.007 " = 0.003103 0.003307 0.003211 0.003188 8" = 0.008 " = 0.003546 0.003779 0.003670 0.003044 9" = 0.009 " = 0.003990 0.004252 0.004128 0.004099 10 " = 0.010 " = 0.004433 0.004724 0.004587 0.004555 20 " = 0.020 " = 0.008866 0.009448 0.009174 0.009110 50 " = 0.050 " = 0.022165 0.023620 0.022935 0.022775 1UO" = 0.100 " = 0.014330 0.047240 0.045870 0.045.550 132 THE MICROSCOPE IN BOTANY. Table I, "Comparison of the Units of Measure, " is in re- spect to its purpose perfect enough, but the calculations involved in its use are somewhat detailed. There will be necessary in each reduction a multiplication at least, and commonly a multi- plication and a division, whereby, on account of the many dec- imal places, an error might easily creep in. By the use of the logarithm tables an addition and subtraction might be substi- o tuted, but the detailed character of the operation would not be altered materially. EXAMPLE : We find in a work the statement that a micros- copic object measures 0.0216 Paris line. We wish to know how much this is in fractions of a millimeter. 1 : 2.2558 : : 0.0216 : x x 0.048725 mm. = 49,* Table II, "Reduction of the Units of Measure toMicromilli- meters," simplifies the calculations quite essentially. In it is represented the value of the different lines to 1-10, 20, 50, 100^ An example will make its use easily understood : 1 have found by my micrometer that the length of an object is 28 ft. In an old botanical work I find the same object given as the 0.011841 of a Paris line. I would compare these two values. I find the value of the 28 /j. to be in fractional part of the Paris line, accoiding to column 1. 20 ti 0.008866 8 AI = 0.003546 28 IL 0.012412 Subtracting the magnitude 0.011841 I get as the difference 0.000571 of a Paris line, which I find, by referring to column 1, is really a difference of something more than 1 //. EXAMPLE 2. In an old work I find it stated that the cells of Nephrocytium Agardhianum var. minus, 10 to be Paris line. I desire to know how great this value is in terms of the frac- tions of a millimeter. 0.005000 Paris line. The value nearest to that in my table, column 1, is 0.004433 = 10 P-. The difference 0.000567 lies between the values of 1 /j. and 2 /* near- est to 1 n as the table shows it, so that i Paris line = 11 /* = 0.011 mm. 10 C. Nageli, Gattungen einzelliger Algen, Zliricb, 1849, p. 80. POLARIZING APPARATUS AND GONIOMETER. 133 Calculations for the English and other lines are made in* the same way, using of course the corresponding columns in the table. We have not considered it necessary to give examples for each kind of line. [The systems in use in micrometry in this country, and in England, are the English and the metric. Of the English inch, vulgar fractions are more commonly used than decimals ; partly, perhaps, because so many of those persons, whose education and habits would lead them to choose decimals, are led to adopt the consistent decimal or metric system, to the exclusion of the inch. The use of the line seems to have been abandoned. It can scarcely be doubted that there is a growing disposition to use the metric units in microscopy, the millimeter (approxi- mately ^ inch) for a large unit, and the micromillimeter (approxi- mately JL inch) for a small unit; and persons who have once learned to think in these units, so as to avoid the trouble of constant computation, find them most convenient and satisfac- tory in themselves, to say nothing of the manifest advantages of harmony with the rest of the world. The table opposite, from the Journal of the Royal Microscopical Society, presents in n most available form the relations to each other of the various English and metric units and their fractions. By following, with this table, the directions of the author (p. 132) for the use of his w Table II," observations in inches and those in millime- ters or micromillimeters can be transposed with great facility. Moreover the table furnishes the necessary information in regard to metric measures of length, to any who may be as yet familiar with only the English system. R. H. W.] IV. POLARIZING APPARATUS AND GONIOMETER. It does not come within the plan of this work to consider critically the process of the polarization of light. Whoever wishes to go into the subject may consult any modern work on physics. For microscopical purposes the subject is developed in the best form by Nageli and Schwendener in " The Micro- scope," 11 and in the work of like name by Dippel, 12 though the 11 II Auflagc (1877) pp. 239-361. " Bel. I, p. 224-227, pp. 407-455. 134 THE MICROSCOPE IN BOTANY. presentation of the matter by the latter is far less satisfactory to me than that by the former. The microscopical polarizing-apparatus, like every other, comprises an arrangement of two polarizing media between which, in the polarized light, the object to be investigated may be examined. The one placed under the object is called the polarizer and the one over it the analyzer. These condi- tions in general characterize the positions of the polarizing ap- paratus of the microscope. The polarizer is most conveniently arranged in connection with the stage. Since it is of a cylin- drical form as at present made it may be put into the place of the cylinder diaphragm. Naturally, the analyzer cannot go be- tween the objective and the object, and it is therefore placed either directly over the objective-system or above the ocular. All [Continental] makers now follow Hartnack in adopting the latter arrangement. As a polarizing medium, the Nicol's prism of Iceland spar is the one universally adopted. As is well known a Nicol's prism consists of two halves ; the joining surfaces run diagonally through the prism and form an angle with the end surfaces of 89 17'. The two halves are o cemented together with Canada balsam. The side surfaces are blackened. If a ray of light running parallel to the side surfaces falls upon the lower end of the prism it is separated into two rays, the ordinary and the extraordinary. The former suffers a total reflection in the layer of Canada balsam and passing to the side of the prism is absorbed in the blackened surface ; the latter, on the contrary, passes directly through the prism into the field of view of the microscope which it illumin- ates. It is known that in the use of the polarizing apparatus the mutual position of the polarizing media must be changed, and it is all the same if the polarizer be turned about while the analyzer remains fixed, or the analyzer be rotated while the polarizer is fast. One or both must be rotated around its longer axis the axis parallel with its sides. The Nicol's prisms, the side edges of which belong to the original rhomboidal calc spar, have a vertical direction, while the artificially ground edges exposed above and below are set POLAKIZIXG APPARATUS. 135 at an angle of 68. [They are set, by means of a soft cork packing, in brass tubes adapted to the instrument for which they are designed, and the exposed ends are by some makers protected by thin cover-glasses. The polarizer, as mounted, is slipped into the substage ring, and in the better class of in- struments can be used in connection with and not in the place of the substage condenser. It nearly always has a rotating move- ment, and in stands designed for chemical or lithological work a graduated arc by which its position can be known and re- corded. It is advantageous to have this prism as large as is consistent with the construction of the stand. The analyzing prism is mounted in a smaller tube. It may be a rather small prism and should be short. It is sometimes mounted in a cap to slide over the top of the ocular, as near as possible to the eye lens, but is more commonly fitted as in Fig. 55 into an FIG. 55. adapter with society screw above and below, to be screwed in between the objective and the tube of the microscope. It is frequently so arranged, by cutting away the sides of the con- taining adapter, that it can be easily rotated when in use. By the addition of a light brass tube, just large enough to contain this apparatus above and to slip over the cap tube of the ocu- lar below, the writer has been able, with great satisfaction, to secure the advantages of both methods of locating the analyzer, which can thus be placed at will in either position. Fig. 55 shows us a common method of mounting the polarizing and analyzing prisms respectively. R. H. W.] 136 THE MICROSCOPE IN BOTANY. Use of the polarizing apparatus. As is well known the po- larizing apparatus is commonly employed to find out if a given object for example a crystal has one or two optical axes, if it be singly or doubly refractive. If the crystal be of mi- croscopical minuteness one naturally uses a microscopical polar- izing-apparatus. On this account it plays an important role in the hands of the crystallographer. But it is frequently of the greatest use to the botanist. For, in the first place it enables him to know the nature of many of the crystals which occur within the plant-cells, and in the second place all tissue struc- tures are doubly refractive and may be examined with polar- ized light. Not seldom, details of structure show themselves when they are illuminated thus in the dark field of view which are otherwise not seen at all, or recognized with the greatest difficulty, and thirdly the polarizing apparatus enables the bot- anist clearly to make out the form of certain microscopic objects. For an experimental observation with the polarizing appara- tus, starch grains from a potato, or a section of the rhizome of Pteris aquilina, in which the ducts of the fibro-vascular bundles alone are doubly refractive, will serve us excellently well for an object. A very good object also is a section of the under- ground stem of Lathraea squamaria for in this we have the starch grains and the doubly refracting fibers together. In the use of the apparatus we proceed as follows. After we have adjusted the object by means of a common ocular and found the best illumination by turning the mirror about, we place the polarizer in position beneath the stage. The field appears to be unchanged, except a slightly weaker illumination. After placing the analyzer also in position, whether above the ocular or .above the objective, we rotate the movable part of the apparatus till we bring the two prisms into such a mutual position that the field of vision appears darkest. If the appar- atus is well constructed the darkening is almost total. The field appears in a quite deep, very agreeable, shade of blue. With the use of low powers there is a certain amount of light falling upon the object from the side which is reflected up- wards, and the consequence is that the field is not perfectly THE GONIOMETER. 137 dark. This difficulty can be remedied by holding the hand so as to shade the object on the stage, or putting round about it for a shade, a piece of angularly folded common blue wrapping paper. The doubly refracting parts of an object are seen on a dark ground, either partly or altogether brightly illuminated, according to their chemical or physical structure. Thus starch grains from the potato appear of a clear bluish shade with a dark cross drawn over them whose radii broaden outwardly toward the circumference. 13 The walls of the ducts from the rhizome of the Pteris appear illuminated partly bluish and partly yeliowish. 1 * As already indicated, we cannot here in the least enter into a discussion of the optical characteristics of organic forms, but must rather recommend the careful study of those parts of Nageli and Schwendener's as well as of Dip- pel's work, which deal with this subject. Rotating selenite plates, which, as is well known, are applied in various ways to the common polarizing apparatus, can be easily added also to the microscopical polarizing-apparatus. [THE GONIOMETER.] [For the purpose of measuring the angles of microscopic objects, the goniometer is frequently added to the microscope. This attachment originally consisted of an elaborately mounted ocular in whose field of view was a line which, by rotating the ocu- lar, could be brought successively into coincidence with the dif- ferent sides of the object, the intervening angles, traversed by the rotating apparatus being read off by means of an attached vernier sliding over a graduated circle clamped to the microscope tube. This accessory may well be combined with thepolariscope, as it is frequently used in connection w r ith studies which require polarized light. Such micro-goniometers are still attached to stands designed for chemical or lithological work, but the in- troduction of the circular concentric stage has rendered them unnecessary except for the use of the specialists. The stand figured in Plate XI, for instance, can be employed for gouiom- 13 The reasons therefor may be found in the before cited works. 14 For this matter see principally Dippel, L c. 138 THE MICKOSCOPE IN BOTANY. etry without addition or preparation ; and any of the circular- stage stands will require for the same purpose only a graduated circle on the edge of the stage. As such graduation is inex- pensive, and is also useful for other purposes, it constitutes, for occasional and incidental use, the most eligible goniometer. For the measurement of angles the stage must be carefully centered, and the angle to be measured brought to the centre of the field of view. Such adjustment is facilitated by placing cross lines at the focus of the eye-lens of the ocular as shown in Fig. 56, but only one line fc is essential. This line may be either a spider's thread drawn across the center of the opening of the diaphragm of the ocular, or a line ruled on a thin cover glass lying in the same,position, one of the lines of an ocular micrometer being often made to answer the purpose. An angle whose plane is parallel to the plane of the stage, and at right angles to the axis of vision, is carefully selected and its apex brought to the center of the field. The line in the ocular is then made to coincide with one of its sides, and the stage is afterward rotated until the other side coincides with the same line, the angle through which the stage and object have moved being readily determined by comparing readings from the stage graduation made before and after the rotation. Should greater precision be required a vernier may be added to give decimals of a degree ; but this is not always needed, all measurements with any micro-goniometer being at best but approximate. It is seldom if ever possible to be certain of the exact parallelism of a given plane of the object with that of the stage, and un- less that parallelism be secured it is evident that the angle will be seen in perspective and will be incorrectly stated in the read- ing of the instrument.] [Mr. Zentmayer combines a goniometer attachment with his cobweb micrometer as shown in Fig. 54, the lower graduated circle being firmly attached to the microscope body, and the ver- THE MICRO-SPECTROSCOPE. 139 nier above it enabling us to read off accurately the extent to which the micrometer with its field crossed by fine lines has been ro- tated. A pair of extra lines, crossing each other in the center, are provided for the accurate centering of the angle to be measured. The analyzing prism, when required with this com- bination, is placed above the objective. R. H. W.] Y. TPIE MICRO-SPECTROSCOPE. In recent times investigations are frequently carried on by means, of a spectroscopic apparatus combined with the micro- scope. The spectro-microscopical apparatus, especially in the hands of botanists, has become an important instrument in the investigation of the coloring matter of plants. Since we have found an adequate. description of the micro-spectroscopic apparatus 15 in no existing work, we shall here attempt to consider it somewhat in detail. We shall found our description on that most perfect construction of it which was first given to it by Sorby and Browning. A. The Prisms. It is well known that when a ray of so-called white light pass- es through a massive glass prism, provided that the re- fracting edge of the prism be perpendicular to fhe ray of light, it will be separated into its elementary colors. Then there is produced a spectrum (solar spectrum) whose colors and their arrangement are well known. Now, if in place of the dispersing prism we substitute a combination of three or five prisms of crown and flint glass alternately, arranged as is shown in Fig. 57 at A 9 the light will pass through Sageli and Schwendener, L c. t p. 39. Frey, Das Mikroskop, p. 36, /. FIG. 57. 140 THE MICROSCOPE IN BOTANY. it in a direction parallel to its axis. This arrangement is here known as a direct 16 vision spectroscope. The prisms are fastened into a brass cylinder M, by means of a cork mounting. The cylinder is closed above by a dull black metal plate K, with a round hole in the middle about 10 mm. wide for looking into. This apparatus is connected with the ocular and is placed above the eye-lens G. Should the ocular have a common circular diaphragm, the field of the microscope, after putting the spectroscopic apparatus in place, should appear to be a small ellipse, the center of which should be quite colorless, and at the points of greatest curvature red and blue respectively. Microscopic objects which do not fill the field seem to be striated in the direction of the longer diameter of the ellipse. If in place of the diaphragm-opening of the spectroscopic ocular we substitute a slit-opening which is so arranged in respect to the prisms that the refracting edge is parallel to the slit B, a spectrum will now appear of the well-known band-form whose brightness and extension are conditioned by the breadth and length of the slit. B. The Slit. To constitute a practical working instrument the slit must be exactly adjusta- ble in the focus of the eye-lens of the ocular, for every eye, and it must be so contrived that the slit may be narrowed or widened, lengthened or shortened at will. The focussing of the slit is easily provided for by having the tube of the ocular made of two parts, one shoving into the other and moved by a rack and pinion. [The contrivances for widening the slit are about as various is This combination was first contrived by Amici in 1803, later applied to the construction of a pocket spectroscope by Hofman (see Schellen, Spectralanalyse, p. 109) and finally proposed for the microscope by John Browning. FIG. 58. THE COMPARISON PRISM. 141 as the ideas of the different makers. It is only essential that the two shutters forming the sides of the slit should steadily approach and finally meet each other in the center of the field, with a perfectly smooth and parallel motion, which is under com- plete and easy control. In Fig. 57 this motion is controlled by a small milled head. Shutters moved by the lever L are pro- vided for controlling the length of the slit. R. H. W.] C. The Comparison Prism. It is often very desirable to compare the spectrum of a body which is being investigated with that of a like body. This may be done by first observing the spectrum of the body under investigation, and after that the spectrum of the body used for comparison. But the spec- FIG. 59. tra, for example the absorption spectra of some colored fluids with many dark bands, are found to be very difficult to carry in the memory so as to make the comparison. Hence a com- parison of the spectra in this way can relate only to the leading features and not to smaller particulars. But in order to make comparison of the finer details with sufficient leisure and exact- ness we must see both spectra very near together, and at the same time the so-called double spectrum. This we can do by means of the comparison prism, an apparatus for which we are indebted to Kirchhofl'. 17 Suppose tl, Fig. 59, 1, be the plate of the drum which bears the 17 Schelling, Spectrum analysis in its application to the substance of the earth and the nature oi the heavenly bodies, Brunswick, 1870, p. 121. 142 THE MICROSCOPE IN BOTANY. slit s. The body to be investigated is at b. The rays from it pass in the direction lib' through the slit and at b' enter the combination of refracting prisms. The slit is not opened along its whole length for the admission of rays from b. For half of its length it is covered by a reflecting prism of the form which we have come to know in the apparatus for microscopical drawing (see p. 114, Fig. 45). The prism in section represents a right angled isosceles triangle, Fig. 59, II, the hypothenuse surface being inclined to the slit at an angle of 45. Now, if we suppose another body to be used for comparison placed so as to send rays from a in a horizontal direction, they fall upon the surface of the prism perpendicular^, and unrefracted pass to the hypothenuse surface and fall upon it at an angle of 45. Here they are totally refracted perpendicularly upwards, and as a' pass through the slit parallel with the rays bb'. If now the refracting prisms are placed over the slit as already described the observer will now perceive not one but two spectra lying next each other whose colors, Fraunhofer lines, etc., exactly coin- cide. We call these two a double spectrum. FIG. GO. Fig. 60 represents a double spectrum which is produced by the rays coming through the free half of the slit, and by those of diffused daylight passing through the comparison prism. I is the spectrum of the rays through the prism, II that of those not reflected. They are separated by a slender black line which one commonly notices but which when one has the eye close over the refracting prisrn has no disturbing influence. We notice in the spectra the strong Fraunhofer lines : those of the under spectrum falling exactly in the prolongation of those of the upper. [The comparison prism, not being required for THE BINOCULAR MICRO-SPECTROSCOPE. 143 FIG. 61. constant use, is mounted in a sliding frame and can be instantly slipped into or out of the field of view by means of the milled head I, Fig. 57. K. H. W.] Laterally, on the largest tube or drum, is a perpendicularly ar- ranged plate E, Fig. 57, provided with spring clamps, which has the form of a microscope stage before which may A be seen a plane mirror F. The plate has a small opening in the middle at (7which shouldbe closed when the comparison plate is not in use. If one wishes to use the comparison prism he pushes the milled head I forward and the prism which has been lying in the side of the drum is shoved into place over one-half of the slit. By proper adjustment of the mirror the neces- sary light can be directed through the open- ing C in the stage E upon the comparison prism. For the production of the comparison spectrum we should bring the comparing body as closely as possible before opening in the plate E. [/>. The Binocular Micro- Spectroscope. The spectroscopic ocular hitherto mentioned is the original form contrived by Mr. H. C. Sorby of London and elaborated by John Browning a distinguished manufacturing opti- cian of that city. It is still the form in most general use. It is shown in Fig. 58 complete and ready to be inserted in any micro -cope in place of the usual ocular. Mr. Sorby has more recently contrived another form known as the Binocular spectroscope. In this arrangement, Fig. 62, and shown in section in Fig. 61, the prisms are transferred from above the ocular to below the H objective, giving greater dispersion as well as fitness for binocular use. The apparatus screws into the tube of the microscope, in place of the objective by the screw B. It consists of a small collecting lens / which when in use should just touch the object under investi- gation ; a slit adjustable by the screw head L and a set of prisms c through which light from /passes upward to the objective A FIG. 62. 144 THE MICROSCOPE IN BOTANY. and thence through the microscope tube and ocular. The com- parison prism G can be made to send up either the absorption bands of a known solution or the interference spectrum from the standard scale H. This apparatus, like the preceding, is of the Beck style, supplied in this country by Wm. H. Walmsley and Co. of Philadelphia. K. H. W.] E. The Measuring Apparatus of the Micro-spectroscope \ It is known that Bunsen and Kirchhoff, in their chemical inves- tigations upon the absorption spectra and the Fraunhofer lines, divided the whole length of the spectrum into 170 equal parts, and determined the position of the Fraunhofer lines in the solar m FIG. G3. spectrum and the illuminated bands in the discontinuous spec- trum according to a scale. In general this scale is fundamental in spectrum analysis. The common spectroscope for the pur- pose of spectrum analysis has a tube, in the front end of which is a gl as s on which is photographed a millimeter scale reduced about fifteen times. It can be illuminated by light admitted for the purpose. The image of this scale is thrown by means of a biconvex lens on the front surface of the refracting prism, this is reflected into the observing telescope and reaches the eye of the observer as an optical image apparently lying on the THE MICRO-SPECTROSCOPE. 145 spectrum. The contrivance, however, suffers from this fault that the thickness and brightness of the division lines are de- pendent on the width of the slit, which is different with differ- ent eyes. But when we undertake to measure the exact distance of the Fraunhofer lines and the breadth of the absorption bands we have to give up this very simple and practical contrivance. The idea of an exact measuring apparatus for the micro-spec- troscope, which we may call really ingenious, originated with Mr. John Browning of London. This not very simple contriv- ance is illustrated in section in Fig. 63. First is rr the per- pendicular brass tube which incloses the five prisms ww, in their cork mounting xx. The upper surface of the prism w Y is inclined to the horizon at an angle of 45. Opposite to it is the hori- zontal tube ran, provided within with a biconvex lens 0. I am not informed \vliat firm, if any, in this country, make or keep, for sale these machines. Doubtless any of the principal optical houses would import them to 01* der. A. B. H. 208 THE MICROSCOPE IN BOTANY. presses too hard against the cutting disk it may be caused to oscillate and break the thin slice of stone. Emery mixed with water should be used as a cutting medium. While turning the wheel with the right hand the other maybe employed in putting the emery on the disk by means of a hair pencil. There is also arranged around the lower part of the disk, a box of sheet metal to catch the ground material and to prevent it also from being scattered about laterally. This was left off the drawing on ac- count of its covering up essential parts of the apparatus. If one wishes he can so modify the construction of this machine as to propel the large wheel by foot-power like a sewing machine. As to the .size of the thin piece it is recommended to take a surface about 15 mm. square at the outset. It will be somewhat reduced in size afterwards, chiefly in consequence of grinding away the edges, so that when finished it will not, perhaps, be more than 12 mm. square, which generally is enough. The next step is 2. Grinding down -the Specimen. By this expression we understand grinding the surface smooth. If the preparation is large enough and of the right consistency it may be held in the hand, otherwise it should be cemented to a small glass plate which serves as a convenient handle. Canada balsam is a suit- able cement. It should be sufficiently warmed to melt it, and the specimen should also be warmed over a spirit lamp or gas burner sufficiently to remove the moisture, then lay it on the balsam and again heat the plate till the balsam boils, being careful not to burn it. After the Canada balsam has cooled and the development of bubbles has ceased, press the specimen down fast. If bubbles yet appear under it, it will be necessary to repeat the process, else one may run the risk of getting the specimen broken. An emery plate serves an excellent purpose for grinding down the preparation. It is first ground on a coarse grained plate and then the surface is polished on a finer one. Since the grinding stone must be kept moist, it may be laid in a suitable dish with a flat bottom and water enough poured in to cover the upper surface of the stone, Fig. 96. Should it not be possible to make the specimen sufficiently smooth on the fine grained stone, PREPARATIONS OF FOSSIL PLANTS. 209 it may be done on a ground-glass disk with emery which con- tains no coarse grains, or, better still, on a whetstone with the use of oil of turpentine. These emery stones have but recently come into the market. x Formerly the grinding was done on a cast-iron plate by means of emery and water. The new method has the advantage that it is simpler, leads more directly to the desired end, and makes greater cleanliness possible. According to the experience of others as well as my own, it may be commended as the best. 3. Grinding the Preparation thin. When one surface of the specimen has been ground and polished, it should be cleaned FIG. 9U. with a hair pencil and plenty of water poured over it. Then it should be left in the air to dry. Do not under any circumstances undertake to hasten the process by rubbing with woollen or linen' cloth, for this procedure unavoidably leaves small fibres upon the preparation which very much damage the microscopic image. It should then be separated from the glass plate by warming the Canada balsam and again cemented down, this time with the smooth side upon the plate, first, however, having dried out any adhering moisture over a spirit lamp. Now begins the real thin-grinding, first again on a coarse grained emery plate, in doing which precautions are to be observed. The second sur- face is to be ground uniform and parallel with the first, but being careful not to grind away the edges. At last on the fine grained 14 210 THE MICROSCOPE IN BOTANY. stone when the preparation has become very thin, special care is to be taken not to grind the section through, or break it, al- though this docs sometimes happen, even with the most skilful. In order to prevent the possibility of its occurrence, Zirkel recommended that four pieces of cover-glass be fastened to the four under corners of the glass plate, and of course the section cannot easily become thinner than those. FIG. 97. In respect to the thinness which the section should be made to attain, nothing in general can be determined, it depending rather in each individual case on the pellucidity of the material. If the material has a high degree of transparency, the prepara- tion may be ground only relatively thin, in order to recognize clearly the structural relations. In other cases, when the material is almost entirely opaque, it is naturally recommended to grind PREPARATIONS OF FOSSIL PLANTS. 211 the section as thin as possible. In general one may consider a section sufficiently thin when one may see through it to read after having moistened it with water. In order to save time and trouble one may fasten several specimens on the glass plate and grind them all at once. To do that, indeed, will require that they should be ground to a like thickness, and about equally withstand the action of the emery stone. The process of preparing the section may be lightened, and time and care saved by the use of a machine which will rotate the disk while the specimen is held stationary instead of the other course as has now been shown. This is recommended by Rosenbusch and others. But, on the contrary, Zirkel in his extensive works does not approve of it. A larger machine has been constructed by the above mentioned firm to be driven by foot power and which can be used for both cutting and grind- ing the section. Fig. 97 gives an illustration of this com- bined machine. The cutting arrangement is essentially the same as described above ; a is the carrier, b the movable axis for this and the weight c, d is the cutting disk, e a small driving wheel which is connected with the balance wheel f, by an endless band. The treadle g moves this wheel, k represents the folding guard box. The cast iron disk m by which the grinding is done runs on the vertical spindle 7, in a depression in the table, by means of a belt from a second wheel i running over the guide pulley k. Probably this contrivance might be so modified as to allow an emery plate to be substituted for the iron one. 4. Mounting the Preparations. Since the glass plate is commonly larger and stronger than the object-slide used for the preservation of microscopic sections, and since also it is much ground and scratched by the process, the removal of the speci- men when the grinding is finished is indispensable. This is often a matter of great difficulty and must be done by observ- ing those precautions which are particularly set forth on page 197 of this work. When the specimen has been successfully embedded in Canada balsam, put on a cover-glass and warm the slide again carefully and press down the cover. After the spe- cimen has become perfectly cold, the superfluous balsam should 212 THE MICROSCOPE IN BOTANY. be removed with a knife from about the edges of the cover and the slide perfectly cleaned with alcohol and water. It still remains to consider the labelling of the specimen, which may be suitably done by strips of paper pasted on above and below as shown in the illustration, Fig. 98. On the upper one write the name of .the fossil and designate in one corner the catalogue or other number, and in the other the direction in which the section is made, using the abbreviations H. for horizontal, R. for radial, and T. for tangential. On the lower label should be the rock formation to which it belongs, the locality, the date of finding, and the name of the collector. Besides this it is advised to write with a diamond, on the back of the slide opposite the label, some inscription by which the section can be identified with the specimen from which it is cut, if possible ; for in case of the loss of the label the engraved note then becomes of the utmost im- portance. For purposes of identification, indeed, it is sufficient to engrave the catalogue number. 5. Preservation of the Preparations. Finally, the preserva- tion of the specimens should be referred to, since it differs from the method applied to other microscopic sections. The latter, so far at least as they are mounted in a fluid, must be kept in a horizontal position, and, if desirable, provided with a protecting ledge. This is not required with ground-sections and so from this fact one may determine what suitable arrangements should be made. Commonly it is done in this way. The specimens are stored in wooden boxes provided with rack supports, whose distance apart corresponds to the size of the slide used. I have recently contrived, for the preservation of microscop- ical ground-sections, for the West Prussia Provincial Museum in Danzig, a tray which rests upon a somewhat different prin- ciple. Its essential point is this, that the slide rests upon its short edge in a sloping position, thus .making it much more con- venient to read the label. Besides, one can thus store in one FIG. THE PREPARATION OF PERMANENT MOUNTS. 213 tray, and have under his eye at once, a very great number of specimens. It is 42 X 46 cm. in size, and exactly fits into the drawers of those cases which are intended for microscopical, palasontological and geological collections. Two to four of these trays, according to the depth of the drawer, may be placed over each other. Thus from eight to sixteen hundred of these ground-sections may be opened for inspection in one drawer. The contrivance is illustrated in Fig. 99. A simplification of this plan might perhaps be made by substituting for each of the forty sloping transverse, cross ledges, in the two rows, two longitudinal ledges with sloping grooves cut in them. FIG. 99. VII. THE PREPARATION OF PERMANENT MOUNTS. Having already learned how an object should be prepared to be examined by the microscope, and having also particularly illustrated the different methods of preparing an object, we will here describe the preparation of permanent mounts. Per- manent preparations are such as are preserved for a long time in a condition to be taken at any moment and subjected to ex- amination. 214 THE MICROSCOPE IN BOTANY. 1. OBJECT-SLIDE AND COVEK-GLASS. It has already been several times mentioned that the prepa- ration to be examined must lie upon a glass plate in fluid, and commonly be covered with a piece of thin glass. In the per- manent preparation both glasses are always required. The under, stronger glass bears the name of the object-carrier (object-slide), the other, the thin plate, the cover-glass. A, TIte Object-slide, is a plate made from pure mim>r glass, often also, of common green window-glass. The most suit- able thickness is from 1 to 1.5 mm. Glass thinner than 1 mm. is so thin as to be easily broken, and such as is over 2 mm. is too thick for this use. Perfectly colorless glass is much to be preferred, though glass with a slight green tinge may be used without harm. It is much more important that the slide, especially where the object lies, should be free of all minute air bubbles, flaws and scratches. The edges should not be rough. They can be ground by the glazier without difficulty, or one can do it for himself on the whetstone with turpentine oil. What form to give the slide is purely a matter of taste, only they should be neither too large nor too" small. Those sizes which have come into most general use are the following : (a) The English form, 76 mm. long X 26 mm. broad, Fig. 100, I. (b) The German or Giessener society form, 48 X 28 mm., Fig. 100, I, II. (c) The Vienna form, 65 X 25 mm., or 70 X 27 mm, Fig. 100, II." The form most used in Germany is form b. We prefer, how- ever, form a to all others. [This is the one by far the most generally used in this country, only one firm offering for sale another size, 60 X 19 mm. A. B. H.] Before using the slide it is necessary to clean it very carefully. This, in most cases, may be satisfactorily done by washing in s* Still other and less useful forms 75X30 mm., 70X35 mm., 70X30mm., 60X^5 mm., 33X33 mm., the latter for stone sections, are also sold by opticians In Germany. THE COVER-GLASS. 215 water and alcohol and rubbing dry with a clean cloth. For very dirty slides, or when one desires to have an absolutely clean surface, the following, although somewhat detailed, pro- cess is recommended. Lay the slide in a porcelain saucer with hot nitric acid. Take it out after a few minutes with the for- ceps and pour distilled water over it. Then for a short time HL FIG. 100. put it in a weak potash solution. Then wash with distilled water till the alkali is entirely removed. Pour over it absolute alcohol and finally cover it with ether and let this evaporate. By this process we get a perfectly clean surface, as bright as a mirror, without the use of a cloth. B. The Cover-glass. The thickness of the cover-glass, as is well known, has its influence upon the microscopic image. Fol- low and medium powers the cover- glass may be from 0.2 to 216 THE MICROSCOPE IN BOTANY. 0.4 mm. thick, for still higher and the highest powers, the thick- ness should never be more than 0.15 to 0.10 mm., or still less than that. The form of the cover-glass is in our judgment of consider- able moment to the usefulness and durability of the preparation. There are circular, square, and rectangular oblong cover-glasses of various sizes. 35 One should make it a rule never to use too small a cover- glass, either in the permanent preparations, or in those which are preserved only for present temporary use. Otherwise, in fresh preparations, one runs the risk of having the fluid em- ployed exude around the cover-glass and come in contact with the objective, and in permanent mounts we find the cement ring around the edge of the glass seriously interfering with the focus- sing in the use of high powers. For common use squares of 18 mm. on the side, and circles of 15 mm. in diameter are per- haps best. We give the circles the preference before all others in mounting permanent preparations, since they are by far th ) easiest to cement perfectly tight. One naturally should employ the largest possible cover-glass in those investigations where reagents are used, which, it' they come in contact with the ob- jective are liable to injure it, or which may develop an injurious vapor upon it. [For cleaning slides and cover-glasses, and for a convenient way to keep the latter so that they will be most easily accessible for immediate use, I know of nothing better than the plan sug- gested by Mr. C. E. Hanaman of Troy, N. Y., some years ago.* It is as follows :] [A solution long in use by photographers for cleaning their negative plates and glass vessels is utilized, as being quite as efficacious as the nitric acid bath and wholly free from its dis- agreeable odors. The mixture consists of a cold, aqueous sat- urated solution of bichromate of potash to which is added about one-eighth of its bulk of strong sulphuric acid. The mixing should be made in a porcelain dish, or a thin glass vessel, as the 35 In the price current of Stender the square ones are quoted of 10, 12, 15, and 18 mm. square, the round ones ol 6, 8, 10, 12. 15, 18, 22 mm. iu diameter, and the oblong, 20X21 > X 16, 18X'2 mm. * American Naturalist, Aug., 1878, p. 573. PRESERVING MEDIA. 217 heat generated might break a glass bottle. The vessel should be set outside for the liquid to cool, after which no more injurious vapors will be given off. Then the liquid may be kept for future use in a glass-stoppered bottle. The slides should be plunged one by one into a porcelain dish containing a quantity of the liquid, till all are put in, then tilt the dish in such a way as to cause the liquid to flow back and forth through the mass for a few moments, and then after pouring off the liquid place the dish under a stream of water from an open tap. They are then wiped dry with soft linen cloths.] [The cover-glasses, after being treated with the cleaning liquid, are thoroughly washed with distilled or filtered water, and then taken out with the forceps one by one, and dried by laying each on one corner of a soft linen cloth on the table and gently rubbing, first one and then the other side with another part of the cloth. The cloths used for this purpose preferably old, worn and soft ones should be first boiled in carbonate of soda and rinsed in hot filtered or distilled water. In order to keep the covers clean, and make them most easily accessible for im- mediate use, as well as to greatly facilitate the selection of one of any desired thickness, the covers are finally arranged edge upwards, in a box or drawer, between strips of thick, white blotting-paper. The strips of blotting paper should be cut two- thirds as wide as the cover, should reach from side to side of the drawer or box, and should be separated at the ends by squares of the same paper, thus forming a rack in which the covers stand edge upward, and from which they can be readily picked out (Hanaman). A. B. H.] 2. PRESERVING MEDIA (Mounting Fluids). The freshly prepared botanical specimen, which is to be sub- jected to microscopical examination, is usually put in a drop of distilled water on the slide and a cover-glass put over it. But we have already seen that water is not the only fluid to be used in examining freshly prepared objects. So likewise in permanent mounts water is scarcely suitable to use, since most objects would in time decay in it. We must choose, therefore, 218 THE MICROSCOPE IN BOTANY. those preserving media which will keep the object from decay j and yet at the same time be sufficiently indifferent to it as not to change the object itself in the slightest degree. A large number of such media have been brought into use, but we shall undertake to enumerate only those deemed most important and most useful. They are either evaporating or non-evaporating fluids or substances, and those which may be applied in a fluid state but which afterwards stiffen. A. Glycerine. For the botanist this is the most important of all the preserving fluids. It may be used with most vegetable preparations, but it preserves starch and chlorophyll grains rela- tively the best. In mounting the red algre, bacteria and diatoms, it should not be used ( Poulsen) . Concentrated glycerine causes much shrinking of the tissue by withdrawing the water from it and a contraction of the primordial utricle. This, however, may be prevented in great part by first placing the preparation for a considerable time in a weak solution of glycerine and distilled water. Glycerine should be employed in mounting, either con- centrated or diluted with water, in various proportions (1:1, etc.). Strong glycerine is often too powerfully clarifying to be suitable for certain kinds of tissue. It should then be di- luted. Frey recommended the use of concentrated glycerine with the addition of a small quantity of pure carbolic acid. For other purposes he adds to 30 g. of glycerine, 2 drops of strong hydrochloric acid or, in place of this, concentrated acetic acid. Certain stain ings require the addition of acid, otherwise the color will fade after a time. According to Dippel, the addition of a little acetic acid to concentrated glycerine modifies the effect of its shrinking properties, and its too powerful clarifying of thin tissue. For the preservation of very delicate objects, green algae, etc., a glycerine mixture invented by Hantsch^ 6 is recom- mended, which Dippel describes as follows : " Three parts pure 90 per cent alcohol with 2 parts water and 1 of glycerine. In order to moderate the effect of this fluid as much as possible at the beginning, the object is put in a drop of water on the slide to which a small drop of the mixture has been added. Then se Hantsch in Reinicke's Beitrngen zur neueren Mikroskopie, Heft III, p. 37, /. PRESERVING MEDIA. 219 the preparation is put in some place free from dust and allowed to stand till the water and alcohol have entirely evaporated. Then add another drop and let it evaporate as before, and con- tinue this process till the object has as much glycerine as it should have to be properly mounted. In this way the good preservation of the object is perfectly assured. It is advised, however, before the mounting is finished, to let the object lie for some days in order to be fully convinced that the preserving medium has no more fluid in it that can be evapo- rated." Objects to be mounted in glycerine should be first moistened in distilled water before they are put into the glycerine. Lay the object in a dish of water, put a drop of the glycerine on the slide and then transfer the object to it with the least possible amount ot* water adhering to it. Then put on the cover-glass. It will often be a matter of some difficulty with the be- ginner to know exactty how much of the glycerine to use in order to fill out the space under the cover-glass and yet not so much as to cause it to run out around the edges. The right quantity may very nearly be determined in the following way. A bottle with a Ion o- stopple, of the form of Fig. 101, ,, ,, i vi i +1 FlG - is partly filled with glycerine, the stopper put in and lifted out : there will always be a drop of the same size hanging to its point. This is put on the slide as an experiment and covered with a glass of the size commonly used. Now, if there is too much or too little fluid on the slide, there should be some fluid taken out of or added to the glyce- rine in the bottle, as the case may be, till it stands at exactly the right place to give a drop of the required size. It should then be kept at the same height. Glycerine should never be shaken because it thus collects small air bubbles which are transferred to the preparation. In order to free commercial glycerine from air bubbles, warm it a 220 THE MICROSCOPE IN BOTANY. little and then filter it through a common filter, or through fine glass wool directly into the glass bottle. B. Glycerine-jelly. At the present time glycerine is being replaced by a fluid that contains glycerine * but which is used warm and stiffens on becoming cold, the so-called glycerine- jelly. We believe that this medium is to be preferred in many cases to glycerine, since it is much more convenient to handle, and, as we have learned by careful experiments, it preserves many botanical objects in a very superior way, always assuming that good glycerine-jelly be used. Kaiser 37 gave a method for preparing glycerine-jelly which we can certify from our own experience is very satisfactory. One part by weight of the finest French gelatine is soaked for about two hours in 6 parts by weight of distilled water. To this is added 7 parts of chemically pure glycerine, and to each 100 g. of the mixture add 1 g. carbolic acid. The mixture should then be warmed with constant stirring for ten or fifteen minutes, till all the flakes which were formed by stirring in the carbolic acid have disappeared. Finally filter while still warm, through glass wool, which has been previously washed and put in the funnel while still moist. Glycerine-jelly stiffens perfectly in ordinary temperature, and so must be warmed each time it is used. For this purpose it should be kept in a thin walled test tube, so that it may be warmed in a moment. Then a drop is taken up by means of a glass rod and put on the slide, the slide itself being gently warmed, and the object which has been previously immersed in a weak solution of glycerine is embedded in it. Then the cover-glass (warmed) is put on and the whole left to cool. The preparation is completed when it afterwards has been pro- vided with a ring of varnish or cement around the edge of the cover-glass. 38 Nordstedt 39 in making glycerine-jelly dissolves 1 part of pure 87 Raiser in Botan. Centralbl., Bd. 1, 1880, p. 25, ff. Vgl. further Brandt in Zeitsch., /, Mi- kroskopie Bd. II, 1880, p. 69, ff. Poulsen, Botanisk mikrok., p. 43 (Translation Am. ed.p. 67). Journal of the Royal Microscopical Soc., London, Vol. Ill, 1880, p. 502. 38 Preparations, Avhosecell contents (protoplasm, chlorophyll, etc.) are to be preserved, must be hardened as much as possible, before they are mounted in jjlycerine jelly. as Nordstedt: Ora anvandandet af gelatinglycerine vid undersokning og preparering af Desmidieer (Botaniska Notiser, 1876, No. 2). Poulsen, I. c., p. 43 (Am. Trans., p. (37). PRESERVING MEDIA. 221 gelatine in 3 parts boiling water, and adds 4 parts of glycerine and (to prevent fungus growth) a piece of camphor. A third method of making nnd using glycerine-jelly is given in the American Monthly Microscopical Journal 40 as follows : "The jelly is made by dissolving transparent isinglass in suffi- cient water so that it makes a stiff jelly when at the ordinary temperature of the room. When the slides are mounted, add one-tenth as much good glycerine, and a little solution of borax, carbolic acid, or camphor water. The mixture while hot should be Avell filtered through washed linen or other fabric, as it will not go through common filter paper, and the subsequent addi- tion of a little alcohol improves its working. Objects, if per- fectly clean, may be transferred from water directly to this medium which should be slightly warmed before using. The cover is adjusted* and the slide put away till a number have accumulated. The cover should not be pressed down too hard, and a liberal amount of jelly used to allow for shrinkage in drying. The slides may be finished as soon as the jelly has set, or may be left for several days. If air bubbles are entangled / ! C they will usually escape while drying, or they may be driven out by warming the slide a little. When ready to finish the slides, take them to a water cooler and let the ice-cold water drop over them, while, with a rather stiff camel's hair brush all the superfluous jelly may be washed away by aid of the flow- ing water which keeps the jelly under the cover hard. The slides are then dried with a towel, or cloth and finished with a ring of cement." For the rest, mixtures of glycerine with gelatine, gum arabic, glue or other stiffening substances have been employed by var- ious microscopists for a long time past. Schacht's glycerine mixture consisted of 1 part gelatine, 3 parts water, 4 parts glycerine. Deane mixed 4 parts glycerine with 2 of water and 1 of gelatine, the latter being dissolved in warm water and the glycerine added. Klebs recommended 2 parts concentrated isinglass solution with 1 of glycerine. Beale softens pure glue o Vol. II, 1881, pp. 4, 5. * The cover should always be adjusted with the slide upon the self-centering turn table, an apparatus to be described farther on, in such a way as to be exactly in its center. A. B. H. 222 THE MICROSCOPE IN BOTANV. in water, dissolves it in a glass vessel in a water bath, adds a like quantity of glycerine and filters through flannel. C. Canada Balsam. In animal preparations this resin comes into most extensive use as a mounting medium, while in vegetable preparations it can seldom be used, because it exercises a quite too powerful clarifying effect on the cell walls of delicate sections, and most vegetable preparations containing water cannot bear the previous dehydrating necessary for mounting in Canada bal- sam. But it is principally applicable to the mounting of diatoms , hard seed coverings, spores, siliceous epidermal cells, and ground-sections of fossil plants. We should use the best to be found in the market, having the clearness of water, and not too thick. Balsam which has become too thick by long keeping may be thinned by turpentine oil, chloroform or ether. In order to prevent evaporation as much as possible, it should be kept in wide-necked, glass-stop- pered bottles. E. Kaiser has recently put into the market Canada balsam in tubes dissolved in spirits of turpentine. It is a very limpid, almost colorless fluid which is put up in me- tallic tubes like oil colors. The balsam in this form is very handy to mannge, dries quickly and very seldom forms air bubbles in the preparation. Mounting in balsam is done as follows : The object to be mounted must contain no trace of water, so it must be perfectly dried in a drying box (like diatoms) , or since all preparations will not bear this kind of drying the water must be removed by putting the specimen in absolute alcohol from which it is transferred to turpentine oil, or oil of cloves. Now put a drop of the balsam on a slightly warmed slide, take the object from the fluid in which it is lying, let all run off that will, and then put it in the balsam, which should cover it on all sides and all over, the slide being kept warm meanwhile by being held over the flame of a small alcohol lamp [or over the short chimney of a small, low kerosene lamp. For all wanning purposes in mounting, such a lamp is quite as good as an alcohol lamp, and naturally much less expensive to keep going. I always use one under my warming table in mounting with glycerine-jelly as well as with balsam. A. B. H.] For mounting in balsam as PRESERVING MEDIA. 223 well as iii glycerine-jelly, it is best to use circular cover-glasses, as there is much less risk of having air bubbles than when using those with corners. Should they occur, however, under the cover-glass, they may be removed by leaving the slide in a warm place for a considerable time, in an inclined position, as on the iron plate of a stove. When the preparation is several days old the balsam will become hard, and all which has exuded about the edge of the glass may be scraped away with a knife, and the last traces of it removed with a cloth, and a little alcohol, or oil of turpentine. It is not absolutely necessary to put a varnish ring around the preparation : however, many microscop- ists take pains to do it. [As a substitute for Canada balsam and other solutions of resinous gums, used as mounting fluids, I have for many years employed with great satisfaction a fluid made after the following formula. (1) Gum mastic 38 g. dissolved in 55 cc. of chloroform. (2) Gum dammar 38 g. dissolved in 55 cc. spirit of terebinth. Mix the two solutions and filter. It may be employed not only as a mounting medium,* but also as a temporary cement for inclosing glycerine mounts with which fluid it will not mingle at all or run in under the cover. After- wards stronger cement should be applied over this to keep all secure. A. B. H.] D. Chloride of Calcium Solution. Chloride of calcium is the oldest preserving medium known for permanent botanical preparations, it having been recommended by H. v. Mohl. 41 It was in earlier times almost the only preserving medium known. According to Harting, Dippel, Xageli and Schwendener it serves an excellent purpose for cell-wall preparations, but not for chlorophyll and starch objects since these swell up in it and are quite destroyed. In recent times chloride of calcium has mostly gone out of fashion, and indeed not without good reason, for it is not easy to find a cement that will hold it. It is used either in a saturated solution or in different degrees of dilution (1 Ca Cl. 2 :4 8 H 2 O, Harting. 1 Ca C1 2 : ^3 H 2 O, 41 Hugo v. Mohl, Mikrographie, p. 335. 224 THE MICROSCOPE IN BOTANY. Dippel), adding to the fluid some drops of chemically pure hydrochloric acid. E. Other Preserving Fluids. Besides those already men- tioned, there is a whole series of mounting fluids to which, however, but a passing word can be given, partly on account of their doubtful value and partly because they are suitable for but comparatively few preparations. (a) /Sugar water, with a little corrosive sublimate added to prevent the growth of fungi, is, according to Nageli and Schwendener, suitable for all such objects as are too much changed by glycerine and calcium chloride. (b) Solution of Corrosive Sublimate. Of this, numerous compounds have been made and recommended, first, that of Goadby (Goadby's Fluid) : Sodium chloride 120.00 g. Alum 60 00 " Corrosive sublimate 0.25 " Boiling distilled water 2.33 1. Pacini modifies the mixture as follows : i ii Corrosive sublimate 1 part Corrosive sublimate 1 part Common salt 2 " Acetic acid 2 " Glycerine 13 " Glycerine 43 " Distilled water 113 " Distilled water 275 " The mixture is left to stand for two months, and is then di- luted one part thereof with three of distilled water and filtered. The botanist, however, finds little use for compositions of cor- rosive sublimate. (c) Creosote Mixture. Harting recommends for some prepa- rations an aqueous solution of creosote, consisting of a saturated solution of creosote in 20 parts of water, to which 1 part of 30 per cent alcohol is added. Beale gives the following formula for a complicated mixture. Mix 180 g. methyl alcohol with 11 g. creosote, then add enough pulverized chalk to form a thick pulp ; then, in a mortar, slowly add with constant trituration, 1920 g. water and put in a few pieces of camphor. After two or three weeks, filter and keep the filtrate in a well closed glass bottle. It is a good preservative for desmids. PRESERVING MEDIA. 225 (d) Topping's Fluid consists of one part absolute alcohol and five parts water, or in place of the water, 4 parts water and 1 part acetate of alum. An equal volume of glycerine is added to the mixture and should be used principally in preserving objects stained with carmine. (e) Potassium Acetate. This salt was first recommended by Sanio, 42 and was afterwards designated as a good preserving medium by Dippel. It preserves the chlorophyll beautifully without a sign of shrinking of the cell wall. Use a saturated solution in distilled water. It is employed as a preservative for bacteria which have been stained with aniline. 43 (/) Preserving Medium for Algce. For preserving conferva, and related forms, including desmitls (fresh-water algae), P. Petit 44 employs the following composition which is filtered after solution : Camphor water 50 00 . Distilled water 50.00 " Glacial acetic acid 00.50 " Crystal copper chloride 00.20 " " " copper nitrate 00.20 " * (g) [King's Fluid for preserving Marine Algaz. Rev. J. D. King finds the following composition entirely satisfactory for mounting and preserving microscopic specimens of marine algae. Powdered alum 62.20 g., corrosive sublimate 0.258 g., dissolved in 2.27 1. of pure sea water and filtered. For several years past I have made much use of a mixture of sea water and glycerine for this purpose. It mny be mixed in various pro- portions, though 5 parts of the former to 1 of the latter, will be found to do well in most cases. It is a good plan to filter the sea water at first and then let it stand tightly corked in a glass bottle for several months, then filter again and mix. A. B.H.] Sanio in Botan. Zeitg. 18G3. p. 359. 43 Poulsen, I. c., p. -U (Translation, p. 69). 44 Brebissonia, Jahrg. Ill, 1880, p. 92. See also Cornu et Rivet, Des preparations mi- croscopiques, Paris, 1*72. Concerning a peculiar method of preparing for examination marine algae, which have been once dried, see C. F. Jones in Northern Microscopist, Vol. I, p. 54-50. Also Jour. Hoy. Micfosoop. Soc., London, Vol. 1., Series II. 1881, p. 530. * Mr. G. W. Morehouse adds to this 100 g. strong glycerine, or if the specilic gravity is too high, less glycerine. See Am. Monthly Microscopical Journal, Dec., 1883, p. 234. A. li. H. 15 226 THE MICROSCOPE IN BOTANY*. (h) Monobrom-Napldhaline , according to the recent experi- ments of Abbe and Dippel, is a suitable mounting medium for objects which are to be examined with very high powers, as, for example, diatoms used as test objects. (/) [tityrax an d Liquid Amber. Dr. H. Van Heurck* has published an account of his very satisfactory experience with these resins, Liquidamber orientalis Mill, and Liquidamber styraciflua L. as a mounting medium for diatoms, and other such objects requiring a medium of a high index of refraction. In this case it is 1.65, Canada balsam, which this is intended to take the place of, being 1.53. The commercial article is first dissolved in chloroform and filtered to purify it from the gran- ular substances which it contains, and the solution thus ob- tained is used in the same manner as a like solution of Canada balsam. It will not form bubbles of air in heating. It is re- commended to expose the liquid amber in a thin layer to the direct light of the sun for several weeks. This will cause it to discharge all of its water and most of its color. It becomes hard and then may be dissolved as before directed in chloroform. It may also be dissolved in benzine or a mixture of benzine and absolute alcohol. A. B. H.]f (&) Colored Mounting Fluid. A complicated preserving me- dium, especially adapted for mounting starch grains, has recently been given by Seiler. 45 He describes its preparation and use as follows : " It is necessary first to have some aniline blue staining fluid which we make after the formula given by Beale : Soluble aniline blue 0.032 g. Distilled water 31. cc. Alcohol 25 drops. A mixture is made of equal parts of glycerine and water, say 15 cc. each, to which are added 2 or 3 drops acetic acid. To this mixture of slightly acidulated dilute glycerine is added the * Bull, de la Soc. Beige de Microscopic. Quoted in Am. Month. Micro. Journal, Apr., 1884, p. 69, 70. I Prof. 11. lj. Smith has discovered two media for the same purpose, the nature of which he has not yet chosen to make public, which have an index of refraction respectively of 2.00 and 2.25. Sec. J. D. Cox. in Am. Monthly Micros. Journal, Apr., 1884, p. 71. 5 Seller's Compend. of Micro. Technology, Phil., 1881, p. 13, /. PRESERVING MEDIA. 227 aniline blue staining fluid until the whole mixture is of a decided blue color. A drop of this mixture is placed on a glass slide and some of the starch to be mounted is dusted over the top. The dusting can be done to the very best advantage by touching the starch with a camel's hair brush and then slightly shaking the brush over the drop of colored glycerine. The starch soon sinks to the bottom of the mixture and the cover is applied. With this method of introducing the starch, air bubbles are avoided. The cover is pressed down quite firmly upon the slide and the excess of glycerine carefully removed. The slide is then transferred to the turn table and a thin layer of dammar or balsam in benzole placed around the border of the cover. This soon hardens, and in a day or two the slide may be finished with a ring of white zinc, Brunswick black or other cement. The effect is this : the grains themselves have not taken the staining in the least, neither will they ever take it ; they retain their natural appearance surrounded everywhere by the blue glycerine and the effect is most beautiful." (?) [Mr. Karl Castelhun of Newburyport, finds the following very satisfactory for preserving sections of lichens : Glycerine 7 parts Alcohol 1 " Water 6 " ] (m) [Strong carbolic acid is highly recommended for preserving vegetable tissue. It should be used with only so much water added as will -keep it from crystallizing. " One great advantage of its use is found in the readiness with which it penetrates a specimen and mixes with the fluids used in mounting, such as water, glycerine and Canada balsam.*] (n) [Preservative for Fungi. Thoroughly mix 1.1341. white wine vinegar with 127.5 g. common salt and 141. 7 g. pulverized alum and keep in a wide-mouthed glass jar. Put the fresh specimens of fungus into it. From time to time the liquid may be strained to take out impurities. f] * Wm. J. Pow. in Am. Month. Micro. Journal, Jan., 1883. p. 8. t Mary E. Banning in Bulletin Torrey Botan. Club, Dec., ISS2, p. 153. 228 THE MICROSCOPE IN BOTANY. (o) [ Wickersheimer's fluid for preserving algag, lichens, fungi, etc., preserves the color of most delicate structures quite perfectly. Alum 100 g. Common salt 25 " Saltpeter 12 " Carbonate of potash 60 " White arsenic 20 " Dissolve by boiling in 3000 cc. of water. Cool and filter and add 1550 g. glycerine and 300 g. methyl alcohol (wood spir- its).*] [This fluid is an excellent preservative for all kinds of land plants ; and plants which ordinarily become stiff and brittle by drying will always retain their natural flexibility if, previously to drying they are immersed for a little time in this fluid, till they become saturated with it. A. B. H.] Besides the preserving and mounting fluids here mentioned, there are a large number of others, formulae for which are scattered through the different works. We have not included them in this work because the greater part of them have been used only by their inventors, their usefulness not having been tested by others. VIII. MOUNTING THE PREPARATION IN PRESERVING FLUID. "We have already pointed out how to proceed in putting the specimen into a preserving medium such as glycerine-jelly and Canada balsam. This is not usually attended with much diffi- culty and the beginner soon learns to do it successfully after a few experiments. It is otherwise in mounting with fluids. In doing this we have carefully to measure out the exact quantity required to fill the space between the glass cover and the slide, and then we must hermetically seal up this fluid in which the preparation is immersed, of the method of doing which we shall speak here- after. * Furnished me by Dr. L. Schouey of New York City. A. B. H.] MOUNTING THE PREPARATION IN PRESERVING FLUID. 229 The little knack by which we may take out each time very nearly the quantity of fluid we need has already been indicated in the case of glycerine .(see p. 219). The fluid may be pre- vented from running out about the edges of the cover-glass by putting a thin rim of varnish, hereafter to be described, upon the slide which is somewhat smaller than the cover-glass itself. Schacht, who invented this process, drew two varnish ledges on the slide corresponding to the two opposite sides of the square cover-glass ; Dippel 46 , three which formed a square with one open side, and Nageli 47 added still a fourth thus closing np the rim of varnish. Each "of these authors held his method to be the best. We leave it to the experienced worker to decide for himself between them. The round cells made by means of the turn table are very pretty, but one must naturally use the round cover- glasses with them. Whether or not the slide be provided with a foundation rinpel. 1. c.. Pxl. I. p. 474. *' Nageli u. Schwdudener, 1. c., p. 297. 230 THE MICROSCOPE IN BOTANY. homogeneous, warm the slide gently till the glycerine-jelly film is melted, when it will mingle freely and perfectly with the other fluid. This process is especially convenient where one wishes to arrange several small sections under one cover-glass. A. B. H.] The following manipulation, putting on the ' cover-glass, must first be taught to beginners who often fail. If angular covers are used, he should take them up in the forceps by one corner, and having breathed upon the side which will come next to the glycerine or other fluid, he puts down the edge opposite to the forceps. Now he lowers the forceps till the drop of mounting fluid touches the middle of the glass. Now, when the skilful manipulator suddenly lets go of the cover-glass it falls into its place so that its edges lie parallel with those of the slide, and the mounting fluid is evenly spread out between the two glasses and there are no air bubbles. If we use slides which have a ring or square of cement already laid on them and put the edge of the cover-glass upon that while it is yet sticky, it will be fixed at once in its place, which is a matter of great importance in the subsequent hermetical sealing of the cell. The cover being now successfully laid on, we examine the object with a magnifying glass or the preparing microscope, to bee if it is still in its place in the middle of the cover-glass, or has been pushed aside. In the latter case (if we have not mounted the object in a closed cement ring as above described), we may replace it again by means of a common hair held be- tween the thumb and finger and thrust in between the cover- glass and slide, or by means of a very fine glass thread which one may make by drawing out a piece of glass tube over the flame. Very delicate preparations, as, for example, sections of the very young parts of flowers, "growing points," etc., are so delicate that the weight of the cover-glass will quite destroy them especially when to this is added a little pressure by the drying of the cement ring which holds the cover. There are several ways of preventing this. One proposed by Purkinje 48 48 Purkinje in Wagner's Handbuch der Physiologie, Artikel Mikroskop. MOUNTING THE PREPARATION IN PRESERVING FLUID. 231 and Hugo v. Mohl 49 consists of laying small wax balls between the cover-glass, which by gentle pressure on the latter can be reduced to the exact thickness of the preparation. This method is in fact a very good one. In extraordinarily thin and deli- cate sections small pieces of the fibres of glass wool, or of the hair from the head, may be substituted for the wax balls to give the cover-glass the right position. But, on the other h.-ind, if the preparation is very thick, a little trough or cell about the height of the thickness of the section must be built up upon the slide, in which it may lie. For this purpose we build up a wall of varnish or shellac, of the size and shape of the cover-glass, by putting on one layer after an- other, letting each layer dry before adding another. AVe may also use wax instead of varnish, and then our cell will soon bo done for that rapidly stiffens. But the wax in thick layers is always brittle and will not without injury bear sudden changes of temperature. On this account the cement Cells are greatly to be preferred. [Making cement cells by the use of the self-centering (or any other) turn table, is a matter of very little difficulty, but of the greatest importance to the microscopist. For all but the very deepest cells they answer the purpose perfectly, and for these metallic or glass rings may be used.] [The strongest cement is the best for cells. Usually some solution or compound of shellac is preferred, directions for making which will be found below. Probably nothing better of this kind can be had than King's amber cement, or white cement. The slide is laid upon the turn table and concentrically clamped. A small hair pencil is dipped in the cement and while the turn table is being somewhat rapidly rotated the brush is carefully brought down upon the rotating slide so as to draw a circular band of cement 3 or 4 mm. wide upon it in such a position that, when the cover-glass is put on, its edge will come about in the middle of the band.] [There are two ways of completing the circular wall of the cement cell. The one is to lay on the first coat of cement so carefully that the circle will have exactly the diameter and < 9 H. v. Mohl, Mikrographie, p. 328, ff. 232 THE MICROSCOPE IN BOTANY. breadth required in the cell. Let this thoroughly dry. Then put on another coat very carefully, exactly on the top of the first. Let it dry and again repeat the operation till the ring is built up high enough. When all is done and the last layer is quite dry and hard, bring the top of the ring to a flat and even surface with a smooth file, or upon a stone. It then may be laid away till wanted.] [The other, and perhaps the preferable because the more rapid, way of making cement cells, is to lay on a considerable quan- tity of the cement at the first, making the inner edge of the circle come as near as possible to the position where it is wanted, but permitting the outer edge to spread out wider than the ring is to be when finished, making it 6 or 7 mm. broad if necessary, and lay on all the cement needful to finish the cell. Then with the point of a knife applied to the slide at the outer edge of the cement ring, while the turn table is rapidly rotating, turn the edge of the ring slowly inward narrowing the band and heaping up the cement until the desired height and breadth of the ring is attained. If the inner edge of the ring should need to be turned and smoothed a little, it may be done in the same way, but it would not be best to move it very far, for unless the work is very nicely done traces of the cement will be found on the slide at the bottom of the cell. If this should happen, the best way to clean it is to let it dry thoroughly, put it again on the tuin table, rapidly rotate it, and with the smooth point of the knife turn off or scrape up the adhering traces of cement. The particles may then be removed with a dry cloth or camel's-hair brush. The top of the ring, when perfectly dry, should be made smooth and flat as in the other case. A. B. H.] But if we wish to make a durable trough quickly, we shall have recourse to the so-called glass cell. It is a perforated glass plate, represented in Fig. 102, I, and is prepared from glass 0.5 to 1.0 mm. thick, rough ground on the underside, and thor- oughly cemented to the slide. [Marine glue is excellent for this, perhaps the best, but the cements mentioned above will answer all purposes.] One can make glass cells for himself without much trouble. Taking a glass tube of about 12 mm. interior diameter and a thickness of wall of about 3 mm., have MOUNTING THE PREPARATION IN PRESERVING FLUID. 233 i it sawed up, at a glass grinder's into rings .5 to 1.0 mm. thick, Fig. 102, II. Then fasten them upon a glass plate with Can- ada balsam in much the same way as fossil wood is prepared for grinding, then with emery or with turpentine oil on a whetstone grind it flat and smooth. Then turn it over, recement it, clean away the Canada balsam and grind the other side in the same way. A still simpler way is to take glass strips 3 mm. broad, 1 mm. thick and pf sufficient length, and by holding them in the flame of a Bunsen burner, bend them into the form given in Fig. 102, III, welding it finally at a. The joint should be made in the middle of the side rather than at the corner. O A FIG. 102. In mounting large specimens in shellac, wax, or glass cells the process is as follows. The cell is filled full of the mount- ing fluid, for example glycerine, and the specimen carefully laid in. When the cover is laid on, it should be fixed at one corner with a small drop of wax or shellac, which should be allowed to stiffen or harden as the case may be. But if some of the fluid has run out and got on the cover-glass it must be carefully removed, a matter sometimes of no little difficulty and labor. The first step toward cementing the cover-glass should be taken only when it and the upper surface of the cell are perfectly cleau and dry. It maybe mentioned in passing, that this method of mounting especially commends itself for those slippery algae which, when we undertake to mount them without a cell, directly we put on a cover-glass slip out from under its edge. In this way only have we succeeded in mounting the gelatinous fresh-water alga Balrachospernum monili/orme, after all other attempts to con- fine it had failed. 234 THE MICROSCOPE IN BOTANY. IX. CEMENTING AND FINISHING THE MOUNT. When the preparation has been embedded in the preserving medium and the cover-glass laid on, the next step is to surround the edge of it with a border of varnish or cement, which when dry will fasten it to the slide, solidly and permanently, hermet- ically sealing up the preparation (see Fig. 100, I, II). Before, however, we describe this process, we should become acquainted with the nature of the cements or varnishes used in it. 1. CEMENTS. 1. Wax. This is used in the form of a little wax candle, the wick of which is lighted and then, after a moment, when the wax is melted around it, we may dip a camel's -hair brush into it and immediately draw the wax rim on the slide around the cover-glass. 2. Asphalt Varnish (Brunswick Black). This consists of a solution of asphalt in like parts of turpentine and linseed oil. It can be had in any drug shop. For microscopical purposes the best only should be used. Really good asphalt varnish renders the very best service in cementing microscopical pre- parations notwithstanding Frey's assertion to the contrary. If it gets too stiff it may be thinned with oil of turpentine. 3. Mastic Varnish. I am not acquainted with the composi- tion of this varnish. The dissolving medium is alcohol. It was first recommended for our purposes by Schacht. The mieros- copist's varnish of E. Kaiser appears to be like it, which from my own experience I can recommend. Both kinds when they become too stiff may be thinned with absolute alcohol. 50 4. Shellac and Sealing-wax Cements. Dr. O. E. R. Zim- mermann has kindly furnished me with the formula of a very useful shellac cement. "Dissolve good brown shellac in abso- lute alcohol, add aniline green and filter. The filtrate should now be allowed to stand protected from dust near a stove in a, 6 Frey, I. c., p. H3.-Dippel, I. c. } Bd. I, p. 473. CEMENTING AND FINISHING THE MOUNT. 235 wide-necked glass bottle till it has become so thick that it will not run when put on a slide with a hair pencil, but makes a sharply defined outline. This varnish never cracks off. When perfectly dry and subjected to frequent changes of temperature, isolated wrinkles will sometimes come in it, but it never loosens up so as to injure the object." Thiersch 51 has given a formula for making a thin shellac cem- ent which can be used with balsam mounts. Thick brown shel- lac cement (prepared by the solution of shellac in alcohol) is evaporated to the consistency of thin mucilage and colored with a concentrated solution of aniline blue or gamboge in absolute alcohol. To 60 g. add at last 2.5 g. castor oil, evaporate still a little further and keep in a well-closed bottle. If it becomes gradually too much concentrated it may be thinned with a few drops of alcohol. Poulsen 52 gives a recipe for a third kind of shellac cement which he names the Gram-Riitzou cement: "50 g. Canada bal- sam, 50 g. shellac, 50 g. absolute alcohol, and 100 g. ether are mixed and evaporated in the water bath to a thick syrupy consistency. An alcoholic solution of sealing wax is often proposed in place of the shellac cements, but I have no great confidence in it. [I am indebted to the kindness of Rev. J. D. King for two formulae, one of a very excellent cement and the other of a "finish" which for its cementing power and its good appearance can scarcely be equalled, I think, by anything yet offered in this line. They are both compounds of shellac.] [King's Amber Cement is made as follows. (1) Dissolve 453 g. of best bleached shellac in half a litre of 95 per cent alcohol. (2) In another vessel dissolve 1 part gum mastic in two parts alcohol and let it stand till perfectly clear. To the shellac so- lution; (1) add 38 g. of the mastic solution, (2) color with "dragon's blood" dissolved in alcohol and filter. Place it in the water bath and stir frequently till it comes to a boil. Filter through flannel, after which, if too thick, bring to a right con- sistency by means of strong alcohol.] 51 Frey, 1. c., p. 143. 5 - Poulsen, Botunisk Mikrokemi. Translation, p. 71. 236 THE MICROSCOPE IN BOTANY. [King's White Cement is made in the same way omitting only the "dragon's blood."] [King's Lacquer Finish.* (1) Dissolve 453 g. Dennison's excelsior sealing wax in one-half litre or more, it' necessary, of alcohol. (2) Dissolve 1 part best bleached shellac in 2 parts 95 per cent alcohol. To every 38 g. of No. 1 add 5 g. of No. 2 and 5 g. of Brown's rubber cement. Let it stand two weeks or more in a warm place, stirring it occasionally. If too thick to flow freely reduce with alcohol.] [The color of this finish will depend of course upon the color of the sealing wax used, and one can thus exercise his taste in ornamenting his slides, at the same time that he secures in the best possible way the permanent safety of his preparations. A. B. H.] 5. Copal Varnish can be employed as a cement in connection with wax and asphalt varnish. I prepare it in this way. I put 5 g. of pulverized copal in a glass retort and pour over it 5 cc. each of absolute alcohol and oil of turpentine and 1 cc. of ether and carefully, slowly and gently warm until the copal is dis- solved. Close the vessel, set it off and pour the clear, transpar- ent varnish into a well closed glass-stoppered bottle. 6. Dammar VarnisJt, used in connection with the foresfoinsr o o is prepared in the following way. The best coarse grained dammar gum should be warmed a long time till all the water is driven out, then pour over it three times its weight of turpentine oil and when dissolved decant the clear, colorless varnish into a glass-stoppered bottle. 7. Gold-size, a cement much used by the English is pre- pared, according to Beale, in the following way. Twenty-five parts of linseed oil are boiled three hours with 1 part vermil- lion and part umber. The clear liquid is then poured off and like parts of well ground white lead and yellow ochre are slowly and gradually mixed in with constant stirring, further boiled and finally turned off and kept in a bottle for use. * These cements and finishes may be had ready made of Rev. J. D. King, Cottage City, Mass. CEMENTING ANGULAR COVER-GLASSES. 237 2. CEMENTING ANGULAR COVER-GLASSES. [Inasmuch as this form of cover-glass is very little used in this country in botanical work, I shall condense what the author has to say upon it into as little space as possible. A. B. H.] The tools to be used are small artists' hair pencils of the form represented in Figs. 103 and 104, the flat one for laying on the cement band and the smaller round one for touching up and finishing off the work, add- ing a little of the cement here and there. They should be kept scrupulously clean and the cement should never be allowed to dry or harden in them. This may be prevented by having little bottles partly filled with the solvent of the cement in which the brush is used, alcohol, turpentine, ether, etc. ; and, having a hole though the cork, put the handle of the brush in it, letting the brush dip into the liquid and there to remain when not in use. The brushes may also be cleaned after each using with the cement solvent, and left dry. If, in placing the cover, a small drop of the mounting fluid, glycerine, etc., runs out upon FIGS. 103 & lot. the slide it should be cleaned off before cement- ing. A hair pencil saturated with oil of turpentine will do this, and the traces of turpentine may be washed away with a brush dipped in ether. When a foundation cell has been made and the cover-glass pressed down upon the soft cement in mounting so as to stick fast all around, the subsequent cementing and finishing are an easy matter. It is only necessary theivto fill the flat brush with the cement and draw it slowly along the edge of the cover-glass, half on that and half on the slide, making the layer not more than 3 or 4 mm. wide altogether. Begin at the corner and go the length of the side at a stroke. So all around. The cem- ent or varnish for the first coat should be of as thick a consist- ency as can be conveniently managed with the brush. The slide should be laid aside for a day or two for the varnish to dry ; 238 THE MICROSCOPE IN BOTANY. then examine it with a magnifying glass to see if it is all tight ; if not, apply more thick varnish. If the first coat is not substan- tial enough, apply a second of a varnish of thinner consistency, making the band extend a little beyond the edges of the first one. After a fortnight, even a third coating may be put on. If no foundation cell has been made and the cover-glass has nothing to stay it but the adhesion of the mounting fluid, a drop of thick varnish should be put at each corner and allowed to harden, then the varnish applied as before, care being taken to use pretty thick varnish so that it will not run in under the cover and spoil the specimen. [If, with glycerine as a mount- ing fluid, a chloroform or benzole solution of Canada balsam or dammar, or the mastic and dammar solution described on p. 223 be used for a first coat, there will be no danger of its running under the cover, and one will find it very convenient to use one or the other of these with all glycerine mounts whether with square or circular glasses. A. B. H.] Several coats of the finishing cement should be applied one after the other as they become dry. Another method is to make the first layer of wax. The wick of a small wax candle is heated over the flame till it is thor- oughly saturated with the melted wax and then drawn carefully along the edges of the cover-glass. It immediately stiffens and the cement may be applied at once. I usually put over a thin layer of wax a layer of copal varnish which very quickly dries, and then over that a third of dammar varnish which dries very slowly but is very durable. When it is dry I put on at in- tervals three layers of asphalt. Balsam and gelatine preparations should have one or two coats of asphalt. Thiersch's shellac cement may be used with the former, after a previous layer of Canada balsam dissolved in chloroform has been applied. [For all finishing processes I know of nothing better than Brown's rubber cement, or King's lacquer finish, the latter being the stronger and therefore the better. The white zinc cement so extensively used in this country is bitterly complained of by some and highly recommended by others. There seems to be no way of accounting for such marked differences of experience and opinion. It has served me well. A. B. H.] MOUNTING WITH CIRCULAR COVER-GLASSES. 239 3. MOUNTING WITH CIRCULAR COVER-GLASSES. The use of circular cover-glasses is very much to be preferred. They are easier to cement, and are more secure, not having the weak points, the corners of the square ones, and it requires much less time to cement them, and they also have a much more elegant appearance than the angular ones. [Mounting with and cementing circular covers requires the use of a turn-table. Those with a device for self-centering are so much better than those without that I would recommend no one to get any other.] [Two forms are herewith represented in Figs. 105 and 106. The construction of them is so obvious that a detailed descrip- tion will not be necessary. The circular brass plate, Fig. 105, FIG. 105. revolves on a central pivot smoothly, and with the least possible friction. It is actuated by a stroke of the hand along its milled margin. The self-centering apparatus consists of two rectan- gular jaws upon the upper surface of the plate which are made to clasp the slide at its diagonal corners. "\Vheu the slide is held by these jaws it is concentric with the plate and with its axis of motion. The jaws are moved toward each other by a spiral spring beneath actuating parallel bars to the ends of which the jaws are attached, and are guided by the screws which hold 240 THE MICROSCOPE IN BOTANY." them, moving in the slots in the plate. The jaws are opened by pressing upon the other end of either of the bars with the thumb beneath the plate, while the forefinger of the same hand holds the plate above. Two spring clips are provided for re- finishing old slides which have been mounted without centering. This turn-table is made by Beck of London and sold in this country by Wm. Walmsley and Co., of Philadelphia.] [The -Bauson and Lomb Optical Company make a turn-table which is provided with a hand rest, which when in use lies down in such a way as to project over the table and the slide, but not touching either, giving the hand a perfectly steady support in the manipulation of applying a ring of cement to the slide.] FIG. 106. [Fig. 106 represents another form of the self-centering turn- table, made by Mr. Zentmayer of Philadelphia. The plan for centering the slide is something quite new. The slide is cen- tered laterally by having its opposite sides brought in contact with two pins projecting from the plate. It is centered longi- tudinally by means of a ring with an oval inner edge, which is fitted to the periphery of the disk in such a way that by turning it, this inner edge of the ring grasps the slide at its diagonally opposite corners.] [A form of apparatus for holding and centering the slide, and the mechanism for actuating it, which it is believed has certain SELF-CENTERING TURN TABLES. 241 marked advantages in manipulation over other forms of self- centering turn-tables, has been contrived by the writer and is represented in Fig. 107. The general construction of the ro- tating plate, pivot, frame, etc., is the same as in Fig. 105. Fig. 107, A and B^ represents an outline of the plate and attached apparatus f the natural size. In A the plate is seen from above, aa are two pins fixed in the plate equidistant from the center, by which the slide is laterally centered, bb are two pins mov- able in the slots cc, and, having a diameter above greater than FIG. 107 A. that of the slots, their shoulders bear upon the upper surface of the plate. These pins center the slide longitudinally. In _Z?is shown the apparatus on the under side of the plate, by which the pins bb are moved in clasping, centering, and releasing the slide. These two pins bb are screwed fast to two short rods oo. The rods are joined to the long bent bars tr tr at nn with a hinge joint. These bars are bent at uu where they work upon pivots made fast to the plate. A pin 10 or 12 mm. long is in- serted in the end of the outer bar at x. The short arms ?v are 16 242 THE MICROSCOPE IN BOTANY. pressed outward at x by the strong steel spring s. This action brings the pins bb firmly against the middle of the ends of the slide, thus centering it longitudinally and holding it fast.] [By grasping the plate at m between the thumb and forefinger of the left hand, the plate is held fast, while the finger presses against the pin at x and moves back the centering pins bb along the slots cc, while the right hand is left entirely free to manip- ulate the slide. An inward movement of x equal to 3 mm. will separate the pins bb a distance of 12 mm. The pin e pre- FlG. 107 B. vents the centering pins bb from coming down to the ends of the slots at cc when no slide is on the plate. As it is screwed into the plate, it may be removed if very short slides are being used.] [In the use of the self-centering turn-table the object itself must be placed in the center of the slide. When a cell is used and that cell has already been made by means of the self-cen- tering turn-table, the object can be mounted and the cover adjusted by that.. But when no cell or ring of cement has been SELF-CENTERING TURN-TABLES. 243 previously put on, which may serve as a guide, there must be something to indicate the central point of the slide. Two things may be done. We may place the slide on the turn-table in the usual way, and as it lies there self-centered, the concen- tric rings cut in the disk will be a sufficient guide for mounting the object and adjusting the cover-glass. Or we may lay the slide its poorest side up if there is any choice, on the turn- table, and with a hair pencil dipped in India ink draw a circle on it as near as possible to the size of the cover-glass to be used. When this is dry it will serve as a guide in placing the object and adjusting the cover, on the other side af the slide, near enough for all practical uses. When the slide goes on the turn-table for cementing if it is found that the cover-glass is not exactly concentric with the motion of the table it should be carefully and gently pushed over to its true place with a dis- secting needle. It may not perhaps need to be moved half a millimeter, but it should be perfectly centered if possible be- fore any cement is applied. A. B. H.] Supposing now we have a preparation on the slide mounted in glycerine and a round cover-glass laid on, the glycerine hav- ing been so carefully measured out that no trace of it can be seen beyond the edge of the cover ; on three or four points at the edge of the cover-glass is placed a drop of thick cement for a stay, as already described in regard to the rectangular glasses. When these are dry the slide is put on the turn-table and clamped and the cover-glass centered on the table. [Our author is not speaking here of the new self-centering turn-tables. With those described above it is obvious that the cover-glass must be made concentric with the turn-table before it is made fast with these temporary stays of cement. And, indeed, if glyce- rine is the mounting fluid, there will be no need of these at all if Canada balsam or the mounting fluid of gum mastic and dammar, described on p. 223, be used as the first layer to enclose the preparation. A. B. H.] We can now proceed to apply the first ring of cement. Fill a small hair pencil with not too much of the cement, which we have chosen to use (there should be no drops of it hanging 244 THE MICROSCOPE IN BOTANY. from the pencil), hold it in a perpendicular direction close over the edge of the cover-glass and set the turn-table in slow motion. Suddenly, but gently, lower the pencil till it touches and the next moment raise it again. The ring is done. On the steadi- ness and accuracy of this motion alone depends the success of the process, and it can easily be attained. One should be careful not to take too much cement in his brush else the ring will not be uniform, particularly in breadth. A second and third ring, each broader than the preceding, should be laid upon the first. If a cement ring was laid upon the slide before the object was mounted and the cover-glass pressed down upon it, we shall not need the stay drops of cement, the glycerine will not be likely to run out, and the final cementing becomes a simple mat- ter. The breadth of the last ring need not be more than 4mm. The author has prepared many slides, which have remained un- changed for years, whose last cement ring with a diameter of 18 mm. was not more than 2 mm. broad. [The ring may be made as narrow as one pleases, by turning in the edge of it from the outside with the point of a knife as described in the paragraph on making cement cells, p. 232.] [I am indebted to Rev. J. D. King for a process of sealing cells with heat that will be found very useful. The cell is made of shellac cement or lacquer finish and completed as already described. Before using, ring the outer half of the flattened top of the cell slightly with shellac cement. When the object is immersed in the glycerine or any aqueous mounting fluid, put on the cover, adjust it and press it down carefully to its bearings all around. Then apply a spring clip which has a gen- tle pressure and pass the slide, cover down, a few times over the flame of a spirit-lamp till the cement shows signs of melting. Remove the clip, press down the cover again at any point nec- essary and then hold it under the cold-water faucet to wash off the glycerine and cool it, after which carefully clean, and com- plete the cementing to fancy with shellac cement and lacquer finish. A. B. H.] LABELING AND CATALOGUING. 245 X. LABELING AXD CATALOGUING THE PREPARATIONS. A. The Label. Every preparation must be carefully labeled in order to be easily found and identified. The proper labels have a rectangular form about as shown in Fig. 100, I, II, IY, or Fig. 98, or as it suits the taste of the preparator to make them. They are fastened on with a thick solution of gum arable, or better still with a solution of brown shellac with absolute alcohol. With the latter, one may cover the whole upper surface of the label and so render the writing indestruct- ible. In beginning a collection of microscopical preparations one should choose different colors for the labels and use each color for a limited group of objects. For example : white for anatomy of the vegetation organs of the phanerogams; blue, anatomy of flowers ; green, vascular cryptogams ; red, algae ; etc., etc. The writing on the labels, of which every slide should have two, should include the following points : 1. Name of plant from which the preparation is made. For example, Pi-unus avium. 2. The part of the plant used in the preparation (stem). 3. The kind of section (transverse, radial or tangential.) 4. The method of preparation (aniline staining). 5. The mounting fluid (glycerine). 6. The date of mounting (15 V, 84). The points 1-3 should be entered on the left hand label and 4-6 on the right : Besides these labels it is well to write on the under side of the slide with a diamond the catalogue number of the specimen in order to identify it in case the left hand label should get lost. 246 THE MICROSCOPE IN BOTANY. [B. The Catalogue. Every collection of microscopical prep- arations should be carefully catalogued. As between the use of a book or a card catalogue I am inclined to prefer the latter ; it seems to allow a somewhat freer classification of the object, with opportunities for throwing out specimens that are no longer desired, together with the card that represents them, without defacing the catalogue, as it would with a book by erasures. The catalogue should furnish a perfect index to the collection, each slide being represented by a card.] [The classification of the preparations in the cabinet should be made in accordance with the natural system, in the main or primary divisions ; and, in accordance with the parts of the plant which they represent, for the secondary division. For example : the primary divisions should distinguish between the phanero- gamic and cryptogamic plants, and of the former between the monocotyledons and the dicotyledons and again, perhaps, of the latter of these between woody and herbaceous plants, and between deciduous and coniferous woods, etc. Under these the secondary divisions should recognize, and put together, sections or other preparations, made severally irom the roots, stems, leaves, flowers and seeds of the plant.] [The number of the slide then, after this arrangement, should represent its place in the cabinet. A Roman capital A, B, C, should represent the compartment of the cabinet in which it be- longs, the Roman numerals I, II, III, etc., the particular box, drawer or tray which contains it, and the Arabic numerals 1,2, 3, etc., the number of the slide in this particular holder. Thus each slide would be numbered in this way, B, XV, 15. The position of the cards in their box should answer exactly to that of the corresponding slides in the cabinet.] [If an alphabetical index is desired in order to get the readiest possible access to any slide representing any plant in the col- lection, it may be had in a supplementary card index catalogue, arranged alphabetically according to genera and species. Let cards be printed with a blank for the proper scientific name of the plant from which the preparation is made, and ruled columns for the five minor heads, under which the slides are all classified, as follows :] STORING PERMANENT PREPARATIONS, 247 SCIENTIFIC NAME OP THE PLANT. ROOTS. STEM. LEAVES. FLOWERS. SEEDS. ^B. XII. 9 B. XX. 6 r B. XV. 23 B. XXI. 21. B. XXL 18. B. XXV. 6. [Thus a single index card could easily be made to represent at least 50 slides if necessary.] ' [What should the catalogue cards contain ? Various answers have been made to this. I will indicate what seems to me mos t important, following mainly the plan proposed by Prof. S. H. Gage* for catalogues of preparations of animal histology.] [1. The scientific name of the plant. 2. The cabinet number of the preparation. 3. The part of the plant from which the preparation is made. 4. The special purpose of the preparation. What it is meant to show. 5. The special method of preparation. Whether it was pre- viously hardened, softened, or cut in a natural state. Whether mounted whole, teased out into its elementary cells or fibers, or cut into sections, and if cut how, free-hand, or by microtome. 6. The bleaching or clarifying agent, if any, and how long a treatment was required. 7. The staining medium and time required. The mounting, cementing and finishing media. Objectives to be used in its study. The date of preparation and name of preparatory General remarks including references to literature and 8. 9, 10. 11, good figures. A. B. H.] XI. STORING PERMANENT PREPARATIONS. Finished preparations should be kept in a box or case, which is so arranged that the slides resting near each other occupy * Prof. S. II. Gage, Proceed. Am. Soc. Microscopists, Chicago Meeting, 1883, p. 169,^. 248 THE MICROSCOPE IN BOTANY. the least possible space. Microscopical cabinets should satisfy the following requirements. By shutting tightly they should protect the preparations from dust. They should not allow the slides to move about, and should permit them to lie in an hori- zontal position, where they may be most easily got at. [For transporting slides special boxes should be used and not those in which they are usually contained. The principal op- tical firms in this country offer an assortment of object cabinets, which for convenience of arrangement and excellence of work- manship leave nothing to be desired. But they are mostly so costly as to be of the nature of a luxury, and most microscop- FlG. 108. ists are obliged to satisfy themselves with the cheaper forms, such as wood or paper boxes with wooden racks, or construct a cabinet for themselves by some contrivance of drawers or trays which has the merit at least of cheapness if not of elegance.] [Recently two adaptations of an old form of object box have been made which make them answer practical ends in a very satisfactory way and at a cost that brings them within, the reach of all.] [One, represented in Fig. 108 is a 26 slide box made of stiff paper board with wooden racks, and a hinged and indexed cover which when shut down holds the slides securely in place, and the whole shoves into a paper-board, cloth-bound case, shown THE EXAMINATION OF LIVING ORGANISMS. 249 beneath the box in the illustration, which makes all secure. A circular blank is shown at the top wherein to write the num- ber of the box for cataloguing. When the. box is placed on end the slides are horizontal. These boxes may be kept on shelves like bound books.] [The other is called " Pillsbury's Cabinet," and is shown in Fig. 101). It consists of a polished cherry cabinet containing twenty wooden-racked slide boxes, each holding twenty-five FIG. 109. slides. In the illustration one box is shown removed from the case, with its cover off and some slides in place. The top end of each box, as placed in the cabinet, is provided with an index and on the bottom of the box inserted under each slide is a cor- responding number. When the boxes are in place the slides lie horizontal and a list of all the slides which they contain is spread out before the eye. A cabinet of this kind capable of storing 500 slides is offered for $3.50. J. W. Queen and Co., Phila., make these two forms of slide holder. A. B. H.] XII. THE EXAMINATION OF LIVING ORGANISMS. There are numerous objects which may be examined under the microscope in a living state, as, for example, microscopic algae and fungi. Some of these we might desire to keep under the microscope for a long time in order to observe their devel- 250 THE MICROSCOPE IN BOTANY. opment or their method of propagation. For this purpose a simple slide and the cover-glass and the object between are not very suitable, for the water quickly evaporates from under the cover-glass so that it frequently has to be renewed ; this gradually increases the percentage of mineral substances held in solution in the water, and the object is soon brought into quite abnormal conditions for its life processes. Many contriv- ances have been invented for retarding or preventing the evap- oration of the water. The older microscopists used a contrivance which consisted of two brass rings one of which screwed upon the other. Into each was fitted a little glass plate, the lower one made somewhat concave. The drop of water with the object was put into this and the other glass screwed down tightly upon it. With the older instruments of Schieck this apparatus had a diameter of 28 mm. and a height of 9 mm. FIG. 110. For a like purpose, a slide about 1.5 mm. thick with a small concavity about 13 mm. wide ground in it answers well. And laterally a thick slide (about 3.5 mm.) has been used in which a ring-like groove is cut about 3 mm. deep and of like breadth. [The "Weber life slide" sold by opticians in this country an- swers the same purpose even better. The bottom of the cell ground in this slide is convex. A. B. H.) But by far the best method of observing living objects, or those under cultivation, is by means of the hanging or sus- pended drop. It may be arranged as follows. A ring of wax is put on the slide making a pretty deep cell THE EXAMINATION OF LIVING ORGANISMS. 251 and wide enough for the cover-glass used. On the cover-glass there is put a drop of water and the object in that. It is then turned quickly over so that the drop will hang suspended on the tinder side. The cover is then placed on the wax ring and put under the microscope. But the water soon dries up and the air also does not come to it freely. But all these objections are obviated by the use of the following little apparatus which is at once a ventilated moist-chamber and a hanging drop. It was Strusburger's 53 contrivance. Cut from common tough pasteboard pieces like those repre- sented in Fig. 110. The hole in the centre should be a little smaller than the cover-glass to be used. Then put the paper, pieces in water and let them soak till they are thoroughly satur- ated. Take them out and lay two or three deep on a slide, and lay the cover-glass with its hanging drop over the central opening. The apparatus is now complete. I have kept a hanging drop unchanged upon such an apparatus for a fortnight by simply putting a few drops of water from the wash bottle upon the side of the paper every evening. By means of this moist-chamber I have been easily able in the spring of the year to observe 54 the Sp'jvogyra form its spores, while with other apparatus I^have in most cases failed. With this apparatus too I have had much success in cultivating the pollen tubes in a 30 per cent solution of sugar, honey and like fluids. 55 Geissler has constructed a very peculiarly formed moist-cham- ber for examining objects in a vacuum or with an atmosphere of carbonic acid or oxygen. It consists of a glass tube which is widened and flattened in the middle so as to form a smooth disk- shaped space, the upper and under walls of which are brought 53 Strasburger, Befruchtnng n. Zelltheilung. Jena, 1878, p. 5. 54 See also Strasburger. L c., p. 5,ff. 53 See also Strasbuvger, 1. c., p. 15-25, especially p. 1(5. Here it is said of the culture of the pollen of Pinus pumilio, "But by the prevalence of bacteria, of yeast cells and mould fungus, the culture will be ruined at farthest in 8 or 10 days. I kept it the longest when I used thyme oil in a thousand-fold dilution with 10 to 30 per cent sugar solution. This ad- dition, however, at first hindered the formation of the tubes; but after about two days when a part of the thyme oil had evapoi'ated they again began to develop (salicylic acid in a 1000 fold dilution kills the pollen grains), while the increase of the lower organizations which were introduced at the same time with the pollen grains was delayed for several days. 252 THE MICROSCOPE IN BOTANY. close together and are of the thickness of common cover-glass. The culture drop is brought between them. The tube allows a current of air to be thrown around it of any desired kind. This apparatus is especially applicable to the cultivation of fungus spores and the like. 53 A modification of the Greissler moist- chamber has recently been devised by Brefeld. 57 [The Gas Slide. For the exposure, under the microscope, of organisms either animal or vegetable to the effect of certain reagents in the form of gases, for the sake of observing the effect of these gases upon the actions of living beings or upon the character of their dead tissues, it is only necessary to have a cell of glass, lying upon the stage, and supplied with tubes for the entrance and escape of the reagent. When constructed of glass cemented together, these instruments are liable to sep- FIG. 111. arate at the joints and are otherwise especially subject to acci- dental injury. The form shown in Fig. Ill, is made almost wholly of brass, lies heavily and firmly upon the stage, and is safe from any considerable injury by breakage. The cover- glasses are easily accessible for cleaning, and if broken are easily replaced. Being metallic it transmits heat promptly, in case of use upon the hot stage. It it is made by T. H. McAllister of New York, at the suggestion of Dr. T. H. Hunt of Brooklyn. It consists of a heavy, slide-shaped brass box with a central, cylindrical perforation 20 mm. wide and 7 deep. This central well is closed at the bottom by a cover-glass cemented to the brass ledge on which it rests, and is covered, after the insertion and arrangement of the object, by another cover-glass which is 56 Nageli und Schwendener, Das Mikroskop, p. 275. 67 Oscai; Brefeld, Culture method for the investigation of fungi. (In Botanische Un- tersuchungen iiber Schimmelpilze. Untersuchungeii aus dem Gesammtgebiete der My- kologie. Heft IV, 1881, p. 1-35). See also Hansen's Chambre lumiide pour la culture di a organismes microscopiques. Avec deux figures dans le texte [Meddelelser fra Carlsberg Laboratoriet, p. 18i-183, Kjobenhavn, 1881.] A LABORATORY TABLE. 253 FIG. 112. held in place and rendered air-tight by a small quantity of par- affine, oil, glycerine, or other available material around the edge. Short brass tubes are provided, at the ends of the apparatus, to be attached to the tube bringing the gas to one side of the box and conveying it away after having passed through to the other. R. H. W.] [A Growing Slide, or moist-chamber and hanging drop, shown in Fig. 112, is sold and used in this country. It consists of two common slides held together with rubber bands, the upper one perforated with a circular hole 10 or 12 mm. in diam- eter, over which the cover- glass with the culture drop is laid, being held by a little grease rubbed on about the edge of the hole. When the slide is not under observation it is laid in a flat dish containing a sufficient depth of water to overflow the lower slide and run in by capillary attraction between the two and so prevent the evaporation of the drop.] [A Laboratory Table. Prof. C. E. Bessey, professor of Bot- any and Horticulture in the University of Nebraska, kindly furnishes me with a plan of the tables used in the microscopical laboratory of that institution. It is represented in Fig. 113 ; w is the window, a b c e represents the form of the table, the breadth of which at a b is 1.5 m., and at c e .6 m. , the perpendicular length 1.8 m. At the points indicated FIG 113> by x are placed the micros- copes, each in such a position as to receive the unobstructed light from the window, without lia- bility of interference from those working at the other micros- copes. A. B. H.] 254 THE MICROSCOPE IN BOTANY. XIII. DRAWING MICROSCOPIC OBJECTS. In the introduction of this work we have already shown how important it is to permanently fix the microscopic image by drawing. Farther along we became acquainted with some appa- ratus by which the image is reflected upon a piece of paper lying near the microscope so that the drawing could be done by simply tracing the outlines. 1. AIDS TO MICROSCOPICAL DRAWINQ. An experienced draughtsman can draw free-hand the sim- ple microscopic images by looking in the microscope now and then, drawing a part of the image which he has especially ob- served, comparing the drawing with the original to see if it fully corresponds, then taking up the next contiguous part and so on. This kind of drawing has this unqualified advantage, that in its use one is compelled to observe the image very exactly in respect to its forms and their respective relations. The prac- tised draughtsman may by this method give to his drawing the greatest perfection. But this chapter is written less for the skilled artist than for those who are but little if at all proficient in the pictorial art. We shall therefore describe those methods which will assist the latter in making graphical representations. To the unskilled it is a matter of the greatest difficulty to bring into proportion the large dimensions of the field of view with the smallness of the object, and, further, to rightly estimate the distance apart of the details of the object and to fix them. These difficulties are overcome by the following means. (a) Small objects, which are considerably extended in one direction like diatoms and other algae, may be drawn very easily with the help of the common ocular micrometer. This method enables us to draw the object in exactly the size in which it appears under a certain definite magnification. An example will immediately make this method clear. I have a diatom, Pinnularia viridis Rabenh., to draw magni- fied 600 times, Fig. 114. I know that my micrometer scale AIDS TO MICROSCOPICAL DRAWING. 255 contains 4 millimetres each divided into 20 parts, and that in relation to my magnification of 600 this length is equal to 0.1 mm. (i.e., 0.1 mm. in the object covers the whole scale in the ocular) ; the scale of the micrometer must also with the 600 fold magnification appear to be 60 mm. long. 58 I draw the scale in this length on a piece of paper, Fig. 114, dividing the whole scale into 8 equal parts, each = 7.5 mm., and these in halves. The long marks 0, 10, 20, etc., correspond to every ten divisions of the micrometer,. the lines between standing for five divisions. I have already ascertained that the diatom measures 0.137 mm. which by 600 fold magnification must give a length of 82 mm. ; by that I can easily fix the position of the points e and f, with the millimeter scale. I now bring the microscopic image, and 70 80 FIG. 114. the scale in the ocular of the microscope into such a position as is shown in the illustration, Fig. 114. Now I can easily fix the outline of the diatom and the position of its separate parts on the drawing of my scale with the greatest exactness. Three markings of the Pinnularia, for example, correspond with one of the divisions of 5 on my scale. The two halves of the inter- rupted middle line are 3 divisions of the scale apart at the center. The^ various dimensions of the picture will be made to corres- pond to the reality since they are all laid out like a chart on a network of fixed lines. This kind of drawing is preferable to that with the camera lucida for diatoms of very delicate frustules since the delicate markings of these forms are seen with great difficulty in the reflected image. 68 This value will naturally differ with every microscope aud for each maguiflcatiou. 256 THE MICROSCOPE IN BOTANY. (b) 111 the drawing of those images that extend uniformly over the field of view, the aid of the micrometer scale is not nearly as suitable. In this case drawing by free-hand is fa- cilitated by the use of an ocular in whose diaphragm are cross threads which divide the field into four parts. One with double cross threads like the form shown in Fig. 115 is still better. Since this can seldom be bought I will explain how it can be made. Prepare a circular rim of brass about the size of Fig. 115, which may be fitted to the ocular tube from above. On this the cross threads may be made fast, which in low power oculars may be made of fine glass threads drawn out by means of the blow pipe flame, or of human hair previously boiled in alcohol to get the oil out. In high power oculars the cross threads should be made of spider's web. The preparation of the latter is by no means so difficult as is generally supposed, only one must not stretch them and the same is true of hairs when the air is dry, since afterwards the fiber by taking up moisture in a humid atmosphere would contract and break. First mark the places on the brass rim where the fibers are to go and then fasten one end on one side by means of wax and draw the thread over to the other side, fastening it in the same way, warming the wax to make it soft. Having made the fibers fast on the rim put some Canada balsam on the lower side of it and drop it carefully into the ocular down iipon the diaphragm where it will stick fast. After the Canada balsam is dry screw on the eye lens and after- wards remove it as seldom as possible. How then shall we apply this contrivance to the drawing of microscopical pictures? Fig. 116 represents a schlerenchyma bundle in a cross section of the root of Pteris aquilina which we will draw by means of the double cross threads magnified 600 diameters. This is easily done when we have first learned the size of the square by direct measurement. 59 In the other drawing with this magnification, the length of our micrometer 69 Lay the crossed threads under the microscope as an object screw on a low power n, and draw it in its natural size with a camera lucida. The length of the side is then meas- ured with the millimeter scale and divided by n; the quotient gives the exact length in millimeters. AIDS TO MICROSCOPICAL DRAWING. 257 scale, 4 mm. as it appeared in the ocular was equal to 0.1 mm. used as an object, and magnified 600 times. We have also ascer- tained by measurement that the length of one side of the mid- dle square is 2.2 mm. Consequently under the same conditions this side will appear to be 33. mm. long, for : 4:0.1 X 600: :2.2:z cc = 33. Now we remark that the upper side of the square ?, Fig. 116, covers three cells. It will be easy to estimate their respective FIG. 116. lengths and to fix the points 5, c, d, e,f, g, also like points on the other three squares, and the uniting points of the cellular network which fall inside the squares i, 7^, k, rr^ are likewise determined without difficulty by comparative estimation. When they are all determined for a given cell then the outline will be drawn, and so on. In this way we are able, without very great errors, to map out a group of cells like these, and, what is of greater importance, we soon attain by this method a certain readiness and skill in estimating sizes and distances under the microscope. 17 258 THE MICROSCOPE IN BOTANY. This readiness will very often be of use to the microscopist. On the right side of the illustration, the network of cells is shown drawn in outline ; the rest of the cells are more fully drawn. (c) In most cases when a microscopic image is to be traced, recourse will be had to some camera lucida already described. The use of this very helpful apparatus is very soon and very easily learned. There is therefore need of saying but a few words about it. The eye should be held close to the opening, provided for seeing in the instrument and look perpendicularly down, for by looking obliquely the image may be considerably distorted. The paper upon which the drawing is to be made should be fastened lying flat, at a standard distance of 25.4 cm. from the camera lucida. It is best to provide a drawing board on which the paper may be fastened and which may be placed at this dis- tance from the camera. In order to draw a picture by means of the camera lucida with- out painfully straining the eyes, it is necessary that the microscopic image, and the paper and pencil be uniformly illuminated. If the image has, in comparison with the paper, too strong a light the pencil will be seen with difficulty if at all. On the contrary, if the paper in comparison to the image be too strongly illuminated, the delicate outlines of the latter will be indistinct. The first usually occurs where the image of the paper and pencil is thrown into the field of view of the micro- scope and the latter when the microscopic image is reflected upon the paper. This difficulty may be remedied by throwing either the image or the paper into a shadow. Both may be done simply with the hand, or by a properly constructed screen of paper,* or by a disk of pasteboard set up at some distance, and the like. Hartnack provides his cameras with a blue glass plate, which partly obscures the light, and this appears in fact to have been applied to the construction of many cameras. 60 A few trials with the microscope with different magnifications will afford the necessary experience for properly managing the light. * See Note, page 116, for description of a handy form. 6 See C. Cramer in Botan. Centralbl. 1881, Bd. VII, pp. 3SXJ91. CONDUCTING MICROSCOPICAL DRAWING. 259 Iu tracing the outlines of the image under the camera, the pencil used should not be too hard and the lines should be very light, and then they will often appear rough, for the position which one has to take in this work is not very favorable to nice drawing. 2. CONDUCTING MICROSCOPICAL DRAWING. How a microscopical drawing should be carried out depends upon what relations, qualities and observations we wish pictori- al ly to express. We have already remarked that a microscopical anatomical drawing should by no means be a mere copy of the image seen, but that it should reproduce the sum of the experi- ences which the observer has had with his preparation. Fur- thermore, in most cases the drawing should show only those relationships which the observer has arrived at by his analysis ; FIG. 117. hence it will often not by far show all of those parts which are in reality seen in the microscopic image. Suppose, for example, that one makes a purely histological study in order to represent the relative position of the cells in the tissue ; certainly he will not need to draw the contents of each single cell, its protoplasm, nucleus, etc., or the finer markings of the cell-wall which may temporarily interest him. Take a concrete example. Some one studies the anatomy of the stem of Richardia africana. He wishes to know the vascular bundles in respect to their form, 260 THE MICHOSCOPE IN BOTANY. their position and their structure. In this case it will be nec- essary to draw only the outlines of the cells, and their respective attachments and perhaps also the relative thickness of their walls. His drawing, Fig. 117, will therefore represent the cell walls with single lines not doubly outlined as they are in fact. He will, perhaps, in order to make it perfectly understandable to another, express the cell walls of the surrounding parenchyma .tissue by the more delicate lines, and the cambiform, by the stronger lines, and the vascular walls by the strongest, or by double outlining. Such a drawing is naturally in the high- est decree diagrammatic, but it perfectly satisfies all demands FIG. us. made upon it, and is to be preferred to any drawing which with a mere photographic fidelity reproduces the microscopic image, in that it does not divert the eye from the principal thing by the presence of unessential accessories. Take another example by which we can see, in the same illus- tration, the distinction between a diagrammatic and a completed drawing. Fig. 118 represents a highly magnified trans-section through the upper part of the stomata, and the adjacent epi- dermis, of the needle of Taxus baccata. The left half of the drawing is diagrammatic, the other is completed. The left teaches us to know only the form and size of the cells, the substance of the cell wall and the casual parting of the protoplasmic cell contents. The right half shows us all these relations, and further CONDUCTING MICROSCOPICAL DRAWING. 261 the extension and form of the cuticle, the structure of the cell wall, the intercellular substance, and the different appearances of the cell contents in the stomata-closing cells, the epidermis and the sub-epidermal layer. The right half shows us all the relations which we are able to deduce from the portion of the preparation examined and with the magnification employed. It is self-evident that one may make a microscopical drawing more or less diagrammatic according to the requirements of each case. Fig. 119 represents the wood cells of a young coniferous plant. I is the most, III the least, diagrammatic. In I the middle III lamella is represented by a simple thin line, while the inward thickening layer is indicated by a heavy single line ; the wood layer between is not designated further. In II the middle lamella is indicated by two delicate lines, the inner layer by a delicate and a heavy line, the latter being the boundary line of the cell cavity while the woody layer is not further exhibited. In III at last the concentric layers of the latter are represented. A quite perfect picture of the cell wall structure is shown in this drawing, as one sees it with a hteh magnification. O O The representation of cell walls in microscopical drawing is by no means difficult since all that is required is uniform con- centric outlines. The difficulty of representation increases when fluid, or semifluid cell contents are to be pictured. If the cell contents are a clear, homogeneous fluid, we must depart from a 262 THE MICROSCOPE IN BOTANY. strictly pictorial representation since a fluid shows itself under the microscope only by its refraction of light. At most it can be (Ungrammatically represented only by shading or laying in with the rubber. But it is otherwise with the protoplasm and its granules and air bubbles, which may be very well and very beautifully reproduced by drawing. It may be done either with the drawing pen and India ink or with the lead pencil. Suppose we are to draw the protoplasmic contents of one of the upper, right hand cells of Fig. 118. We first make the outlines of the granular substance with fine points. Then we fill in the space pretty uniformly with very delicate points. Then in those places which in the microscope appear to be more densely gran- ulated we add a corresponding quantity of points, so that they stand thicker and here and there touch each other. And finally the larger and largest grains are represented by minute circles or irregular outlines according to the appearance of the object. If the protoplasm is very thick and cloudy it will not do to make the background of delicate little points, but it should be made of many finely entangled lines running through and through each other as is seen in the lower cells on the right hand side of Fig. 118. In the examples thus far treated we have dealt with the re- production of images which are seen with a single adjustment of the microscope, but we shall often be required to combine several adjustments, and so make a drawing which at least in places represents corpo- real thickness. As is well known, this can be accomplished only by the addition of shading, and nowhere will there be more faults committed than right here, as Nageli has well said. In botanical literature there are many drawings of this kind, in which the body is represented as at the same time transparent and untransparent, as flat and wavy, and as round and angular. In order to draw, at least npproximately right, we must think always of a definite direction from which the liof the outflow may be perfectly regulated by the pressure upon the bulb. The bur- ette is filled by dipping the end of the tube, c, in the fluid and sucking at the opening in e. The tube, b, has the graduation in cubic centimeters (holding from 40 to 60) and fractions of the same to about 0.2. The value refers to the interior diameter of 5, and the tube c, which dips into the fluid. MOHR'S SPRING COMPRESSOR BURETTE. 277 Mohr's Spring Compressor Burette consists of a long tube, 7^, divided into whole and fifths of a cc., open above at i, and below at k. The lower end is much narrowed and a piece of rubber tube about 27 mm. long is drawn over it. This is provided with a small glass tube having its point drawn out as at L The rubber tube is closed by means of a spring compressor, k, and the burette is supported at g, on the stand,/, in a perpendicular position. The tube is filled by means of a small funnel and the opening at top is afterwards closed by a small glass globe, with a peg upon it, which excludes the dust. The outflow of the fluid to be measured is effected by partly opening the spring compressor. Mohr's spring compressor burette is used chiefly with those indifferent fluids which would not affect the rubber tube, while those which would damage it should be put into the bulb burette where they will come in contact only with glass. 4 The measuring vessels here described allow us, first, to measure any desired volume of fluid, but they can also, secondly, be applied to measure quantities of fluids by weight when they have been changed into equivalents in volume. Take water for instance. It is known that 1 cc. of water, at 4 C., weighs 1 g. So if one has 30.5 cc. to weigh out he would get the right weight by measuring, at 4 C., 30.5 cc. of water. If he has to perform the operation at the common temperature of the room (say 17 C.) then the 30.5 cc. of water would weigh somewhat less than 30.5 g., since the water has a less degree of density at 17 than at 4, but this difference is so inconsiderable that it may be disregarded in our work. Accord- ing to the investigations of Despretz, if the volume of a gram of water at 4 C. be 1 cc. at 17 C. it will be 1.00120 cc. Hence the volume of 30.5 g. at 17 C. would be 30.5366 cc. But the excess of 0.036 cc. is far less than the error one would make in reading off the scale. 4 In regard to getting the caliber of measuring vessels, cf. Bunsen, gasometric method (Brunswick, 1857), pp. 26-38. Concerning testing and correcting the same, cf. F. Mohr, Lehrb. d. Chem-analyt., Volumetric Method (4 Aufl., Bi swg., 1874). pp. 1-50. further, Fresenius Anleit. z. quantitative chem. analyse (6 Aufl., Brschwg., 1875), pp. 36-46. In the last two books are exact directions concerning all quantitative analytical determinations ami the cautions to be observed in reading off the same. 278 THE MICROSCOPE IN BOTANY. It is clear, therefore, that quantities by weight, of other fluids, may be converted into equivalents of volume when we know their specific gravity. If, for example, one has 30 g. of gly- cerine to weigh out and knowing that the specific gravity of glycerine is 1.264, he needs to divide 1.264 by 30 to get the num- ber of cc. of glycerine which will weigh 30 g. The division gives 23.7 cc. The following table will be useful in the re- duction of weight measures to equivalents of volume, for some of the fluids used as microscopical reagents. SPECIFIC GRAVITY OF FLUIDS AT 15 C. Ethyl ether 0.736 Ammonia saturated 0.884 Alcohol absolute 0.794 Carbolic acid " 1.066 " 90 per cent 0.823 Chloroform 1.480 " 50 per cent 0.919 Acetic acid 1.055 " 40 per cent 0.940 Glycerine 1.264 Nitric acid 1.526 Hydrochloric acid 1.210 Carbon disulphide 1.271 Sulphuric acid 1.842 Turpentine oil 0.870 1 Small variations of the temperature of the room may be en- tirely disregarded in the calculations. III. APPLICATION OF THE VOLUMETRIC METHOD IN THE PREPARATION OF MICROSCOPICAL REAGENTS. The volumetric 5 method may be successfully employed in the rapid preparation of certain reagents as Frey 6 has rightly as- serted, and, on the other hand, it offers the possibility of deter- mining with ease the per cent value of certain simple fluid reagents. We may here briefly set forth the few cases which concern the microscopist from the wide field of quantitative analysis. For the rest he may consult the work of Mohr al- ready cited on page 277. 1. Standard solution of oxalic acid. Good commercial oxalic acid is pulverized and dissolved in a little warm water, 5 Mohr, I. c. 6 Frey, Mikroskop, p. 90. STANDARD POTASSIUM SOLUTION. 279 so that still a considerable part of the acid remains behind on the bottom of the vessel. Then filter and crystallize by rapid cooling. The crystals should be drained ofi* in a filter and dried at common temperature between blotting papers. To test their dryness press a piece of smooth paper upon them. If they are perfectly dry none of them will stick to the paper. 7 Oxalic acid which has two molecules of water bound up with it has the chemical formula : C O . OH | + 2H 2 CO. OH The atomic weight is 126 (C=12, O = 16, H 1). Oxalic acid combines with two molecules of potassium hydroxide H K O to form potassium oxalate, C 2 O 4 K 2 + H 2 O, a salt which contains one molecule of water, after this formula : . ( V CO. OH | CO.OH CO. OK +H 2 0) + 3H 2 C O. O K Oxalic acid -f- 2 potassium hydroxide = potassium oxalate + 3 water. To form this salt the two molecules of potassium hydroxide, K 2 O, contributes what corresponds to an equivalent of 94 (K = 39, O=16). If now we reduce both numbers (126 and 94) to one atom of potassium by dividing by 2 we shall have the respective numbers 63 and 47. We now weigh out 6.3 g. of pure oxalic acid on the chemical scales and dissolve it in exactly 100 cc. of distilled water. Each cubic centimeter of the solution will contain 0.063 g. of oxalic acid. With 0.047 g. of potassium added to each cubic centi- meter of the oxalic solution we shall be able to transform it to potassium oxalate. 2. Standard potassium solution. It is necessary also to have a standard solution of potassium, that is, a solution of caustic potash in water, which contains in each cc. of the fluid 0.047 dipped first in hydrochloric acid and then into the solution turns rapidly and intensely violet. In this way one gets a brown liquid which smells like camphor. 33. INDOL N C 8 H 7 . Xiggl 46 has recently recommended this very unpleasant smell- ing stuff 47 as a reagent for lignified cell membranes. It cannot be used in an alcoholic solution since it spoils in a few days. It is but little soluble in water and one can work for months with a few little crystal plates for a solution. The best way of mak- ing the solution is by warming the water. It should be em- 44 To be had of Dr. Theodor Schucharclt, Gb'rlitz, Schlesien Germany. Also of J. T. Brown, cor. Washington and Bedford Sts., Boston, Mass. Mr. Browu also will keep on hand all the micro-chemical reagents. v. Hohnel, 1. c., p. 685. 46 M. Niggl. Indol a reagent on lignified cell membranes. Microchemical investigations (Flora. 1881. pp. 545-559, pp. 561-5G6). 47 Beyer in Ann. Cheui. Pharm., Bd., CXL, p. 1, ff, p. 295,/. 304 THE MICROSCOPE IN BOTANY. ployed with sulphuric acid of the specific gravity of 1.2, which consists of 1 vol. English sulphuric acid diluted with 4 vol. water (cfr. p. 236). 34. EOSIN C2oH 8 B 4 O 5 . In a weak aqueous solution this reagent, which may be had free from arsenic in the market, is used according to Poulsen 48 , to color bacteria, also according to this author it colors dead protoplasm a rose-red excellently well. It has recently been recommended for double staining of the tissue of the higher plants, the methods of which require further testing. 49 35. HJEMATOXYLIN C 16 H, 4 O, + 3 H 2 O. (Extract of Logwood.) This reagent may be had perfectly pure in the market. Boh- mer introduced it into histology. Frey 60 gives the following form- ula for the preparation of hsematoxylin in solution. Dissolve 1 g. of the coloring matter in absolute alcohol. Then prepare an alum solution of 0.5 to 1 g. in 30 cc. distilled water. Into this drop the alcoholic solution till it has attained a deep violet color. The fluid should now be allowed to stand some days in the air and then filtered ; also, afterwards it must be filtered from time to time. Duration of staining process from 5 to 30 minutes. Wash with distilled water. Over colored preparations may be bleached by putting them in a solution of alum. According to Poulsen 51 0.35 g. hsematoxylin should be dis- solved in 10 g. water and to this should be added a few drops of an alum solution which consists of 3 g. alum and 30 g. of water. It may be added that hsematoxylin colors the more intensely the more alum it contains, but at the same time the section is made more brittle. 48 Poulsen, Om nogle mikroskopiske Plantorganismer; Nat. Foren. vidensk, Medd' Kobenhavn, 1876-80, p. 235 (separatabz, p. 7). Botanisk Mikrokemi, p. 89 (Trans, p. 57). * Anicr. Monthly Microscop. Journal 1880, p. 81, ff. eo Frey, 1. c., p. 99. ei Poulsen, L c., p. 98, translation, p. 56. ORGANIC COMBINATIONS. 305 Poole 52 makes a double staining of vegetable tissue with hae- matoxyliu and a dilute aniline solution. For animal tissue Frey, 53 after Stralzoff, recommends a double staining with haematoxylin and an ammoniacal carmine solution (see p. 306), which method may be applied, probably un- changed, to vegetable tissue. The section should be stained with haematoxylin washed with distilled water, and then laid in the carmine fluid ; after that it should be again washed, and finally subjected to the influence of a weak solution of alum. It does not keep well in glycerine. According to Schmitz, 5 * preparations hardened in picric acid are especially adapted to staining with hsematoxylin. HaBtnatoxylin colors nuclei a deep blue, 55 and may be applied to the staining of bacteria. 56 36. COCHINEAL EXTRACT. An aqueous extract of the pulverized insect prepared with heat contains carmine acid (C 17 H 18 O 10 ) , and is useful in staining vegetable tissue. It is very liable to mould and so must be protected by a few drops of carbolic acid. Before using, a few drops of acetic acid or solution of alum should be added. Czokor 57 has recently recommended a cochineal carmine so- lution which is notable for being capable of preservation un- changed for a long time. Triturate 1 g. cochineal with 1 g. burnt alum to a fine powder. Then add 100 cc. of distilled water and boil till it is but 60 cc., cool, add a few drops of car- bolic acid and filter several times. The resulting solution is a beautiful carmine color and may be kept without change for six months. Then add again a little carbolic acid and filter. The cochineal colors bast elements, also many wood cells, proteid bodies and cell nuclei. 62 Poole, in Quart. Jour, of Micros. Science, New Series, Vol. XV, 1875. p. 375, ff. 53 Frey, I. c.. p. 101. Cf. lurther Brandt in Biolog. Centralbl., 1881, p. 2O2. ff. 64 Sclimitz in Sitsungsber. der niederrh. Gesellsch., Bonn, 1880, Jahrg. XXXVII, p. 160. M Johow, The cell nuclei of the higher monocotyledons, Bonn, 1880, p. 9. 66 Koch in (John's Beitr. z. Biol. d. Pfl., Bd. II. p. 421. Poulsen, I. c. 57 Joh. Czokor in Archiv fur mikrosk., Anatomic, Bd. XVIII, 1SSO, p. 412. jf. 20 306 THE MICROSCOPE IN BOTANY. 37. CAKMINE SOLUTIONS (Carmine red C U H 12 O 7 ). Commercial carmine is the coloring matter with which it was first attempted (by Th. Hartig) to stain anatomical prepa- rations. Hartig is also the inventor of the method of stain- ing as mentioned on p. 267. Since the introduction of this staining medium, many naturalists have employed various mix- tures of which the most important are the following. 1. Hartig 's ammoniacal carmine. 5 * Commercial carmine is mixed with water and ammonia fluid is added in drops till a perfect solution results. The solution should then be filtered, and by very gentle heat evaporated to dryness. This carmine ammonia thus prepared may be dissolved in water and can be kept for years in good condition as an aqueous solution. 2. Gerlach's ammonium-carminate. 59 According to Frey, 60 this is prepared best in the following way. Take 0.2 to 0.4 g. of carmine, mix with 30 cc. of water and add a few drops of ammonia. Thus a part of the carmine will be dissolved and the fluid should be filtered. The rest which remains behind may be kept over for future use. If the filtrate smells at all strongly of ammonia it should be permitted to evaporate for half or a whole day under a glass bell. If after the lapse of time grains of carmine begin to be deposited a drop of ammonia will restore the solution. In order to get any desired color from this mass it should be transferred to water, drop by drop, the color growing of course from a light to a darker and more intense red. 3. Frey's glycerine carmine.* 1 Dissolve 0.2 to 0.4 g. of car- mine in the required amount of ammonia and add 30 cc. of distilled water. To the filtered fluid add 30 g. of good glycer- ine and 8 to 11 g. of strong alcohol. The tincture should be O o used unmixed, or with a further addition of glycerine. 4. TltierscJi's oxalic acid carmine. 6 ' 2 One g. carmine is dis- 6 8 M. Hartig, Entwick'ungsgesch. d. Pflanzenk. p. 154. Dippel, I. c. } Bd. I, p. 284. Poulsen, 1. c., p. 3(>, trans, p. 49. 6 * Mikrosk. Studien aus d. Gebiete der menschl. Morphologic, Erlangen, 1858. eo Frey, 1. c., p. 9:5. ei Frey, J. c., p. 94. ea Frey, I. c., p. 94. Dippel, I. c., Bd. I, p. 285. Poulaen, I. c., p. 36, /., trans, p. 50. Baclmianu, I. c., p. 62. ORGANIC COMBINATIONS. 307 solved ill 1 cc. of ammonia, and mixed with 3 cc. of distilled water. Also dissolve 8 g. crystallized oxalic acid in 175 cc. of distilled water. Then mix the solutions, add 16 cc. of absolute alcohol and filter. If the solution has an orange color which comes from a predominance of the oxalic acid, it can be cor- rected by carefully adding drops of ammonia. If there are in addition deposits of crystals of ammonia oxalate in the filtrate, which may happen from the addition of the ammonia or the alcohol, the fluid must be filtered the second time ; subsequent occasional filtering is also beneficial. The tincture stains very quickly. The coloring matter which adheres to the object should be washed off with 80 per cent alcohol. If the color becomes too dark or diffuse, soak the preparation out in an alcoholic solution of oxalic acid. 5. Thiersch's borax carmine. Dissolve 2 g. borax in 28 cc. distilled water and add 0.5 g. pulverized carmine. To the red solution thus produced add 60 cc. of absolute alcohol and filter. (If on the filter paper there remains a mixture of undissolved carmine and borax it may be dissolved in distilled water and kept over for future use.) For soaking out use an alcoholic solution of oxalic acid, or boracic acid. The mixture colors somewhat slowly but very beautifully. According to Frey (I.e. ) one gets the most beautiful coloring when one lays the prep- aration for a moment in the solution after it has been previously impregnated with boracic acid. Studies of vegetable nuclei are very essentially facilitated, according to Stras burger, by the use of this carmine mixture, the color making the form of the nucleus come out most beautifully. The preparations should be ex < mined in glycerine and mounted in that or in glycerine 6. BeaVs carmine solution.^ Put 0.6 g. of pulverized car- mine in a test tube, pour over it 2.3 cc. of concentrated am- monia fluid and heat. After the solution is completed let it stand for an hour and pour the red fluid into a mixture which is made of 60 cc. of water, 47.5 cc. of concentrated glycerine, Frey, 1. c., p. 64. Dippel, I. c., Bd. I, p. 285. Strasburger, Zellbild. u. Zelltheilung, 1880, p. 9. 64 Frey, I. c., p. 95. Poulsen, L c., p. 37, trans, p. 52. 308 THE MICROSCOPE IN BOTANY. and 19 cc. of absolute alcohol, stir up with a glass rod, let it stand for some time and then filter. For the study of the protoplasmic contents of the cells in filamentous algae (Spirogyra) one should, according to Stras- burger, 65 lay the fronds in a 1 per cent solution of chromic acid, for at least four hours. Then repeatedly wash in distilled water and lay in a mixture of Beal's carmine and camphor, diluted with 8 parts water, 1 part glycerine and 1 part alcohol. There will follow after some time a rosy coloring of the protoplasmic cell contents which makes its fine structural relations very distinct. 7. Grenadier's alum-carmine.* The alum-carmine represents an exceptionally fine staining fluid for cell nuclei, which, accord- ing to Grenadier is prepared in the following way. Dissolve in 100 cc. of distilled water, 0.5 to 1 g. pulverized carmine and 1 to 5 g. potassium alum or common alum. Tangl fi7 recommends a like composition which colors nuclei as well as cellulose membrane. "Dissolve alum in water to saturation, mix the solution now with any desired quantity of carmine, boil about ten minutes and filter after cooling." The staining re- quires from 5 to 10 minutes. The preparation keeps very Avell in glycerine, and the use of this stain gives very instructive specimens. In order to obtain clean specimens it is very much recommended to previously harden the part of the plant which is to be stained in absolute alcohol. This not only facilitates the imbibition of the stain but also has the further advantage that the staining capacity of the substances in the cells remains unchanged. 8 . The Schweigger-Seidel acid carmine volution , 68 A common ammoniacal carmine solution is mixed with an excess of acetic acid and filtered. This tincture stains diffusely at first. Then put the colored preparation in glycerine to which has been added ^
5, thence in Zeitschrift f., Mikroskopie, Jahrg. II, 1879, p. 55. 67 E.Tangl in Pringsheims' Jahrb., Bd. XII, 1880, p. 170. 68 Frey.f, c., p. 96. ORGANIC COMBINATIONS. 309 cleus. In order to mount in glycerine one should wash the preparation in water containing acetic acid. All carmine mixtures are used principally for staining nitro- genous, most of all protoplasmic, substances. The nucleus takes the stain with particular avidity, and mostly in greater quantity than the surrounding protoplasm. All these substances absorb carmine but never till after death. Cellulose (with the exception of ^some modifications), starch and other cell elements without nitrogenous contents, do not absorb the coloring mat- ter of most of the carmine compounds. 38. PlCRO-CAHMTKTATE OF AMMONIA. (Picro-Carmine.) A rapidly staining medium is recommended by Treub for the investigation of nuclei and by Weigert for studying bacteria, and which was first introduced into zoo-microscopy by Ran- vier, viz. picro-carminate of ammonia. According to Frey 69 it is thus made. To a concentrated aqueous solution of picric acid add to saturation, drop by drop, an ammoniacal carmine solution. Then evaporate to one-fifth the original volume. The cold solution deposits a small sediment of carmine. Then filter and evaporate to dryness when a red, ochre-yellow powder will be obtained. Dissolve portions of this in the preparation of the reagent 1 g. in 100 cc. water. Filtering from time to time is indispensable. Baber (1. c.) mixes 1 g. of carmine in 4 cc. of concentrated ammonia, and 200 cc. of water, then adds 5 g. picric acid and shakes up and decants so that the undissolved excess of the picric acid remains behind. After the red liquid has stood for several days with frequent shaking, it is put in a shallow dish in the air to dry. The red powder should then be dissolved; 2 parts to 100 of water, and after some days filtered through two layers of paper. The fluid should now be yellowish red without smell of ammonia. A drop on white filter paper gives on drying a yellow, red-bordered fleck. A couple of drops of carbolic acid keeps the stain from decomposition. 6 Frey, 1. c., p. 96. 310 THE MICROSCOPE IN BOTANY. Weigert's 70 formula is as follows. Pour 4 g. common am- monia over 2 g. of carmine and let it stand for twenty-four hours ; now all that is soluble is dissolved. Then add the small- est possible quantity of acetic acid till the first faint traces of precipitation are seen. After standing another twenty-four hours add some ammonia, drop by drop. According to Baber the mounting fluid which is best for specimens stained with this reagent is one made with 10 drops glycerine, 10 drops water, and 1 drop of the reagent itself. 71 39. ALCANNA TINCTURE. This reagent is recommended by N. J. C. Miiller in an aque- ous alcoholic solution as a test for resins and essential oils. A preparation of sufficient thinness, from some part of the plant that has little drops of resin in the cells, should be put on the slide and with it a clean fragment of alcanna, and to both add a drop of dilute alcohol. In a few minutes, two or three, the resin drops in the cell will be stained a lively red, and strangely enough, more intensely stained than the surrounding aqueous- alcoholic pigment solution. Protoplasmic masses without the resinous substances require from one-quarter to one-half hour in a like concentrated pigment solution before a distinct color is perceived. If the stained section is treated with alcohol and the colored drop disappears, no more treatment of the samo drop with the same reagent will make the color perceptible to him. The following is the best process for preparations in water. Break from the alcanna a thin, even scale of perhaps about the size of the section to be tested. Rub it between the clean fingers with a drop of water to remove all attached pow- dered parts, and lay it on the section which is covered already with water. Then put 011 the cover-glass and at the edge of this a drop of alcohol. After two or three minutes remove the 70 C. Weigert, Zur Tecknik rter Mikroskop, Bacteriemintersuchung. Virchow's Archiv f. pathol. Anatomie, Bd. LXXXVIII, Heft 2, 8, Folge, Bd. II, Kelt 2, 1881, pp. 275-315. 71 Double stainings with this reagent and carmine, aniline, osmic acid, and picric acid have been frequently prepared. (Cf. Jour. Anat. and Phys., Vol. XV, 1881, pp. 349-351 Jour. Roy. Microscop. Society, 1881, p. 528, etc.) ORGANIC COMBINATIONS. 311 fragment of alcanna, and with sufficient magnification you will find the drops in question stained a beautiful red. This will all happen as indicated if the alcanna is rich in coloring matter, but a very poor article is now often sold in the market. It is self-evident that if we apply the above mentioned test of washing out the stained section with alcohol we may easily distinguish between the drops of resin and essential oils, and those of fatty oils. 72 72 N. J. C. Miiller, Untersnchung iiber die Vertheilungder Harze, etc., in Pflanzenkorper (Pringsheim's Jahrb., Bd. V, pp. 387-421). 312 THE MICROSCOPE IN BOTANY. CHAPTER Y. MICROSCOPICAL INVESTIGATION OF VEGETABLE SUBSTANCES. FOR such a complete presentation of the microscopical inves- tigation of the elementary substances of plants as is possible fiom the present standpoint of scientific microscopy, we must first of all begin by making such a classification of the very elab- orate materials at hand as may be practically and scientifically justified. Since, as we have already remarked, a comprehensive presentation of this matter does not at the present time exist, we must here build from the very foundation. As we survey the rich amplitude of material which the investigations of the vegetable histologist and physiologist as well as those of the chemist have provided for us, it is not difficult to see, that the manifold substances which compose the organs of plants may be divided according to the frequency of their occurrence into two groups. First, we recognize a large series of plant substances which have a very wide distribution in the vegetable kingdom. We need refer here only to albuminous bodies which no plant lacks, or to cellulose, which, except in the very lowest plants, occurs likewise in all. So also starch, plant mucilage, sugars, chlorophyll combinations, etc., are widely distributed throughout the plant kingdom, and the instances in which they fail are comparatively very few. In contradistinction to these widely distributed bodies stands a series of substances whose occurrence is either limited to small groups of plants, or which are produced only by certain growths, substances, at all events, which are of but secondary importance in the building up of plants. Belonging to this group, to mention some bodies of which microscopical analysis has already taken possession, are the coloring matter of algae, tannic acid, resin, balsam, terpene, essential oils, coniferiii, chrysophanic acid, and many others. SUBSTANCES OF UNIVERSAL DISTRIBUTION. 313 It is also not to be forgotten that many substances which are placed in the latter category have, perhaps, a much wider and more general distribution in nature than the present state of our science would justify us in assuming. Thus, for example, some physiologists have supposed that coniferin occurs in all woody growths. So also the investigations of Weinzierl (see p. 302) have shown that phloroglucin which hitherto had only been prepared synthetically by the chemist (benzole, on which for the 3 atoms of hydrogen are founded 3 groups of hydrox- ides of equal value, trioxhydro-benzol), or the so-called xylo- philin of v. Hohnel (see p. 302), which, according to Weisner's investigations represents a mixture of phloroglucin and pyro- catechin, constantly occurs not only in woody but also in many herbaceous plants. But perhaps as a parallel to such statements, the objection may be raised that such a presentation as the one in hand must adapt itself exactly to the present state of our knowledge of these matters, and thence can be useful but for a limited period of time, and must necessarily become unser- viceable with the further progress of science. TTe* now first consider those plant substances which are of universal distribution and of such we present the following. 1, Cellulose and its modifications; 2, Starch; 3, Dextrine; 4, Vegetable mucilage ; 5, Gums; 6, Inuliii ; 7, Grape sugar; 8, Cane sugar; 9, Albuminous substances; 10, Chlorophyll; 11, The coloring matter of flowers; 12, Asparngin ; and 13, Inor- ganic vegetable elements. The question now lies before us, according to what points of view we might arrange these substances into a continuous series, and according to which of these points of view we should arrange them so as to be both practically and scientifically justified. Three points of view offer themselves to us. These substances maybe grouped' according to their morphological, physiological or chemical characteristics. In the first case we should have to place those things together which have a like or similar 'ap- pearance : thus, in the first place, the materials forming the cell Avail, then the solid, and finally the fluid and semi-fluid cell contents. ^Ye should then also distribute into one and the same group, starch and proteid grains, chlorophyll, calcium 314 THE MICROSCOPE IN BOTANY. crystals, etc., joining things which are altogether different. A classification of vegetable substances in accordance with their physiological functions is impracticable, simply on the ground that we know very little of what part many substances take in the vital processes, and of many others we know nothing at all. There remains to us, therefore, only the classification of these substances according to their chemical nature. And this is also, in fact, the most suitable, and the following chapter makes it its principal purpose to furnish directions for determining the chem- ical nature of the substances occurring in the interior of plants. We make the classification upon the inorganic elements, and the absence or presence of nitrogen gives us the first character- istic for a wide classification. The first division, combinations containing no nitrogen, we designate collectively carbo-hydrates, and the second division nitrogenous combinations. The car- bo-hydrates include the cellulose and the rest of those isomeric vegetable substances in which the formula C 6 H 10 O 5 occurs. To the carbo-hydrates also belong the different kinds of grape sugars, C 6 H 12 O 6 , and the cane sugars, C 12 H 22 O n . The nitrogenous combinations include the well-known albuminous substances (proteid bodies), vegetable coloring matter, of which chloro- phyll may be designated as the most important, and finally as- paragin as an amido-combination. According to the point of view which we have here developed we get the following arrangement of those vegetable elements which have the widest distribution. CARBO-HYDRATES. Cellulose Group C 6 H 10 O 3 Sugar Group. 1. Cellulose. 4. Vegetable mucilage. 7. Grape sugar C 6 H 12 O 6 2. Starch. 5. Gums. 8. Cane sugar C^U^O^ 3. Dextrine. 6. Inulin. NITROGENOUS COMBINATIONS. 9. Albuminoids (proteid bodies). 10. Chlorophyll. > 11. Coloring matter of flowers | Vegetable coloring matter. 12. Amido combinations, asparagin. 13. Inorganic vegetable elements. CELLULOSE AND ITS MODIFICATIONS. 315 It should be remarked in reference to this arrangement that the substances designated w matter " (Stoffe) are by no means such in the chemist's sense. A chlorophyll grain, for example, consists as is known of a large number of bodies which on their part again are not to be considered as simple chemical combinations. We should regard the present arrangement as nothing more than a grouping of important physiological in- dividuals. In the following analysis we shall first make ourselves ac- quainted in general with the qualities of each of the substances, and then describe the methods of microscopical reaction to be employed with each in order to recognize it with certainty. Very unimportant reactions, or those whose value has not yet been sufficiently established, will either not be mentioned, or will be but incidentally referred to, and on this account we once for all refer the reader to the " literature " to be found be- fore each section. A. SUBSTANCES OF UNIVERSAL DISTRIBUTION. I. CELLULOSE AND ITS MODIFICATIONS. Cellulose or cell-substance (C 6 H 10 O 5 or C ]2 H^ O 10 ) presents in its pure state a solid, colorless transparent body. Cellulose appears in plants in the form of cell cuticle or cell wall. It is isomeric to the other members of the group of cell substances enumerated on p. 314, but in part varies much from them in its chemical and physical behavior. The isomerisin explains the fact that many of these substances during the vital pro- cesses in the body of the plant can be easily changed into one another, as, for example, a transformation of starch into cellu- lose and of cellulose into gum often takes place. Under the physiological conception of cellulose we are how- ever to understand, in accordance with the science of to-day, not only pure cellulose, but also certain related substances, which result from the metamorphosis which cellulose undergoes in the life processes of the plant, and which in general are dis- 316 THE MICROSCOPE IN BOTANY. tinguished by containing more carbon and less oxygen than cellulose in the strict sense. These modifications some nat- uralists regard as different chemical individuals (Fremy), while the majority of chemists and physiologists see in them, as already mentioned, only modifications (Payen, Fromberg, Mulder). Thus Fremy specifies as chemical cellulose individuals of this sort, essential cellulose, paracellulose, vasculose, fiberose and cutose. 1 * If we can at present form no definite idea of the nature of the modifications of cellulose, it still appears prob- able, in opposition to the view of Fremy, and in accordance with the chemical investigations of Payen and later of Schultze, 2 as well also as in accordance with the studies of the botanists, that the change in the cellulose is produced by the molecular intercalation during the process of growth, of certain other substances into the cell-wall which originally consisted of pure cellulose. For example, we have the phenomenon of lignifica- tion, whereby the cellulose, as is well known, is transformed into lignin. This takes place by the intercalation of a sub- stance, which Payen had already recognized and designated by the name of " incrusting substance," and which Schultze after- wards believed himself to have prepared pure. The modifications of cellulose which have been distinguished with sufficient distinctness, are wood substances (lignin) ; middle lamella, intercellular substance, which very much re- sembles wood in many respects; cork substance (suberin) out of which is also composed the corky layer known as cuticle which regularly covers the epidermis (cutin), and the inclosing layer of the pollen grain (pollenin) ; finally, fungus cellulose. The latter to which one might suitably give the name of fun gin, if this had not previously been used in another sense, was, till a short time ago looked upon by botanists of repute 3 as an 1 Fremy, Comptes rend., t. XLVIII, p. 667, ff., p. 862, ff. * Fremy at present classifies the constituents of vegetable tissues under the following seven heads, the characters being derived from their chemical constitution. 1, Cellulose substances (cellulose, paracellulose, and nietacellulose); 2, Vasculose; 3, Cutose; 4, Pec- tose; 5, Calcium pectate; 6, Nitrogenous substances; 7, Mineral elements (see at large, Ann. Sci. Nat. XEII.1882, pp. 360-382, condensed in Jour. Roy. Micro. Soc., Vol. HI, 18*53, pp. 232-5). A. JB. H. 2 Schultze in Chem.Centralbl., 1857, p. 321. 8 De Bary, Morphologic d. Pilze, Flechten u. Myxomyceten (Bd. II, von Hofmeister's Handb., p.7,2F.). CELLULOSE AND ITS MODIFICATIONS. 317 isomeric substance to cellulose in the sense of Fiemy. But according to the latest investigations of Richter, 4 it appears that this also is nothing else than common cellulose with foreign admixtures, mainly albuminous substances. On the contrary, it remains questionable if the medulin of the chemist forms a like sharply pronounced modification of cellulose. Omitting now these modifications, we have still to describe, under the true cellulose, those conditions which arise from the disorgani- zation of cellulose and lead to isomeric combinations, as amy- loid, plant mucilage, caoutchouc, arabin, bassorin, etc., and which we may suitably designate by the expression rnuculent cel- lulose. Classified according to their characteristic qualities, cellulose and its related substances may be arranged in the following manner : 1. Essential. Cellulose, cell substance, soluble in cupram- monia, concentrated sulphuric and chromic acid. It colors blue or violet with iodine and sulphuric acid, or with chlor- iodide of zinc. It has no admixture of foreign substances. 2. ^Incident Cellulose, frequently soluble in cupraminonia as well as in concentrated sulphuric acid and chromic acid. It seldom colors blue with iodine and sulphuric acid, or with chlor-iodide of zinc, but mostly yellow or yellowish, or remains quite colorless. It is distinguished from all other forms of cellulose by its swelling. 3. Wood Cellulose, Lignin, insoluble in cuprammonia, soluble in concentrated sulphuric and chromic acid ; is col- ored almost always yellow with iodine and sulphuric acid, or with chlor-iodide of zinc. With phloroglucin and hydrochloric acid rose red (distinguished from all other kinds of cellulose) ; possesses less oxygen than pure cellulose. 4. Middle Lamella, Intercellular Substance, insoluble in cuprammonia, insoluble in concentrated sulphuric and chromic acid, colors yellow with iodine and sulphuric acid, or with chlor- iodide of zinc. Richter in Sitsungsber.K. K . Acad. d. Wiss. Wien, Bd. LXXXIII, I AUh., 1881, p. 510. 318 THE MICROSCOPE IN BOTANY. 5. Cork Cellulose, Suberin (including cutiu, po'llenin), in- soluble in cuprammonia, insoluble in concentrated sulphuric acid and chromic acid (or in the latter very slightly soluble), colors very seldom yellow, mostly brown with iodine and sul- phuric acid ; gives cerinic acid reaction with Schultze's mixture. It contains an admixture of certain nitrogenous substances. 6. Fungus Cellulose, insoluble in cuprammonia, very slightly soluble in concentrated sulphuric acid. It very sel- dom takes a blue color with iodine and sulphuric acid, or with chlor-iodicle of zinc. It occurs only in fungus (and lichens) and appears to contain an admixture of albuminous substances. From these varieties of modified cellulose, may be obtained the pure cellulose which will give a true cellulose reaction with iodine and sulphuric acid, or chlor-iodide of zinc, if they, ac- cording to their nature, be treated with water, or with alcohol or ether, dilute acids, nitric acid, together with potassium chlo- rate, or caustic potash. 1. CELLULOSE IN THE NARROW SENSE. (Cell substance.) The most important reagents for testing cellulose are those iodine solutions particularly described on p. 285 ; in contra- distinction to these the other methods for testing cellulose are of a very subordinate nature. They are commonly employed only when by the reactions of the iodine solutions, we are not able to determine with definiteness whether we have pure cel- lulose before us or one of the more closely related modi- fications of it. In this case one must observe its behavior when treated with mineral acids, alkalies, cuprammonia, alum car- mine, copper sulphate and potassium, or in a secondary way by its negative behavior towards phenol-hydrochloric acid, ani- line sulphate, phloroglucin and indol, which will demonstrate its distinction from other cellulose modifications. CELLULOSE AND IODINE REAGENTS. 319 A. Behavior of Cellulose to Iodine reagents. Literature. J. B. Eead, On the chemical composition of vegetable membrane and fiber (Lond. and Edinburgh, Phil. Magazine, Vol. XI, 1837, p. 421, f.) Schleiden, Einige Bemerk. iiber die sogen. Holzfaser der Chemiker (Wiegmann's Archiv, Jahrg., IV, 1838, Bd. I, p. 49, /I) Schleiden, Einige Bemerk. iiber d. vegetabil. Faserstoft' und sein Verhalten z. Starkmehl (Poggendorff's Annalen, Bd. XLIII, 1838, p. 391). Schleiden, Beitrage, etc., 13, 160, 164, 172, u. a. and O. Schleiden, Noch einige Bemerk. iiber d. veget. Faserstoff, u. sein Verb. z. Starkmehl (Flora, 1840, Bd. II, p. 737, ff., p. 753, ff.) Mohl, Einige Beobacht. iiber d. blaue Farbung d. veget. Zellmembran durch Jod (Flora, 1840, Bd. II, p. 609, f., p. 625, ff.) Payen, Mem. Stir la Compos, chim. du tissu propre des veget. phanerog. (Ann. des sc. nat. 2e ser., t. XIV, 1840, pp. 73-100.) Lantzius-Beuinga, De evol. sporid. muse. Gott., 1840, p. 7. Mohl, Verm. Schriften. Tub- ing., 1845, p. 337,/*., u. daselbst a. v. a. O. Mohl, Bildet d. Cellulose d. Grundlage sammtl. veget. Membraneii? (Bot. Zeitg., 1847, p. 497, ff.). Mohl, Die veget. Zelle, p. 30, etc. (c/*., auch Wagner's Handworterbuch, Bd. IV, p. 189, etc.). Dippel, Beitr. z. Losung der Frage, etc. (Bot. Zeitg., 1851, p. 409, ff.) Schacht, D. Pflanzenzelle a. v. O., z. B. p. 143, ff. Pringsheim, Algologische Mittheilungen (Flora, 1852, p. 470, ff. ) Hof'meister, Ueb. die zu Gallerte aufqnell. Zellen der Aussenflache v. Samen u. Perikarpien (Ber. Kon. Sachs. Ges- ellsch. der Wiss. Bd. X, 1858, p. 21, ff.) Frerny, Recb. chim. sur la compos, des Cellules veget. (Comptes rendus, t. XLVIII, 1859, p. 202,/".) Fremy, Caracteres distinctits des fibres lign., des f. corticales et du tissu cellulaire qui consiste la moelTe des arbres (1. c., t. XLVIII, 1859, p. 275, ff) - Payen, Differents etats de la cellulose dans les pi. (I. c. , t. XLVIII, 1859, p. 772, /".) Mulder, Physiol. cheni., p. 475. Kabsch, Unters. lib. d. chem. BeschaiFenh. d. Zelhvande (Pringsheim's Jahrb., Bd. Ill, 1863, p. 357,/".) Nageli, Ueb. d. Verhalten d. Zellhuut zum Jod. (Sitzungsber. d. bayer. 320 THE MICROSCOPE IN BOTANY. Acad. d. Wiss., 1863, Bd. I, p. 383, /*.) Nageli, Uber die Reactionen von Jod auf Starkekorner u. Zellmembr. (L c., p. 483,/ 1 .) Hofmeister, Handb. d. physiol. Bot. Bd. I, p. 252, /*., etc. Sach's Handb. d. Experi mental phys. d. Pfl., p. 433, ff. Hofmeister, D. Lehre v. d. Pflanzenzelle, Lpz., 1867, a. v. O. Dippel, Mikroskop, Bd. II, p. 6, ff. Sach's Lehrb. d. Bot. p. 19,^. Nageli und Schvvendener, Mikrosk., p. 474, p. 517, /., p. 549, etc. Strasburger, Zellbild. u. Zellthiel. III. Aufl. 1880, a. v. O. Ponlsen, Botanisk Mikrokemi, p. 49,/*. (Trans, p. 75). Richter, Beitr. z. genaueren Kenntniss der chem. Beschaffh. der Zellmembranen bei den Pilzen (Sitzungs- ber. K. K. Acad. d. Wiss. Wien, Bd. LXXXIII, 1 Abth., 1881, pp. 494-510). When, as appears from the investigations of Strasburger, 5 in the dividing of a cell, the primary cell plates form between the connecting fibres of the separated nuclei, there will be intro- duced into these by intercalation very fine granules, which by their behavior towards iodine solutions (see below) will. demon- strate themselves to be grains of starch of the utmost minute- ness. 6 But as soon as the granules are transformed into the substance of the outer cell wall they will then give no reaction 7 whatever, either by the addition of chlor-iodide of zinc, or iodine and sulphuric acid. These primary cell plates are either transitory, that is to say, are absorbed and give place to cellu- lose plates or they coexist with these and are employed in their formation. . The completed but very young cell membranes of the meres- tematic tissue are not often colored (Dippel), or if at alt yellow (Solla) 8 by the use of iodine and sulphuric acid or chloi -iodide of zinc. If, however, they are previously treated with muriatic acid, or potash lye or have lain for a short time in water in which the process of fermentation is taking place they would be col- ored blue by a brief subjection to the influence of the iodine reagent. 9 6 Strasburger, Zellbild. und Zellth. Ill, Aufl., 1880, p. 1, ff. 8 Strasburger, I. c.. p. 16, Table I, Fig. 6-9. 7 Strasburger, L c., p. 13. Dippel, Mikrosk., Bd. II, p. 7,/. Solla in Oesterr. Bot. Zeitschr. Jahrg., 1879, p. 351. Richter in Sitzungsber. der K. K. Acad. d. Wiss. Wien, Bd. LXXXIII, 1 Abth., 1881, p. 498. CELLULOSE AND IODINE KEAGENTS. 321 The older cell layers, which consist of pure cellulose, are not colored at all, or only with a yellowish or brown -yellowish or reddish tint by the addition of freshly prepared iodine water. 10 But if the iodine water contain traces of hydriodic acid a blue or violet color will be produced (Nageli). But if the prepar- ation which has been impregnated with iodine water he treated to a drop of sulphuric acid or caustic potash, there immediately appears an intense blue color, while neither muriatic nor nitric acid will produce this staining (Meyer, Schleiden). Chlor- iodide of zinc solution colors pure cellulose blue under all circumstances and is the most important reagent for it. Zinc chloride causes the cell walls to swell very quickly and so disturbs their natural relations to each other. This can, how- ever, be prevented for a considerable time by suitably diluting the fluid with water or potassium iodide of iodine solution. Chlor-iodide of zinc is almost always to be preferred to iodine water and sulphuric acid, since the sulphuric acid very quickly destroys the whole tissue. The intensity but not the shade of the blue or violet color which is produced by the iodine solution is conditioned upon the quantity of the intercalated iodine in the membrane. ^Nageli 11 who subjected the behavior of pure cellulose mem- brane towards iodine to a very searching investigation reached the following principal results. The quantity of the intercalated iodine determines in general not the character but only the intensity of the color. Each tint (yellow, orange, red, violet, blue) may be made bright by less iodine and intense by the use of a greater quantity. One may observe in single cases a transition from bright yellow to dark blue when during the reaction of the iodine hydriodic acid is formed. In other cases the absorption of more iodine changes the color from blue to brown when the membrane consists of 10 The walls of the spore utricle of the lichens are an exception to this, however, as they are colored blue by the use of iodine water alone. (Xageli in Sitzungsber. Bayer. Acad., 18C3, Bd. I, p. 485, ff.}, the blue color becomes, however, more intense by addition of sulphuric acid (Kichter, I. c., p. 496). For the rest see also Mohl in Flora, 1840. II Bd. p. 614, and G. Dickie in Annals of Nat. Hist., 1839, p. 165. Concerning the reactions of the cellulose walls of spores of algae, see Pringsheim in Flora, 1852, p. 470, /. 11 Xiigeli, concerning the reactions of iodine on starch grains and cell membranes (Sit- zungsberichte der Bayerischen Acad. 1863, Bd. I, pp. 524, 530, 532, 535, 539, 541, 543). 21 322 THE MICROSCOPE IN BOTANY. a mixture of two different materials which are acted upon differently by the iodine. Cell membranes which are permeated by water, and have received some one color by iodine, retain this color when the water is drawn out of it at the common temperature, and when otherwise no chemical or physical change has occurred. But, on the contrary, if some substance is dissolved by the interpenetrat- ing water which is again concentrated by the evaporation, it may so effect the arrangement of the molecules of iodine as to cause a greater or less change of color. Membranes colored by iodine, which may become 'uncolored either in the moist or dry condition, frequently change their color more or less. These transformations always proceed in the direction from blue through red to yellow. When a cell membrane will not immediately stain by iodine and water it may be colored by the action at the same time, of hydriodic acid (which is formed by the prolonged action of iodine on different organic combinations as well as by drying them with iodine), or of potassium iodide, or ammonia iodide, zinc iodide, phosphoric acid or sulphuric acid, in other cases also by sulphuric acid after a more or less energetic treatment by caustic potash or nitric acid. The treatment with hydriodic acid, potassium iodide, am- monium iodide, with sulphuric and phosphoric acid, caustic potash and nitric acid removes without doubt a less or greater quantity of foreign substances contained in the membrane which are soluble in that particular combination. This purifying of the cell membrane may in many cases facilitate the bluing, but it is in no case the only determining condition of it. Treatment by the above named reagents causes a greater or less swelling of the membrane but this loosening up of the tis- sue is in no case the cause of the bluing. For the bluing of the cell membrane with iodine and water (except in the case of the lichen tissue) there is required the presence at the same time of an assisting combination, hydriodic acid, potassium iodide, ammonium iodide, zinc iodide (or an- other metallic iodide), sulphuric acid, phosphoric acid, zinc chloride (?). But, perhaps, the sulphuric and phosphoric acid CELLULOSE AND MINERAL ACIDS. 323 do not act directly but indirectly by favoring the formation of hydriodic acid, through the decomposition of alcohol, or of or- ganic combinations of the cell, so that thence the blue color is almost exclusively conditioned by the presence of a definite quantity of an iodine combination. B. Behavior of Cellulose towards Mineral Acids. Of these, concentrated sulphuric and chromic acid are partic- ularly adapted to give the wished-for demonstration. In opposi- tion to hydrochloric acid which leaves the cellulose almost perfectly unchanged, and to nitric acid which when cold causes only a swelling of the cell membrane and dissolves it only by boiling, the other acids named dissolve the cellulose at ordinary temperature and in a very short time. On the contrary the modifications of cellulose, middle lamella, suberin, together with cutin are not soluble in them (see below). However, it should be stated that (probably all) wood membranes share with cellulose in the narrow sense, its solubility in sulphuric and chromic acid. If a section through the stem of a plant be put into concentrated sulphuric acid all that part which consists of lignin and pure cellulose will be dissolved out, while suberized layers and cuticularized combinations, as well as the middle lamella of the woody tissue, will remain behind intact. Cellu- lose is changed by sulphuric acid into an isomeric body to which Schleiden gave the name amyloid, because while it in itself remains unchanged it effects changes in other bodies by con- tact, or "catalysis" as the chemists say. This amyloid stands very near to starch as we infer from the fact that by the application 1 of iodine water it takes on a very intense blue color. On this circumstance rests the above many-times-mentioned reaction of iodine and sulphuric acid on cellulose, and is conducted in the following waj'. Put the section to be examined in freshly pre- pared iodine water for a short time. Take it out and remove as much as possible of the adhering fluid, lay it on a slide, put on a, cover glass, put the preparation under the microscope, and add at the edge of the cover-glass a large drop of concentrated 324 THE MICROSCOPE IN BOTAN1T. sulphuric acid. Now look quickly in the microscope and see how all the true cellulose membranes take on an intense blue color, while all the modifications of cellulose, lignin, middle lamella, and suberin are stained yellow or brown. After a few moments, however, the blue reaction becomes indistinct because of the destruction of the tissue which goes rapidly forward. C. Behavior of Cellulose toiuards Alkalies. In comparison with the destructive effect of mineral acids upon cellulose, the alkalies take but little if any hold upon it. Ammonia itself even in a concentrated state, and when the sec- tion is boiled in it for a short time, docs not alter the substance of the cell walls, while concentrated or nearly concentrated potash lye will only cause them to swell. By washing out the section treated with potash, and putting it in absolute alcohol the cell walls will resume their original form, on which account Hanstein's method of bleaching is recommended (see p. 199) ; compare also what is said concerning Russow's potassium alco- hol. D. Behavior of Cellulose towards Cuprammonia. Schweizer 12 found in 1857 that cotton laid in cuprammonia very quickly dissolved and assumed a jelly-like consistency, thence changed into a mucilaginous fluid which on being diluted with water was filtered. On applying muriatic acid a precipitate was thrown down which was colored brown by potassium iodide and chlorine water, showing that the cellulose was not changed to starch by the process. Cellulose is distinguished from all its modifications by the characteristic of its being soluble in cu- prammonia. Put a drop of the freshest possible preparation of the reagent upon a damp section lying under the cover- glass and then observe how the cellulose walls gradually swell up. Afterwards the outlines become indistinct and the 12 Schweizer, Jour, prakt. Chem., Bd. LXXII, p. Ill see also above, p. 244, ff\, then the treatise of Fremy and Payen cited on page 268. CELLULOSE AND CARMINE. 325 cess ends with a perfect solution of the whole cellulose struct- ure. It may be remarked in this connection that the modi- fications of cellulose may be dissolved in cuprammonia when the intercalated incrusting substance has previously been re- moved. This is most readily accomplished by Schnitzels macer- ation process, with potassium chlorate and nitric acid (see p. 163). Boil the section with this mixture in a test tube, wash out the imdestroyed portion with water and put it on the slide with a drop of cuprammonia solution. E. Behavior of Cellulose towards Alum Carmine. Literature. E. Tangl, Concerning the open communication between the cells of the endosperm of some seeds (Pringsheim's Jahrb., Bd. XII, 1879-81, p. 170, f.) According to Tangl, Grenadier's alum carmine (p. 308) offers a superior means of distinguishing pure cellulose membrane from that which has been cuticularized or changed to' cork. For while the former eagerly takes up the coloring matter and after five to ten minutes becomes an intense red, the latter remains uncolored. The color keeps very well in glycerine, and gives us a very instructive specimen and particularly so in respect to the vascular bundles. In order to get a beautiful stain of the nucleus and plasma one should previously harden the prepara- tion in absolute alcohol (see above p. 308). 13 F. Behavior of Cellulose towards Potassium- Copper Sulphate. Literature. Sachs, Concerning some new methods of micro- chemical reactions (Sitzungsberichte der K. acad. d. Wiss.,Bd. XXXVI, 185 ( J, p. 1-22). Sachs, Micro-chemical investiga- tions (Flora, 1862, p. 288, /".). Sachs, Concerning the sub- stances which furnish the material for the growth of the cell wall (Pringsheim's Jahrb., Bd. Ill, 1863, p. 187, ff.). 13 Tangl states, I. c., p. 173, also that the cellulose membrane of cambium and older parenchyma cells absorbs the blue coloring matter from an aqueous decoction of logwood in insoluble modification if this contains an addition of sulphate of iron and is applied cold. However, the same thing may happen in modified cellulose membrane so that it will not serve as a test of cellulose. 326 THE MICROSCOPE IN BOTANY. Sachs has given a process of bluing membranes which con- tain a certain kind of cellulose by treatment with copper sul- phate and potassium solution. The method is as follows. A very thin section, if possible thinner than the thickness of a layer of cells, should be laid for a long time in a concentrated solution of copper sulphate. It should remain in this from five to ten minutes, or several hours, or a day even, according to its nature. Then remove the section and lay it for a few minutes in water in order to wash off the salt solution. For this purpose it is necessary to put the section in a considerable quantity of water and not merely in a drop on the slide. The better way is to take the section in the forceps and move it back and forth sev- eral times in pure water. Then in a porcelain saucer which holds 8 or 10 cc. make a strong potassium solution of 1 part by weight of water, and 1 part caustic potash, and put the sec- tion in it for a short time, till in certain of the cell walls there appears a blue tint and in others a yellow color, while others still remain colorless. Lying in a drop of the fluid the section can then be examined. Then put the section in a small porce- lain cup and boil it for a few minutes in the potash solution. This intensifies the color and makes it appear now for the first time general. If the color is too transparent one must take a section sufficiently thick to make it distinct and charac- teristic. By this method many but not all cell walls consisting of cell- ulose will be colored an intense blue, while others will remain colorless. Those cell walls which are colored yellow with iodine are not cellulose in the strict 'sense and are colored yellow or orange yellow by this method. So, according to Sachs, the peripheral layer of parenchyma of the germ-root of the horse bean ( Vicia faba) takes on a beautiful blue stain ; also parts of the parenchyma, the very } r oung vascular bundles, wood cells, and bast cells of the germ-root of Phaseolus multiflorus, the subepidermal layer from the blooming branch of the gourd, and generally all collenchyma cells, young bast cells and wood cambium become blue, while commonly the thick walled par- enchyma cells remain uncolored. The walls of older bast cells color yellow or yellow orange, and all lignified elements of the MUCILAGINOUS CELLULOSE. 327 wood bodies. 14 Since a characteristic reaction will not take place in all cellulose walls by this method, its application there- fore is not a sufficient test without verification by iodine reac- tions. 2. MUCILAGINOUS CELLULOSE. Literature. Meyen, Die Secretionsorgane der Pflanzen, Ber- lin, 1837, p. 36, etc. Schleiden in Wiegmann's Archiv, 1838, p. 145. Deciasne, Sur la structure des poils qui couvrent le pericarpe de certaines composees (Ann. des Soc. Nat. lie, Ser. t. XII, 1839, pp. 251-254). Meycn, Pflanzpathologie, Berlin, 1841, p. 235. Schleiden, Beitr. z. Botanik (1844), p. 137. Mohl, Einige Bemerk. fiber den Ban der vegetal). Zelle (Botan. Zeitung, 1844, p. 323, /.). Mohl, Yermischte Schr. a. a. O. Kippist, On the existence of spiral cells in the seeds of AcanthncejB (Trans, of the Linn. Soc. of London, 1845, Vol. XIX, pp. 65-76). Kutzing, Grundziige der philosoph. Botanik, Leipzig, 1852, Bd. I, p. 195, ff. Cramer, Botan. Beitrage, Zurich, 1855, p. 1, ff. Unger, Anat. und Physiol. der Pflanzen, Pest, 1855, p. 78, 119, Taf. IV. Karsten, Ueber die Entstehung des Harzes, TVachses, Guminis, und Schleinies durch die assimilirende Thatigkeit der Zellmem- branen (Bot. Zeitg., 1857, p. 313, ff.). Mohl, Ueber die Entstehungsweise des Traganthgummi (Bot. Zeitg., 1857, p. 33, ff.) Hofmeister, Uber die zu Gallerte aufquell. Zellender Aussenflache von Samen und Perikarpien (Berichte der Sachs. Gesellschaft zu Leipzig, Bd. X, 1858, pp. 18-36). Trecul, in Comptes rendus, 1860; Journal de 1'Institut, 1862, p. 241.- Wigand, Ueber des Desorganisation der Pflanzenzelle, insbes. fiber die physiol. Bedeutung von Giunmi und Harz (Pringsheim's Jahrb., Bd. Ill, 1863, pp. 155-182). Frank, Uber die anat. Bedeut. und die Entstehung der veget. Schleime (Pringsheim's Jahrb., Bd. Y, 1866, pp. 161-200). Hofineister, Handb. d. physiol. Botan., Bd. I (1867), p. 258, ff. Hanstein, Ueber die organe der Harz- und Schleimabsonderung in den Laub- knospen (Botan. Zeitg., 1868, p. 697, ff.) Behrens, Unters. fiber den anat. Ban des Griflfels und der Narbe, Gott., 1875, p. " Sachs, in Sitznngsber. Wein, 1. c., p. 18, 19, Flora, 1862. p. 295. 328 THE MICROSCOPE IN BOTANY. 28, jf. Prillieux, Etude sur la formation do la gomme, etc. (Ann. des sc. nat. Vie ser, t. I, 1875, pp. 176-200). Reinke, Beitr. zur Anat. der an Laubblattern, besond. an den Zahnen ders. vorkoram. Sccretionsorgane (Pringsheim's Jahrb., Bd. X, 1875, pp. 119-178). Capns, Anatomic du tissn conductenr (Ann. des sc. nat. Vie ser., t. VII, 1878.) Behrens, Die Nectarien der Bliiten (Flora, 1879, p. 118, f., 144, /"., 233, jf., 440, ff.). Dalmer, Ueber die Leitnng der Pollensehlauche bei clen Angiosperrnen (Jenaische Zeitschr., /., Naturwissen- schaft, Bd. XLV, 1880, a. a. O.). Meyen and linger have already observed the fact that cer- tain cells and cell groups whose walls originally consisted of true cellulose were gradually transformed by a process of degen- eration into isomeric carbo-hydrate combinations which in their chemical but principally in their physical behavior are more or less different from cellulose. Although the finished product of the mnciparous process cannot be any longer regarded as cel- lulose (it will be treated more in detail under the section "vegetable mucilage"), yet in the first stages of the transfor- mation of cellulose to mucilage it is so much like cellulose that it should be described in this place. The cellulose walls of the inuciparous cells and cell groups have quite generally the ca- pacity of swelling to several times their original volume by an unusual power of imbibing water; thus often, the whole cell, and always the inuciparous tissue, loses its original form and is changed into a quite thin jelly. If the muciparous cell walls are thickened and stratified as are those of the Astragalus spe- cies whose medullary rays furnish the gum tragacanth, the strat- ification will gradually become indistinct till at last the resulting mucilaginous mass will appear to be very nearly structureless. 16 The whole of the cell wall or only its outermost layer may be drawn into sympathy with this process and it ends with its transformation into a jelly more or less soluble in water. 17 In other cases a middle portion of the wall, which is often strongly developed, is dissolved into a fluid amyloid (Collagen) as for example in epidermal cells where, by the absorption of water, Mohl, in Botan. Zeitung, 1857, p. 33,^". 16 Compare Prillieux in Ann. sc.Nat., Vie Ser., 1. 1, pi. V. fig. 1. "Mohl,Z. c., p. 42, /. MUCILAGINOUS CELLULOSE. 329 they swell out and become a mucilaginous jelly raising up the cuticle into blisters which finally burst. 18 In such cases there remains commonly besides the cuticle a thin layer of cellulose not participating in the process of disorganization, which sepa- rates the mucilaginous complex from the interior of the cell ; but it is always a part of the strongly thickened cell wall which swells up to become mucilage, the process itself being much diversi- fied in details. The swollen substance sometimes agrees with cellulose both in its anatomical behavior and its chemical reactions. The jelly under discussion is colored, for example, blue (Hofmeister) with iodine water and iodine alcohol in the seeds of Salvia horminum and Teesdalia nudicaulis or pale blue (seeds of Linum usitatis- simum). Iodine in connection witj) sulphuric acid of a certain definite concentration for each individual case colors it blue. The swollen layer of the seeds of Salvia horminum blued by iodine becomes reddish 19 by the addition of dilute sulphuric acid. In other cases the muciparous cellulose tissue behaves quite dif- ferently towards iodine reagents, often not taking any color whatever from any kind of iodine combination (Collagen, Plan- stein). Where, as in the formation of gum tragacanth, the muciparous cell walls originally showed color with chlor-iodide of zinc solution, this reaction becomes weaker in the mass as the muciparous process goes on till finally it altogether ceases (Mohlj. Other mucilaginous substances take a yellow color with iodine and sulphuric acid and with chlor-iodide of zinc; 20 only rarely do the older stages of muculent complex give the reaction of cellulose, viz., bluing by the two well-known re- agents. 21 So the reagents show with their different effects, that in the process under discussion we have detected all transition from true cellulose to true mucilaginous matter. As the most successful method of investigating muciparous cells the following may be mentioned. According to the statements of Fenzl 22 sections through muciparous seeds are best made by " Hanstein in Bot. Zeit., 1868, p. 697 ff. Behrens in Flora, p. 118, ff. t p. 232, ff. 19 Hofmeister iu Ber. Sachs, Ges. Leipzig, Bd. X, p. 30. 2 Frank in Pringsheim's Jahrb., Bd. V, pp. 163, 165, 167, etc. 21 Kutzing. Grundz. d. philos. Bot., Bd. I, p. 195. Frank, 1. c., pp. 168, 181. 22 Feuzl in Dcnkschr. d. Wciner Acad. VIII, Erkl. zu. Taf. I. 330 THE MICROSCOPE IN BOTANY. embedding them in stearine ; having cut the section wash in al- cohol to remove any shavings of stearine. The embedding me- dium described on pp. 186-7, given by Koch, may be employed. The section should be first studied dry or better still in alcohol, sometimes also in essential oils, because the use of water causes a rapid swelling. Since very delicate mucilaginous substances refract the light quite like alcohol and therefore can with diffi- culty be recognized in it, a small quantity of coloring matter, which will not be absorbed by the mucilage, should be added to the alcohol 23 in such cases. For this purpose nothing is bet- ter than the aniline dyes. If, after having studied the prepara- tion in alcohol, water is added, the muciparous cells rapidly expand in a radial direction, at the same time the mucilaginous complex begins to swell very quickly, the swollen substances immediately separate in the water and directly disappear from observation. The addition of potassium solution will produce a swelling in the dry preparation. 3. WOOD SUBSTANCE. (Lignin.) Wood or lignin (C 19 H 24 O 10 ) 24 forms the walls of all those cells which are changed into wood. 25 Lignified cells are commonly found in wood bodies; other lignified cells are, however, found isolated in the parenchyma tissue, as for example, the stone- cells which occur in the pith of numerous woody plants ; cells of like name in the pulp of the fruit of the Pomacece and similar cell tissue in the bark layer, etc. In all lignified cell walls, as mentioned on p. 316, there is inter- calated an incrusting substance, which contains relatively more hydrogen and carbon than does cellulose in the strict sense. According to the later investigations of Singer and the earlier o o o ones of v. Hohnel there are four substances which constantly 23 Hofmeister, 1. c., p. 21. 24 Particulars in Burgerstein in proceedings, d. K. Acad. Wein, Bd. LXX, 1 Abth., 1871, p. 338, Anm. SB F OI . g enera i ij s t s of literature see, Sachsse, Chemie mid Physiol. der Farbstoffe, Car- bo-hydrates u. Proteinsubst. Lpz., 1887, p. 144, ff. Niggl, Ueber die Verholzung d. Zell- membranen (Jahresber. Pollichia, 1881). Ebermeyer, Physiol. Chem. der Pflanzen, Berlin, 1882, Bd. I, p. 174, JT. Singer, Beitrage ztir naheren Kenntniss d. Holzsubstanz, u. d. ver- holzt. Gewebe (Silzuiigsber. d. K. Acad. Wien, Bd. LXXXV, 1882, 1 Abth., p. 345, JO LIGNIN AND IODINE EEAGENTS. 331 accompany lignified tissues, namely, Vanillin, Coniferin and a kind of gum which stands near to Arabin and perhaps repre- sents a modification of the wood gum of Thompson, and fin- ally a body which takes a yellow color with muriatic acid but whose chemical nature is still quite unknown. All these ele- ments may be extracted by boiling the wood in water for a longer or shorter time ; they are that which gives the character- istic reactions of wood-substances. In what relation they stand to lignin cannot yet be made out, but they indicate that that which we call lignin represents a mixture of several chemical individuals. As the most important reagents for lignified cell walls, iodine solutions, aniline sulphate, phloroglucin, indol, and phenol- hydrochloric acid should be named. As a distinction from pure cellulose, lignin does not dissolve in cuprammonm. It is, on the contrary, soluble in potash lye (more easily than cellulose) concentrated 26 nitric acid, sulph- uric and chromic acid. Concentrated sulphuric acid blackens it in dissolving. Schulze's maceration mixture dissolves lig- nin very easily. 27 A. Behavior of Lignin towards Iodine Reagents. Literature. See the treatises cited on p. 319. Also Mohl, Einige Bemerk. liber die Ban, der vegetal). Zelle (Botan. Zeitg., 1844^ p. 307, /*.). Schacht, Lehrb. d. Anat., etc., Bd. I, p. 16, etc. Fremy, Kecherches sur la comp. chim. du bois (Comptes rendus de Paris, t. XLVIII, 1859, p. 862, /".) Payen, Compos, de Fenveloppe des pi. et des tissus ligneux (Comptes rendus de Paris, t. XLVIII, 1859, p. 893, /".) Sa- il io, Einige Bemerk. liber d. Ban des Holzes (Botan. Zeitg. 1860, p. 193, ff.) Sanio, Vergl. Unters. liber die Elementar- organe d. Holzkorpers (Botan. Zeitg., 1863, p. 85,^.) Sanio, 26 Weak acids frequently make the layers of the cell wall come out very distinctly. Dilute sulphuric acid colors the young layers of lignifled membranes a beautiful i-ose red (Hartig, But. Zeit., ISon. p. 213). 117 According to S:inio (Dot. Zeig.. 1860, p. 204), the wood substance is disintegrated into a granular mass which finally comes into the cell cavity, and if potash be now added this granular mass will be dissolved in it with yellow color. 332 THE MICROSCOPE IN BOTANY. Yergl. Unters. iiber die Zusammensetz. des Holzkorpers (Botan. Zeitg., 1863, p. 358, ff. Mulder, Physiol. Chcrn.^ Bd. I, p. 209. Dippel, D. Mikroskop, Bd. II, p. 96, ff. Sanio, Zur Anatomie der gem. Kiefer (Pringsheim's Jahrb., Bd. IX, p. 65,^".). Sachs, Lehrbuch d. Botanik, p. 35. Lignified cell-walls are colored yellowish, yellow or brownish by the application of any of the iodine reagents, potassium iodide of iodine, chlor-iodide of zinc, and iodine and sulphuric acid. The last-named shade is produced only by iodine and sulphuric acid, or very rarely by chlor-iodide of zinc ; in this case, however, it shows only a brownish-yellow. The brighter and darker layers which are commonly perceptible in lignified walls either show no difference on the application of iodine and sulphuric acid, or else a distinction is apparent in the alternating stronger and weaker yellow color. Pure yellow is seen in the perfectly lignified portion of the wall, while such parts as have been imperfectly lignified are shown by the mixed colors. They are either a transition be- tween yellow and blue (blue-green or yellow-green) or reddish. The yellow color, produced by iodine reagents, which indicates lignification, shows itself through the whole extent of the cell- wall, or the wall will be but partially colored yellow. In this case it is commonly the peripheral layer of the wall which has the highest degree of lignification, while the inner portion next to the cell space is often less lignified. The layer of the lignified cells which lies naked about the cell-cavity, and which is commonly an optically distinguishable thickening layer, called by Sachs "an inner shell," and by Dip- pel a tertiary membrane, behaves very differently. This is colored yellow by the use of iodine and sulphuric acid or chlor- iodide of zinc quite infrequently (Sanio) 28 , but also reddish yel- low, commonly violet, bluish or quite blue, consisting in these cases of slightly lignified or quite unlignified cellulose. Thus, for example, the innermost thickening layer of the wood cells of Pinus sylvestris consists of pure cellulose. The incrusting substance may be removed from all lignified membranes by maceration, so that the cellulose which is left 28 Sanio in Botan. Zeit.,1860, p. 202. LIGNIN AND ANILINE SULPHATE. 333 will give the characteristic reaction with iodine reagents (see p. 317 and p. 319, /*.) Method. Prepare the thinnest possible section of the wood- tissues to be examined, and impregnate it if one is to produce the reaction with iodine and sulphuric acid (see p. 323) with potassium iodide of iodine or alcohol iodine, by laying it in a dish filled with these solutions for a longer or shorter time. The adhering solution should be washed off with distilled water, and the section laid upon the slide and a cover-glass put over; then a drop of concentrated sulphuric acid added and the reaction is quickly observed. In most cases this reaction is preferable to that by chlor-iodide of zinc. In the use of the latter reagent the moist section must sometimes remain in the solution several hours before the wished-for reaction will take place. B. Behavior of Lignin toicards Aniline Sulphate. Literature. Runge, in Poggendorfs Ann., Bd. XXXI, 1834, p. 65. Schapringer in Wochenschr. d. niederosterr. Gewerbevereins, Bd. XXVI, p. 326. Wiesner in Karsten's Botan. Unters., 1866, Bd. I, p. 120. Wiesner in Sitzungsber. derK. Acad. Wien, Bd. LXII, 1. Abth., 1870, p. 202 (Sep- aratabdr., p. 32). Wiesner, Die Rohstoffe des Pflanzen- reiches. Burgerstein, Unters. iiber d. Yorkommen u. die Entsteh. des Holzstoffes in den Geweben d.Pfl. (Sitzungsber. d. K. Acad. Wien, Bd. LXX, 1. Abth., 1874, pp. 338-355). Hohnel, Ueber Kork. mid verkorkte Gewebe Uberhaupt (id., Bd. LXXVI, 1. Abth., 1877, p. 527). Hohnel, Histochem. Unters. iiber d. Xylophilin u. d. Coniferin (id., p. 663, ff., u. a. a. O.). Sachs, Ein Beitr. z. Kenntniss des aufsteigenclen Saftstromes in transpirirenden Pflanzen (Arb. d. Botan. Insti- tutes zu Wurzburg, Bd. II, Hft. 1, 1878, p. 150, f.) Gau- nersdorfer, Beitrage z. Kenntniss der Eigenschaften mid Entstehung des Kernholzes (Sitzungsber. d. K. Acad. Wicn, Bd. LXXXY, 1882, 1. Abth., pp. 9-41;. Singer, Beitrage z. naheren Kenntniss der Holzsubstanz und der verholzten Ge- webe (id., pp. 345-360). 334 THE MICROSCOPE IN BOTANY. It was formerly supposed that chlor-iodide of zinc and iodine with sulphuric acid would always produce a yellow color in lignified cell-walls ; but it was afterwards found out that though this was generally the case there were a few excep- tions to the rule. Wiesner 29 was the first to point out that aniline sulphate was a reagent which in an acid solution (see p. 301) could be used as a test of wood substance in every kind of vegetable tissue. Already Runge and Schapringer had microscopically demonstrated that wood treated with this substance assumed an intense yellow color. . Later Burgerstein subjected the action of aniline sulphate upon lignin to a very exact investigation and found Wiesner's views confirmed in all points. That aniline sulphate (Wiesner's reagent) is a positive reagent upon lignin follows from this, that wherever this substance is chemically traceable in any tissue it shows the yellow color, but in all tissue from which the lignin has been withdrawn, by powerful oxidizing agencies such as chromic acid and Schultze's mixture, the reagent leaves no color. 30 Method* 1 Put the tissue to be tested in a drop of distilled water and let a drop of the concentrated solution flow in from the edge. In tissue full of sap the reagent may be used without water. Potassium, sodium, and ammonia destroy the yellow color, but acid restores it again. The color is a pure gold yellow, yet the shade will depend upon the quantity used. Burgerstein has investigated the different systems of tissue and their lignification, by means of aniline sulphate, and has arrived thereby at the following principal results. Among thalophytes, only certain lichen tissue shows a slight lignitication. The tissue of algse and fungi is never lignified. In the vascular plants all the tissue systems are partially ligni- fied (epidermal tissue, tissue of the vascular bundle, and funda- mental tissue) . 29 Wiesnev in Karsten's Botan. Unters., Bd. I, p. 120. so Vesque (Comptes rendus de Paris, t. LXX:, p. 498) criticises the aniline sulphate, since it colors other membranes not Hgnified yellow. ( ??; Hohnel came to the contrary con- elusion (Sitzungsber. d. K. Acad. Wien, Bd. LXX VI, 1 Abth., p. 528.) 31 Burgerstein in Sitzungsber. der K. Acad. Wien, Bd. LXX, 1 Abth., p. 349. LIGNIN AND ANILINE SULPHATE. 335 A. Epidermal Tissue.* 2 Epidermis, according to Schacht and Dippel, is never lignified. Burgerstein confirms this. He found this tissue lignified only in the seed wings of Pinus and Abies. The cuticle as well as the membranes of the stomata cells are never lignified. Hairs sometimes are and sometimes not. The collenchyma tissue which supports the epidermis is never lignitied (Dippel asserts the contrary). 33 B. Tissue of the Vascular Bundles.^ The vessels in the xylem are, with few exceptions, always lignified (slightly so in the submerged parts of water plants and in very sappy laud plants). Wood-cells are always lignified, both in the thicken- ing cell-walls and in the middle lamella, us all naturalists ad- mit. 35 The tertiary membrane, "innermost shell," is, according to Sauio, 86 . usually lignified, but according to Sachs, Schacht and Dippel it is not. Burgerstein agrees with Sanio. The wood parenchyma is as Sanio 37 has already shown always lig- nified. The bast cells are, according to Sachs and Schacht, sometimes lignified and sometimes not. Burgerstein distin- guished : a, bast cells lignitied uniformly in all the layers of the membranes with the exception of the middle lamella, which appears always to be the most lignitied (fully lignified bast cells) ; 6, bast cells in which the primary and older secondary layers are becoming lignified, while the younger secondary and tertiary layers remain unlignified (partially lignified bast cells) ; c, bast layers whose whole substance is unlignified. The lignitied bast cells are of most frequent occurrence. The sieve tubes are not lignified. The vascular bundle layer is always more or less lignified. C. Fundamental Tissue. 38 The pith cells are for the most part lignified, especially those lying next the vascular bundles, likewise the cells of the medullary rays. The parenchymatous fundamental tissue is mostly not lignified, the leaf parenchyma never. Sclerenchyma cells are always lignified. 32 Bnrgerstein, L c., p. 344, ff. as Dippel, Mikroskop., Bd. II, p. 155. * Burgerstein, 1. c., p. UU ? ff. as Sanio in Pringsheim's Jahrb., Bd. IX, pp. 50-126. as Sanio in Prings. Jnhrb., I. c., Bot. Zeit., I860, p. 202. a- S.'iiiio in Bot. Zeitg., 183, p. 98. 38 Burgenstein, 1. c., p. 350, ff. 336 THE MICROSCOPE IN BOTANY. The lignifying process begins very early and advances very rapi lly forward. First the vessels lignify, then the wood cells, and the wood parenchyma, very soon thereafter the bast cells, and relatively later lignification begins in the pith. C. Behavior of Lignin towards PJdoroglucin. Literature. Hohnel, Histochem. Unters, iiber d. Xylophilin u. d. Coniferin. I, Ueber d. Xylophilin (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. LXXVI, 1. Abth., 1877, pp. 663- 698). Hohnel, Ueber den Kork, etc. (id., p. 528). Wies- ner, Note iiber das Verhalten des Phloroglucins uud einiger verwandter Korper auf verholzte Zellmembranen (id., Bd. LXXVII, 1. Abth., 1878, pp. 60-66). Singer, Beitrage z. naheren Kenntniss der Holzsubstanz undcler verholzten Gewebe (id., Bd. LXXXV, 1. Abth., 1882, pp. 345-360). Poulsen, I. c., p. 34 (Translation, p. 46, f.). 39 This reagent was discovered by Wiesner. In an aqueous or alcoholic solution of even no more than 1 per cent, with the addition of muriatic acid, it stains lignin an intense red violet color. Method. Put the section to be examined under a cover-glass and add a drop of the aqueous or alcoholic reagent according to circumstances, and no color will be produced. Then at the edge of the cover-glass put a drop of concentrated or somewhat dilute muriatic acid, and directly a very delicate violet color will begin to enter the lignified tissue, which becomes more and more intense till the whole shows a uniform, beautiful violet red color. By reversing the process and adding the acid first and the rea- gent afterwards the result is the same ; the muriatic acid does not color the tissue noticeably. Vary the experiment by putting a drop of the reagent on a moist section and evaporating it al- most all away and then adding the acid and the reaction takes place almost instantaneously. According to v. Hohnel we operate with the extract of cherry 89 References to the literature belonging to this subject may be found in the works of Borne of the older writers. There is a list of the related literature in v. Hohnel, I. c., Bd. LXXVI, 1 Abth., pp. 693-698, besides which see Weiss und Wiesner, id., Bd. XL, p. 276. LIGNIN AND PHLOROGLUCIX. 337 wood mentioned on p. 303, in a similar manner. Add to the fresh section a little quantity of the fluid letting it mostly evaporate and then add the acid. v. Hohnel found that in a transverse section of the stem of the Anthericum liliago, by this treatment the epidermis and the soft parenchyma lying directly beneath the young pith ancl the soft bast cells remained perfectly color- less. The vessels and the middle lamella of the woody tissue became dark violet ; the more imperfectly lignified thickening layers of the wood cells and the elements of the sclereuchyma sheath bright violet. 40 A transverse section through the stem of Rumex obtusifolius treated with an alcoholic solution of phloroglucin and concen- trated muriatic acid showed the following. The epidermis and the strongly developed collenchyma layer lying beneath as well as the next following thin bark parenchyma remained perfectly colorless. In the vascular bundles all the woody parts inclusive of the less numerous wide vessels were colored a dark red violet. At the beginning of the reaction the color began to become distinct in the middle lamella first ; afterwards it ex- tended itself uniformly through the whole of the lignified walls. The outer layers of the pith are likewise strongly lignified corresponding to their red violet coloring. Towards the center of the section the pith cells become gradually a brighter violet and at the center remained quite colorless since they are not in the least lignified. If the above described section were put in distilled water the violet red color would be changed to a brick red which would grow paler by degrees till at last the section would be quite colorless. It behaves the same way in alcohol or ether. The water dissolves the phloroglucin because the latter forms no chemical combination with the wood substance, it being only mechanically absorbed and intercalated in the cell walls. A section was carefully w r ashed in water for a long time (five hours) and in spite of that took a faint violet color when muriatic acid was added, showing that there still remained a small quan- tity of the phloroglucin to produce the reaction. But if one removes the adhering acid from the section by passing it through V. Hohnel, I. c., Bd. LXXXI, 1 Abth., p. 686. 22 338 THE MICROSCOPE IN BOTANY. water and then adds ammonia the color will change instantly to yellow and cloudy orange ; the parts which before were tinted the intense violet will now show the deep shade of yellow. If now the alkali be washed out and acid again applied the violet color will be restored and of the same intensity as before. Ammonia (sodium or potassium lye, a basic salt) produces the decolorization of the ^phloroglucin stain. Muriatic acid (sul- phuric acid, nitric acid, acid salts) restores the coloring again (see also v. Hohnel, I. c.). D. Behavior of Lignin towards Indol. Literature. Niggl, Das Indol ein Reagenz auf verholzte Zellmembranen. Mikrochernische Untcrsuch. (Flora, 1881, pp. 545-559, 561-566 ; also separate as a dissertation. Regensburg, 1881, 22 pages). Singer, Beitrage zur naheren Kenntniss der Holzsubstanz uiid der verholzten Gewebe (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. LXXXV, 1 Abth., 1882, pp. 346-360). Indol, according to the recently published investigations of Niggl, produces a stain quite like that of the phloroglucin re- action. 41 By means of an acid it colors lignified membrane from a cherry red to red violet. Method. The section to be examined is put on the slide with a drop of the aqueous solution of indol and a cover-glass laid over it. Then by means of a piece of blotting paper draw out a part of the solution and let flow in 1 to 2 parts of the dilute sulphuric acid mentioned on p. 304, whereupon the reaction immediately takes place. The specimen thus prepared keeps its beautiful color a long time. If concentrated acid be used, or the superfluous quantity bo not drawn off, the color of the lignified membranes in a few weeks will be changed to brown- red. To prevent this let the acid work for an hour or two and then draw it out with filter paper and replace with glycerine. Not only does the phloroglucin and indol reactions show the 41 Still several other substmces have been proposed for the same purpose in recent times, asPyrol (Niggl), Orcin (Lippmann), Resorcin (Molisch, Wiesner), Pyrogalin (Wies- ner), Hydrochinon (Niggl). 1 have not tested the working of all these substances, on a'ccount of the scarcity of some of them. On the contrary, I have carefully followed out Niggl's statements regarding indol and find them to be correct in all cases. LIGNIN AND INDOL. 339 greatest agreement in respect to coloring, but also as I have found in the behavior of the colored membranes towards bases and acids. Wash out the acid from the indol-stained section and substitute ammonia and the violet color wilt disappear and in its place will appear a yellow to ochre colored tint. If this again be washed out and sulphuric acid be added the violet color will be restored. To distinguish it from phloroglucin it is to be mentioned that the indol stain does not, like that of phloroglu- cin, disappear by prolonged treatment with water. Sections stained with indol retain their color with undiminished intensity when they have lain in water for twenty- four hours. This makes the indol reaction preferable to that of phloroglucin. Just as Btirgerstein tested the effect of aniline sulphate on the different systems of tissue, Niggl has studied that of indol. We give in the following a brief resume of his results. For the purpose of a more easy comparison with those of Burger- stein on p. 335 they are presented in the same consecutive order. THALLOPHYTES. In algse there appears to be no lignification except in the stout, warty, thickened membranes of some Cosmarium species. In the greater part of the fungi no kind of coloring appears. The exceptions are Polyporus fomentarius (a glimmer of red), Ochrolechia pallescens and Trametes suaveolens (distinct red). The thallus of the lichens behaves variously. 42 VASCULAR PLANTS. A. Epidermal Tissues. The epidermis is not colored by indol and sulphuric acid. The exception to this is the epi- dermal cells of the leaves of Cinnamomum Culilawan, Cycas revoluta suidflexuosa, and of the needles of several Coniferce. The cuticle is generally not lignified. The young sprouts of ^Esculus hippocastanum, Acer pseudoplatanus and Hippuris vulgaris are an exception to this. The cuticle consists of numerous scales, and these often show a difference of behavior, the inner ones being sometimes reddened by indol. The mem- 41 Niggl, 1. c., separatbdr., p. 5, /. 340 THE MICROSCOPE IN BOTANY. brane of the hairs is as frequently lignified as not. The sto- mata cells of the Ooniferce and the Cycadeaz are frequently colored red by indol. Collenchyma tissue is not lignified. The collenchyma of the stem and leaves of Sapindus laurifolius is an exception to this rule. B. Tissue of the Vascular Bundles. The vessels, are al- ways lignified, the wood cells also, middle lamella and thick- ening layer always; tertiary membrane (inner shell) remains uncolored in Astragalus, Caragana, Robinia and Cytisus. The cells of the wood parenchyma are always lignified and indeed all three layers ; in its younger state the innermost is not fully colored. The bast cells show considerable variation. Niggl observed, as did Burgerstein, bast cells perfectly lignified and others totally lacking in that; frequently, however, they were partially lignified. The outer layer, especially in the younger stages, is commonly reddened, while the inner one remains uncolored. Later, the middle part shows the reaction on the lignin. The sieve tubes are not lignifijed. The sheath of the vascular bundles is always at least partially lignified. 43 C. Fundamental Tissue. Pith cells are commonly ligni- fied ; the cells of the medullary rays always, except in Aristo- lochia sipho. The hypoderm is sometimes liguified, but rarely the leaf parenchyma (Cycas revoluta, C. flexuosa). In the sclerenchyma cells the lignification can always be demon- strated. The lignifying process begins earliest in the vessels. E. Behavior of Lignin towards Phenol -muriatic Acid. Literature. Tiemann und Haarmann, Ueber d. Coniferin seine Unwandlung in das aromat. Princip der Vanille (Ber. Deutsch. Chem. Gesellsch., Bd. VII, 1874, p. 608,/*.) Tangl, Vorlauf. Mitth. liber die Verbreitung des Coniferin (Flora, 1874, p. 239, /".). Rud. Mtiller, Ueber Coniferin (I. c., p. 399). v. Hohnel, Ueber den Kork und verkorkte Gewebe iiberhaupt (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. LXXVII, 1 Abth., 1877, p. 700, ff.).v. Hohnel, Histochem. * 8 Particulars in Niggl, I. c., pp. 12-14. LIGNIN AND PHENOL-MURIATIC ACID. 341 Unters. iiber d. Xylophilinu. d. Coniferin. II, Ueber d. Conif- eriu (id. p. 699, ff.). Singer, Beitrag. z. naheren Kennt- niss d. Holzsubstanz und verholzten Gewebe (id. Bd. LXXXV, 1 Abth., 1882, p. 347, ff.) It has long been known to the chemist that a pine shaving passed through carbolic and muriatic acid becomes blue. After- wards it was proved by Tiemann and Haarmann that this coloring depended upon a substance existing in wood discovered by Th. Hartig and called coniferin. Tangl showed about the same time that a like reaction took place not only in coniferous wood, but also in Sambucus niyra, Populus bahamifera, Frax- inus excelsior and Vitis vinifera. v. Hohnel afterwards ad- vanced the hypothesis that coniferin is an element of all wood tissue and that therefore the carbolo-rnuriatic reaction would serve as a test for wood tissue in general. Singer confirmed the views of v. Hohnel in a recently published investigation. While it was at first supposed that the reaction took place by first moistening with carbolic acid and then with muriatic acid, v. Hohnel first observed that the action of direct sunlight was o also necessary. Method (v. Hohnel). 44 Use the phenol-muriatic acid de- scribed on p. 302. With the perfectly clear solution we may obtain very clean and beautiful preparations. The section should not be too thin, and being moistened the least possible with the reagent it should be put under a cover-glass and set in the direct sunlight. An exposure of from one-half to one minute will be sufficient. A short time after this the section will have an intense color. If the exposure is for a longer time the strength and vividness of the color slowly fade and it becomes a sea-green or a yellow-green. The beautiful green color is characteristic of the really lignified membranes. All cells which would be colored blue with chlor-iodide of zinc, the epi- dermis, bark which is destitute of wood and cellulose, remain uncolored or colored yellowish by the muriatic acid in the re- agent. 45 The section must be immediately examined because 4 V. H6hnel,.Z. c., Bd. LXXVI, p. 700, ff. 45 Muriatic acid colors all wood tissues and also some others a more or less intense yellow. The color is, however, mostly very weak, the addition of water readily destroy, ing it. 342 THE MICROSCOPE IN BOTANY. the green color is not durable. In lack of direct sunlight, con- centrated artificial light may be substituted, though with poorer results. Very beautiful preparations are made from the aerial roots of orchids, stems of monocotyledonous plants, woods of Evonymus, ^Esculus and the Coniferce. According to Tommaso and Donato Tommasi 46 the "conifer! 11 reagent" is more distinctly effective if the section to be treated is first moistened with a mixture of carbolic acid and potassium chlorate, and then with the muriatic acid. By this means the blue stain comes out in diffused light, more rapidly and more intensely, and the preparation will not lose its color in a day. For the purpose of determining the relative sensitiveness of lignin reactions, Singer 47 has experimented with solutions of like concentration of phloroglucin, indol, pyrol, aniline sul- phate, resorciu, paratoluidin, pyrogalic acid, etc., by observing the effect of each upon tissue uniformly lignified, and has arrived at these results : " That with a 1 per cent solution of phloroglucin, indol and pyrol, we are able to produce vivid colors, quite uniform in their intensity. But also much weaker dilutions of the last named reagents carried were able to produce coloring, and in a 0.001 per cent concentration the limit of the effectiveness of phloroglucin was reached (see also p. 303), while the indol reduced to a 0.0007 per cent dilution colored coniferous wood, particularly after several hours. Py- rol is effective only when used in a stronger dilution. Indol is thus the most sensitive reagent which we possess for testing lignification. But it does not prove to be the most use- ful ; for, not to mention its great costliness (1 g. costing 70 M.) [in America $45], it will not keep well and requires the greatest caution in working with sulphuric acid which in a concentrated form destroys all vegetable tissue. In consideration of this and in respect to the fact that pyrol is very difficult to make and changes its nature after a few 46 Tommaso and Donato Tommasi, Ueber d. Fichtenholzreaction zur Entdeckung des Phenols im Urin. (Ber. Deutsch. chem. Gesellsch., 1881, p. 1834, JT.) Cf. auch Singer, I. c., p. 353, ff. Singer, 1. c., p. 358. INTER-CELLULAR SUBSTANCE. 343 hours, we must give the preference to phlorogluciu in combina- tion with muriatic acid over all other lignin reagents." After my experiments with aniline sulphate, phloroglucin and inclol, I have to add to this that I agree with Singer in respect to the sensitiveness of indol ; but that it appears to me as if the indol deserves the preference over the phloroglucin. Prep- arations stained with it seem to keep far better than when treated with phloroglucin, if one will carefully wash out the acid with distilled water and preserve in glycerine. The use of sulphuric acid of a dilution of one to four sufficiently insures the specimen against harm. And, finally, in respect to the de- composition of indol I must remark that I have an aqueous solution which now for more than seven months has perfectly preserved its efficiency and also its pungent smell. 4. MIDDLE LAMELLA, Intercellular Substance. Literature. Mohl, Verm. Schr., p. 314 ff., etc. Mohl, Die veget. Zelle, p. 196. Wigand, Intercellularsubstanz und Cu- ticula, Brschwg., 1850. Schacht, Lehrb. d. Anat. u. Phys. d. Gew.,1856, Bd. I, p. 108. Sanio, Ueber Intercell. im Holz (Bot.Zeitg., 1860, p. 208-213). Vogl, Ueber d. Intercellulars. u. die Milchsaftgef. in d. Wurzel des gem. Lowenzahns (Sit- zungsber. der K. Acad. d. Wiss. Wien, Bd. XLYIII, 2 Abth., 1863, pp. 668-690) Wiesner, Unters. liber d. Auftretenv.Pec- tinkorpern in den Geweben d. Runkelriibe (id. Bd. L, 2 Abth., 1865, pp. 442-453) Hofmeister, Lehre v. d. Pflanzen- zelle, 1867, sec. 31. Wiesner, Einl. in d. techn. Mikroskopie, Wien, 1867, pp. 62, 244, 246, etc. Dippel, Die Intercellulars. und deren Entstehung. Rotterd., 1867. Dippel, Mikroskop, Bd. II, p. 99 ff. Sachs, Lehrb., p. 72. Dippel, D. neuere Theorieiiberd. feinereStructurd.Zellhiille,etc. (Schr. d. Senck- enbergischen Gesellsch., Bd. X, XI, 1875-78, p. 41 ff.). Solla, Beitr. z. naheren Kenntn. der chem. und physikal. Bes- chaffenh. der Intercellulars. (Oesterr. bot. Zeitschr. 1879, pp. 341-353). v. Hohnel, Notiz iiber d. Mittellamelle der Holz- elemente, etc. (Bot. Zeitg, 1880, p. 450^".) See also, in part, the writings cited on pages 319 and 333. 344 THE MICROSCOPE IN BOTANY. Under the conception of the middle lamella we are to under- stand the homogeneous, sharply-defined partition lying between two contiguous cells and which appears to be transformed from the primary cellulose membrane by chemical metamor- phosis 48 and then often assumes certain conditions of solubility. It is always recognizable in wood tissue by being easily seen. With many anatomists the term " intercellular substance " is used as synonymous with "middle lamella." But Sachs and Wiesner distinguish between the two expressions and apply the former term to the jelly or pectin-like substance which sometimes lies between the cells and which is formed by a chemical metamor- phosis in which more or less of the cell wall becomes homoge- neous (endosperm of Ceratonia siliqua, the tissue of the Fnci, etc.). The views of botanists concerning the production and chem- ical nature of the middle lamella or intercellular substance have greatly differed. Schacht 49 supposed the intercellular substance to be a binding cement between the cells distinct from the cellu- lose. According to Dippel 50 the middle lamella (which he calls the primary cellulose covering) is not homogeneous, but consists of two corresponding layers of cellulose and of one of intercellular substance lying between, which latter proceeds from the cambium walling of the tissue cells, which is formed rom a combination essentially different from cellulose but iso- meric with it, tind, indeed, of the daughter and not the mother cells of the tissue, which latter, as soon as they have fulfilled their function, are dissolved and reabsorbcd. That combination essentially favors the dissolving of the contiguous coverings of the cells and allows, in consequence, transformations which are not peculiar to cellulose. According to Wigand, 51 the intercellular substance arises from the intimate commingling of the primary cell walls, which view was also advanced by Sanio 52 who added that the intercel- 48 A strict distinction must be made between the primary membrane and the middle la- mella, which in some botanical hand books is not expressly done. * 9 Lehrb. d. Anat. u. Physiol. d. Gew., Bd. I, p. 129. 50 Dippel, Mikroskop, Bd. II, p. 105 /. 61 Wigand, Botan. Unters., p. 79. 62 Sanio in Bot. Zeitg., 1860, p. 210 ff. INTER-CELLULAR SUBSTANCE. 345 lular substance was altogether or partially lignified, and, indeed, this lignification takes place sometimes before that of the secon- dary cell layer. It colors yellow with chlor-iodide of zinc, but if the intercalated wood substance is removed, by boiling in potash the reactio'n of chlor-iodide of zinc will be that of cellu- lose. The views of the two last-named naturalists are current to-day. The more recent investi orations of Sol la 53 teach that the inter- cellular substance or middle lamella, in the course of the devel- opment of the tissue, enters into different chemical as well as physical transformations. It is molecularly distinct from the adjoining layers of cell wall. The first foundation of the inter- cellular substance is either pure cellulose (cambium) or (at the point of the stem), a substance in which cellulose is afterwards traceable in the young permanent tissue. The intercellular sub- stance of the young permanent tissue consists, as a rule, of cellulose. In perfectly formed permanent tissue cellulose is but rarely traceable (in many kinds of bast) ; commonly it enters into many metamorphoses and then exhibits toward the reagent a very different behavior. These metamorphoses lead finally sometimes to the complete separation of connected cells. Reactions. Of the iodine reagents, 54 iodine and sulphuric acid, or chlor-iodide of zinc, produce in most cases a yellow coloring of the middle lamella : after previously boiling it in potash lye these reagents give a blue or violet tinted color (cellulose reaction) , K This latter color, however, always appears at the outset when the middle lamella consists of cellulose. Par- tially lignified middle lamella shows a corresponding mixture of colors between yellow and violet. Boiling nitric acid in combina- tion with ammonia often gives a strong yellow color to the middle lamella (Solla, v. Hohnel). Phloroglucin and indol behave toward the middle lamella very much as toward the lignified thickening layers of the cells. When the reaction takes place gradually the middle lamella colors before and more intensely than the adjoining layers. (See p. 337.) 53 Solia in Oesterr. Bot. Zeitschr. 1879, pp. 341-353. Sanio, I. c., Taf. VI, Figs. 10-12, 15. 65 Sanio, I.e., Taf. VI, Fig. 16. 346 THE MICROSCOPE IN BOTANY. Dissolving reagents, on the contrary, behave very differently toward the intercellular substance and its solubility is not proportionate to its age. 56 The rule is that cuprammonia, concentrated sulphuric acid and dilute chromic acid ap- plied cold will not dissolve it (see p. 317), while concentrated chromic acid will do so with difficulty and Schultze's maceration mixture, easily. 57 The intercellular substance of very delicate tissue will sometimes be dissolved partially or altogether by the action of boiling water (for example in the parenchyma of the beet root, Wiesner). Acetic acid dissolves the intercellular substance of the potato after a long time, 58 tartaric and oxalic acid very slowly (Wiesner, Solla). Potash lye, nitric acid and muriatic acid dissolve it very rapidly (potato, pith of jSambucus). The middle lamella of wood is most rapidly dissolved in boiling nitro-muriatic acid and strong chromic acid. Potash lye slowly dissolves the intercellular substance of certain bast fibres as in the parenchyma of the beet root (Wiesner) . Intercellular substance may, in certain cases, undergo a pec- tose metamorphosis. Mulder 59 and Kabsch 60 first showed that pectose occurs in many cell walls ; the latter also showed that in the boundary layer of the cells it is most intimately mingled with cellulose and appears as intercellular substance. Yogi 61 further found that in the root of the dandelion the intercellular substance is produced by the transformation of cellulose into pectose. Wiesner studied the appearance of the pectose bodies in the beet root and found, in agreement with Kabsch and Vogl, that the intercellular substance is the seat of the pectose which is principally a product of the transformation of the outer layer of the mother cell, but that not only parenchyma tissue, but also cambium, vascular and wood cells and likewise peridermal 6 Wiesner in Sitzungsber. d. K. Acad. d, Wiss. Wien, Bd. LXH, 1 Abth. p. 201. w According to Wiesner (Einleit. in d. techn. Mikrosp. p. 47) chromic acid will always dissolve the intercellular substance (see also Wiesner in Proceed. Imperial Acad. of Science, Vienna, Vol. LXII, part 1, p. 200). According to H. MUller, the intercellular substance of wood is dissolved by bromine water (official report of the Vienna World's Exposition, 1873, Brunswick, 1877, Vol. Ill, 1 part, 2nd half, p. 27^). 68 Solla, 1. c., p. 344. 5 9 Mulder, Physiol. Chem., p. 514. e Kabsch in Pringsheim's Jahrb., Bd. Ill, p. 3G7. i Vogl, Sitzungsber. d. K. Acad. Wiss. Wien, Bd. XL VIII, 2 Abth. 1863, p. 668 ff. CORKY CELLULOSE. 347 cells may contain pectose. 62 This pectose metamorphosis may lead in certain cases to the formation of jelly and to the loosening or separating of the individual cells. It maybe mentioned also that pectic acid salts are detected in many plants ; according to Fremy, calcium pectate is, in many of the tissues, the cementing medium of the cells. According to Maudet it is an element of the pith of Aralia papyrifera. According to Gireaud pectic acid is found in large quantities in gum tragacanth. 63 The presence of pectose substances is demonstrated by the cell walls swelling in boiling water and potash lye, and dissolv- ing in the latter. According to Poulsen 64 cuprammonia will pre- cipitate in tissue containing pectose a copper pectinate, which in thin sections will still remain after the entire disintegration of the rest part of the membrane. The methods of investigating intercellular substances with reagents have been sufficiently given in treating of lignin. Should we wish to dissolve these substances by heating the re- agent, the operation may be conducted with a watch-glass which should not be heated over a free flame, but, following the process of Sanio, 65 should be placed upon a thin plate of iron and this heated till the contents of the glass boil. 5. CORKY CELLULOSE, SUBERIN. (Including Cutin, Pollenin.) Literature. Kroker, De plantar, epidemide observ. Yratisl., 1833. Mohl, Unters. iiber d. Entwickl., des Korkes u. d. Borke auf der Rinde der baumart. Dikotyl. (Verm. Schr., pp. 212-232 ; auch Diss. aus d. Jahre, 1836). Mohl, Unters. tiber d. Lenticellen (id., pp. 233-245; auch Diss. vom. Jahre, 1836). Mohl, Ueber d. Cuticula der Gewachse (id., pp. 260- 368; auch Linnaea, 1842). Fritsche, Ueber den Pollen, Petersbg., 1837. Nageli, Entwicklungsgesch., d. Pollens, etc., Zurich, 1842. Cohn, De Cuticula. Yratisl., 1850. Schacht, 2 Wiesner, ibid, Bd. L, 2 Abth, 1864, p. 450. 63 Husemann, Pflanzenstoffe, Bd. 1. 1882, p. 186. "Poulsen, Botan. Mikrochem., p. 57, Trans, pp. 15, 91. e 5 Sanio in Bot. Zeit., 1860, p. 211, Anm. 348 THE MICROSCOPE IN BOTANY. D. Pflanzenzelle, p. 239. Hanstein, Ueber d. Ban u. d. Ent- wickl. d. Baumrinde, Berlin, 1853. Fremy, Recherches chim. sur la cuticule (Comptes rendus de Paris, t. XLVIII, 1859, p. 667, ff.). Sanio, Ueber d. Bau. u. die Entwickl. des Korkes (Pringsheim's Jahrb., Bd. 11,1860, pp. 39-108). Schacht, Ueber d. Bau einiger Pollenkorner (id., pp. 109-159). Pol- lender, die Chroms., ein Losungsmittel fur Pollenin u. Cutin (Bot. Zeitg., 1862, p. 405). Faivre, Sur les plaies d'ecorce par incis. annul, et sur leurs effets, etc., Paris, 1864. Fllicki- ger, Lehrb. d. Pharmakogn. d. Pflanzenreiches, Berlin, 1867, p. 336. De Bary, Ueber d. "VVachsuberziige der Epidermis (Bot. Zeitg., 1871, p. 128, ff.) Pfitzer, Beitr. z. Kenntn. d. Hautgewebe d. Pfl. (Pringsheim's Jahrb., Bd. VII, p. 532, ff. 9 Bd. VIII, p. 73, ff.) Hegelmaier, Ueber d. Bau u. die Entwickl. einiger Cuticulargebilde (ed.,Bd. IX, p. 286, ff.). Haberlandt, Ueber d. Nachweisung der Cellulose ira Kork- gewebe (Oesterr. bot. Zeitschr., 1874, pp. 229-234). Miiller, E-., Die Rinde unserer Laubholzer. Bresl., 1875. Tschistia- koff, Ueber d. Entwicklungsgesch. des Pollens v. Epilobium angusti folium (Prings. Jahrb., Bd. X, p. 7-45). ^-v. Hohuel, Ueber den Kork und verkorkte Gewebe iiberhaupt (Sitz- ungsber. d. K. Acad. d. Wiss. Wien, Bd. LXXVI, 1 Abth., 1877, p. 507-562; cf. auch Bot. Zeitg., 1877, p. 783, ff.). v. Hohnel, Ueber d. Cuticula (Oesterr. Bot. Zeitg., -1878, No. 3, u. 4). Niggl, D. Indol, eiu Reagenz auf verholzte Zellmembranen, p. 9, f. Corky or cuticularized cellulose is distributed through the well-known cork layer, which is often solid, free from inter- cellular spaces, mostly consisting of not very much thickened cell layers and their derivatives, also in the endodermis, and in that fine continuous coating which is drawn over the outer walls of the epidermis cells, and finally in the outer inclosing sheath of pollen grains and many spores. According to De Bary's 66 and especially v. HohnePs 67 investigations the suberization does not seize upon all portions of the cork cell region, but it is De Bary in Bot. Zeitg., 1871, p. 128, ff. v. Hohnel in Sitzungsber. d. K. Acad. Wiss. Wien, Bd. LXXVI, 1 Abth., p. 507. CORKY CELLULOSE. 349 limited to certain definite, often sharply marked, zones. Ac- cording to v. Hohnel, almost eyery cork cell wall (exclusive of many young cork cells of the Coniferaz) , which belongs to two neighboring cells, consists of the following five lamella: (1) of a middle strongly lignified plate, which is not distinguishable from the middle lamella, or is only partially lignified ; (2) of two suberized layers which lie on the two sides of this one ; (3) of two cellulose layers which lie next to the two last and also to the cell space, and which are more or less strongly lignified. The cork substance forming the suberin layer is as little known in respect to its chemical nature as is lignin. Accord- ing to Mitscherlich, Dopping and others, cork substance is dis- tinguished by containing 1.50 per cent to 2.3 per cent of nitrogen, while according to v. Hohnel there are no grounds for supposing it to contain nitrogen, since albuminous sub- stances have never anywhere been detected in suberin. But suberin contains from 73 to 74 per cent of carbon and 10 per cent of hydrogen (by which it follows that it must contain from 16 to 17 per cent of oxygen). It is insoluble in boiling alcohol and stands in its chemical as in its physical nature be- tween wax and cellulose. De Bary 68 has shown that frequently, perhaps always, in the formation of cuticle, a molecular interca- lation of wax takes place. A very characteristic physical quality of the cork lamella is that it is almost entirely imper- meable by diosmosis, as Sanio, 69 by a series of very striking experiments, has already proved. As in other modifications of cellulose, so in the suberine lamella, pure cellulose may often be detected when the "in- crusting substance" has been removed by a process already described on pages 318 and 331. The first who directed at- tention to this characteristic were Mohl 70 and Hofmeister 71 . The former noticed the cellulose in the cork of a flask after 8De Bary in Dot. Zeitg., 1871, p. 593, ff. 69 Sanio in Pringsheim's Jahrb., Bd. II, p. 54, /.See also De Bary, I.e.; Hanstein in Bot. Zeitg., 1868, pp. 70S, 748; Behrens in Flora, 1879, p. 374, /. TO H. v. Mohl in Bot. Zeitg., 1847, p. 497. "Hofmeister in Ber. d. K. Sachs Gesellsch.d . Wiss. Leipzig, Bd. X (4858), p. 21. 850 THE MICROSCOPE IN BOTANY. maceration in potash lye. The latter showed that the lami- nated cuticular layer of epidermis cells of Hoja carnosa gave a very distinct blue coloring with iodine reagent when it had been excluded from the air and treated for two or three weeks with concentrated potash lye. The same reaction appears in the cuticle of the leaves of Orchio morio after previous treatment with concentrated sulphuric acid. "The principal reasons for including cuticle among those membranes which are essentially different from cellulose falls to the ground with this proof." 72 According to De Bary 73 the cuticular substance of the leaves of Klopstockia is very easily destroyed by a warm ten per cent solution of potash whereof the pure cellulose walls remained behind. Haberlandt, who has made the most searching inves- tigation of the occurrence of cellulose in cork found that the test may be made by maceration in chromic acid, and Schultze's mixture, but preferably by boiling the section to be tested in potassium chlorate and nitric acid, and then before the section quite falls apart treat it for some moments with boiling potash lye. Then after washing it out with water the membranes of the separated tissue will be colored an intense blue by cblor- iodide of zinc and be dissolved by cuprammonia* 74 The suberized parts of the membrane may be easily recog- nized as such by some characteristic reactions. Like the middle lamella it is perfectly insoluble in cuprammonia and con- centrated sulphuric acid. The latter often colors the cuticular- ized extine of many pollen grains usually a beautiful rose-red, 75 seldom yellow. Acetic acid causes the extine of many spores of ferns to swell. 76 Concentrated chromic acid itself will not dissolve suberin, or if at all with the greatest difficulty ; v. Hohnel uses this therefore as a test of suberin (chromic acid reaction) : 77 use a pure quite concentrated solution. It causes the suberized membrane to stand out clear and distinct while the rest part of the tissue first 72 See also v. Mohl, Vermischte Schr., p. 263. 73 De Bary, 1. c. p. 578. 74 Haberlandt, in Oesterr. Bot. Zeitsch., 1874, p. 232, /. 76 Schacht in Priugsheim's Jahrb., Bd.II, p. 134, etc. Taf. XVIII, Figs. 10, 11, 14, 15, 31, 32. 76 Fischer v. Waldheim in Priugsheim's Jahrb., Bd. IV, p. 375. 77 v. Hohnel, I. ti. t p. 526. CORKY CELLULOSE. 351 becomes gradually more indistinct and then entirely disappears. Suberized membrane is dissolved by chromic acid, as previously noted only with much difficulty but after treatment by it for eight or ten hours it becomes transparent. Very strongly sub- erized membrane holds out, however, for a week, but finally becomes transparent. Wash out the acid and again it becomes dark and distinct as before. Cuticularized membranes have the same characteristics. Iodine with sulphuric acid as well as chlor-iodide of zinc col- ors suberized cellulose yellow or brown or deep brown itself. According to Mohl 78 if the cuticle is impregnated with iodine it is colored a deep yellow or brown. If the specimen is treated with sulphuric acid the cuticle is dissolved off and can be seen very well. Sometimes by this process it becomes a still darker brown. 79 The extine of the pollen grain colors with iodine and sulphuric acid quite the same way. 83 So also do many spores, as for example, fern spores. Indol with sulphuric acid (see p. 339) leaves suberized cells, as in the cuticle, quite uucolored, 81 at least the suberized lamellae never show any coloring, while in the walls of old cork cells a red coloring is noticeable after this treatment. But, as can be shown in very thin sections, only the middle lamella is stained. Young cork cells show no reddening. Concentrated potash lye produces no noticeable change in cork tissue except a very faint yellow coloring. But by holding the slide over a small flame, slowly heating it but not quite to the boiling point, the color becomes darker, the membrane itself a little swollen and at least a definite layer of the wall assumes a granular appear- ance (pure cellulose membrane only swells but for the rest maintains a smooth surface) . By boiling the granulization be- comes more pronounced and in most cases the granular and 78 v. Mohl, Verm. Schr., p. 261. For particulars concerning the coloring of cuticle see pp. 260-207, and compare with illustrations in Tables IX and X. 7 According to Hofmeister (Ber. Sachs, Gesselsch., Leip.Bd. X, p. 21), the cuticle of the seed of Linum usitatissimum behaves in a characteristic manner. By treatment with iodine and dilute sulphuric acid it is colored blue the blue bordering on the black. Add- ing concentrated sulphuric acid changes the color to yellow. Washing out the acid with water restores the blue color. * Schacht, 1. c., p. 103-168. si Xiggl, 1. c., p. 9. 352 THE MICROSCOPE IN BOTANY. variegated substances protrude from the membrane. If now the section be washed with water the granular masses will be for the most part destroyed. It now becomes evident that every cell wall of cork, however thin it may be, consists of three mem- branous lamellae (see p. 348), a middle one in common and two which belong to the adjoining cells, which lamellae are often separated from each other by a wide space between. These spaces were originally filled with this granular mass. Suber- ized cells treated with a cold concentrated solution of potash take on a yellow color while all other cell membranes re- main almost or altogether uncolored. By heating, the yellow color of the first becomes more intense, and that of the latter when there is any, more pale (potassium reaction, v. HOhnel). 82 By boiling the section under investigation in Schultze's mix- ture (see p. 163) the suberized membrane becomes very in- distinct, while the rest, even strongly lignified tissue itself, gradually becomes transparent. Cuticle and cuticularized tissue behave in the same way. Warmed under a cover-glass a violent development of gas takes place and then the suberized tissue alone remains. , Now wash out the Schultze's mixture and add alcohol and then ether and the whole becomes hyaline. But if the heating is carried still further the membranes suddenly swell and melt together into masses which finally become quite glob- ular, consist of eerie acid, and are soluble in hot alcohol, ether, benzole, chloroform and dilute potash lye. To recognize slightly suberized tissue, lay the section a short time in cold Schultze's mixture wash out and add potash lye. By the former the suberized membrane becomes somewhat more distinct and by the latter it takes an ocher yellow color and becomes crumbly. If this does not immediately happen slightly warming commonly helps it on. At the same time the potash lye produces a fur- ther clarification of the suberized membrane (eerie acid re- action, v. Hohnel). 83 82 V. Hohnel, pp. 522-524. 83 v. Hohnel, I. c., pp. 524-526. FUNGUS CELLULOSE. 353 6. PUNQUS CELLUT,OSE. Literature. Schacht, Die Pflanzenzelle, p. 13. Dippel, D. Mikroskop, Bd. II, p. 7, /. De Bary, Morphology der Pilze, Flechtenund Myxomycetin (Hofmeister, de Bary, Sachs, Hand- buch d. Physiol. Botan., Bd. II, p. 7, /). Poulsen, Bot. Mikrochemi, p. 51 (Trans, p. 79). Richter, Beitrage z. Gen- aueren Kennta. derchem. Beschaffeuheit d. Zellmembranen bei den Pilzen (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. LXXXIII, 1 Abth., 1881, pp. 494-510). [Ber. Deutsche Bot. Gesellsch. 1(1883), pp. 288-308 (1 pi.), Jour. Roy. Mic. Soc., Vol. Ill, part V, p. 676.] The membranous substance which forms the walls of the hyphae of fungi and lichens, fungus cellulose, was, until the recent investigations of Schacht, Dippel and De Bary, looked upon as an entirely different substance to cellulose, since it had not been possible before that by any known medium of macer- ation to remove the incrusting substance so as to produce by iodine reagents the cellulose reaction. According to Schacht and De Bary the fungus walls themselves were not colored blue with iodine and sulphuric acid after boiling in potash lye, nor indeed after treatment with Schultze's mixture or chromic acid. For the rest it was already known by De Bary and others that many plant membranes become blue by iodine or chlor-iodide of zinc without any other previous treatment. In Mucor it was demon- strated that in the young state the cell walls were colored blue with iodine and sulphuric acid, but in the older stages they re- mained colorless. In later investigations Richter opposed the view that fungus cellulose is essentially different from true cellulose. He suc- ceeded in removing the incrusting substance from the fungus Polyporus, after long and numerous treatments of the fungus tissue with water, hot potash lye, acetic acid, alcohol, ether and again water, and then in producing the cellulose reaction with chlor-iodide of zinc. The same results were produced with fungi and lichens by macerating the tissue continuously 23 354 THE MICROSCOPE IN BOTANY. fur two or three weeks in potash lye frequently changing the macerating liquid. 84 The fungus membrane thus purified was colored a rose red to a violet by chlor-iodide of zinc ; it also appeared to be dissolved by cuprammonia but this could not be definitely verified. Richter 85 did not succeed in demon- strating any lignification of these membranes by means of aniline sulphate or phlorogliicin, not even in the lichen itself. 86 On the other hand, he demonstrated the most distinct suberiza- tion in Daedaka quercina by means of the eerie acid reac- tion (see p. 351). The purification of the membranes of fungus cells so as to get the cellulose reaction is generally accomplished only with the greatest difficulty, and the utmost patience is necessary. Richter 87 found by his studies that the fungus cellulose is but common cellulose with an admixture of foreign matter, perhaps albuminous substances ; that a fungus cellulose in the sense of De Bary does not exist. In its natural state fungus cellulose is distinguished by its extraordinary resistance to the different reagents. It is perfectly insoluble in cuprammonia, can be scarcely touched by cold potash lye, muiiatic acid and Schultze's mixture. Concen- trated sulphuric acid destroys it only with the greatest difficulty. On the contrary, it should be distinctly stated that many fungus membranes are soluble in muriatic acid. [Pringsheim has further investigated the peculiar granules long since observed by him in the fertilizing tubes and oogonia of the Saprolegniese and described by Zopf as amoebae. They are found in the fertilizing tubes at all ages. While young they are flat disk-shaped or polyhedral plates with rounded corners composed of a dense homogeneous substance. They vary greatly in size and form. They gradually become stratified and at last as regularly and completely as starch grains. They are abun- dant also in the oogonia and a few grains occur in other parts of the plant.] si Of lichens those must be selected which have the least possible amount of lichenin in them since this gives the same reaction as cellulose (Richter, 1. c., p. 503. See above p. 270). 85 Richtev, 1. c., p. 505, f. so On the contrary, see Burgerstein above p. 2S3, and Xiggl above, p. 287. Richter, I. c.. p. 510. FUNGUS CELLULOSE. 355 [The structure, mode of development and chemical properties of these substances show that they are neither organs of re- production nor independent parasitic organisms but are a special modification of the cell contents. The stratification indicates a close resemblance to other bodies of this character. They are, however, not colored blue by iodine nor do they take any other color but that of the iodine itself. They are completely in- soluble in all ordinary solvents of oils and resins, even in abso- lute alcohol and ether. Nitric acid either with or without ammonia or potash produces no effect on them nor does Millon's reagent. They have no power of taking up coloring substances except under special circumstances. Caustic alkalies cold produce no visible effect on these bodies and very little change is effected by dilute or concentrated nitric or hydrochloric acid at common temperature. In moderately concentrated sulphuric acid they dissolve rapidly and completely, at the ordinary tem- perature, as also in solutions of zinc chloride when not too dilute. They do not dissolve in cuprammonia even after long treatment.] [These reactions show that the bodies in question belong , neither to the proteinaceous cell contents, nor to the series of oils and resins, but that they are composed of a substance closely al- lied to cellulose which has been separated from the protoplasm in a granular form. It is perhaps identical with so-called "fungus cellulose" and with the "fibrose" of Fremy, and Pringsheim pro- poses for it the term "cellulin." Its special chemical characteris- tic is its remarkable solubility in dilute sulphuric acid, and in an aqueous solution of zinc chloride.] The stratification of the cellulin grains is concentric around a nucleus of denser substance. They grow, however, to a con- siderable size before any stratification is evident. Compound grains are not uncommon. A common mode of multiplication is by a kind of budding not dissimilar to that of torula. [When the oospores are formed out of the protoplasmic con- tents of the organism, an unused residue remains behind, which is the substance out of which the cellulin grains are subsequent- ly developed. This substance is morphologically identical with 356 THE MICROSCOPE IN BOTANY. 1 M to M 1 l! f ?5 F ^ s cr2 2l 00 rf CD II ill g? | S p Iff C" H< ' 3 ^ 1 o* ' 3 ^ Iodine water. SCO - 2 s ^ f g 5* 5* r Chlor- iodide of ziuc. W S s H a g- H 2 ? Iodine and sulphuric acid. 05- II II &s fi Cupram- monia. fillftf |fi CD Is 03 Potassium h v drox- ide JO M 00 trt- ;p, Sachs, Sitzungsber., Bonn, 1863, p. 178, 1864, p. 10. 12 Sachs, I c., p. 78. 121 Sachs, I. c., p. 85. GRAPE SUGAR, GLYCOSE. 377 inated structure becoming more distinct (Fig. 133). crystals become much larger, often breaking through the tissue, if we put large organs con- taining inuliu for a long time, days and weeks, in alcohol and glycerine and then prepare the sections from them. Also by allowing such organs to dry, one can detect inulin in them in the form of spherical crystals, after the preparation of the sections . According to Sachs the external appear- ance of the inulin crystals is sufficient to identify them as such, but, according to Prantl, a more exact testing of their qualities as given above is indispensable. The FIG. 133. VII. GEAPE SUGAR, GLYCOSE. Literature. Sachs, Ueber eiuige neue mikrosk.-chem. Re- actionsmethoden (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. XXXYI, 1859, p. 5, ff.). Sachs, Mikrochem. Unters. (Flora, 1862, p. -289, ff.). Sachs, Ueber d. Stoife, welche d. Material z. Aufbau d. Zellwand liefern (Pringslieirn's Jahrb., Bd. Ill, 1863, p. 183, jf.)- Grape sugar, also called glycose or starch sugar, C 6 H^ O 6 , very frequently occurs in plant tissues and almost without ex- ception in aqueous solution, and mostly in connection with solutions of cane, or other kinds of sugar. It arises as a trans- formation product of other carbo-hydrates chiefly indeed of starch (starch sugar) and in this form appears to travel through the interior of plants. The single method for the testing of grape sugar, as is that of cane sugar and dextrine, is based on the reaction of copper sulphate and potash discovered by Trommer and modified by Fehling. It was introduced into microscopy by Sachs. Impregnate .the tissues containing the glycose with copper sulphate solution and add thereto cold potash lye in excess so as to produce a beautiful blue fluid. Directly, if cold, sooner Nach Sachs, I. c., Taf. II, fig. 5. 378 THE MICROSCOPE IN BOTANY. by boiling, there appears a copious display of reduced copper oxide which is colored a beautiful brick red. Under the mi- croscope the precipitate appears like that of dextrine ; the grains, however, are larger and collected in many large flocculent masses which is not the case with dextrine. The manipulation to be followed in this reaction is the same as that described on page 365, for dextrine; for this reason it should be remarked here, that, as in the case of dextrine, the section to be tested for grape sugar should have a thickness greater than that of a single layer of cells, else will the fluid cell contents escape and the desired reaction will not take place. In place of a pure copper sulphate solution one containing a tartaric acid salt may be employed (Fehling's solution). The latter is prepared by dissolving one part by weight of copper sulphate, and five parts of potassium sodium tartrate in eight parts of water afterwards keeping the solution in the dark. VIII. CANE SUGAR, SACCHAROSE. Literature. The same as in the grape sugar. As is the grape sugar so is the cane sugar or saccharose, C 12 H^ O u , a widely distributed plant substance which presents itself in the cells likewise as a clear solution. The method for microscopical testing as given by Sachs is the same as that for grape sugar. Treat a section containing cane sugar with Tronimer's or Fehling's solution and then add potash lye and there will appear a beautiful blue coloring, but no re- duction of copper oxide takes place even after a short boiling. The appearance of the blue coloring in the cold liquid is quite characteristic and is indeed definitely perceptible even with quite thin sections. The reduction of copper oxide shows always the presence of grape sugar or of dextrine, but with cane sugar a reduction never occurs. Concerning testing for a mixture of these substances with each other or with albuminous matter see Sachs in the proceedings of the Imperial Academy of Sciences, Vienna, Vol. XXXVI, p. 10, f. ALBUMINOUS MATTER. PROTEIDS. 379 IX. ALBUMINOUS MATTER. PROTEIDS. Among nitrogenous substances in plants, albuminous or pro- teid matter holds the chief place. It is lacking in no living cell and all vital processes are intimately connected with it. Chemically it is characterized by its components of carbon, oxygen, nitrogen and hydrogen, with a less but constant quan- tity of sulphur. As to the rest a satisfactory chemical formula has not yet been made. Perhaps this is in general im- possible, since the albuminous substances met with in plants are probably not individuals in the chemical sense. Such a formula has been long desired for protoplasm for^instance. The recently published chemical analyses of the protoplasm of ^Ethalium septicum 123 by Rodewald show it to consist of a large number of different organic and inorganic chemical elements. Albuminous substances occur in cell contents ; they are solid or plastic-viscous, often indistinguishable from a fluid. They are insoluble in absolute alcohol, soluble or insoluble in water. They are mostly colorless, rarely yellowish, and still more 'rarely reddish or bluish. As universal qualities of albuminous substances, which make their recognition easily possible under the microscope, are the following : that they take a yellow or dark yellow, or brown color from any weak solution of iodine, the color being more intense than that of the surrounding iodine solution ; thai they enter into a dark-yellow combination with nitric acid which was invested by Mulder with the name of xanthroproteid acid ; that they take a beautiful violet color with copper sulphate and potassium ; that they give a beautiful rose-red with Millon's reagent ; that they become red with a solution of sugar and the aid of sulphuric acid ; that they are unchanged by Hanstein's aniline solution. Dead but not living albuminous matter gener- ally may be stained with coloring substances, as carmine solu- tions, haematoxylin, etc. "3 Reinke, Ueber d. Zusaramensetz. d. Pro topi. v. JEthal. sept. Gottingen 1880. Reinke n. Rodewald, Studien iiber d. Protoplasraa, I, Die Chem. Zusamniensetz. des Protopl. v. sept. (Unters. aus d. Bot. Laborat. d. Uuiv. Gottingen, Heft II, 1881, pp. 1-75). 380 THE MICROSCOPE IN BOTANY. A chemical classification of proteid substances has indeed frequently been attempted, but heretofore with no very satis- factory results. Ritthausen, 124 one of the best of those who know these substances, divides them into albumin (plant albu- min), plant casein (legumin, conglutin and glutencasein) and paste-like proteids (gliadin, mucedin and glutenfibrin). Pfeff- er 125 provisionally holds every chemical classification to be in- sufficient and with A. Mayer 126 classifies albuminous bodies as reserve and functional proteid substances. This classification is the foundation of the following representation. I. RESERVE PROTEID SUBSTANCES. (Proteid grains, Aleuron, Gluten Meal.) Literature. Hartig, Ueber das Klebermehl (Bot. Zeit. , 1855, p. 881). Hartig, Weitere Mittheil. d. Klebermehl (Aleuron) be- trefiend (id., 1856, p. 257, ff.). Hartig, Entwicklungsgesch. d. Pflanzenkeims, Lpz., 1858, pp. 108-134. v. Holle, Bei- trage z. naheren Kenntniss d. Proteinkorner im Samen der Gewachse (Neues Jahrb. f. Pharm., Bd. X, 1858, pp. 1-24). Cohn, Ueber Protein krystalle in den KartofFeln (37, Jahresber. d. schles. Gesellsch. f. vaterl. Cultur, 1858, pp. 72-82). Trecul, Des formations vesiculaires dans les cellules veget. (Ann. des sc. nat. 4e ser., t. X, 1858, p. 20, ff.). Maschke, Ueber d. Ban u. d. Bestancltheile der Kleberblaschen in Ber- tholletia, deren Entwicklung in Ricinus (Bot. Zeitg., 1859, p. 409,^".). Radlkofer, Ueber Krystalle proteinartiger Korper pflanzlichen u. thierischen Ursprungs, Leipz., 1859. Nageli, Ueber d. Krystallahnl. Proteinkorper u. ihre Yerscheidenh. v. wahren Krystallen (Sitzungsber. d. K. Acad. d. TViss. z. Miinchen, Jahrg., 1862, Bd. II, pp. 120-154). Cramer, Das Rhodospermin, ein krystalloidischer, quellb;irer Korper im Zellinhalt verschiedener Florideeu (Vierteljahrsschr. d. uatur- forsch. Gesellsch. in Ziirich, Jahrg., VII, 1862, pp. 350-365). Nageli, Pflanzenphysiol. Unters. Sachs, Zur Keimungs- 124 Husemann, Pflanzenstoffe, 1882, Bd. I, p. 233, ff. 125 Pfeffer in Prin^sheim's Jahrb., Bd. VIII, p. 491. 188 A. Mayer, Lehrb. d. Agricul. Chem., 1877, p. 191. ALBUMINOUS MATTER. PROTEIDS. 381 gesch. d. Dattel (Botan. Zeitg., 1862, p. 241, jf.). Sachs, Ueber d. Keiraung des Samens v. Allium cepa (id., 1863, p. 57, j^*.). Gris, Recherches anatom. et physiol. sur la germi- nation (Ann. des sc. nat. 5e ser. t. II, 1864, pp. 1-123). Cohn, Beitr. z. Physiol. d. Phyeochromaceeu und Florideen (Schultze's Archiv f. mikrosk. Anat., Bd. Ill, 1867, pp. 1- 60.) Dippel, D. Mikrosk., Bd. II, 1869, p. 29, /".Klein, Ueber d. Krystalloide einiger Florideen (Flora, 1871, pp. 161-169. Klein, Zur Kentniss des Pilobolus (6 Gefonnte In- haltskorper) (Pringsheim's Jahrb., Bd. VIII, 1872, p. 337, f.). Pfeffer, Unters. iiber d. Proteinkorner u. d. Bedeutung des Asparagins beim Keimen der Samen (id., pp. 419-574). Sachs, Lehrbuch, pp. 53-59. Prillieux, Sur la coloration et le verdissement du Neottia Nidus-avis (Ann. des sc. nat. 5e ser., t. XIX, 1874, pp. 108-118). Van Tieghem, Nouvelles re- cherches sur les Mucorinees (Format, et role des cristalloides de inucorine) (id., 6e ser., t. I, 1875, pp. 24-32). Schimper, Unters. liber d. Proteinkrystalle d. Pfl. Strassb., 1879. Klein, Pinguicula alpina als iusectenfr. Pfl. u. in anatom ischer Bezeihung (Cohn's Beitrag. z. Biol. d. Pfl., Bd. Ill, 1880, pp. 163-184). Klein, Neuere Daten iiber d. Krystalloide Meeres- algen (Flora, 1880, p. 65, ff.). Vines, On the Chemical com- pos, of Aleuron-grains (Proceedings of the Royal Soc. of London, Vol. XXX, 1880, p. 387, f.). Proteid grains (v. Holle, Pfeffer), also called alenron or glu- ten meal (Hartig), were discovered in the year 1855 by Theo- dore Hartig and thoroughly investigated first, by him, and later by Sachs, but principally by Pfeffer. They occur in the endo- sperm or in the parenchyma of the cotyledons of the seeds of a number of plants, 127 and indeed, "often in company with starch, embedded in the fatty protoplasmic substance of the cells. They are formed in the last stages of the ripening of the seeds and change again at the beginning of the sprouting. They are represented by very small, small or large granules. Sometimes there are found in the same cell many small granules and one great proteid grain (Solitar, Hartig) . They consist altogether of 127 Compare, concerning their occurrence, Hartig, Entwicklungsgeschichte d. Pflanz- keims, pp. 1-20-124 and the above cited treatises of Pteffer. 382 THE MICROSCOPE IN BOTANY. proteid matter, perhaps being mingled with a small quantity of other substances. The statement of Sachs that they consist in part of fatty matter has been, recently, refuted by Pfeffer, The mass of the granules is either amorphous, or a part of the proteid matter assumes a crystal-like nature (proteid grains with crystalloids). Still others contain inorganic substances. The latter are either true crystals of calcium oxalate or crystalline roundish bodies, the so-called globoids which consist of calcium and magnesium phosphates. We will, therefore, describe in their turn: (a) amorphic proteid grains, (b) proteid grains with crystalloids, (c) proteid grains inclosing inorganic matter. A. Amorphic Proteid Grains. All amorphic proteid grains are insoluble in absolute alcohol and ether (both must be absolutely anhydrous), in chloroform and benzole, in fatty and essential oils. But benzole especially dissolves the fat of the surrounding fundamental substance, and in oily seeds the proteid grains appear more distinctly after its application (for the rest see below). If water be gradually added to a preparation lying in. alcohol many proteid grains show a concentric lamination. This appearance often occurs (Pceonia, endosperm) when alcohol containing a little sulphuric acid is employed and the section examined in water. 12 * The lamination, however, appears only in the peripheral part of the grain, the center remaining amorphic (Fig. 134, after Pfeffer) Many proteid grains are quite soluble in water, others partly, still others not at all. All are soluble in water which contains a trace of caustic potash, likewise in acids and alkalies, many in glycerine and sugar solution (in this often slowly). Those grains which are soluble in water must be ex- amined in absolute alcohol or oil ; their presence is best dem- onstrated by iodine in glycerine, iodine with a little potassium iodide dissolved in glycerine, see page 286. i 28 Pfcffer, 1. c., p. 499. ALBUMINOUS MATTER. PROTEIDS. 383 Pfeffer has given a method by which proteid grains which are soluble in water may be transformed into an insoluble 1 modifica- tion. He says, 129 "I make use of the property of proteid matter to become insoluble in water by corrosive sublimate and be transformed into an achlorate mercurial combination. In order to get this without disorganizing the proteid grain, I digest the section of the seed for at least twelve hours in a small flask with a solution of simple mercuric chloride in absolute alcohol, of the concentration of which it is not necessary to be very particular, though I find in most cases about a 2 per cent solution to be best. Then wash the section in alcohol and carry it into water in which it is now quite insoluble. It is not rec- ommended to wash out the section very carefully in alcohol. I may also remark that in taking the section from the fluid no needle or scalpel of steel should be used, since iron makes a precipitate with metallic mercury which would contaminate the surface of the section coming in contact with it. On this ac- count one should use a glass rod or most conveniently a needle or small scalpel made of platinum, which latter one can make for himself for this purpose by cutting a piece of thin platinum and fitting it into a glass tube by melting the glass. The pro- teid grains thus prepared will indeed swell in water but will return to their original volume after a time by treatment with alcohol." They are, however, soluble in dilute acids and al- kalies and give the same reactions as the fresh proteid grains. The process especially fits them for the study of the effects of acid reagents upon them. By this method of making the proteid grains insoluble, proof is at the same time afforded that gums, pectinaceous substances or cane sugar, occur, if at all, only in very small quantities in proteid grains. These substances enter into no insoluble combinations with corrosive sublimate, so that they would be dissolved out after this process when subject to the action of water and the granule would change in form and mass which it does not in fact. 130 If proteid grains, which are or have been made insoluble in Pfeffer, 1. c., p. 141. i3o pfeffer, 1. c., p. 442. 384 THE MICROSCOPE IN BOTANY. water, be boiled with water or treated with alcohol or ether, they will coagulate and are then scarcely soluble in dilute alka- lies and acids at common temperatures, but gradually dissolve in concentrated alkali. Every proteid grain is surrounded with a delicate envelope. If by the means indicated the fundamental substance of the proteid grain be dissolved this envelope remains behind and the application of reagents shows that it consists of proteid sub- stances. The principal microscopical reactions of proteid grains are the following. Iodine will in all solutions give a brown or yellow-brown color (use a neutral solution where the granules have not been modified by coagulation) ; numerous other col- oring substances are also absorbed by the grains. Pfeffer 131 used principally for this purpose the aniline blues dissolved in water, solutions which remain unchanged for a long time. The pro- teid grains absorb therewith, in small quantity, a much more striking color than with cochineal extract. Nitric acid and potash give the yellow color proceeding from xanthoproteid acid ; sugar and sulphuric acid a rose red (p. 299) ; Millon's re- agent a brick red (p. 296). The latter, according to Pfeffer, is not worthy of much commendation, likewise copper sulphate and potassium should be rejected, which Nageli and Schwendener 132 have very highly recommended for this purpose. Cupram- monia does not dissolve the proteid granules. All amorphic proteid grains are not doubly refractive and therefore are not illuminated on a dark field in the polarizing apparatus (Caspary, Hartig). 133 A few words may be added concerning the protoplasmic foundation of the cells which contain proteid grains. 134 It is protoplasm whose water is replaced by oil, the latter occurring in the form of small or large drops. Benzole or ether easily dis- solves it away leaving behind the fine, granular, often very scanty fundamental mass. The oily contents of the fundamental sub- stance is made very beautifully visible by means of alcanna red 1 31 Pfeffer, 1. c., p. 444. 1 32 Xageli and Schwendener, Mikrosk., p. 530. 3 Hartig, Entwicklungsgesch. d. Pflk., p. 109. Pieffer, I. c., pp. 478-4S5. ALBUMINOUS MATTER. PROTEIDS. 385 (Pfeffer). Use a deeply colored, about 70 or 80 per cent, al- coholic extract of alcanna root (see p. 310). Move the seed- section back and forth in this a few times, wash it off in weak alcohol, and put it immediately in strong glycerine for examin- ation. The coloring matter dissolved in alcohol does not pene- trate into the proteid granules in the short space of time in which it comes in contact with them. But the alcoholic solution of the coloring matter adhering to the section, is quickly dissolved by the oil in the fundamental mass with which it comes into such intimate contact.. By its distribution through the funda- mental mass, sections of very oily seeds are soon colored a beau- tiful blood red. When the fundamental substance itself is very deeply colored the proteid grains appear to be isolated, 135 quite colorless. If the oil is very easily dissolved in alcohol, as in the seeds of Hicinus, the alcanna tincture should be diluted with an equal quantity of glycerine. If the cells containing proteid grains be treated with potash solution or potash water after the oil has been removed with benzole or ether, the grains will be dissolved away and the fundamental mass, as well as the envelopes of the granules, will ,be left as a delicate network, which looks often not unlike parenchyma tissues. 136 Iodine solutions color it brown-yellow, Millon's reagent red. It consists of albuminous matter. The nucleus lies shrunken up within. B. Proteid Grains with Crystalloids. 187 A great number of proteid grains contain formed proteid matter, which appears in the form of crystal-like bodies. Har- tig called them "white granules," Nageli named them crystal- loids. They are surrounded by the envelope of the granule (Fig. 135, A, B,) , which may moreover often be almost entirely wanting. Sometimes several crystalloids are found united in one grain (Fig. 135, C, D, after Pfeffer). In oil and alcohol the crystalloids are not usually visible in consequence of the like IBS pfeffer, 1. c., Taf. XXXVIII, Fig. 21. no Pfeffer, L c,, Taf. XXXVIII, Fig. 2. 7 Pfeffer, I. c., pp. 450-iGi. 386 THE MICROSCOPE IN BOTANY. refracting power of the crystalloids and the envelope. In order to make the former visible the grains must be put into water. This either dissolves the surrounding mass of the envelope or makes it swell thus causing the crystalloids to appear. The enveloping mass has the same qualities as the substance of the proteid grains without crystalloids. Of the characteristics of the crystalloids we mention the fol- lowing : The crystal systems are not very thoroughly known. The crystalloids of Bertholletia should, according to Nageli, be clinorhombic ; others belong, according to Hartig, to the tesseral and rhombic systems. Sorauer found, besides these, four-sided columns. All crystalloids, although but slightly, are double refractive. 138 According to Radl- koferthis latter characteristic is increased by coagulation. The angles of the crystalloids are very inconstant ; the ad- dition of water changes them 2 or 3 degrees, swelling about 15 or 16 degrees. According to Nageli the crystalloids, like the starch grains, consist of two distinct substances (see p. 360), according to Maschke, of casein and a little albumen ; according to PfefFer both views are groundless. All crystalloids are insoluble in water ; they can, however, be freed from the fundamental mass by water alone or with the addition of sodium phosphate. They are insoluble likewise in absolute alcohol but, on the contrary, are soluble in glycerine containing potash, in dilute potash as well as in muriatic and acetic acid. After solution every crystalloid leaves behind a little envelope (Fig. 135, E and F, after Pfefier). The crys- talloid should be dissolved by concentrated glycerine with a trace of potash. The envelope gives the same reaction as that of the grain itself. By boiling, the crystalloids are transformed into the insoluble modification of proteid matter. They are then insoluble in dilute potash but will swell considerably in it. 8 Maschke in Bot. Zeitg., 1839, Tab. XV, Figs. 95, 96, 98, 99, 101, 102. ALBUMINOUS MATTER. PROTEIDS. 387 Proteid Crystalloids without an Inclosing Mass. In the proteid grains containing crystalloids the inclosing mass is often quantitatively considerable, but sometimes the crystalloids are inclosed only by a very thin layer. These pro- teid grains are closely related to those crystalloids which are quite uncovered. In the latter case the proteid crystalloids lie free in the cells or in the plasma. A sharply defined boundary between the free lying crystalloids and those inclosed in an integumentary mass can scarcely be drawn. Free proteid crys- talloids frequently occur in resting seeds as well as elsewhere, in phanerogams as well as in cryptogams. We limit ourselves here to the presentation of some carefully studied cases. 1. Crystalloids of the Potato tuber (Cohn). In the cuticular layer of the potato tuber occur numerous cubic crystalloids measuring from 0.007 to 0.013 mm. on a side. Cohn 139 gives the following reactions for these forms which are feebly double refractive. Iodine colors them yellow to deep golden brown y sugar and sulphuric acid a peach blow red, Millon's reagent a brick red, carmine with the least possible ammonia a dee[> red, cochineal extract with water and a subsequent addition of acetic acid (Maschke) intense burning red. They are slowly soluble in glycerine, soluble in ammonia from outward toward the center, in acetic acid from the center outward, and in dilute potassium lye (but not in concentrated ; in this they swell and are stained yellow) . Sulphuric, nitric and muriatic acid dissolve most of the crystalloids or make them swell up to a globular* drop. By boiling in water the crystalloids in general remain unchanged but become more easily visible and show a laminated structure. 2. Crystalloids in Bertholletia excelsa (Hartig, Maschke, Radlkofer, Nageli) . The crystalloids in the endosperm of the Para nut have been frequently described, but the statements con- cerning the reactions produced on them by the several naturalists are wide apart, which Nageli claims is chiefly caused by using the reagents in very different degrees of concentration by which " 9 Colin, 58 Ber. d. Schlea. Gesellsch. f. vaterl. Cultur, 1859, pp. 74-77. 388 THE MICROSCOPE IN BOTANY. the tested crystalloids would present themselves in a very dif- ferent manner. Hartig held the crystalloids to be hexagonal, Maschke tesseral ; Nageli finally made it very probable that they are clinorhombic (Fig. 136, A, B, after Nageli). According to Nageli they do not dissolve in water. Hartig had stated that they were soluble in water, Radlkofer that it took but slight hold of them. By boiling in water they coagulate and are not then soluble in weak alkali (Radlkofer). Alcohol and ether do not alter them even when boiling hot. According to Nageli FIG. 136. glycerine does not dissolve them, only increases their volume. JRadlkofer says that they dissolve in it very slowly. Acetic acid will not dissolve them even in the presence of glycerine. Very weak acids do not change the crystalloids, stronger dissolve them gradually or quickly ; first, however, making them swell up ,(Fig. 136, C, D). In nitric acid they become round and full of vacuoles and in time yellow. Ammonia dissolves them with .less swelling, likewise weak potash, while the concentrated does not dissolve them, only rounds them up. Iodine colors them brown or yellow brown, Millon's reagent red ; coloring matter is energetically absorbed by them. 140 3. Crystalloids in Pilobolus (Klein, Van Tieghem), In the fruit bearers of Pilobolus occur likewise many small crystalloids which were in- vestigated in Pilobolus crystallinus by /$i IA \] ^^ Klein, and in P. roridus by Van Tie- ghem (Fig. 137, after Van Tieghem FIG. 137. and Klein). They are colorless and appear to be octahedral or quadratic pyramidal, and have sides Radlkofer, Krya- Nageli, Sitzungsber. Bayer. Acad., 1862, Bd. II, pp. 128-13 talle proteiuartiger Korper, pp. 65-69. ALBUMINOUS MATTER. PROTEIDS. 389 which are not quite plane. Potash either swells or dissolves them. Iodine colors them yellow or brown. If the iodine is dissolved in alcohol they are shrunken at the same time ; alcohol alone contracts them. Sulphuric acid alone colors them a rose red. It colors the plasma of Pilobolus likewise the same. The long continued action of concentrated nitric acid colors them a pale yellow. 141 4. Crystalloids in Floridia. (Rhodospermin.) (Cramer, Klein, Cohn) . Crystalloids of proteid bodies have been observed in different algae, as, for example, Bornetia secundiflora Thuret, Callithammon caudatum Ag., C. seminudum Ag., Griffithsia barbata Ag., Gr. Nepolitana Nag., Gongocerus pellucidum Ktzg., and designated by Cramer Rhodospermin, since they have commonly been stained by the red coloring matter of the algae and are rose red. Cohn and Klein demonstrated afterwards that the crystalloids in the living plants are mostly colorless. The crystalloids are either simply refractive and belong then to the hexagonal system (needles or plates of from 0.004 to 0.050 mm. long), or they are double refractive, clinorhombic (octa- hedral-like forms of from 0.01 to 0.04 mm. long). Cramer distinguished therefore hexagonal and clinorhombic Rhodosper- rnin. (a) Hexagonal Rhodospermin: insoluble in water, and absolute alcohol (cold or boiling) , glycerine and acetic acid, cold muriatic acid (boiling slowly destroys it), sulphuric acid like- wise and nitric acid. Nitric acid alone does not color it, but does with the addition of ammonia. It is insoluble in dilute and concentrated potash lye, ammonia and cuprammonia. These substances, however, cause it to swell and in boiling potash lye it is slowly destroyed. Iodine colors it first gold-yellow then brown-yellow ; ammoniacal carmine solution colors it red but not essentially different from the surrounding solution ; carmine solution with the addition of cooking salt, on the contrary, an intense red. Sugar with sulphuric acid gives no reaction, (b) Clinorhombic Rhodospermin: Iodine, nitric acid with ammonia behave as with the hexagonal Rhodospermin. In potash and 141 Klein in Pringsheim's Jahrb., Bd. VIII, p. 337. _/F.,uncl 376. Van Tieghemiu Ann. des sc. nai. 6e Ser., t. I, p. 25,/. 390 THE MICROSCOPE IN BOTANY. ammonia it swells but contracts again with nitric, sulphuric or muriatic acid. Millon's reagent colors it a brownish yellow. 142 5. Colored Crystalloids in the pulp of the fruit of Solatium Americanum Mill (Nageli). These occur in the form of thin plates and rhombs (Fig. 138, A, after Nageli) which are fre- quently united (B), belong to the rhombic system and possess an intense violet color. The crystalloids consist partly of albu- FlG. 138. minous substances which are permeated with the violet coloring matter. Water does not alter them but if it be slightly sour or alkaline the tone of the color is changed. Alcohol bleaches them from the inside outward and dissolves most of them, likewise ether. Iodine colors them a brown-yellow. Very weak acids color the crystalloids bright red, strong acids bleach and disintegrate them into separate pieces and finally dissolve them. Potash and boiling water behave alike; essential oils and chloroform are without effect upon the dry crystalloids. 143 C. Proteid grains inclosing Inorganic Substances. As already briefly mentioned there are proteid grains which contain within themselves inorganic substances. These are either globoids, spheroids (Fig. 139, A, after Pfeffer), or crystals (B). The former are a combination of magnesia and lime with a little phosphoric acid, the latter consists of calcium acetate. Frequently spheroids and globoids occur with crystal - 142 According to Cramer, I. c., Cohn in Schultze's Archiv, Bd. Ill, p. 24, /. Klein in Flora, 1871, pp. 161-169. 143 Nageli, Sitzungsber. d. Bayer. Acad., 1862, Bd. II, pp. 147-154; Vgl. auch Nageli in Pflanzenphysiol. Uiiters., Bd. I, p. 6. ALBUMINOUS MATTER. PROTEIDS. 391 loids iu the same proteid grains, but more seldom globoids with crystals. (a) Proteid grains with globoids. The globoids (Kranz- korper of Hartig) have a roundish or cluster-like form (Fig. 139, A) . They occur in almost every seed that, contains reserve proteid matter. The largest (Vitis) attain a diameter of 0.01 mm. In order to investigate globoids (as crystals) first remove the oil from the section of the seed and then dissolve away the proteid substance with water or very dilute potash (Pfeffer) . The globoids are singly refractive, insoluble in cold and boiling water and alcohol, soluble in all mineral acids and acetic acid (without effervescence). They take no color from iodine or aniline blue. They gradually dissolve in an ammo- niacal chlorine-ammonia solution, like- wise in alcohol which contains a little sulphuric or oxalic acid. In the latter case after a considerable time one may find in place of the globoids tiny needle crystals of calcium and magnesium oxalate. Concentrated potassium and am- monia dissolve a substance out of the globoids from the outside inward. They then appear as a finely granulated, feebly re- fractive mass with a cuticular layer which may be stained like proteid matter with iodine and aniline blue. No swelling is produced by the action of the potash. (b) Proteid grains with crystals. The crystals occur as clinorhombic plates, etc., or as a cluster of crystals grown to- gether. They are insoluble in water, and acetic acid ; calcined, the residuum dissolves in the latter with effervescence. They are insoluble in not too concentrated potash lye. (For fur- ther reactions see under section XII, Inorganic Vegetable Ele- ments.) If the crystals are carefully dissolved in muriatic acid, there remains behind a delicate skin consisting of proteid matter, also in the middle something like a nucleus is found. Both can be recognized with certainty when a little iodine is added to the dilute muriatic acid used in the solution. 392 THE MICROSCOPE IN BOTANY. 2. FUNCTIONAL PROTEID MATTER. (Protoplasm and Cell nucleus.) Literature. V. Mohl, Einige Bemerk. iiber d. Bau d. veget. Zelle (Bot. Ztg., 1844, p. 273, /".). V. Mohl, Verm. Schr. Tubing., 1845, a. v. O. V. Mohl, D. Veget. Zelle, Brschwg., 1851, p. 198, ff. Schacht, D. Pflanzenzelle, Leipzig, 1852, a. v. O. Hiirtig, Ueber d. Verf. b. Behandl. d. Zellkerns mit Farb- stoffen (Bot. Zeitg., 1854, p. 877, ff.). v. Mohl, D. Primordi- alschlanch (Bot. Zeitg., 1855, p. 689, /.). Schacht, Lehrb. d. Anat. u. Physiol. d. Gew., 1856, a. v. O. Hartig, Eutwick- lungsgeschicte d. Ptikeims, Lpz., 1858, a. v. O. Maschke, Pig- mentlosung als Reagenz bei mikrosk. physiol. Unters. (Botan. Zeitg., 1859, p. 21, ff.). Raxllkofer, Ueber Krystalle proteinar- tiger Korper, Lpz., 1859, p. 1, ff. Sachs, Ueber einige neue mikrosk.-chem. Reactionsmeth. (Sitzungsber. d. K. Acad. d. Wiss. Wieu, Bd. XXXVI, 1859, p. 9, f.).Ve Bary, Ueber d. Bauu. d. Wesender Zelle (Flora, 1862, pp. 243-251). Sachs, Mikrochem. Unters. (Flora, 1862, p. 297, /".). Schultze, Ue- ber d. Bau d. Nasenschleimhaut (Abh. d. naturf. Gesellsch. zu Halle, Bd. VII, 1863, p. 92). Sachs, Zur Keimungsgesch. d. Graser (Bot. Zeitg., 1862, p. 145, ff.). Sachs, Zur Keimungs- gesch. d. Dattel (id., p. 241, ff.). Schultze, D. Protoplasma d. Rhizopoden u. d. Pflanzenzellen, Lpz., 1863, p. 39, ff.). Cienkowsky, Zur Eutwicklungsgesch. d. Myxomyceten (Pring- sheim's Jahrb., Bd. Ill, 1863, p. 325, ff.). Cienkowsky, D. Plasmodium (id., p. 400, ff.). Sachs, Beitrage z. Physiblog. d. Chlorophylls (Flora, 1863, p. 193, ff.). Sachs, Ueber d. Keimung d. Samens von Allium cepa (Bot. Zeitg., 1863, p. 57,^".). Sachs, Ueber d. Stoffe, welched. Material z. Aufbau d. Zellhaute liefern (Pringsheim's Jahrbuch, Bd. Ill, 1863, p. 185, ff.). De Bary, D. Mycetozoen, Leipz., 1864, p. 41, ff. Kiihne, Unters. iiber d. Protoplasma, Leipz., 1864. Sachs, Handbuch d. Experimentalphysiol. d. PfL, Leipz., 1865, p. 309, ff. De Bary, Morph. u. Physiol. d. Pilze, Flechten u. Myxomyc., Leipz,., 1866, p. 103, /. Nageli u. Schwen- FUNCTIONAL PROTEID MATTER. 393 dener, Mikrosk., p. 529, ff. Hanstein, Ueber d. Organe d. Harz- u. Schleimabs. an d. Laubknospen (Botan. Zeitg., 1868, p. 697, ff.). Dippel, Mikrosk., Bd. II, Brschwg., 1869, pp. 9-18. Schroder, Beitr. z. Kenntn. der Friihjahrsperiode des Ahorn (Pringsheim's Jahrb., Bd. YII, 1869, pp. 283, 314, 325). Sachs, Lehrb., p. 39, ff. Strasburger, Stu- dien iiber Protoplasma. Jena, 1876. Tangl, D. Protoplasm* d. Erbse (Sitzungsber. d. K. Acad. d. AViss. Wieu, Bd. LXXVI, 1877, Decemberheft, Bd. LXXVIII, 1878, Juniheft.) Treub, Quelques rech. sur le role du noyau dans la divis. des cellules veget., Amsterd., 1878. Behrens, D. Xect. d. Bliiten (Flora, 1879, a. v. O.) Schmitz, Unters. iiber Structur d. Protopl. u. d. Zellkerne d. Pflzellen (Sitzungsber. d. uiederrh. Gesellsch. zu Bonn, 1880, p. 159, ff.) Strasburger, Zellbild- ung u.Zelltheilung, Jena, 1880, a. v. O. Johow, Uuters. iiber d. Zellkem der hoheren Monokot. Bonn, 1880. Hanstein, D. Protopl. als Trager d. pflanzl. u. thier Lebensverricht. Heid- elbg., 1880. Reinke, Studien iiber Protoplasma, Berlin, 1881. Tangl, Ueber offeue Communu. zwischeu d. Zellen d. Endosp. einiger Sameu (Pringsheim's Jahrb., Bd. XII, 1881, p. 170, ff.). Detmer, D. Wesen d. Stoffwechselprocesse im veg. Or- ganismus (id., p. 253, ff.). Poulsen, Botan. Mikrochem., p. 52, /. (Trans, p. 63) 144 [E. Pfitzer in Bericht Deutsch. Botan. Gesellsch. I (1883), pp. 44-77.] Functional proteid substances, protoplasm and cell nucleus are found in all living cells, and on account of their constant occurrence are also generally known. Functional proteid sub- stance either fills the cell and has no constant form as a whole (protoplasm), or it has a form and is localized (lying in the protoplasm) and is enclosed in a delicate cuticular membrane (cell nucleus). Protoplasm is not always enclosed in the cellu- lose walls of surrounding cells ; it may also exist in a living form by itself (Amoeba, Plasmodia, Myxomyceta, swarm spores, etc.) It frequently exhibits (free or in cells) charac- teristic appearances of motion. It is seldom represented by a 144 A perfect list of the literature concerning this subject is really impossible. In the present list only those treatises are quoted which describe microscopical methods of re- action. All others, for instance those which treat of the appearances of the movement of protoplasm, are not referred to. 394 THE MICROSCOPE IN BOTANY. horny, hard mass as in resting seeds. In most cases it is per- meated by a greater or less quantity of water and then is plastic, soft and often very like a fluid. In it are almost always small or very small granules (oil drops) by which it gets a granulated ap- pearance. The protoplasmic body is commonly surrounded out- wardly by a solid hyaline ungranulated layer (cuticular layer). Protoplasm is composed chemically, first of all, of the (frequent- ly prevailing) albuminous substances, also of a great number of other combinations (Sachs, Reinkeand Rodewald) ; and lastly, of a small quantity of inorganic incombustible substances. There are often found, temporarily or otherwise, in the proto- plasm, other substances which are afterwards employed either for building the wall of the cell (cellulose builder, Sachs), or which are separated out as substances of secretion (meta- plasm, Hanstein). The nucleus, which, as has been established by the recent investigations of Strasburger, Hanstein, Treub, Schultz and others, plays an important part in cell division, consists of several elements, concerning which one must consult the authors named. All reactions to be hereafter referred to show both in the protoplasm and the nucleus, since in both albuminous sub- stances predominate. Both will be described separately, care being taken that in treating of the nucleus, the methods already given with reference to protoplasm be not repeated. A. Protoplasm, Epiplasm, Metaplasm. 1. Protoplasm in the narrower sense. Substances which absorb water, as absolute alcohol, concentrated glycerine, solu- tion of common salt, absorb the water from protoplasm and cause it to shrink up or contract. It thus draws itself away from the cell wall and commonly assumes an irregular outline. Absolute alcohol, osmic acid, solution of picric acid kill proto- plasm and cause it to stiffen. The more quickly this stiffening or hardening takes place, as, for example, in boiling alcohol, the more perfectly is the original structure preserved (Strasburger, see p. 178). A solution of common salt does not kill the shrunken protoplasm. A cell so treated is " plasniolized." FUNCTIONAL PROTEID MATTER. 395 Plasma contracted by means of alcohol appears to be far less soluble in acids and dilute alkalies than when fresh (v. Mohl). A characteristic quality of dead protoplasm is its ability to absorb a large number of coloring substances. Living proto- plasm does not possess this power as already proved by Hartig, who grew Algce, Lemma, Cham, Hydrocharis in carmine so- lution. The growth was not particularly hindered by the col- oring matter, but the protoplasm and nucleus absorbed not the least trace of the pigment. 145 Furthermore the nucleus has the power of absorbing the coloring matter in a much higher degree than the protoplasm. Grenadier's carmine solution (p. 308) and most of the other carmine solutions as well as the extract of cochineal (see p. 305) can be commended. The resistant enveloping layer of the protoplasm behaves quite negatively towards most coloring substances. 146 Hanstein's aniline solution will be taken up by unchanged protoplasm as a blue-violet. Iodine in the familiar solutions (potassium iodide of iodine, chlor-iodide of zinc, glycerine iodine, and iodine and sulphuric acid), will give a yellow or brown-toned color. The brown shades are the most common and in many cases are very dark. , The alkalies behave toward protoplasm differently according to their degree of concentration, those most in use being potash and soda lyes whose action is much like that of the rest. Con- centrated solution of potash leaves protoplasm entirely un- changed, neither dissolving nor swelling it. M. Schultze 147 therefore recommends strong potash lye as a mounting medium for protoplasmic preparations. Dilute potash solutions on the contrary make protoplasm first transparent and then soon per- fectly dissolves it. Concentrated ammonia fluid clears it up very soon and dissolves it, though sometimes not perfectly till after several hours. Concentrated mineral acids, for example, concentrated sul- phuric acid, have the greatest dissolving power. This acid does not commonly color protoplasm, but if the protoplasm be anhy- drous it becomes rose-red to brown. Sulphuric acid with con- " Hartig in Bot. Zeitg., 1854, pp. 576, 877. i46Tangl in Pringsheim's Jahrb., Bd. XII, p. 174. * 47 M. Schultze in Abhandl. d. naturf. Gesellsch. z. Halle, Bd. VII, p. 92, f. 396 THE MICROSCOPE IN BOTANY. centrated solution of sugar colors every kind of protoplasm rose-red, and moreover this reagent is quite sensitive. First lay the preparation in the sugar solution, then put on a cover- glass and let the acid flow in from the edge. Phosphoric acid changes protoplasm but little ; acetic. acid makes it opaque. Nitric acid, used warm or cold, colors it yellow or brown with the formation of xanthoproteid acid ; by adding potassium or ammonia there will be formed the related xanthoproteid salt which is distinguished by its positive, mostly brown, color. Millon's reagent colors protoplasm brick-red, still it is on the whole not very sensitive. Indol with sulphuric acid colors it a feeble rose-red if at all (Niggl). Copper sulphate with potash (method p. 365) colors all albuminous substances violet, which color will not be changed by continued boiling. By reflected light it is uniformly a dark violet ; by transmitted it plays more into the wine-red. 148 The Plasmodium of the Myxomycetc^ becomes rose-red by sugar and sulphuric acid, and Millon's reagent with iodine yel- low. Alcohol and nitric acid cause coagulation ; in acetic acid the substance becomes colorless and transparent. It liquefies in dilute potash solution, likewise in potassium carbonate which first often somewhat shrinks it. Alcohol, glycerine, chlorate of zinc, iodine and dilute chromic acid leave the marginal layer at first unchanged, but on the other hand quickly contracts the remainder of the plasmodium. 2. Epiplasm. De Bary 150 designates by this name the protoplasmic residuum in the spore sacs of the Ascomycetm which still remains after the spores are formed. It is more strongly refractive than the common protoplasm, has a charac- teristic, homogeneous sparkling appearance, and is very sensi- tive toward iodine solutions, the most dilute of which colors it a beautiful red to a violet-brown. 3. Metaplasm. Under this designation of Hanstein 151 we are to understand protoplasm in which are contained con- siderable quantities of carbo-hydrates, the most important "8 Sachs in Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. XXXVI, p. 9. " 9 De Bary, d. Mycetozoen, p. 41, /. is" De Bary, Morphology und Physiol. d. Pilz. Flectin und Myxonwc., p. 103, /. i Hanstein in Bot. Zeit., 1868, p. 710. FUNCTIONAL PROTEID MATTER. 397 of which are the amyloid-like substances, which sooner or Liter will be separated from it to be applied to the construc- tion of cell walls or as secretions. In many organs of secretion the albuminous substances of the metaplasm are so far thrust into the background that it is with the greatest difficulty they are detected by the reagents commonly used. 152 In the remain- der the albuminous substances are detectable by the previously described methods of reaction. Metaplasm behaves character- istically toward Hanstein's aniline mixture as has already been shown. It is not colored by it, like common protoplasm, blue- violet 153 , but scarlet-red, this color being more fuchsin-red when tannic acid is present in it (see below). B. Cell Nucleus. As has been previously indicated, the cell nucleus gives the same reactions as other proteid*substances. For a long time a considerable number of histological reagents have been employed to make the nucleus itself more clear and bring out all the fine structural relations which had heretofore been indistinct. These reagents are again in use, and especially since the epoch- % making investigations of Strasburger. The unusual activity in the study of the nucleus has brought to light a great number of these reagents. WQ can, therefore, in the following, name but a fe\v of the more important, and for the rest may refer to those works which treat of these matters, and which every phytotomist who would be conversant with the questions of the day must study. Hartig first attempted to stain the nucleus with a carmine solution in water which had absorbed ammonia from the air. He added some drops of metallic quicksilver or iodine solution to it in order to make it keep. 154 He found, furthermore, 155 that the nucleus with nitrate of silver was colored almost black under the influence of light, and that when it was laid first in a dilute solution of ferrocyanide of potassium, then carefully washed out and treated with a dilute solution of ferric chloride it IBS Behrens in Flora, 1879, p. 444, ff. is 8 Hanstein, I. c., Taf, XI, Figs. 17, 23. 4 Hartig in Bot. Zeitg., 1854, p. 877. "6 Hartig, I. c., p. 878. 398 THE MICROSCOPE IN BOTANY. was colored a deep blue. But if we apply Berlin blue direct, the nucleus becomes not blue but a pale, smutty, reddish color. If in addition to this we note that it was known that the nucleus became distinct in acetic acid, and thus was first made visible, but that by the use of concentrated acid it was made to swell, we have exhausted pretty nearly all the histological nucleus- reagents of the older authors. In later times we aim at two things in the use of these rea- gents : (1) fixing the nucleus; (2) rendering it visible by staining. We will particularly consider both. A. FIXING THE STRUCTURE OF THE NUCLEUS. The fixing (or "setting") is done by very dilute organic or inorganic acids. In almost all cases the fixing takes place very quickly so that the preparation fteed remain but a short time in the reagent. Acetic acid works very well and a solution of not higher than one per cent should be used. It then produces no shrinking of the nucleus but its stringy framework comes out very distinctly. By the use of stronger acid the swelling produced makes it again very soon quite indistinct. In place of the acetic one may use formic acid (Retzius). For other cases chromic acid gives excellent service in a |- to per cent solution, some- times even as strong as a one per cent solution. 156 Picric acid may also be employed in different degrees of dilution. Nitric or picro-sulphuric acids are less worthy of commendation since they occasion a considerable shrinking, the preparation becoming much less beautiful than in chromic acid. On the other hand osrnic acid is a very excellent fixing medium used in a one per cent solution. 157 It makes the structure of the nucleus very distinct but in many cases indeed causes it to swell. Strasbur- ger used it, for example, in following out the division of the nucleus in the mother cell of the pollen. He emptied the pollen sack into a 3 per cent solution of sugar and added a drop of 1 per cent solution of osmic acid, when all the relations 8 Strasburger, Zellbildung u. Zelltheilung, pp. 172, 173, etc. 167 Strasburger, I. c., p. 39 und anderwarta, STAINING THE NUCLEUS. 399 came out sharply after some minutes. 158 Fleisch 159 has proposed for a like purpose a mixture of chromic and osmic acid, which according to Fleming fixes it well enough but the structure appears pale and is stained with difficulty. But this objection according to Fleming 160 is obviated if one adds a little acetic acid to the mixture. Then a very beautiful staining with hem- atoxylin, picro-carmine, and gentiana is obtained. Fleming's mixture consists of chromic acid, 0.25 per cent, osmic acid 0.1 per cent and acetic acid 0.1 per cent in distilled water. [Absolute alcohol fixes the protoplasm without contracting it. The section or the whole organ may be plunged into it. Strasburger by putting Spirogyra orthospira in absolute alcohol at different hours of the night succeeded in fixing the division of the nuclei of this alga in their various stages of development so they could be studied the next day very easily. The same observer also retarded the development of the nuclei till morn- ing by placing the plant in a room without heat in November and so could watch the development by daylight and fix them at the most suitable moment. A. B. H.j B. STAINING THE NUCLEUS. By the use of these means the structure of the nucleus is fixed, that is, becomes distinct. We now proceed to stain it. This may be done with the aniline dyes (p. 299,^*.), haematoxy- liu (p. 304), cochineal extract (p. 305), or carmine solutions (which see). Those most worthy of commendation are the fol- lowing. (The methods of preparing these staining media have already been given.) 1 . Staining with Borax-carmine (Strasburger) . 16] The sec- tion commonly needs to lie in the mixture, described on p. 307, but a short time. Concerning the examination and preservation of the specimen see that page. 158 Strasburgev, 1. c., p. 21. For a like purpose Hartig, earlier, treated pollen grains with carmine glycerine twelve to twenty-four hours (Dot. Unters. herausgeg. v. Karsten, 1866, HeitS, p. 249). 9 Fleisch in Arch., f. mikrosk. Anat., Bd. XVI, p. 300. 160 Fleming, Zellsubstancc, Kern und Zelltheiluug, Lpz., 1882, p. 381. 161 Strasburger, 1. c , p. 9. 400 THE MICROSCOPE IN BOTANY. 2. Staining with Beale's carmine (Strasburger) . Com- mended for filamentous algoe. For particular directions see p. 307. 3. Staining with Acetic acid carmine (Fleming). This fluid is suitable only for fresh sections, which sometimes be- come very beautiful in it. 4. Staining with Picro-carmine (Fleming, Treub). Alike commendable for animal and vegetable tissue. The section needs to lie in the fluid but a very short time. Mount in glycerine. 5. Staining with Hcematoxylin (Frey, Strasburger, Flem- ing). Those sections which have been fixed in osmic acid and have been freshly washed are especially to be commended for this staining medium, as they then take up the coloring matter well. If they have lain in alcohol for a long time they color badly. Staining may be done in either a strong or dilute solu- tion ; in the latter case it will require from twenty-four to forty- eight hours. If the section becomes over-colored, alum water or dilute muriatic acid will clear it up ; in the use of the latter the nucleus will be slightly swollen. Make use of the hsematoxylin solution given by Frey or one of the new things recommended by Grenadier. 162 6. Staining with Picro-hoematoxylin. 1 This as I can tes- tify from some experiments of my own is a very superior method and is described by Schmitz as follows. The section of the fresh plant is put in a concentrated solution of picric acid and remains for a shorter or longer time, even over night if neces- sary. In this picric acid solution the protoplasm immediately hardens. By longer continuance in the solution the plasmic part of the cells contracts a very little, but that is in many cases an advantage to the investigation, for by this means the cell membrane becomes much more transparent to the coloring matter of the plasma without itself becoming stained. As a coloring matter for the plasmic body I now almost always use hsematoxylin in an aqueous solution without the addition of 162 Prepare a saturated solution of crystallized haematoxyliu in absolute alcohol and a like one of ammoniacal alum in water. Mix 4 cc. of the former with 150 cc. of the 'lat- ter. Let it stand for a week in the light, filter and add 25 cc. of glycerine and 25 cc. of mythel alcohol. After all the free precipitate has settled the reagent is ready for use. 16 Schmitz, in Sitzungsb. der niederrhein. Gescllsch. zu Bonn, 1880, p. 160. STAINING THE NUCLEUS. 401 alum. I lay the object in water, it having been freed from every trace of picric acid by repeated and careful washings, and add a small quantity of hsem-itoxylin which has absorbed am- monia from the air and so is partly changed into hiBinatein-am- monia. The coloring matter dissolves rapidly in pure water with a red color and gives a solution which gradually darkens and after some time decomposes. After remaining for some time (from one to several hours) in the solution, whose degree of concentration must be chosen, according to the special pur- pose in view, the object should be taken out and washed in water till the wash water remains quite colorless. Then the object will, according to the quantity of the coloring matter used and the length of time given to its effect (this must be tested for each case), be colored blue, in more or less intense shade, either the eliminate bodies of the nucleus alone, or these and the rest of the substance of the nucleus, as also the thicker plasmic bodies, as for example the crystalloids, or the whole of the plasmic elements of the cell ; but the whole cell membrane, starch grains, oil drops and crystals remain almost colorless. The color is best preserved when the preparation is mounted in glycerine, but one must be absolutely sure that not a trace of free acid remains in the specimen. The least particle of acid will infallibly destroy the color in time and very provok- ingly render the most excellent specimens useless. 7. 'Staining with Methyl green. (Strasburger, Fromann, Fleming.) Strasburger 164 uses a one per cent solution of acetic acid which he dilutes with methyl green (commended by Mey- zel). In order to see the form of the nucleus of the pollen mother cell, he puts a young anther in the acetic methyl green solution and breaks it open by pressure. The outcoming con- tents are immediately fixed and the figure of the nucleus be- comes at once beautifully stained by the methyl green. Alas ! that a preparation so made should not keep. 8. Staining with other Aniline coloring substances. Among the many coloring substances here commended may be specially mentioned Saffranin, Dahlia, Gentiana violet, the latter with 164 Strasburger, 1. c., p. 141. 402 THE MICROSCOPE IN BOTANY. acetic acid, as affording very beautiful colors. Aniline prepara- tions are best preserved in dammar varnish or Canada balsam, but they shrink if they are for this purpose previously put in oil of cloves. Fleming 165 , therefore, recommends mounting them m resinous turpentine oil after previously passing through dilute and then absolute alcohol with which one may gradually mix the turpentine oil. \_Pfitzer's Reagent for simultaneous Staining and Hardening. E. Ptitzer has reported a fluid which both hardens and stains vegetable protoplasm.- It consists of the coloring matter, nurrosin, dissolved with picric acid in water or alcohol.] \_(a) To a concentrated solution of picric acid is added a small quantity of an aqueous solution of nigrosin. If the .object to be studied contains much water some crystals of the acid should be added to maintain the strength of the liquid.] [The deep olive-green fluid kills with great rapidity. After some hours' immersion of the object which is to be examined it may be transferred to alcohol, especially if it be desired to dissolve out the chloiophyll, or if the object has to be kept some time. By this means the denser masses of protoplasm are stained pale violet, the chroinatophores darker, while the pyrenoid, nucleoli and other colored parts of the nucleus come .out deeply stained ; thin films of protoplasm and ordinary cellulose membranes are scarcely, if at all, stained; starch .grains not at all. By washing the objects in water after stain- ing instead of in spirits, a gray-blue color is obtained ; trans- ference to strong glycerine makes the color purer. The color comes out best however alter washing in alcohol, treating with oil of cloves and mounting in one of the resins, dammar or Canada balsam] . [To avoid contraction the clove oil may be diluted with alcohol and allowed to concentrate upon the object by evaporation of the alcohol. The watery solution is especially adapted for rapidly killing and staining objects already under the micro- scope.] [(&) Nigrosin and picric acid may also be used in solution in alcohol. The solid acid and nigrosin are left for some time in "* Fleming, L c,, p. 384. IMPORTANT REACTIONS FOR FLUID CONTENTS OF CELLS. 403 ^ ! * ea "* 1 10 | t p X .2 ^ C jfi SS 1 B js 5-1 a tt 3 , s si 3 S * o 3 s c r a h ^ t ^ "o n M li- 1 1 lk 8g| 1 s B B Si) o cs "o c ft| | M c sfg P5 i "3 lit '* K o *" g ft,* P p * w 5 jt So l^p 2 = o "X-S-H c"5 a ^ a 5*2 3 ^^ = S-2 B| MH 5 2 %-s i o 1 35 | "" 5 fi 1 I |! 3 |L O 2 s*s S J2 .z "* pq W Q P -E '5s * 2 g i M C ^ 411 ="? o r^ - P5'3 5 I S f Blue fl CV. |lf [o || IP 5 icolored Ll colored fill S |3 5 ^r a V I s 1 i B '3 a S i 1 2 M a 5 Vegetable (9 1 p-t i ^ J 5, J CO ^ 1 I 404 THE MICROSCOPE IN BOTANY. absolute alcohol ; by this solution the chromatophores and pyr- enoids are less deeply stained ; the colored contents of the nucleus very deeply so.] [Quoted from Jour. Roy. Microscop. Soc., Vol. Ill, No. Ill, pp. 445-6. A. B. H.]. Under the headings III, IV, Y, VI, VII, VIII and IX we have treated those frequently-occurring substances, the contents of cells, which appear to be colorless fluids or very like such. As with the solid framework of the cell (see p. 356) so with these we also tabulate the principal reagents used in their identification (p. 403). X. CHLOROPHYLL (LEAF-GREEN). Literature, v. Mohl, Vermischte Sch. Tub., 1845, p. 352, ff. (Unters. iiber d. Anat. Verhalten d. Chlorophylls). v. Mohl, Ueber d. Bau d. Chloroph. (Bot. Zeitg., 1855, p. 89, ff. ). Bohm, Beitr. z. naheren Kcnntn. d. Chi. (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. XXII, 1857, pp. 479-512). Gris, Recherch. microsc. sur la chl. (Ann. des sc. nat., 4e ser., t. VII, 1857, pp. 179-219). Hartig, Entwicklungsgeschichte d. Pflankeims., Leipzig, 1858, pp. 79-82. Morren, Dtss. sur les feuilles vertes et colorees, 1858. Sachs, Ueber d. Ergeb- nisse einigeneu. Unters. iiber d. Chl. (Flora, 1862, p. 129,^".). Sachs, Ueber d. Einfl. d. Lichtes auf d. Bild. d. Arnylums in d. Chlkornern (Bot. Zeitg., 1862, p. 364, /.). Sachs, Beitr. z. Physiol. d. Chl. (Flora, 1863, p. 193, /.). Sachs, Ueber d. Auflos. ti. Wiederbild. d. Amylums in d. Chlk. (Bot. Zeitg., 1864, p. 289, ff.). Sachs, Handbuch d. Experimental- phys. d. Pfl., Lpz., 1865, pp. 309,/"., 313, ff. (Nageli u. Schwendener, Mikros., Lpz., 1867, p. 496). Micheli, Quelq. obser. sur la matiere col. de la chl. (Arch. d. sc. d. la bibliothe. univ. de Geneve, Mai, 1867). Dippel, Mikrosk., Bd. II, p. 32, ff. Kraus, G., Einige Beob. ttber d. Einfl. d. Licht. u. d. Warme auf die Entsteh. d. Starkeerzeugung im Chlorophyll (Pringsheim's Jahrb., Bd. VII, p. 511, ff.). CHLOROPHYLL (LEAF-GREEN.) 405 Wiesner, Chi. in Xeottia Nidus-avis betr. (Bot. Zeitg., 1871, p. 619). Kraus, G., Z. Kcnntu. d. Chlorophyllfarbst. u. ihrer Verwandten., Sttittg., 1872. Wiesner, Unters. iiber d. FarbstofFe einiger fur chlorophyllfroi gekaltenen Phanerog. (Pringsheim's Jahrb., Bd. VIII, 1872, p. 575, ff.). -Kraus, G., Einige Bemerk. iiber d. Erscheiimngd. Soinmerdiirre unser- er in Baura- und Strauchblatter (Bot. Zeitg., 1873, p. 401, ff.). Briosi,Uebernormale Bild. v. fettart. Subst. im Chlrophyll. (id., p. 529, ff.). Drude, D. Biol. v. Neottia Nidus-avis u. Monotropa hyp. Gottingen, 1873. Kraus, G., Ueber d. Ur- sache d. Farbung d. Epidermis vegetat. Organe d. Pfl. (Flora, 1873, p. 316, /.). Treub, Z. Chlorophyllfrage (id., 1874, p. 55, /.) Wiesner, Bemerk, iiber d. angelb. Bestandth. des Chi. (id., p. 278,^.). Prill ieux, sur la color, et leverdiss. du Neottia Nidus-avis (Ann. des sc. nat., 5eser., t. XIX, 1874, p. 109, ff.). Batalin, Ueber d. Zerst. Chloroph. in d. leb. Organen (Bot. Zeitg., 1874, p. 433, ff.) .Wiesner, Vorl. Mitth. iiber d. Einfl. d..Lichtes auf Entsteh. u. Zerstor. d. Chi. (id., p. 11 6, ff.). Sachs, Lehrb., p. 47, ff. Wies- uer, Welche Strahlen des Lichtes zerlegen bei Sauerstoffzutritt d. Chloroph.? (PoggendorflTs Aiinalen, Bd. CLIl, 1874, p. 496, ft.). Pringsheim, Ueber d. Absorptionsspeetra d. Chloro- phyllfarbst. (Monatsber. K. Acad. Berlin, 1874, pp. 628-659). Pringsheim, Ueber d. natiirl. Chlorophyllmodificationen, etc. (id., 1875, pp. 745-759). Askenasy, Ueber d. Zer- stor. d. Chi. leb. Pfl. clurch d. Licht. (Bot. Zeitg., 1875, p. 457, /*.). Haberlandt, Ueber d. Einfl. d. Frostes auf d. Chlk. (Oesterr. Bot. Zcitschr., 187(5, p. 249,^.). Haberlandt, Ueber d. Entsteh. d. Chi. in d. Keimbl. v. Phaseolus (Bot. Zeitg., 1877, p. 3(U,jf.).--Sachsse, Chem. u. Phys. d. Farbstotto, Kohlehyclrate, etc., Lpz., 1877. Dippel, Einige Bemerk. iiber d. Gemength. d. Chlorophylls, etc. (Flora, 1878. p. 11, ff.). Hoppe-Seyler, Ueber d. Chloroph. d. Pfl. (Bot. Zeitg.. 1879, p. 815,^.). Pringsheim, Ueber d. Lichtwirk. u. Chlorophyll- function in d. Pfl. (Bot. Zeitg., 1879, p. 789, ff.).p Y i^. sheim, Ueber Lichtwirk. u. Chlfunction in d. Pfl. (Monatsber. K. Acad. Berlin, 1879, Juli, 17 pp.)- Pringsheim, Ueber d. Hypochlorin u. d. Bedingungen s. Entst. in d. Pfl. (id, 1879, 406 THE MICROSCOPE IN BOTANY. Nov., 21 pp.)- Flahault, Stir la presence de la mat. verte dans les org. actuellem. soustraits a 1'infl. de la him. (Bull, de la Soc. bot. de France, t. XXYI, p. 249, /.). Pringsheim, Z. Kritik d. bisherig. Grundlagen d. Assimilationstheorie d. Pfl. (Monatsber. d. K. Acad. Berlin, 1881, pp. 117-135). Prings- heim, Ueber d. primare Wirk. d. Lichtes auf d. Veget. (id., pp. 504-535). Pringsheim, Ueber Lichtwirk. u. Chl- funct. in d. Pfl. (Pringsheim's Jahrb., Bd. XII, 1881, p. 288, ff.). Hansen, Gesch. d. Assimilationstheorie u. Chlorophyll- function., Lpz., 1882 (auch Arb. Bot. Inst. Wiirzbg., Bd. II, Heft. 4). Tschirch, Unters. liber d. Chloroph. (Botan. Cen- tral bl., Bd. XI, 1882, p. 107, /".). Wiesner, Bemerk. liber d. Natur d. Hypochlorins (id., Bd. X, 1882, p. 260, /*.).- Piingsheim, Ueber Chlorophyllfunct. u. Lichtwirk. in d. Pfl. (Pringsheim's Jahrb., Bd. XIII, Hett 3; 116 pp.). Schim- per, Ueber d. Gestalten d. Starkebildner und Farbkorper (Botan. Centralb., Bd. XII, 1882, p. 1 75, ff.). Meyer, Ueber Chlorophyllk., Starkbildner u. Farbk. (id., p. 314, ff.). tipectroscopic behavior of Chlorophyll. Brewster, on the color of natural bodies (Transact. Roy. Soc. of Edinburgh, t. XII, 1834, p. 538, ff.) Angstrom, Ueber d. grtine Farbe d. Pfl. (Poggendort's Annalen, Bd. XCIII, 1854, p. 475, jf.), also (Ofversigt af. K. Ventesk. Acad. Forhantll, 1853, p. 246, 2f.). Stockes, Ueber d. Verand. d. Brechbark. d. Lichtes (Poggendorff's Ann., Erganzungsbd. IV, 1854, pp. 217-228). Harting, Ueber d. Absorptionsvermogen d. reinen und un- reinen Chlorophyll fur die Strahlen der Sonne (id., Bd. XCVI, 1855, p. 543,/l). Askenasy, Beitr. z. Kenntn. d. Chi. u. einigerdass. begleit. Farbst. (Bot. Zeitg., 1867, p. 225,/.). Sorby. On a definite method of qualit. analysis of aniin. and vegetable coloring matters by means of the spectro-microscope (Proceed, of the Roy. Soc. of Loud. Vol. XV, 1867, p. 433- 436). Hagenbacb, Unter?. liber d. opt. Eigenschaften des Blattgruns (Poggendorff's Ann. Bd. CXLI, 1870, p. 245-275). Gerland et Rauwenhoff, Rcch. sur la Chlorophyll et quelques- uns de ses derives (Arch, neerland., t. *VI, 1871, p. 97, ff.). Sorby, Various tints of autumnal foliage (Quarterly Journ. of Science, No. XXIV, Jan. 1871, p. 64-77). Kraus, Zur CHLOROPHYLL (LEAF-GREEN). 407 Kemitu. tier Chlorophyllfarbstoffe, Spectralunalyt. "[Inters. Stuttg., 1872. Sorby, On comparative vegetable Chromatol- ogy (Proceed. Royal. Soc. of Loud., Vol. XXI, 1873, p. 442- 483). Pringsheim, Uebcr d. Absorptionsspectra d. Chlfarb- stoffe (Mouatsber. d. K. Acad. d. Wiss., Berlin, 1874, p. 628-G59) . Pringsheim, Ueber natiirl. Chlorophyllmodifica- tionen, etc. (id., 1875, p. 745-759). Pringsheim, Ueber Lichtwirk. u. Chlomphyllfunction in d. Pfl. (Pringsheim's Jahrb., Bd. XII, 1880, p. 408, jf.). 166 [C. Timiriazeff, Coinptcs Kendus XCVI, 1883, pp. 375-6.] Chlorophyll or leaf green very seldom occurs in cells in a pure state, that is, as a dissolved green pigment (Hildebraud, Weiss, Trecul ; the cases are, however, still doubtful because they have not been exactly investigated), but mostly in combination with proteid substances. The latter commonly form grains, more rarely spiral bands (Spirogyra), or star-shaped forms (Zyg- nema). They represent the colorless fundamental substanc3 which is covered or penetrated by the green coloring matter. On account of their prevailing granular form we commonly speak of chlorophyll grains. But the chlorophyll can easily be separa- ted from the fundamental substance (by alcohol, benzole, etc., in which it dissolves) the colorless mass being left without having its form perceptibly changed. It consists as already mentioned in great part of albuminous substances but contains also small quantities of fats, oils, tannic acid and sugar. As is well known the chlorophyll grains arc the seat of the process of assimilation. In them starch is formed, in a manner still unknown, from the elements of carbonic acid and water, as the first visible product of assimilation (in many cases fat-like sub- stances in place of this, Sachs, Briosi) . Till very recently starch has been considered the first visible product of assimilation, but according to the more recent investigations of Pringsheim the first product is a body containing oil, called Hypochlorine, which may be separated by muriatic acid and soon assumes crystal-like strata. It penetrates the whole porous proteid framework of the grain. Sachs, Hansen, Tschirch deny the "6 See also note 144 on p. 334. A perfect list of the chemical literature concerning chlo- rophyll may be found in lliiseinann, p. 24, ff. 408 THE MICROSCOPE IN BOTANY. existence of hypochlorine, asserting it to be the product of the effect of the acid upon the chlorophyll. The investigations of this subject are not yet concluded. That starch may easily be made visible in chlorophyll grains we have already shown (p. 364). In the microscopical investigations of chlorophyll grains they should be so treated as to study either the colorless ground- substance, or the coloring matter itself. We will consider both in their turn. 1. THE FUNDAMENTAL SUBSTANCE OF CHLOROPHYLL GRAINS- The fundamental substance imy be investigated in the gran- ules which are permeated with coloring matter, also and indeed much better in those which have been freed from the , chlorophyll. For the latter purpose the portion of the plant to be investigated or a section of it should be laid in at least 90 per cent alcohol or in ether which will dissolve out the coloring matter and bleach the object. It is not recommended to boil the specimen in water as in preparing a chlorophyll solution since this coagulates the fundamental mass. The bleached grains remain behind quite unchanged in the cells. Those chlo- rophyll grains which contain no starch are best adapted to this investigation (for example, those of Allium cepa Sachs), other- wise one would naturally get the characteristic reaction of starch. Many reactions, however, are uninjured by the presence of starch. Of the microscopical reactions the following may be referred to. 167 The bleached grains take a brick red color from acetic acid cochineal extract (Maschke) : alcoholic iodine solution colors them yellow or dark brown with contraction. Put a section containing the bleached grains in a concentrated solution of copper sulphate for about half an hour, wash and transfer to a strong solution of potash and the grains become a distinct violet. Similar sections somewhat warmed in nitric acid, washed out with water, and treated with potash solution, the 167 Mostly according to Sachs, Flora, 1863, p. 195, ff. THE FUNDAMENTAL SUBSTANCE OF CHLOROPHYLL. 409 chlorophyll grains will either retain their form or be changed into a formless mass. In the former case each chlorophyll grain becomes a distinct orange yellow ; in the latter case the cell is filled with an orange yellow, amorphous mass. Green leaves laid in a concentrated solution of potash for about an hour, the chlorophyll grains remain green and unchanged ; wash with water and the cells will contain a homogeneous mucilage; neu- tralize with acetic acid and add alcoholic iodine and the cells will appear to be filled with a fine granular brownish mass. If the leaves lie for several days in the potash the chlorophyll will run together into a homogeneous layer. Bleached chloro- phyll grains behave under this treatment quite the same only that they are more resistant to the action of the strong al- kali than the green. Ammonia leaves the form in fresh gran- ules quite distinct, the substance only becoming somewhat filled with spaces and vacuoles. After washing they are still almost exactly the same green ; neutralize with acetic acid and add alcoholic iodine and the granules appear sharply defined, some- what contracted with vacuoles, brown. In other cases they are less resistant toward ammonia. Phosphoric acid makes fresh chlo- rophyll grains yellow, but does not change the bleached grains at all. The gi-een as well as the bleached grains are much more resistant toward sulphuric acid than is either protoplasm or the cell nucleus ; the green ones become either verdigris or blue green. Cold acetic acid colors green chlorophyll grains clear yellow but leaves their form unchanged ; by boiling in it they be- come knotty. We infer from all these reactions that the fundamental sub- stance of chlorophyll grains belongs to nitrogenous matter. It is a "protoplasmic form" (Sachs). According to Pringsheim, 168 treating chlorophyll grains with dilute muriatic acid (the best is one part acid to four parts water) for a long tinie they become a yellow-green, gold yel- low or brownish. After a longer time (several hours) their dark, reddish-brown or rust-colored periphera separates from the rest of the substance of the sharply defined mass of the chlorophyll grain. This becomes afterwards distinctly angular, , las Pringsheim's Jahrb., Bd. XII, p. 294. 410 THE MICROSCOPE IN BOTANY. pointed and forms a more or less extended scale or nest of in- distinct crystal-like forms which throw out oblique and pointed projections. They are Hypochlorin and correspond to a mix- ture of oil and resin substances. They are insoluble in water and dilute acid ; on the other hand they are soluble in ether, benzole and sulphuretted carbon, and vaporize at about 50. 2. CHLOROPHYLL COLORING MATTER. 'Notwithstanding the many investigations into the nature of the coloring matter of chlorophyll it is not yet satisfactorily known. By the latest investigations it appears to be established that crude chlorophyll (Rohchlorophyll, Wiesner) consists of at least a yellow and a green coloring matter (chlorophyll in the strict sense). The chemical composition of the green col- oring matter is, according to Gautier, C = 73.97, H = 9.80, O = 10.33, N = 4.15, incombustible elements = 1.75. With this essentially agrees the analysis of Rogalski, 169 while those of others are very different. Essential chlorophyll is accord- ing to Gautier 170 a crystal I izable body ; he obtained clinorhombic crystals about cm. long of soft consistency and intense green color which in the light became yellow-brown, yellowish or brownish and afterwards quite colorless. In the study of chlorophyll coloring matter we must investi- gate its chemical reaction, as well as its optical (spcctroscopic) behavior. A. Behavior toward Reagents. With the exception of a yellow or greenish decomposition product 171 which crude chlorophyll often forms in water and is soluble in that, it is insoluble in cold or boiling water as well as in dilute acids or alkalies. On the other hand it is soluble in alcohol, sulphuretted carbon, ether, benzole (Kraus), in many oils and in turpentine (Wiesner). In order to prepare an alcoholic solution of crude chlorophyll the part of the plant 169 Cf. Husemann, 1. c., p. 251. 170 Gautier in Comptes Kendus, LXXXIX, p. 861, ff. 17 1 Pringsheim inHouatsber. d. K. Acad. d. Wiss. Berlin, 1875, p. 748. CHLOROPHYLL REACTIONS. 411 to be used, preferably leaves, should be (according to Kraus) 172 put in hot water and boiled once or twice, the water poured off and boiling alcohol of 95 per cent (sp. w. 0.816) poured on. If alcohol of 83 per cent be used cold, the parts of the plant containing oils, wax, etc., will not enter into the solution (Gautier). The alcoholic crude chlorophyll extract should be fresh when used in investigations, although it is much less decomposable if the leaves have been previously boiled ; apparently this manipulation removes the salts and other im- purities from the leaves (Stockes, Kraus). The chlorophyll solution thus prepared is of a beautiful green color and has a dark red luster. It represents a mixture of colors which can be easily separated as Kraus 173 has indicated into a green and a yellow part. Add to an alcoholic extract of crude chlorophyll a like quantity of benzole, vigorously shake it up and leave the mixt- ure a short time to itself, and the alcohol and benzole will again separate. The under fluid is now a yellow-colored al- cohol, the upper a green-colored benzole. 174 By this process the crude chlorophyll is separated into a yellow alcoholic part, xanthophyll (Kraus) and a green benzole part, kyanophyll (Kraus). According to "\Viesner, 175 instead of the benzole one may use fatty oils (linseed oil, nut, poppy, olive oil) essen- tial oils (turpentine, rosemary, gaultheria oil) or sulphuretted carbon. Kraus and others therefore held that the yellow coloring matter (xanthophyll) and the blue green (kyanophyll) together represent chlorophyll ; that they are both components of the same green coloring substance. According to the investigations of Pring- sheim and Wiesner it appears, however, that the kyanophyll of Kraus is relatively pure chlorophyll, but that the xantho- phyll of Kraus consists of yellow modifications of chlorophyll 172 Kraus, Chlorophyllfarbstofle, vj.23. 173 Kvaus, I. c., p. 87, ff. The objections raised by Konrad (Flora, 1872, p. 396, /.) rest on insufficient experiments and have already been duly confuted by Wiesnev (Flora, 1874. p. 284, /.) 174 Concerning the behavior of benzole toward alcohol of various percentages, see Pringsheim in Monatsber. K. Acad. Berlin, 1874, p. 648, /. Wiesner in Flora, 1874, p. 282, /. 412 THE MICROSCOPE IN BOTANY. whose relations to crude chlorophyll are not fully established, but which as such occurs independently in it. (a) Benzole Chlorophyll (Kyanophyll, Kraus). The chlo- rophyll procured from alcoholic solution of crude chlorophyll by the use of benzole is a beautiful green with a distinct shade of blue. It has a strong red luster, a much stronger, more car- mine red luster than the crude chlorophyll solution. It is very sensitive to acids, the least trace being sufficient to change the beautiful green to a smutty yellow brown or bronze-green (Kraus). If the separation is produced by sulphuretted carbon or the above named fatty or essential oils the solution is full green and has a strong red luster. A saturated solution of chlorophyll in pure olive oil or sulphuretted carbon is a deep, almost a black-green color, and appears dark-red by reflected diffused daylight. It will keep a long time in the light if the oxygen is excluded from it ; also in the dark, even if oxygen is admitted ; but by the admission of oxygen to it in the light it rapidly loses its color (Wiesner). (b) The Yellow, Alcohol Part (JKantliopliyll, Kraus) is according to Kraus a pure gold yellow and shows no trace of fluorescence (also Gerland and Rauwenhoff, Filhol). Accord- ing to Pringsheim it has a touch of green shade and is distinctly fluorescent if one examine it with a condensing lens in direct sunlight. Evaporated to dryness there remains a deep yellow- brown, sticky hydroscopic mass, which may be dissolved again in alcohol, ether, benzole and carbon sulphate, but not in water. If sulphuric or muriatic acid be added to the yellow solution it will remain yellow for a short time, then become emerald green, verdigris green and finally a beautiful indigo blue. Organic acids do not apparently alter the solution. In the sunlight it gradually bleaches out after several days. Ac- cording to Kraus the gold-yellow alcohol solution is identical with the yellow coloring matter of etiolated plants. According to the investigations of Pringsheim 176 and Wiesner it is very probable that the yellow alcohol portion of a solution of crude chlorophyll is a mixture of one or more yel- i7Q Pringsheim in monthly report of the Imperial Acad. Berlin, 1874, p. 628, ff. THE SPECTROSCOPIC BEHAVIOR OF CHLOROPHYLL. 413 low modifications of chlorophyll with a little chlorophyll. It first appears independently in the crude chlorophyll and is no preexisting component of it. Pringsheim investigated three yellow modifications of chlorophyll, viz., etiolin, xanthophyll and anthoxanthin. Etiolin is the coloring matter which is formed by etiolated growths breathing in the darkness. It is a yellow modification of chlorophyll having a red fluorescence, soluble in alcohol, ether, benzole, and carbon sulphate but insoluble in water. Its solu- tion becomes by the addition of muriatic or sulphuric acid, first verdigris green and afterward blue. Xanthophyll (in Pringsheim's sense) is the yellow coloring matter of autumn leaves. It behaves towards the before men- tioned dissolving media quite like etiolin, but becomes emerald green not a bine, by the addition of muriatic or sulphuric acid. The yellow coloring matter of autumn leaves arises from a pro- cess of decomposition in the crude chlorophyll. If the coloring mutter of the yellow xanthophyll grains be extracted by alcohol the grains remain behind in their original size. These are but gradually affected by concentrated sulphuric acid but boiling potash changes them to a greasy brown mass. 177 Anthoxanthin is the yellow coloring matter of yellow flowers and fruit. It will be described further on. Which of the two first yellow modifications of chlorophyll occurs in the crude chlorophyll, whether etiolin or xanthophyll, or both together, cannot be previously determined. B. Spectroscopic Behavior of Chlorophyll. The optical characteristics of a solution of chlorophyll have been frequently investigated, since Brewster first directed atten- tion to the subject, and himself observed its most important phenomena, which led him to views that proved the incorrect- ness of some of the statements of Newton conceining the nature of light. Not to mention that he first observed the fluorescence of a solution of leaf green, he discovered its dichromism also, 177 In many cases, however, before the appearance of the xanthophyll the chlorophyll grains pass into a beautiful green amorphous mass (Sachs, Flora, 1603, p. 202.) 414 THE MICROSCOPE IN BOTANY. that is, the quality by which a thin layer of it gives an absorption color of green and a/ thicker one of red. He first also observed the dispersive power of chlorophyll for red light (carefully investigated, later, by Stockes), and finally the pe- culiar absorption spectrum of leaf green. Of the latter, to which we here exclusively devote our attention, special and exact investigations were subsequently made by Askenasy, Kraus, Pringsheim, and others. It has been shown by these naturalists that the chlorophyll spectrum may be employed under all circumstances for the recognition pf chlorophyll and its modifications, that also a spectro-analytic investigation of chlo- rophyll is possible, which has been much employed in the most important studies of later times. B C D E TJ j 100 \ 200 300 \ \iOO 500, 600 700 800 \ 900 1000 VL Vff The absorption spectrum of chlorophyll shows seven dark bands, which correspond to the places of maximum absorption. The seven absorption bands are counted progressively from the red towards the blue and are designated by the Roman numerals I to VII. I to IV lie in the anterior part of the spectrum in the region of least refraction, between the Fraunhofer lines A and E (bands of the first half of the spectrum), V to VII in the posterior part or region of most refraction (bands of the second half of the spectrum). Bands I to IV are easily per- ceived in solutions of chlorophyll of medium concentration. Bands V to VII often give a continuous absorption, but they always appear with a sufficiently weak concentration of the solution. Fig. 140 represents an absorption spectrum with all THE SPECTROSCOPIC BEHAVIOR OF CHLOROPHYLL. 415 the bands, Fig. 141 a spectrum with a continuous absorption in place of bands V to VII (after Kraus). The visibility, the intensity, the relative distance apart (with- in certain limits) of the band, are dependent on certain different factors, namely : The concentration of Ike solution, or what is equivalent the thickness of the layer of coloring matter the optical concen- tration, conditions the number and the intensity of the bands, and secondarily also their position. The dissolving medium conditions at the same time lesser variations iu the relative distance apart of the bands, as well as the rapidity or tardiness of the development of the absorption. The kind of chlorophyll modification has no influence upon the position of the maximum and minimum of the absorption, no. Hi. but rather upon the slower or more rapid development of the absorption within the single absorption bands. We will consider next the spectrum of a normal alcoholic solution of chlorophyll. The anterior half of the spectrum is produced by a medium, the posterior by a weaker concentration of the solution. Fig. 140 after Kraus. Band I, deep black, both edges sharply defined ; lies between the Fraunhofer lines B and C in the red. Band II, less black, very dark brown however abruptly shad- ing out toward both sides, exactly in the middle between C and D in the orange. Band III, for the most part much less dark than II, penum- brated toward both sides, in the yellow close behind the sodium line D. Between II and III a slight lessening of the light. 416 THE MICROSCOPE IN BOTANY. Band IV, very slender, weak, often scarcely visible lying before E in the green, the green behind it obscured. Band V, broader than I, almost black in the middle, both sides shading out, lying in the light blue portion exactly be- hind F. Band VI, broader than V, almost black in the middle, both sides broadly shading out, lying in the indigo, beginning in the middle between F and G and ending at G. Band VII, corresponding to the whole of the remaining violet end of the spectrum. FJG. 142. That the spectrum of the alcoholic solution of the chlorophyll, here described, appertains to the coloring malter as such, that the chlorophyll extract has not been subjected to a fundamental decomposition before it is applied to the investigation, have been demonstrated by Kraus, by the fact that chlorophyll within the living plant produces similar or identical spectra. The spectrum of a single chlorophyll grain (Fiir. 142) has the appearance of a luminous spectrum through which a dark 33 C T> looo FIG. 143. line is drawn, which is interrupted in the red and yellow. The darkening between B C corresponds to Band I. That which begins behind b and runs through the whole posterior part of the spectrum corresponds to the total absorption of the bands v-vn. The spectrum of a living leaf (Fig. 143 of Deutzia scabra THE SPECTROSCOPIC BEHAVIOR OF CHLOROPHYLL. 417 after Kraus) is not essentially unlike the spectrum of the solu- tion. The leaf to be investigated is put on the microscope stage and the objective shoved down till it touches it. By suit- able magnification one may now recognize bands I to IV very distinctly not altered in their relative position. Band V is also sharply visible, while VI and VII are mingled in a single ab- sorption. If a double layer of leaves is used, band VI will blend with the total absorption of the posterior half of the spectrum. If an alcoholic solution of crude chlorophyll, of whose spectrum we have hitherto been speaking, be mixed with ben- B C E b i n in iv FIG. 144. zole, the benzole portion (pure chlorophyll, kynnophyll of Kraus) will give a spectrum quite like the other, with this dif- ference that the relative distances apart of the bands and the relative breadth of the same will have undergone some alter- ation. Kraus held this to be a characteristic of his kyanophyll, but according toPringsheim, the reason for it is to be sought in the different influences of the media of solution. 178 If by the effect of light or oxygen or of other agents (acids, etc.) a decomposition of the chlorophyll takes place, the pro- "s For particulars see Pringsheim (Monatsber. Berl. Acad., 1S74, p. 62S. ff.} especially also the simple and double dividing of band I of the benzole solution of chlorophyll in certain degrees of concentration. 27 418 THE MICROSCOPE IN BOTANY. duct of the decomposition gives a very different spectrum from that of the original coloring matter (see concerning this, Kraus, I.e., p. 68, /I)- By the investigations of Pringsheim it has been established that the spectrum of chlorophyll solutions of different thick- nesses shows certain highly characteristic changes which may be best seen in Fig. 144, copied from Pringsheim. The horizontal divisions, a to i, represent the spectrum of a single degree of dilution (or thickness of layer) of an alcoholic chlorophyll solution ; a is a layer of tlis solution 10 mm. thick, i that of a like concentration 374 mm. thick. The other values are apparent from the illustration. The spectrum is divided, accord- Ing to the fundamental scale of Sorby and Browning, into 100 or 1,000 parts. B, (7, A etc., give the position of the Fraun- hofer lines ; I to VII designate the absorption bauds. 1000 FIG. The spectrum i shows the bands I to IV very clearly, while V to VII run together into a single absorption. The spectra h, <7, /"are like this, only that bands I, II and IV become more narrow, and the absorption of the second half 'of the spectrum is drawn back more toward F. Spectrum e is distinguished by the almost total disappearance of baud III and the coming out distinctly of band V. In d, band III has altogether disap- peared, and II and IV become almost entirely clear, and V, VI and VII clearer. In c of the anterior bands only I is still to be clearly seen ; but V, VI and VII are coming to be more distinctly perceptible. Finally, in b and a, all the bands but I appear no more. Baud I is thus the most persistent, and, ex- cept that it becomes gradually narrower it remains quite uu- THE SPECTROSCOPIC BEHAVIOR OF CHLOROPHYLL. 419 changed. It has, therefore, a special importance for the recog- nition of very dilute or much modified solutions and may be designated as the characteristic chlorophyll band. The spectrum of etioliu is apparently very different from that of chlorophyll in weaker concentrations. It shows no absorp- tion bands in the anterior half (Fig. 145, -after Kraus), while beyond the line F are seen three absorption bands correspond- ing to V, VI and VII of the chlorophyll spectrum, the spaces between which are shaded. It was formerly supposed that the coloring matter of etiolized plants would not generally pro- E b iv FIG. 146. dace the bands I to IV, but Pringsheim has shown that a layer of etiolin sufficiently thick would afford a spectrum which es- sentially agrees with that of chlorophyll (Fig. J46 is con- structed in the same manner as Fig. 144). The essential distinction lies in this, that bands I to IV are not so strongly pronounced, and appear only when using thicker layers of the solution. So also number II is divided into two bands, a, b, and the position of the bands in the blue is somewhat altered. Etiolin stands therefore optically very near to chlorophyll. The spectrum of xanthophyll in Pringsheim's sense is much more variable. It shows only the three bands in the blue, and it 420 THE MICROSCOPE IN BOTANY. is still uncertain if even with a thickness of 370 mm. band I in the red really exists. But if the solution be much concentrated by evaporation and then tested in that thickness, there appears quite a distinct dark, but slender band I from the lithium line to near (7, and one beginning at E and from b on becoming a very dark absorption. On the other hand bands II, III and IV have never been made to appear. The absorption appearances in the spectrum, especially as they are produced by layers of fluid of different thicknesses, may be graphically expressed by the form of curves, the so-called ab- sorption curves. Askenasy 179 first employed them. He drew a curve from the spectrum of a layer whose ordinates stood in relation to the intensity of the darkening, so that the maximum of the curve corresponded with the maximum of the darkening. Without photometric apparatus this method must, however, lead to very arbitrary, or at least subjective results. It has, therefore, been but little used. Another method introduced by Pringsheim 180 aims at the graphical representation of the maxima and minima of absorp- tion. It brings the whole course of the absorption before the eye. See Figures 144 and 146. Theabscissa axis is divided into 100 or 1,000 parts, correspond- ing to the scale of the spectroscopic measuring apparatus, when the Browning scale is the standard, or directly in wave lengths in hundred thousandth parts of a mm. when the scale of Ang- strom 181 is the standard. The ordinate axis gives the optical concentration (height of fluid layer in millimeters). There is obtained in this way a coordinate system in which the observed absorption bands may be directly registered. For the better locating of points, the position of the Fraunhofer lines, B, (7, Z), JE, b, jp, 6r, should be designated above the abscissa line, [Distribution of energy in the chlorophyll spectrum.] [C. Timiriazeff* points out the intimate relationship between the absorption of light by chlorophyll and the intensity of the 179 Askenasy in Botan. Zeitg., 1867, Taf. V. iso Pringsheim in Monatsber. d. Berl. Acad., 1875, p. 795. i Cf. Nebelung in Bot Zeitg., 1872, Taf. XI. * In Comptes Rendus, I. c., pp. 375-6. THE COLORING MATTER OF FLOWERS. 421 chemical phenomena produced, the curves of absorption of light and of the decomposition of carbonic dioxide presenting an almost exact concurrence. This last function may be consid- ered as dependent on the energy of radiation, as measured by its effect on the thermo-pilo. Langley has definitely fixed the position of maximum energy in the solar spectrum to be in the orange, exactly in that part which corresponds to the character- istic band of chlorophyll between B and (7.] [It follows, therefore, that chlorophyll maybe regarded as an absorbent specially adapted for the absorption of those solar rays which have the greatest energy, and its elaboration by the vegetable economy is one of the most striking examples of the adaptation of organized beings to the conditions of their en- vironment.] [Under the most favorable conditions 40 per cent of the solar energy, corresponding to the rays of light absorbed by the characteristic chlorophyll bands (see pp. 153 and 413^*. ) is trans- formed into chemical work. Chlorophyll therefore constitutes an apparatus of great perfection capable of transforming into useful work 40 per cent of the solar energy absorbed.] [Quoted from the Journal of the Royal Microscopical Society, Vol. Ill, No. Ill, p. 390. A. B. H.] XL THE COLORING MATTER OF FLOWERS. Literature. Marquart, Die Farben der Bliiten, Bonn, 1835. Bohm, Physiol. Unters. ii. blaue Passiflorabeeren (Sitzungs. d. K. Acad. d. Wiss., Wien, Bd. XXIII, 1857, p. 19, /.) Wigand, Einige Satze iiber die Bedeut. d. Gerbstoffe u. d. Pflanzenfarben (Botan. Zeitg., 1862, p. 121, /".). Wiesner, Einige Beobacht. iiber Gerb- und Farbstoffe d. Blumenbl. (id. p. 389, ff.). Hildebrand, Auat. Unters. iiber d. Farben d. Bliiten (Pringsheim's Jahrb.,Bd. Ill, 1863, p. 59,/".). Weiss, Unters. iiber d. Entwicklungsgeschichte d. Farbstoffes in Pflzellen (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. LIV, 1 Abth., 1866, p. 157,/ 1 .). Nageli u. Schwendener, Mikrosk., p. 500, ff. Kraus, Zur Kenntn. d. Chlorophyllfarbstoffe, etc., Stuttg., 1872. Kraus, D. Entsteh. d. Farbstoffkorper in den 422 THE MICROSCOPE IN BOTANY. Beeren von Solanum Pseudocapsicum (Pringsheim's Jahrb., Bd. VIII, 1872, p. 131, /*.). Wiesncr, Unters. iiber d. Farb- stoffe einiger fiir Chlorophyll frei gehaltenen Phanerog. (id. p. 575, ff.). Pringsheim, Ueber d. Absorptionsspectra der Chlo- rophyllfarbstoffe (Monatsber. d. K. Acad. d. Wiss., Berlin, 187|, p. 628, ff.). Pringsheim, Ueber natttrl. Chlorophyll- mocnficationen, etc. (id. 1875, p. 745, ff.). Borscow, Notiz iiber d. Polychro'ismus einer alkohol. Cyaninlosung (Bot. Zeitg., 1875, p. 351). Holstein, D. Schicksal d. Anthoxanthin- kornerin abbliih. Blumenkr. (Bot. Zeitg., 1875, p. 25, ff.). Flahault, Sur la form, des matieres colorantes dans les vege- taux (Bull, de la Soc. bot. de France, t. XXVI, 1879, p. 268, ff-)' The coloring matter of floral leaves and colored pericarps is still much less perfectly known than chlorophyll and its related substances. Like these it always appears as a part of the cell contents, never united with the membranes. Either it is dis- solved in the cell sap and so represents a fluid, or it is united with variously formed granular structures, of probably proto- plasmic nature. Dissolved it constitutes chiefly the blue violet and rose red colors ; united with granules it is yellow, orange and green. To both cases there are, however, exceptions. These colors together often produce mixed colors. 182 Since the in- vestigations by Marquart of the colors of flowers we desig- nate the dissolved blue and red pigment as Anthocyan, 183 the yellow and orange colored as Antboxanthin. Frerny and Cloez assumed three kinds of flower coloring matter, viz. Cyanin, the blue pigment, probably identical with Marquart's Antho- cyan, red coloring matter is a modification of it. Secondly, Xanthein, a yellow coloring matter soluble in water, thirdly, Xanthin, a yellow coloring matter insoluble in water, which contains a considerable quantity of fatty matter and is solu- ble* in ether and alcohol. Identical with Xanthein is probably the Lutein of Thudichum and the Anthochlor of Prantl. Ac- cording to Pringsheim, the separation of the yellow pigment is inadmissible both being according to him Anthoxanthin, a * 82 For this see Hildebrand, I c. 183 The red might also be especially set off as erythrophyll. THE COLORING MATTER OF FLOWERS. 423 modification of chlorophyll similar to etiolin and xanthophyll (see p. 413,/.). A. Anthocyan, Cyanin, soluble in alcohol and ether. If the dissolving medium be evaporated, the blue coloring matter may be again taken up with water. Acids color the pigment red or violet, alkalies change the red again to blue, violet or yellow green. According to Wiesner 184 anthocyan never is col- ored green by the use of alkalies. Where this is the case the green color is given by the presence of tannin which is colored yellow with alkali and gives the green color with the blue an- thocyan (Nageli and Schwendener, and Sachsse controvert this). Spectroscopically anthocyan has not been very carefully tested The blue modification shows an absorption beginning at D and continuing to F, the violet a weak one at />, and a larger one tit the blue end of the spectrum, the red an absorption band in green and blue to F, and an end absorption beginning at G. B. Anlhoxanthin (including xanthein, lutein, anthochlor). It occurs mostly in connection with proteid substances (rarely in oily substances). It is not distinguished from the colorless fundamental substance of the chlorophyll granules. With few exceptions it is insoluble in water, soluble in alcohol, ether, benzole and other media for dissolving chlorophyll. These solutions are colored blue with acids and are faintly fluorescent. According to Hanstein anthoxanthin granules are changed by the fading of the floral crown by being transformed into a yel- low quite homogeneous mass. Pringsheim 185 has shown that the spectrum of anthoxanthin itself is not essentially different from that of chlorophyll (Fig. 147). An alcoholic solution of coloring matter in thin layers shows only bands V, VI, VII, which soon run together into a continuous terminal absorption. By further increasing the con- tents of the coloring matter, first, band I, then II and IV, and at last baud III, make their appearance. Anthoxanthin shows spectroscopically all the essential marks of chlorophyll and is to be regarded as a modification of it. is* Wiesner iti Bot. Zeitg., 1862. p. 389. iw Monatsber. d. K. Acad., Berlin, 1874, p. 638, ff. 424 THE MICROSCOPE IN BOTANY. If the yellow coloring matter of the flowers occurs as a solu- tion in the cells, it may be extracted by water, and colored brown yellow by the addition of potash lye. Those peculiar coloring substances which occur in pericarps and elsewhere, mostly connected with granules, and which have been investigated by Bohm, Weiss and others, are probably to be regarded as flower pigments. The blue granules in the fruit of the Passiflora are soluble, according to Bohm, in water, alcohol and ether, both cold and boiling. Potash changes their color to yellow brown. Acids and alkalies dissolve thorn. B C II III IV FIG. 147. Weiss has given the reaction of numerous granules of that kind, the most important of which are the following : Orange colored granules. Iodine colors them green ( Oucurbi- ta, Aeschinanthus, Canna, Gazania, Lilium, Hemerocallis, Cap- sicum) , or blue green ( Gaillarda) , potash does not alter the color (Cucurbita, Aschinanthus, Gazania) but dissolves them, some- times when applied dilute (Aeschianthus) , sulphuric acid colors green-yellow (Lilium), nitric acid first light blue then the color disappears (Lilium). Yellow granules. Adonis: iodine does not alter them, like- wise benzole, potash bleaches them somewhat. Tydaea: iodine colors yellow-green, potash is without effect. ASPAKAGIX. 425 Carmine red granules. Lycopersicum : iodine colors them green to yellow-green, potash leaves them unchanged. Col- umnea: iodine as well as sulphuric and muriatic acid leaves them unaltered, but coagulates them. Nitric acid makes them a vermillion red but does not coagulate them. Chlorine water is without effect. Violet granules. Convallaria : iodine colors red, acids destroy the color. Solanum melongena: iodine colors gold yellow, potash blue. Blue granules: iodine does not color them, but potash does a beautiful green (Delphinium) . These coloring substances arise from the transformation of chlorophyll. Plasmic forms are their fundamental substance. They give double refraction. XII. ASPAEAGIN. Literature. Piria, Rech. sur la const, chim. de 1'asparagine, etc. (Ann. de chim. et de phys., 3e ser., t. XXII, 1848, p. 160, f. ; abgedruckt aus II Cimento, Jan., 1846). Pasteur, Noirvelles rech. sur les relations qui peuvent exister entre la forme cristalline, la comp. chirn. etc., (id., 3e ser., t. XXXI, 1851, p. 70, ff.) Hartig, Eutwicklungsgesch. d. Pflkeims., Leipzig, 1858, p. 128, /. Pfeffer, Unters. fiber d. Protein- korner u. d. Bedeut. d. Asparag. beim Keimen d. Samen (Pringsheim's Jahrb., Bd. VIII, 1872, p. 530, jf.). Pfeffer, Ueber d. Bezieh. d. Lichtes z. Regen. v. Eiweissst. aus demb. Keiniungsprocess gebild. Asparagm (Monatsber. d. K. Acad. d. Wiss., Berlin, Dec., 1873, auch Bot. Zeitg., 1874, p. 249, ff. ; ubersetz in Ann. des sc. nat., 5e ser., t. XIX, 1874, p. 371,- /I) Pfeffer in Tagebl. d. 46. Yers. dtsch. Naturf. u. Aerzte z. Wiesbaden, Sect. Bot. (Bot. Zeitg., 1874, p. 236). Pfeffer, D. Bild. stickstoffhalt. Subst. in d. Pfl. (Jahrb. f. Land wirthsch., Bd. Ill, 1873, p. 437, /".). Gorup-Besanez, AVeitere Mitth. liber d. Auftret. v. Leucin neben Asparagin, etc. (Bot. Zeitg., 1874, p. 379, f.). Borodin, Ueber d. phy- 426 THE MICROSCOPE IN BOTANY. siol. Rolle u. d. Verbreit. d. Asparagins imPflreiche (id., 1878, p. 801, jf.)- 186 Asparagin (C 4 H 8 N 2 O 3 ) is, it would seem, a widely distrib- uted substance in the vegetable kingdom. It was first pre- pared from shoots of asparagus and was named from that plant- It is found in the milk-sap of the sprouts, stems, root-tubers, fruit and seeds. Examples of its occurrence are furnished by Convallaria, Paris, Ornithogalum (roots, weeds), germs, seeds, roots and the stems of numerous Papilionacece which have been grown in the dark, potatoes, althea root, seeds of Castanea, small shoots of the hop, sprouts of Tilia, Syringa, Sambucus, Quercus (Borodin). Especially in the germs of Lupinus lu- teus it occurs very plentifully. It is found in the living plant always in a state of solution. It was first microscopically ob- served by Th. Hartig and given the name "gleiss" (glister). In respect to its physiological function there exist two oppo- site and incompatible theories. According to one (Pfeffer) asparagin is a transitional form between the proteid-reserve substances of the seed and the living albumen of the developing plant which aids in the transference of the nitrogen. It is produced from proteid substances and is again transformed into them. But its disappearance stands in intimate connection with the disappearance of sugar, and the presence of that is necessary also to its formation. According to the other theory (Hartig, Borodin) asparagin is the form under which generally albuminous substances pass from cell to cell. ("The ' gleiss ' crystal is to a certain e'xtent the sugar of aleuron," Hartig). Proteids, not carbo-hydrates, are decomposed in the formation of asparagin which again is changed back into albumen. This ex- plains the fact that the plant is always poor in albuminous sub- stances when asparagin occurs. Asparagin crystallizes easily with one atom of water in limpid, transparent orthorhombic columns, twin forms often appearing. Fig. 148, A^ JB, represents two forms of asparagin crystals which have been many times observed (macroscopic according to Pasteur) (7, D, E, F, some more abundant microscopic forms. It is soluble in water, acids and alkalies, but insoluble 186 The chemical literature in Husemann, 1. c., p. 261. ASPARAGIX. 427 in absolute alcohol (not in dilute), ether, fatty and essen- tial oils. The microscopical 'investigation "should be conducted with absolute alcohol (or oil) (Hartig, Pfeffer Borodin). Add a .drop of absolute alcohol to a section lying under the cover- glass. Then after some minutes one sees crystals shoot out, partly in and on the section, partly on both glasses and partly about the evaporating edges of the fluid. The crystals may be preserved for several days by coyering the section on the slide with a superficial layer of oil. 187 In order to test the presence of asparagin in the cells, take, according to Pfeffer, 188 a section which is thicker than a layer of cells and lay it in a watch-glass in absolute alcohol and move it quickly about. Sections with little asparagin in them should be put on a slide and the alcohol added. This may either peiie- FIG. US. trate into the cells too quickly and so prevent the outward diosmosis of the asparagin, or it may be too dilute in the neigh- borhood of the section so that no asparagin can then be separated out. Alcohol should therefore be applied to the preparation 187 Hartig, Entwickhingsgeschichte cl. Pflkeiras, p. 127. iss Pfeffer in Pringsheiru's Jahrb., Bd. VIII, p. 533. 428 THE MICROSCOPE IN BOTANY. several times. According to Borodin 189 this is not a suitable thing to do. Alcohol should be applied to the section but once ; lay on the cover-glass and allow it to dry. In this way a much smaller quantity of asparagin may be detected than by adding alcohol several times. Borodin 190 gives two methods for the more certain detec- tion of crystallized asparagin as such. Warm it to about 100 and the crystal will give up its water of crystallization and be transformed into a clear, homogeneous, strongly refrac- tive drop, which has an outward appearance like oil and which is easily soluble in water. From this solution it may again be crystallized by means of alcohol. But if it be heated to 200 it will be so decomposed that it appears as a brown drop ap- parently filled with gas bubbles and no longer soluble in water. But one may dilute the crystallized asparagin with a satu- rated aqueous solution of asparagin which is not colder than the object to be tested. It is best to take a cold section with a slightly warmed saturated solution and observe under the micro- scope the effect of a drop of it on the doubtful crystals. All other crystals soluble in water will dissolve the same in this as in pure water, but asparagin remains unchanged. In the same manner Borodin tested tyrosiu which frequently occurs in company with asparagin. XIII. INOKGANIC VEGETABLE ELEMENTS. Literature. Raspail, Mem. de la Soc. d'hist. nat. de Paris, Sept., 1828. Sanioin Monatsber. d. K. Acad. d. Wiss. Berlin, 1857, pp. 53, ff., 253, ff. Hanstein, UeBer ein noch nicht bekanntes System schlauchartiger Getasse, etc. (id. 1859, p. 705, ff.). v. Mohl, Ueber d. Kieselskelett lebender Pflanzen (Botan. Zeitg., 1861, No. 30, ff.). Wicke, Ueber d. York, u. d. physiol. Verwend. d. Kieselerde (Bot. Zeitg., 1862, p. 76). Sachs, Ergebnisse einiger neuer Unters. iiber d. in d. Pfl. enth. Kieselsaure, I (Flora, 1862, p. 33, ff.). Sachs, do., II (id. 1883, p. 113, ff.). Holzner, Ueber d. Kryst. in d. 189 Borodin, I. c., p. 804. o Borodin, I. c. } p. 805. INOKGANIC VEGETABLE ELEMENTS. 429 Pflzellen (Flora, 1864, p. 273, /".). Holzner, Ueber d. phys- iol. Bedeut. d. oxals. Kalkes (id. 1867, p. 497, /I). Holzuer, D. Krystalldrusen in d. Bl. d. weissen Maulbeerbaumes (id. 1867, p. 470,/*.). Rosanoff, Ueber Krystalldrusen in den Pflan- zenzellen (Bot. Zeitg., 1867, p. 41, ff.). Hilgers, Ucber das Auftr. der Kiyst. von oxals. Kalk im Pareuchym. einiger Mon- okot. (Pringsheim's Jahrb., Bd. VI, 1867, p. 285, ff.). Dip- pel, D. Mikroscop., Bd. II, 1869, p. 37, /".Graf Solms- Laubach, Ueber einige geformte Vorkomann. oxals. Kalkes in leb. Zellmembranen (Bot. Zeitg., 1871, p. 509, ff. ) . Pfitzer, Ueber d. Einlagerung v. Kalkoxalatkryst in d. pfl. Zellhaut. (Flora, 1872, p. 97, ff.). Vesque, Obs. sur les crist. d'oxalate d. chaux dans les plants, etc. (Ann. des sc. nat. 5e ser., t. XIX, 1874, p. 300, f.). Sacbs, Lehrb., pp. 38-66. Penzig, Z. Verbreit. d. Cystolithen im Pflanzenreicb (Bot. Centrabl., Bd. VIII, 1881 p. 393). Also numerous statements scattered through various treatises. The inorganic elements occurring in plants are as heteroge- neous as they are wide spread. They occur in every cell mem- brane, in the cell contents, cell sap and protoplasm, and are the indispensable components of the body of the plant. Their pres- ence may be detected by the incineration of a portion of the plant, after which process they remain as ashes, often indeed very minute. Commonly they are not to be recognized by means of the microscope. More seldom they appear as crystals or crys- talline forms, or also as amorphic masses in the cell membrane or cell contents, and can then be discovered and investigated by means of the microscope. Only the latter therefore come within the province of our inquiry. The elements having an inorganic basis which are visible in the cells are either silicic acid or lime (or magnesium salts) What physiological role they play is indeed a question often discussed, but as yet almost altogether unsolved. Doubtless, indeed, they have the biological function, to give to plants and to parts of plants a higher degree of solidity and a greater re- sistance to outside influences, especially toward the assaults of animals (silex layers in Equisetum stems, and grass blades, cell walls of diatoms, etc.). The salts of calcium are, on the con- 430 THE MICROSCOPE IN BOTANY. trary, often to be regarded as excretions or more exactly as cell excrement. The forms to be described here are insoluble in water, but in strong mineral acids, mainly muriatic or nitric acids, either sol- uble or insoluble. Siliceous secretions are insoluble in mineral acids, but calcium salts are soluble in them. A. /Silex. It occurs in the cell membranes of numerous plants, in the stalk and leaves of many grasses and Bam- busce, in the sparkling outer layer of the Catamites, in the epidermis of the Equisetoe^ in the cell walls of the Bacillaria. It is insoluble in acids and alkalies, and is incombustible, and in this is distinguished from every other vegetable element with an inorganic basis. We may best obtain the siliceous incrusta- tion as a complete skeleton, by calcining the part on the plati- num slip, after having first withdrawn the other inorganic salts by means of muriatic or nitric acid (for method see p. 164). In order to obtain the siliceous frustules of diatoms beautiful and free from impurities the material should be first separated from the larger impurities by a fine metal sieve. 191 Then boil with muriatic acid with the addition of calcium chlorate, where- by the cell contents and cellulose membranes will be destroyed and the frustules will be separated. The mixture is then poured with a considerable quantity of water into a high, nar- row test-tube ; let it settle, pour off the fluid and replace it three or four times with pure water. There is now with the diatom frustules a small quantity of impurity in the form of yellow or colorless flakes which may sometimes be removed by boiling the material in water to which is added a piece of clean soap. B. Calcium salts. By far the most frequently occurring in- organic element belongs to the calcium salts, and indeed the prevailing forms are calcium carbonate and calcium oxalate, very rarely calcium sulphate or phosphate (see p. 391). Calcium oxalate, which forms most of the microscopic crys- tals, crystallizes in quadratic or clinorhombic, monoclinic forms. Some of the forms of crystals most frequently found are repre- sented in Fig. 149 (A, B, quadratic, C-F, monoclinic forms). 181 They are to be had of dealers in microscopic objects. PLANT SUBSTANCES OF LIMITED DISTRIBUTION. 431 When the frequently-occurring monoclinic pfisms with ortho- doms are very much developed in the direction of the longer axis and very little in the direction of the transverse axis, the formation of crystalline needles takes place (raphides) which are com- monly united into bundles, the needles lying parallel and near to each other (bundles of raphides). Aggregations of crystals of calcium oxalate frequently occur. Crystals of calcium oxalate are insoluble in water, potash lye and acetic acid, soluble without the development of gas \r\ A FIG. 149. in dilute muriatic acid. If the crystals have been previously calcined they dissolve in acetic acid with the formation of gas. Crystals of calcium carbonate are soluble in dilute acetic and muriatic acid with the development of gas. They occur now and then as the so-called cystoliths. Crystals of calcium sulphate which have sometimes been ob- served are soluble in cold water. B. PLANT SUBSTANCES OF LIMITED DIS- TRIBUTION. Few of the vegetable substances of limited distribution were at first included in the domain of microscopical analysis. Whole groups, as for example, of the most important chemical as well as technical vegetable bases, are, microscopically, almost totally unknown. Others have indeed been better studied, but in respect to these also many questions still remain unsolved. 432 THE MICROSCOPE IN BOTANY. The substances described below are separated into groups which (up to the last one) correspond to their chemical be- havior. They are therefore directly connected with those of the preceding section. They are : 1 Glycoside, 2 Tannic acids, 3 Alkaloids, 4 Fatty oils, 5 Essential oils, 6 Stearoptine, 7 Resin, 8 Phanerogamic coloring matter, 9 Cryptogamic coloring matter. XIV. GLYCOSIDE. Literature. Hartig, Ueber d. Zucker u. einem dem Saliciu ah nl. Korper aus d. Cambiumsafte der Nadelholzer (Bot. Zeitg., 1863, p. 413, /.). Nageli und Schwendener, Mikrosk., p. 494^ f t Franchimont, Rech. s. 1'origine et la const, chim. des resines de terpenes (Arch, neerland, t. VI, 1871, p. 426, ff.). Tiemann u. Harmann, Ueber d. Coniferin, etc. (Ber. Deutsch. Chem. Gesellsch., Bd. VII, 1874, p. 608 ,/".). -Tangl , Vor- lauf. Mitth. liber d. Verbreitung d. Coniferins (Flora, 1874, p. 239, /".). Miiller, Ueber Coniferin (id. p. 399.) Borscow, Beitrage z. Histochemie der Pfl. (Bot. Zeitg., 1874, p.17,/1). Pfeifer, Hesperidin, e. Bestandth. einiger Hesperideen (id. p. 529, ff.). v. Hohnel, Ueber d. Kork u. verkorkte Gewebe iiberhaupt (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. LXXVII, 1 Abth., 1877, p. 700,^.). v. Hohnel, Histocheni. Unters. liber d. Xylophilin u. d. Coniferin (id. p. 699, ff.). Schwartz, Chem-botan. Studien liber d. in den Flechten vor- komm. Flechtensauren (Cohn's Beitrag. z. Biologic d. PH., Bd. Ill, 1880, p. 249, /I). Singer, Beitr. z. nalieien Kennt- niss d. Holzsubstanz u. verholzt. Gewebe (Sitzungsber. d. K. Acad. d. Wiss. Wien, Bd. LXXXV, 1 Abth.', 1882, p. 347, J.). Under this designation is included that series of vegetable substances which are produced by the action of dilute alkalies or acids on cane and grape sugar and their relatives (not includ- ing others produced therefrom by dividing the sugar molecule) . Most of these are in a pure state crystallized and soluble in water ; many also in alcohol ; others are insoluble in the latter GLYCOSIDES. 433 and may be separated by means of this. Of the numerous bodies belonging to this group the microscopical investigation of the following has been attempted: Coniferin (abietin), vanillin, salicin, hesperidin, frangulin, syringin and chrysophonic acid (of the latter it is still doubtful if it belongs to this group). Most of the following statements require still further verifi- cation. 1. CONIFERIN (Abietin) C ]6 H^CV It crystallizes in white or yellow needles, soluble in water, not easily soluble in alcohol and ether. With concentrated sulphuric acid it gives a violet-tolue color which by the subse- quent addition of water becomes blue. According to Franchi- mont, coniferin can be detected in the cells by means of sulphuric acid, giving them a purple-violet coloring. It be- comes green with muriated carbolic acid (more particularly as to this substance as well as its occurrence in lignified membrane, see pp. 340-1, /*.). Hartig's abietin is probably identical with this. It is with difficulty soluble in water and ether, easily soluble in dilute al- cohol. It occurs in the cambian sap of many Conifer ce and can be detected by treating a section of this wood with concentrated sulphuric acid. It shows itself in a characteristic violet blue color in the whole region of the bast ring. 192 2. VANILLIN. C 8 H 8 O 3 White crystal needles soluble in much water, alcohol and ether, becoming yellow with concentrated sulphuric acid (with iron chloride, dark violet). According to Singer it constantly occurs in lignified cell membranes and affords the well-known reaction oi wood substance (see p. 3. SALICIN. C 13 H 18 O 7 . Likewise crystallizes (orthorhombic) ; insoluble in ether, sol- M Hartig in Bot. Zeitg., 1863, p. 414. 28 434 THE MICROSCOPE IN BOTANY. thrin (red;. 5. Phykoxanthiu (yellow). 5. Phykocyau (blue). 3. Diatomiii (yellow-brown). 6. Palmellin (red). 7. Phykophaein (brown). 1. Floridia green is extracted from the Floridia by means of alcohol, represents a variety of chlorophyll, and is really very like it. 2. Phykoxanthin. Yellow coloring matter in kelp and nu- merous fresh water algae in connection with protoplasmic bodies. It is easily extracted from the former by means of 40 per cent alcohol which will not dissolve true chlorophyll. Evaporate the solution and the coloring matter remains behind a slimy, amor- phous, brown mass. Alcoholic solutions of phykoxanthin are made blue green by acids but are not changed by alkalies. 29 450 THE MICROSCOPE IN BOTANY. 3. .Diatomin (endochrom). The yellow to brown-yellow coloring matter of the Diatomacece. It becomes greenish by application of acids and alkalies and with concentrated sulphuric acid a beautiful verdigris geeen. It consists of phykoxanthiu and chlorophyll. 4. Phykoerythrin (Floridia red). The red coloring matter of the Floridia appears the same when dry, is soluble in water but not in alcohol or ether. The aqueous solution loses its color in the light. Alkalies color it pale olive green (almost color- less) , acids restore the red color again. Concentrated sulphuric acid does not alter the aqueous extract. 5. Phykocyan (Phykochrom). In blue-green alga3. The blue-green or indigo blue coloring matter is soluble in water but not in alcohol, becomes yellowish brownish or yellow green with alkalies ; with muriatic acid, orange red or smutty orange. 6. Palmellin. A red coloring matter of PorpJiyridium which is soluble in water and becomes blue with alkalies. 7. Phykophaem. A brown coloring matter from the Fuca- cece, soluble in water and dilute alcohol, but insoluble in absolute alcohol, ether and benzole. It is intercalated in the protoplas- mic grains in connection with chlorophyll and phykoxanthin. The aqueous solution is intense brown red but is not fluorescent. Absolute alcohol, cold, produces a cloudiness in the solution. By warming, the coloring matter is thrown down as a partly flaky brown precipitate. A like effect is produced by muriatic nitric and sulphuric acids. Concentrated alkalies bleach the solution somewhat. The above described coloring substances commonly occur in connection with chlorophyll and its optical effect mixes, in the living plant, with that of chlorophyll. Spectroscopic behavior. Some of the above named coloring substances of the algre have been subjected to critical spectro- scopical studies, while others are in this regard quite unknown. The following have been exactly tested spectroscopically. Floridia-green and Floriaia-red (Pringsheim). 206 The Phy- koerythrin (Florida-red) shows a spectrum which possesses all aoe Pringsheim in Monatsber. d. K. Acad., Berlin, 1875, pp. 749-751. COLORING MATTER OF CRYPTOGAMIC PLANTS. 451 the essential marks of the chlorophyll spectrum (Fig. 150, constructed after Pringsheim's method I. c., with Angstroms scale for a standard ; 0, 10, 20, etc., gives the optical concentra- tion of the solution under investigation ; the dotted line is the absorption curve of the phykoerythrin, the full line that of the Floridia-green). But the chlorophyll bands III, IV and IV (see p. 413, ff.) appear to be considerably strengthened in the phykoerythrin, bands I and II being very much weakened while the bauds in the blue and violet remain unchanged in their intensity. With some coloring substances which belong B C so FIG. iso. to the phykoerythrin group, there appear to be some minor differences in the weakening of bands I and II. But on the whole the maxima and minima of the absorption curve coincide with those of the absorption curve of chlorophyll. The green alcoholic extract of the Floridia (Fig. 150) is spectroscopically somewhat different from chlorophyll. Its spectrum differs from that of chlorophyll by a slight weakening of bands I, II and III, and by a considerable strengthening of 452 THE MICROSCOPE IN BOTANY. band IV, and of the bands in the blue and violet, which in a medium optical concentration flow together to make an end absorption, and finally by a new maximum of absorption which includes the wave lengths 51 and 49 (unit 0.00001 mm.). By comparing the spectra with each other (Fig. 150) there is seen to be a very exact coincidence of the absorption maxima and minima, from which it becomes apparent that Floridia-red is a modification of Floridia-green, and not a direct modification of the phanerogamic chlorophyll (Pringsheim). 75 70 C3 i ! r'l 1 ' I . i !,i ' I' ' ' 1 aB C E.I) G H FIG. Phykocyan (Reinke). The clear blue, aqueous extract of Oscillaria, which has a red fluorescence, gives in a layer 15 cm* thick a spectrum with four absorption bands (Fig. 151 after Reiuke), of which III is very weak* If the solution be boiled only the bands at F and H remain visible, likewise it loses its fluorescence. If after the extraction of the phykocyan, the Oscillaria be again subjected to extraction by alcohol, and the filtrate shaken 75 70 I i 1 ! 1 .--''.-' ' 1 ': ' 1 , I i I U j -;.,! aB JD Eb FIG. 152. H up with benzole, phykoxanthin is retained in the alcohol as an amber yellow fluid. This gives a spectrum like that of chloro- phyll (Fig. 152 after Reinke), with this difference that band II shows a not unimportant broadening towards the red end of the spectrum. It even divides sometimes into two bands ; band III is also broadened towards the red side. Of the bands of the COLORING MATTER OF CRYPTOGAMIC PLANTS. 453 second half of the spectrum, VI and VII coincide with the corresponding chlorophyll bands, IV and V differ from them 207 . Concerning the older spectroscopical investigations of the coloring matter of algae, which were commonly made without specifying the optical concentration and have therefore only relative value, one may compare the above cited writings of Cohn, Kraus, Millardet, Rosanoff, and Askenasy. It may be remarked by way of appendix that Nageli 208 mentions two membrane coloring substances of algae, viz., Gloeocapsin and Seytonemin, Gloeocapsin occurs in the membranes of Gloeo- capsa and some other algae, and is a red or blue coloring mat- ter which is colored rose, red orange, or brown red by muriatic acid, and with potash lye blue or blue-violet. Sc}^tonemin is u yellow or dark brown coloring matter in the walls of the Phy- cromacecB which becomes verdigris green with muriatic acid, and with alkalies yellow, often almost gold yellow. 2. FUNGI CoLOKiNQ MATTER. Literature. Schroter, Ueber eininge durch Bacterien ge- bildete Fermente (Conn's Beitrag. z.,Biol. d. Pfl., Bd. I, 1872, p. 109, ff.). Klein in Quart. Journ. of Microsc. sc., 1875, p. 381,^. Sadebeck, Durch mikrosk. Organismen rothgef. TTnss- er (Verf. d. bot Ver. d. Prov. Brandenburg, Bd. XVII, 1876, p. 77,/.) Cugini, Sulla materia colorante del Boletus luridus (Gazetta chimicu, Vol. VII, 1877, p. 209,/".). In the group of fungi coloring substances of very different nature seem to occur in great numbers all of which have nothing whatever to do with chlorophyll. Alas ! that these substances have not been at all studied. We can therefore give here only some altogether superficial statements. First. Coloring matter frequently occurs in the Schizomyce- tece. The color producing bacteria show different colored pig- ments (red, yellow, green, blue, brown) of intense shades. They are insoluble in water, alcohol and ether. 209 Alcohol and 207 Reinke in Pringsheim's Jahrb., Bd. X, p. 406, ff. 209 Nageli u. Schwendener, Mikr., p. 507. 209 According to Sadebeck, 1. c., the red pigment of micrococcus is partly soluble in water. 454 THE MICROSCOPE IN BOTANY. ether remove the red from the bacteria pigment, potash solu- tion makes it transparent. The red coloring matter of Micro- coccus is according to Helm 210 aniline red. With muriatic acid it becomes rose colored, likewise with sulphuric acid (violet with the addition of more acid), with alkalies yellow. In this case acids restore the red color. The coloring matter of Agaricus atrotomentosus dissolves in alcohol and acetic acid with rose red color, becomes yellow with alkalies and is insoluble in water and benzole. According to Phipson aniline-like coloring matters occur like- wise in Boletus luridus and B. cyanescens. Cugini controverts this and gives the following characteristics of the "acid-like" coloring matter of Boletus luridus. It is soluble in water and alcohol, acids color it a beautiful yellow (chromic acid, yellow brown), ammonia blue, potash-lye red. Ferric chloride gives it an intense green color. Spectroscopically the fungi coloring substances, with the exception of the bacteria pigment, have not been investigated. The spectra have not the remotest likeness to those of chloro- phyll coloring matter. i O. Helm in Avch. f. Pharm., 1875, p. 19, #. INDEX. Abbe condenser, 89. Abbe's binocular ocular, 41. Aberration, chromatic, correcting, 28. " " spherical and chromatic, 5. " " " correcting, 26, 27. Aberration, spherical, illustrated, 25. Abrus pectorus, red coloring matter of, 448. Absorption spectra, 153. ' " comparison of, 154. ' " measuring, 154. " of chlorophyll, 415. " represented by curves, 420. Accessories, microscopical, 100. Acetic acid, 297. " " to prepare a 1 per cent solu- tion of. 282. Acetic acid carmine for cell nucleus, 400. Achromatic lenses, set of. 23. " microscope first made by Van Deyl, 6. Achromatic triplet, 101. Adjustment, the fine, 77. Agaricus atrotomentosus, coloring matter of, 454. Aids to microscopical drawing, 254. Air pump, a home made, 197. Alantin, 375. Albuminous matter, 379. Alcanna tincture, 310. ' and proteid matter, 385. Alcohol. 297. Alcoholic solution of chlorophyll, 411. " and nitric acid as a bleaching medium, 202. Aleuron, 380. Algae, coloring matter of, 448-9. " and fungi examined without pre- paration, 161. Alkalies and cellulose, 324. Alkaloids, 438. Alum carmine and cellulose, 325. Amber cement, King's, 235. Amici's aplanatic lens, 6. Ammonia, 291. Amorphic proteid grains, 382. Amyloid mucilage, 368, 370. Angle of aperture. 24. " " large in lens systems, 27. Aniline coloring matter, 299. " colors for cell nucleus, 401. " fuchsin solution, 300. " green and violet, 301. " method of staining with, 300. " mixture, Hanstein's, 299. *' sulphate, 301. " " and lign'm, 233. " " reaction on epidermal tissue, 353T Anthocyan, 422-3. Anthoxanthin, 413, 422, 423. " spectrum of, 423, 424. Aplanatic lenses, 6, 28. Apparatus for the preparation of "re- agents, 272. Arabin, 371, 374. Asaron, 441. Asbestos for filtering, 273. Asparagin, 298, 425. " crystals, 427. " " testing, 428. " method of investigating, 426-7. physiological function of, 426. Asphalt varnish for cement, 234. B. Baber's picro-carmine, 309. Balsam. 442. Canada, 222. Bands of chlorophyll absorption spectra, 415. 416. Band I, characteristic of chlorophyll, 418. Barberry root, coloring matter of, 448. Bark of Lonicera stained bluish, 269. Bassorin, 371, 374. Bast of Lonicera colored reddish, 269. atracheospermummoniliforme,mounting. 233. Beale's carmine solution, 307. " " for cell nucleus, 400. " " for spirogyra, 308. Behren's cuprammonia, 294. Bell-glasses, 175. Benzole chlorophyll, 412. (455) 456 INDEX. Berthollctia cxcelsa, crystalloids in, 386, 387. Bessey's laboratoiy table, 253. Betula-resin acid, 445. Binocular micro-spectroscope, 143. " ocular, 38. " " Abbe's, 41. " " Nachet's, 39, 41. " Stephenson's, 40. " " Tolles', 39. " " Wenham, 40, 41. Bleaching with alcohol and nitric acid, 202. " bacteria with carbolic acid, 201, 202. - Bleaching with calcium chloride, 202. with chloride of lime and car- bonate of soda, 200. Bleaching with chlorine gas, 201. " cork cell walls with chromic acid, 202. Bleaching with potassium alcohol, 200. " " " hydroxide, 199, 200. Bluing cellulose, 321, 322. " starch with iodine, 360. Blue vitriol, 293. Boletus luridus, coloring matter of, 454. Borel's account of Janssen, 3. Botanical dissecting microscope, Zent- mayer, 107. Bb'ttoher's cuprammonia, 294. Bottle for glycerine, 21!). Box for slides, 248. Brewster's achromatic globe, 6. Brewster's measuring apparatus for mi- cro-spectroscope, 145-148. Browning's micro-spectroscopic measur- ing apparatus, use of, 148, 149. Brown's rubber cement, 238. Brack's compound dissecting microscope, 107. Bulb-burette, 275. Bull's-eye condensers, 93. Bunsen gas burner. 273. Burette, the bulb, 275. " Mohr's spring compressor, 277. " use of, 277. Butterflies' scales as test objects, 57, 58, 59, 60, 61. Butter plates used for porcelain dishes, 173. c. Cabinets for slides, 249. Calcining plants, 164. Calcium carbonate, 431. " incrustations, to re- move, 164. Calcium chlorate dryer, 175. " swells starch, and chlo- rophyll grains, 158. Calcium chloride for bleaching, 202. " oxalate, 431. " salts, 430. " sulphate, 431. Caldwell's automatic microtome, N., 195. Cambium zone of Lonicera, 269. Camera lucida, drawing with, 258. " Grunow's, 118. " as measuring appara- tus, 127. Camera lucida, Nobert's, 117. " " the Wollaston, 115, 116. Camphor, 441. Cane sugar, 299, 377. Canada balsam for mounting, 222. " method of using, 222. Carbo-hydrates, 314. Carbolic acid, 302. " for bleaching bacteria, 201. " " for preserving plant tissue, 227. Card catalogue for preparations, 246-7. Carmine and protoplasm, 207. " solutions, 306. " stains for protoplasmic sub- stances, 309. Castlehun's preserving fluid for lichens, 227. Cataloguing preparations, 264. Caustic potash, 288. Cell nucleus, 397. " fixing, 398. " " staining, 399. " contents, table of reactions of, 403. Cellulose, 313. " and alkalies, 324. " " alum carmine, 325. " cork, 318, 347. " and cuprammonia, 324. " dissolved in sulphuric and chromic acid, 323. " essential, 317. " fungus, 318, 353. " and iodine reagents, 319, 323. " " mineral acids, 323. " " its modifications, 315. " mucilaginous, 327. " muculent, 317. " in the narrow sense, 318. " and potassium copper sulphate, 325, 326. Cellulose reactions, table of, 356. " reagents for, 318. " and the sulphuric acid and iodine reaction, 323, 324. Cellulose, wood, 317. INDEX. 457 Cement, asphalt varnish. 234. " Brown's rubber, 238. " cells, method of making, 231-2. " copal varnish, 236. " dammar varnish, 236. " gold size, 236. " King's acquer finish, 6. 11 " white, 236. " mastic varnish, 234. " shellac and sealing wax, 234. " wax, 234. " white zinc, 238. Cementing angular cover-glasses, 237. " cells by heat, 244. " and finishing the monnt, 234. " glycerine mounts, 238. Cerasin, 374. Chemical reactions, 268. Cherry gum, 372. Cherry wood contains phloroglucin, 303. Chevalier's aplanatic lens, 6. Chloride of calcium for mounting, 223. " " lime and carbonate of soda for bleaching, 200. Chlor-iodide of zinc, 286, 287. " " swells cell walls, 158. Chlorophyll, 403. " absorbent of solar energy, 421. " coloring matter, 410. " decomposed, changes spec- trum, 417. Chlorophyll grains, drawing, 263. " " fundamental substance of, 408. Chlorophyll grains and starch, 364. " " under the spectro- scope. 413. Chromatic aberration, 5. " " correction of, 28. Chromic acid, 292. " " for bleaching cork cell walls, 202. Chromic acid, testing solution of, 282. Chromogen, tannin a, 436. Chrysophanic acid, 4:>5. Circular cover-glass, mounting with, 239. Clarification of the preparation, 198. Classification of plant substances, 312, 313. Cleaning the lenses, 97. " slides and cover-glasses, 216. Cobweb micrometer, 127. Cochineal extract, 305. Cocldington lens, the, 101. Colored granules in flowers and fruit, 424, 425. Colored mounting fluid, 226. " wood, 447. Coloring matter of cryptogamic plants, 448. Coloring matter of flowers, 421. " " " flowering plants, 447. Comparison prism of micro-spectroscope, 141. Comparison table of English and metric scales, opposite p. 133. Compound dissecting microscope, 107,103, Compressor, parallel, 176. Condenser, 87. the Abbe, 89. bull's eye, 93. " described, 88. " stops, 90. " the Webster, 89. Conducting microscopical drawing, 259. Congress nose-piece, 75. Coniferin, 433. Coniferous wood section, 55. Copal varnish, a cement, 236. Copper sulphate, 293. J^rv+JUU)**. y 7 O Cork cellulose, 318. 347. " cell walls, bleaching, 202. " layer, chemical nature of, 349. " in section cutting, 184. " reactions of, 350, 352. Correction system, 31. Corrosive sublimate, 295. " " for mounting, 224. Cover-glass, the, 215. " correction for, 33. " damage to image by, 32. " putting on the, 230. " supports for, 231. " cementing angular, 237. " mounting with circular, 239. Creosote mixture for mounting, 224. Cross thi-eads for drawing, 256. Cryptogamic plants, coloring matter of, 448. Crystals, asparagin,427. " of calcium carbonate, 431. " " " oxalate, 431. " drawing, 264. " in proteid grains, 391. Crystalloids of Bertholletia, 386, 387. " in Floridia, 389. " in Pilobultts, 388. " in proteid grains, 385. " Solanum Americanum, 390. " without inclosing mass, 387. Culpeper & Scarlet invent the mirror, 4. Cupramrnonip, 293. " and cellulose, 324. Cupric acetate, 298. Cutting out fossil specimens, 207. D. Dahlin, 375. Dammar and mastic for mounting, 223. 458 INDEX. Dammar varnish cement, 236. Definition, testing the, 54. Dextrine, 365. Diaphragm, cylindrical, 85. " the iris, 86. " position of, 84. " the revolving, 84. " stops, special, 86. " " Ward's, 87. Diatomin, 450. Diatoms in Liverpool coal, 204. " method of cleaning, 430. " mounting for test objects, 62. " as test objects, 61. " " " Fritsch & Miiller, N., 64. Dissecting microscope, compact, 106. " " handy, 103. Distilling reagents, 273. Distribution of energy in spectrum of chlorophyll, 420. Divini's ocular, 4. Dolland's achromatic telescope, 5. Double staining, 305. Doublets as objectives, 5. Drawing apparatus for the microscope, 110. Drawing with camera lucida, 258. " chlorophyll grains, 263. " conducting microscopical, 259, 260. " controlling the light in, N., 116. " with cross-threads, 256. " crystals, 264. " fluid drops, 264. granular masses, 262. With India ink, 262. materials, 265. microscopic objects, 11, 254. by ocular micrometer, 254, 255. protoplasm, 262. " section of Pteris aquilina, 257. " spiral tissue, 262. " starch grains, 263. " wood cells, 261. Drawings coloring microscopical, 265. Draw tube, 71. " " use of, 72, 73. Drebbel, supposed inventor of the micros- cope, 3. E. Elder-pith, embedding and cutting sec- tions in, 183-5. Embedding in glycerine jelly, 186. " . " gum and glycerine, 186. " media, 186-7. " for Providence microtome, 190. Embedding in tallow and paraffine, 187. Emery plate for grinding sections, 208. Engravers' glass, 10-2. Eosin, 304. Epidermis, to examine without prepara- tion, 160. Epi plasm, 396. Essential oils, 441. Ether, 297. Etiolin, 413. " spectrum'of, 419. Euler's achromatics, 5. Examples of volumetric method, 281-3. Exponent of refraction, 158. Eye shade, Ward's, 43. Faber's pencils for drawing, 265. Facility nose-piece, 75. Farrant's medium, N., 186. Fasoldt's nose-piece, 76. Fats, 439. Fatty oils, 440. Fehl ing's solution, 378. Ferric chloride, 292. Ferrous salts, tannin test, 268. Field of vision, requirements of, 43. Filter of asbestos, 273. " " glass wool, 273. " preparing a, 273. Fine adjustment, 77. " " Bausch & Lomb, 79. " " Bulloch, 78. " " Zentmayer, 78. Finishing mounts on turn-table, 243, 244. Fixing the cell nucleus, 398. Flask for measuring, 274. Flax, mucilage of, c74. Flea glasses, 2. Floridia, crystalloids in, 389. " green, 449. " " spectrum of, 451. " red, 450. Flowers, coloring matter of, 421. Flowering plants, coloring matter of, 447. Fluid drops, drawing, 264. Fluids, table of specific gravity of, 278. Focussing, coarse and fine, 7. " the objective, 98. Fontana, supposed inventor of compound microscope, 3. Forceps and scissors, 172. Fossil plants, preparation of, 203 Fossil wood, grinding thin, 209. Frangulin, 434. Fraunhofer, achromatic lenses " lines in spectrum, 142 Free hand section cutting, 179. Freezing microtome, Taylor's, 191. INDEX. 459 Freezing mixture, Hartig's, 163. Freezing mixtures, other, 192. " seeds to separate cells, 163. Fremy's classification of plant tissue, N., 316. Frey's fuchsin solution, 300. " glycerine carmine, 306. Fritsch and Miiller's diatom tests, N., 64. Fuchsin, Frey's, 300. Fundamental substance of chlorophyll grains, 408. Fundamental tissue and aniline sulphate, 335. Fundamental tissue and indol, 340. Fungi, coloring matter, 453. Fungus cellulose, 318, 353. ' " theories of, 353-4. Fuss' achromatic microscopes, 5. G. Galilei, supposed inventor of compound microscope, 3. Gas slide. Hunt's, -252. Gerlach's ammonium carminate, 306. Glass cells, making, 222, 233, " rods, 173. " wool for filters, 273. Globoids in proteid grains. 391. Glycerine, bottle for holding, 219. " a clarifying medium, 198. '' a mounting fluid. 218. " jelly, Kaiser's. 220. preparing, 221. Norstedt's, 220. embedding medium, 186. for mounting, 220. an aid in glycerine mounts, 229. Glycose, 377. Glycoside, 432. Gold-size, cement, 236. Goniometer, 133, 137, 138. Gb'ppert's, studies of fossil wood, 204. Grammatophora marina, 67, 68. " oceanica, 68. Graphical representation of absorption, 420. Grape sugar, 377. " <; testing, 377. Grunacher's alum-carmine, 308. Grinding down rock specimens, 208. " preparation thin, 209. Ground sections first made by Sorby, 206. Growing slide, 253. Grunow's camera lucida, 118. Gum, 371. " arabic and glycerine for embedding 186. Gum, cherry, 372, 374. Glim mastic and dammar for mounting, 223. Gum mucilage, 368, 371. " tragacanth, 372, 373. Gummy resin, 445. H. Haematoxylin, 304. " " for cell nucleus, 400. " double staining, 305. Hair pencils, 173. " " for cementing, 237. Hairs of plants, to examine, 160. Hanaman's, C. E., method of cleaning slides and cover glasses, 217. Handy dissecting microscope, 103. Hand rests, Ward's, 108, 109. Hanging drop, 251, 253. Hanstein's aniline mixture, 299. " method of clarifying sections 199. Hardening material with alcohol, 178. " with chromic acid and potassi- um bichromate, 178. Hardening with perosmic acid, 178. " tissue by freezing, advantages of, 194. Hartig, colors protoplasm with carmine, 267. Hartig, founder of microscopical analysis, 270. Hartig's ammoniacal carmine, 306. " freezing maceration process, 163- " mixture, 163. " maceration by boiling, 163. Hartnack, inventor of immersion lenses, 30. Helenin, 375. Hervey's turn-table, 241. Hesperidin, 434. Hipparchia janira, scale of, 58, 60, 61, 69. Histological dissecting microscope, Beck's, 108. Honing the razor, 169. Hooke, Robert, 4. How to judge a microscope, 53. Hydriodic acid, how formed, 287. Hydrocharis Morsus-rance, circulation in root hairs, 160. Hydrochloric acid, 284. Hunt's gas slide, 252. I. Illuminating apparatus, 82. ' combinations, 90. Illuminator, Beck's, 94. " opaque, 93. " Ward's iris, 91, 92. 460 INDEX. Immersion system, 30. Incinerating plants, 164. India ink, drawing with, 262, 265, 266. Indol, 303. " and lignin, 338. Inorganic matter in proteid grains, 390. " plant substances, 428. Intercalation in cellulose walls, 316. Intercellular substance, 317, 343. " the theory of, 344. Instruments for making sections, 165. Inulin, 375. " method of testing, 376. Iodine alcohol, 266. " and cellulose, 318-323. " early use of, 267. " and glycerine, 286. " " lignin, 331. " potassium iodide of, 286. " solutions of, 285. " " kept in the dark, 288. and starch, 360. " water, 285. Iron plate for grinding sections, 209. J. Janssen, Hans and Zacharias, inventors of compound microscope, 3. Jones, 7. K. King, Rev. J. D., Providence microtome, 189. King's amber cement, 235. " lacquer finish, 236. " method of sealing cells by heat, 244. " fluid for marine algae, 225. " white cement, 236. Knife for cutting sections, 195. Koch's embedding medium, 186. Kyanophyll, 412. spectrum of, 417. L. Labelling preparations, 245. Laboratory table, 253. Lancets and needles, 171, 172. Leeuwenhoek's microscopes, 2. Lens holder, 103, 104. Lichenin, 368, 371. Life cells, making, 251. Light, regulating the, for drawing, N., 116. Lignin, 317, 330. " and aniline sulphate, 333. " " indol, 338. " " iodine reagents, 331. Lignin and phenol-muriatic acid, 340. " " phloroglucin, 336. " reactions, relative sensitiveness of, 342. Living objects, examination of, 249. Lonicera, staining sections of, 268, 269. Lyccena, scale dots of, 57, 58. Maceration of plants, 162. " " " by boiling, 162. Machines for making rock sections, 207, 210. Magnification, the highest, 66. ' " measuring, 44. " " produced by objective, 44, " " tables of, 45, 46, 47, 48. Manipulation in preparing reagents, 273. Marginal rays, 26. Markings on butterflies' scales, 58, 61. Marsh's bleaching process, 201. Martin, 7. Mason, N. N., originator of Providence microtome, 188. Mastic varnish for cement, 234. Matter (Stoffe), the term how used, 315. Measuring cylinder, 275. 14 flask, 274. " by camera lucida, 127. Mercuric chloride, 295. " nitrate, 296. Metaplasm, 396. Methyl-green aniline, 301. " " for cell nucleus, 401. " violet aniline, 301. Method of aniline sulphate reaction on wood, 334. Method of cleaning diatoms, 430. " " investigating asparagin, 426, 427. Method of iodine reactions on wood, 333. " " phenol-muriatic acid reaction, 341. Method of phloroglucin reaction, 336. *' " testing asaron, 441. cane sugar, 378. dextrine, 365. grape sugar, 377. resin, 444. tannin, 437, 438. Micro-chemistry, not a valid term, 269. Micrometer, the cob-web, 127. " the ocular, 122. " ocular-glass, 122-3. " " screw, 126. " objective, 121. " " glass, 121. " " screw, 121. INDEX. 461 Micrometric tables, 131. Micrometry, 121. " in general, 128-133. ' unit in, 130. Micron unit in microraetry, 130. Microscope accessories for the, 100. " botanical dissecting, 107. " compact dissecting, 106. ' the compound described, 14. invention of, 3. " dissecting, 107, 108. " foot, 95, 96. " magnifying power of early, 5. " " " modern, 43. " handy dissecting, 103. " histological, dissecting, 108. " history and name of, 1. " how to judge a, 53. the mounting, 100. u stand described, 15. ' the Acme, 18. " " Biological, 21. " Histological, 20. " " Illustrator's, 20. " " Model, 17. " " Physicians', 18. " " New Student, 18. " " Student, 16, 19. " " Universal, 21. " testing optical powers of, 53. * tube, 71. " " like telescope, 5. " use ol to be learned, 9. " " Sachs on, 10. u u " rules for, 96-99. Microscopic image drawing, 110. " " projection of, 110, 112. " " reflected by mirror, 113. Microscopic image reflected by prism, 111. " ' requisites for produc- ing, 15. Microscopic preparations of fossil plants, 203. Microscopic preparations, historical sketch of, 157. Microscopic preparations should always be transparent, 157. Microscopic sections, cutting, 177. Microscopical analysis, 2(38. ' founded by Hartig' 270. Microscopical analysis not concluded, 270. " measuring, 120. reagents, 267, 268. " ' an experimental sci- ence, 270. Microscopical technique, 156. Microscopist, personal qualities of, 12. Micro-spectroscope, 139. " " the binocular, 143. ' *< description of, 140-3. * " measuring apparatus of, 144. Microtome, Caldwell automatic, N., 195. " the common, 188. " Providence, 189. ** " Taylor freezing, 191-3. " " Thoma sliding, N., 195. Middle lamella, 317, 343. " " its pectose metamorphos c. 346. Middle lamella, how produced, 344. " " reactions on, 345. " layer, 56. Milk-saps, 446. " of Euphorbia, 446. Millon's reagent, 296. Mineral acids, reactions on, cellulose, 323. Mirror, the, 82, 83. " use of, 83, 84. " introduced by Culpeper and Scarlet, 4. Modifications of cellulose, 315, 318. Mohl, Hugo v, 8. " " on microscopic preparations, 157. Mohl, Hugo v, section making, 165. Mohr's spring compressor burette, 277. Moist chamber, 251, 253. Molecular intercalation, 269. Monobrom-naphihaline lor mounting 262. Mounting fluid, 217. " " for algae, 225. " " Canada balsam, 222. " chloride of calcium, 223. " " a colored, 226. " " corrosive sublimate, 224. " " creasote mixture, 224. " " glycerine, 218. " jelly, 220. * " gum mastic and dammar, 223. Mounting fluid monobrom-napthaline, 226. " " potassium acetate, - 2'25. " in fluid, process of, 228, 229. " fluid Styrax and liquid amber, 226. " " sugar water, 224. " " table of refraction of, 159. " " Topping's, 225. Mucilage, characteristic, 36S..369. " of flax, 374. " vegetable, 367. Mucilaginous cellulose, 327. " " reactions for, 329. Muculent cellulose, 317. Muriatic acid, 284. 462 INDEX. N. Nachet's binocular ocular, 39, 41. Nageli and iodine reagents, 270. Naturalist, the, must use simple tools, 165. Needles and lancets, 171, 172. Neubauer's cuprammonia, 294. Neutral tint reflector, 115. Nicol, W., first ground sections of fossil wood, 206. Nicol's prism, 134. Nitric acid, 284. " " and alcohol for bleaching, 202. Nitrogenous combinations, 314. Nitzschia linearis, 68. Nobert's camera lucida, 117. ' test plate, 70. Nose piece, the Congress, 75. Facility, 75. Fasoldt,76, " " " Triple, 74. " " " Zentmayer, 75. Nucleus of the cell, 397. " stain ing the, 268, 399, 403. " structure of the, 398. O. Objects always mounted in fluids, 158. Objects for immediate observation, 160. Object table, 80. 44 slides, 214. " ' different forms of, 215. Objective, the, 22. " condenser, 94. " micrometer, 121. " glass-micrometer, 121. " protector, 31. " screw-micrometer, 121. 4 ' system, 23, 29. " " requirements of, 24. " " screw collar adjustment of, 34. Objectives, table of American, 50, 51. Observation by artificial light, 94, 95. Ocular, Divini's, 4. " Huygenian, 35-38. " Kellner's. 38. 4> llamsden's, 38. " glass-micrometer, 122. " " " use of, 124-5. " screw-micrometer, 126. " micrometer used iu drawing, 254, 255. Oil crayons for coloring drawings, 266. " stones for razors, 168. Oils, essential, 441. " fatty, 440. Opaque objects not used, 158. Optical powers, testing the, 53. Osmic acid, 296. Over-corrected lenses, 28. Oxalic acid, 298. " " standard solution of, 278. P. Palmellin,450. Paraffins and tallow for embedding, 186, 187. Parallel compressor, 176. Parnassus pahistris, hairs of, 264. Pectose metamorphosis of the middle lamella, 346. Permanent preparations, 157. making, 213. Petrified woods, treatment of, 205. Pleffer's method of making proteid grains insoluble, 383. Pfitzer's reagent for hardening and stain- ing cell nucleus, 402. Phenol, 302. Phenol-muriatic acid and lignin, 340. Phloroglucin, 302. " and lignin, 336. Phosphoric acid, 285. Photo-micrography, 119. Phykocyan, 450. ' spectrum of, 452. Phykoerythrin, 450. PhykophaBin, 450. Phykoxanthin, 449. " spectrum of, 452. Physical reactions, 268, 269. Physiological functions of asparagin, 426. Physiological functions of silicates, 429. Picro-anilme, 301. Picro-carmine, 309. " " for cell nucleus, 400. " haematoxylin for cell nucleus, 400. Pieris brassicce, scale of, 59. Pillsbury cabinet, 249. Pilobulus, crystalloids in, 388. Pinularia nobilis and viridis, 62. 4i viridis, drawing, 255. Pipette for handling unicellular plants, 161, 162. Pipette for measuring, 274, 275. Plant substances of limited distribution, 431. Plant ^substances of universal distribu- tion, 313. Plasmodium of Myxomycetae, 396. Plossl, 8. Pleurosigma angulatum, 64, 69. with high pow- ers, 65. INDEX. 463 Pleurosigma angulatum, low powers, 64. " medium " 65. balticum, 63. " species lor test objects, 63. Polarizing apparatus, 133, 134. < use of, 136. Porcelain crucible, 174. " dishes, 173. Potassium acetate, for mounting, 225. alcohol, 290. " for bleaching, 200. " bichromate, 291. ' chlorate, 291. " copper-sulphate and cellu- lose, 325, 326. * Potassium ferrocyanide, 298. " hydroxide, 288. t , " bottles for, 289. < " for clarUying sec- tions, 199. Potassium hydroxide, preserving, 289. as a tauniu reagent, 437. Potassium nitrate, 291. " standard solution of, 279. " solution of definite concentra- tion, 282. Potassium, to find percentage of solution, 281. Potash, caustic, preparation of solution, 288, 289. Potato tuber crystalloids, 387. Poulsen, author of micro-chemistry, 271. Poulsen's shellac cement, 235. Preparation of microscopic objects, 156. " of objects without cutting, 160. Preparation of permanent mounts, 213. Preparations clarified with glycerine, 198. cataloguing, 246, 247. labelling, 245. " storing, 247-9. Preparing microscope, 100. Preserving fluid, carbolic acid, 227. ' " for fungi, 222. . " " lichens. 227. " " Wickersheimer's, 228. " media. -217. Primordial utricle shrinks in glycerine, 158. Prism, illuminating, 89. " the Nicol's, 134. " polarizing and analyzing, 135. Proteids, 379. Proteid grains, amoi-phic, 382. " coloring with alcanna, 385. < " with crystalloids, 385, 391. " " discovered by Hartig, 381. " " with globoids, 391. Proteid grains inclosing inorganic matter, 390. Proteid grains made insoluble, 383. " matter, functional, 392. " " with starch, 363. Protoplasm, 392, 393. " drawing. 2G2. " in the narrow sense, 394. " reactions of, 395. Providence microtome, 189. Prussiate of potash, 298. Pteris aquilina, section of rhizome, 257. " " fibro-vascalar bundles, in polarized light, 136-7. Rack and pinion, 7. " for holding rock sections, 213. Radlkofer's chlor-iodide of zinc, 287. Razor for cutting sections, 166. " different forms of, 166. < k honing, 169, " how to sharpen, 168, 170. " the J. R. Torrey flattened, 167. " strops, the J. R. Torrey, 169. Reactions of cell contents, table of 403 " " cell nucleus, 397. " of chlorophyll coloring matter, 410. Reactions of essential oils, 441. ' ' fatty oils, 440. milk-saps, 446. mucilaginous cellulose, 329. proteid grains, 384. protoplasm, 395. resin, 443, 444. " table of cellulose, 356. " of tannin, 437, 438. " " wax, 440, ' Reagent bottle, 272. Reagents, preparing, 273. microscopical, 267, 268. " volumetric method of preparing, 278. Refraction, exponent of, 158. Refractive index of fluids, table of, 159. " power of fluids, value of in investi- gations, 159. Removing the air from the section, 196, 197. Reserve proteid substances, 380. Resin, 442. " betula, 445. " drops stained with alcanna, 310. " meal, 443, 444. " " test for, 444, 445. " production of, 443. Resolving power, 44. " testing the, 57. 464 INDEX. Rhodospermin, 389. Kiddell, J. L., inventor of binocular mic- roscope, 39. Rock sections grinding down , 208. " " " thin, 209. " " labelling, 212. " " machines lor making, 207, 210. " " mounting, 211. " " preserving, 212, 213. Rubber cement, Brown's, 238. Jtiubia tinctorum, coloring matter of, 447. Russow's bleaching process, 200. Rules for the use of the microscope, 96. S. s' method of incineration, 164. Sachs on microscopical drawing, 11. "' " microscopical preparations, 156. " " use of microscope, 10. Salicin, 433. Salix purpurea, contains phloroglucin, 302. Sambucus nigra elder pith, 183. Scalpels, 171. ' different forms of, 170. " sharpening, 171. Schultze's maceration mixture, 163. ' mixture and cork tissue, 352. " warming stage, 82. Schweigger-Seidel acid carmine solution, 308. Schweitzer's reagent, 293. Scissors and lorceps, 172. Screw-collar adjustment, shown, 34. Screwing ou the objective, 98. Sealing cells with heat, 244. Section cutting with cork, 184. " " . "% elder pith, 183. " in embedding media, 185. " " free hand, 179. " " longitudinal, 181, 182. " with a microtome, 187. " " transverse, 180. " further handling of, 196. " now to handle, 181. " instrument, 165. " knife, 195. " lifter, 196. ' machines the common, 188. " removing air from, 196-7. ' under preparing microscope, 197. Sections, cutting microscopic, 177. " of fossil wood, 'ground, 20(5. Self-knowledge of the microscopist, 13. Sharpening razors, 168, 169, 170. " scalpels, 171. Shellac cement, Foulsen's, 235. " ** Thiersch's, 235. Shellac and sealing wax cement, 234. Silex, 430. Silicious skeleton of plants, 430. Simplex, the.100. Single-celled plants, 161. Slides, how to clean, 216. Slit, influence of, ou spectrum, 151-2. Society screw, 71, 73. Sodium chloride, 291. " nuro-prusbiate, 298. Solanum Americanuiti, crystalloids in, 390. Solar energy and chlorophyll, 421. Sorby's tubes for the uiicro-speotroscope, 101. Specific gravity of fluids, 278. Spectra, absorption, 153. " of chlorophyll, 415. Spectroscopic analysis ot alga;, 45u, 451, " behavior of chlorophyll, 413. Spectrum of Anihoxanthm, 42ii-i. " etiohu, 418, 4ia. " Jbloridia green , 451. *' " leaf, 41(3, 417. " " phycocyau, 452. kt phycoxanthm, 452. Spherical aberration, 5. Spiral spring clip, 116. " tissue, drawing, 2(52. Spirit lamp and tripod, 175. Spirogyra, Beale's carmine lor, 308. Spring clip, spiral, 17U. Stage, 7, 80. " the circular, 81. " mechanical, 81. " warming, si, 82. Staining the nucleus, 399. Stand, the early, of wood, 7. *' of brass,, 8. the microscope, 15. Standard potassium solution, 279. ' solution of oxalic acid, 278. " " sulphuric acid, 280. tables of equivalents for, 281. Standard scale, 148. Starch, 357, 359. " in chlorophyll grains, 364. " grams, drawing, 263. " colored by iodine, 267. " two elements of, 362. " and iodine reagents, 360. " first visible product of assimila- tion, 358. Starch in proteid plasmic matter, 363. " reactions of, 360. " solubility of, 362. " and tannin, 436. Stearoptene, 441. Stepheuson's binocular ocular, 40. INDEX. 465 Storing permanent preparations, 247-219. Strops, razor, the J. R. Torrey, 169. Styrax and liquidamber in mounting, 226. Suberin, 318, 347. Sugar, cane, 378. " grape, 377. Sulphuric acid, 284. " " to dilate to 20 per cent so- lution, 282. Sulphuric acid and iodine reaction on cellulose, 323, 324. Sulphuric acid, standard solution of, 280. Surirella gemma, 66, 68. Synapta, anchors of, 55. " plates of, 54. Syracuse watch glass, 174. Syringin, 434. T. Table of American objectives, 50, 51. " " apertures, Prof. Abbe. " " cellulose reactions, 356. " " comparison of English and met- ric scales, opp. p. 133. Table of equivalents for standard solu- tions, 281. Table of magnification, of Bausch and Lomb lenses, 48. Table of magnification of Hartnack lens- es, 45. Table of magnification of various combi- nations, 47. Table of magnification of strongest lens- es, 46. Table of magnification of lenses of vari- ous makers, 46. Table, micrometric, 131. " of reactions on cell contents, 403. " " refraction of fluids, 159. " " specific gravity of fluids, 278. " test objects, 70. " " vegetable substances, 314. a laboratory, 253. Tannic acid, 435. Tannin, 435. " as a chromogen, 436. " reactions of, 437, 438. " and starch, 436. " tested by ferric oxide, 268. Taxus baccata, drawing cells of, 260. Taylor, the freezing microtome, 191. " how to use, 193. Taylor, the freezing microtome, modifi- cation of, 194, 195. Technique, microscopical, 156. 30 Test objects, 53, 71. " ' table of, 70. Testing the defining power, 54. " " resolving power, 57. Thalophytes tested with indol, 339. Thiersch's borax carmine, 307. " oxalic acid carmine, 307. " shellac cement, 235. Thoma sliding microtome, N., 495. Tissue colorless in natural state, 267. " epidermal and aniline sulphate, 335. Tissue epidermal and indol, 339. " of vascular bundles and indol, 340. Tolles' binocular ocular, 39. Topping's fluid for mounting, 225. Torrey's flattened razor, 167. " razor strop, 169. Tradescantia, circulation of, protoplasm in, 160. Triple nose-piece, 74. Trommer's copper sulphate and potash test, 377. Turn-table, self-centering, Bausch and Lomb's, 240. Turn-table,self-centering, Beck's, 239. Hervey's, 241. " Zentmayer'8,240. use of in making cement cells, 231. Turn-table, use of in mounting, 242, 243. Turpentine, 442. U. Under-corrected lenses, 28. Unit in micrometry, 130. Units of measure, comparison table of. 131. Universal accessory, 90, 91. Use of the burette, 277. V. Van Deyl, made first achromatic micro- scope, 6. Vanillin, 433. Vascular bundles of Lonicera, 269. " tested by aniline sul- phate, 335. Vascular plants tested with indol, 339. Vegetable substances, classification of, 312, 313. Vegetable substances of universal occur- rence, 313, 314. Vegetable mucilage, 367. Veratrum, 438. Volumetric method in preparing re. agents, 278. 466 INDEX. W. Walmsley's photo-micrographic appara- tus, 120. Ward's eye shade, 43. " hand rest, 108. " iris illuminator, 91. Warming the microscope in winter, 99. Wash bottle, 175. Watchglass, the Syracuse, 174. " glasses, combination of, 174. " " a set of, 174. Water, 283. ' colors for drawings, 265. Watman paper for drawing, 266. Wax, 440. " cells for mounting, 238. " cement, 234. Weber life slide, 259. Webster condenser, 89. Weigert's picro-carmine, 310. Weighing scales, 274. Wenham's binocular ocular, 40, 41. " button condenser, 88. White zinc cement, 238. Wickcrsheimer's fluid, 228. Wiesner's cuprammonia, 294. Wolf, 4. Wollaston's camera lucida, 115, 116. Wood, bituminous, 205. " cells, drawing, 261. " cellulose, 317. " petrified, 205. " substance, 330. Xanthein, 422. Xanthin, 422. Xanthophyll, 412, 41S-. " spectrum of, 419. Zeiss' binocular ocular, 41. Zentmayer's nose-piece, 75. Zirkel, an early German petrologist, 206. EKRATA, ETC. Page 19, line 8, before A insert ]. Page 32, line 20, for J read T. Page 43, Fig. 17 is printed upside down. Page 52, line 11, for opposite read oQth. Page 91, in Fig. 31, the lower middle figure should show a black center-stop instead of a central aperture. Page 92, line 7, for if, is hoped will soon be, read has lately been. Page 108*, line 4, for cm read mm. Page 109, line 6 from bottom, after The insert dotted. Pages 156-7 for Microscopical objects and preparations read Microscopic, etc. Page 158, 2nd paragraph, 10th line, read primordial utricle. Page 282, first line, read, " To prepare a potash solution of a definite concentration." Page 296, 2nd paragraph, 2nd line, read fuming for foaming. 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