a Cornell University Library OF THE Rew Work State College of Agriculture Ie 26.07 lati ve t "B08 ornell Universi ‘i PLANT ANATOMY STEVENS PLANT ANATOMY FROM THE STANDPOINT OF THE DEVELOPMENT AND FUNCTIONS OF THE TISSUES AND HANDBOOK OF MICRO-TECHNIC BY WILLIAM CHASE STEVENS PROFESSOR OF BOTANY IN THE UNIVERSITY OF KANSAS SECOND EDITION, REVISED AND ENLARGED os ‘ WITH 152 ILLUSTRATIONS PHILADELPHIA P. BLAKISTON’S SON & CO. 1012 WALNUT STREET 1910 Ag. 207: CopyRIGHT, 1910, BY P. BLAKISTON’s Son & Co. Printed by The Maple Press York, Pa. - PREFACE TO THE SECOND EDITION In getting ready the second edition it became evident that a chapter on reproduction should be added. Because of its promise in helping to solve the problem of evolution and its great importance for plant and animal breeding the subject of repro- duction and heredity has come to the forefront of biological research; and especially under the great light that has shone from Mendel’s laws has eager investigation been directed toward the details of cell behavior in reproduction. It cannot yet be said that these investigations have arrived at undisputed achievement, but their results, however tentative, are so suggestive of important possibilities as to justify their survey in a text-book for students in colleges and agricultural schools. Necessarily that part of the chapter on reproduction dealing with an interpretation of observed nuclear behavior that has frequently been suggested in current literature is a fit subject for critical examination and debate, and as such it will serve its purpose of marking a present-day view arising from a con- templation of observed facts of structure and behavior. The theory of pangenes and unit characters may or may not stand as our knowledge advances, but it is serving the purpose in biology to-day that the atomic theory has so long and honorably fulfilled in chemistry. Of great aid to me in the preparation of Chapter XIII have _ been Coulter and Chamberlain’s Morphology of Angiosperms, Mottier’s Fecundation in Plants, Mendel’s Principles of Heredity, by W. Bateson, Strasburger’s Die Stofflichen Grundlagen der Vererbung, Lotsy’s Vorlesungen iiber Deszendenztheorien, de Vries’s Die Mutationstheorie, Species and Varieties, and v vi PREFACE Plant Breeding, Bailey’s Plant Breeding, and the Proceedings of the American Breeders’ Association. It is a pleasure to me to express my thanks here to Dr. Mc- Clung and Dr. Billings for suggested improvements in Chapter XIII, to Mr. Peace for improved processes in microtechnic, and to Miss Eugenie Sterling for the drawings in Chapter XIII. W. C. STEVENS. UNIVERSITY OF KANSAS. October, 1910. PREFACE TO THE FIRST EDITION To one interested in biology the study of plant anatomy affords a rich and alluring field, since it reveals how plants, under conditions the most exacting, have met and solved the problems of their existence by achieving the power and habit of cell differ- entiation and cell association into tissues adapted to carry on the different physiological functions; and when the study of plant anatomy is directed to reveal the process of cell differentiation and the steps by which the mature tissues are made fit for their functions, the student cannot fail to see at once its high biological significance. , The ontogeny and physiology of the tissues is in fact so illumi- nating to their mature form and structure that the student of anatomy works to a distinct disadvantage if he is not constantly reverting for enlightenment to questions of origin and function; and whatever motive may incite him to the study of plant anatomy, whether it be purely intellectual curiosity, or the recognition of the necessity of a knowledge of plant anatomy to the scientific pursuit of pharmacognosy or agriculture, he will find the outcome more worthy of his efforts if he has sought out the physiological and ecological interpretation of his anatomical findings. It is not nature’s way to evolve cells and tissues at random, with no problems to be solved by their evolution. The tissues are not an aimless expression of the power of variability. Rather they represent the means of the triumph of living organisms over the conditions and forces which make up their environment. This book attempts to point out in a brief and elementary way how plants arrive at this achievement by the evolution of the different physiological tissue systems from a primitive, undifferentiated embryonic tissue, and how the tissue systems vii viil PREFACE are adapted by their character and relation to each other to carry out the plant’s vegetative functions. It seeks to answer in some measure questions about what kind of organisms plants are; how they wrest their living from the inorganic world; and how they are equipped to make satisfactory terms with their environment. At the close of each chapter are given directions for obser- vations that will afford a good foundation for critical discussion. In carrying out the work as there outlined the student will become familiar with the most important practices in microtechnic, and he will at the same time get training in independent work that will prove a significant part in his education. Chapters dealing in sufficient detail with microtechnic and microchemistry are given to help the student to pursue the subject beyond the limits of this book, and to undertake practical work in pharma- cognosy and pure food and drug investigations. In the illustrations much use has been made throughout the book of generalized diagrams. I have found these very helpful in my teaching, and I offer them here in the hope that they may prove suggestive to students in correlating and interpreting the details of the isolated sections with which the histologist has to deal, and of service in throwing light on the operation of the physiological functions. I must make acknowledgment of especial indebtedness for substance and point of view to Strasburger’s Botanisches Prac- ticum, and Leitungsbahnen, Haberlandt’s Physiologische Pflan- zenanatomie, Pfeffer’s Physiology of Plants, Zimmermann’s Microtechnic, Czapek’s Biochemie der Pflanzen, Meyer’s Grund- lagen und Methoden fiir die Mikroscopische Untersuchung von Pflanzenpulvern, Chamberlain’s Methods in Plant Histology, and Winton’s Microscopy of Vegetable Foods. The Bausch and Lomb Optical Company have kindly supplied the cuts for Figs. 127, 133, 135 and 136; and the Spencer Lens Company the cut for Fig. 131. I am indebted to Miss Eugenie Sterling for preparing the drawings for most of the illustrations, and to Mrs. Marguerite PREFACE ix Wise Sutton for some of the drawings. Mr. Alban Stewart pre- pared the camera lucida drawings of tissues of Avicennia, Psidium and Mangrove, from materials collected by him in the Galapogos Islands. Mr. L. M. Peace made the photomicrographs appearing here, prepared many of the sections from which the drawings were made, and gave me many valuable suggestions for the chapters on microtechnic. My thanks are due my colleagues, Dr. M. A. Barber, Dr. C. E. McClung, Dr. F. H. Billings, and Prof. Chas. M. Sterling for reading and critizing different parts of the book, and to Ada Pugh Stevens for reading all of the proof sheets. W. C. STEVENS. UNIVERSITY OF KANSAS. CONTENTS CHAPTER I THE PLANT CELL The Protoplast.—Plasma Membrane or Ectoplasm.—General Cytoplasm.—The Nucleus.—The Plastids.—Cell-divi- sion.—Cell Differentiation.—Sizes of Cells—The Cell- wall.—The Chemical and Physical Nature and Physio- logical Powers of the Protoplast.—Illustrative Studies. CHAPTER II DIFFERENTIATION OF THE TISSUES General Survey.—The Primordial Meristem.—The Pro- toderm.—The Procambium.—The Ground Meristem.— The Primary Permanent Tissues.—Illustrative Studies. CHAPTER III SECONDARY INCREASE IN THICKNESS Dicotyledons and Gymnosperms.—Growth of the Vascular Bundles.—Increase in the Cortex.—Monocotyledons.— Unusual Growth in Thickness.—TIllustrative Studies . . CHAPTER IV PROTECTION FROM INJURIES AND Loss OF WATER The Epidermis.—The Epidermis as a Protective Tissue.— The Epidermis as a Waterproof Covering.—The Radial xi PAGE I-23 24-45 46-60 xii CONTENTS and Inner Walls of the Epidermis.—The Cell Contents of the Epidermis.—Outgrowths and Excretions of the Epidermis——The Multiple Epidermis.—The Cork.— Cork as a Protective Tissue—Cork as a Waterproof Covering.—Use of Cork in Healing Wounds.—Other Means. of Protection.—Illustrative Studies . CHAPTER V THE PLANT SKELETON The Making of the Skeleton.—The Tissues of the Skeleton.— The Collenchyma.—The Bast Fibers.—The Wood Fibers.—The Stone Cells. aera of the Skeleton. —lIllustrative Studies i CHAPTER VI THE ABSORPTION OF WATER AND MINERALS Roots in the Soil—The Root Hairs.—Method of Intake of Water and Solutes.—Effect of Temperature of Soil, and Character and Amount of Solutes upon Absorption.— Absorption of Water and Solutes " wide = Illustrative Studies . . . |. ; 5 CHAPTER VII TRANSPORT OF WATER AND SOIL SOLUTES The Need of a Transporting System.—Tissues Devoted to the Transport of Water.—The Tracheal Tubes.— Course of Tracheal Tubes through the Stem.—The Tracheids.—Relation of the Tracheal Tissues to the Medullary Rays and Wood Parenchyma.—The Ring of Annual Growth.—Relation of Rings of Growth to Growth in Length.—Relation of Annual Rings to the Leaves.—Distribution of Water and Solutes throughout the Leaf.—The Power Concerned in the Ascent of PAGE 61-74 75-89 go-I00 CONTENTS xili PAGE Water.—Path of Water Ascent.—Influence of Environ- ment on the Water Conducting Tissues.—TIllustrative Studies... .. ee des 0 G8 Sopa s, s( WOrre2 CHAPTER VIII INTAKE AND DISTRIBUTION OF GASES Oxygen and Carbon Dioxide Necessary to Plants——The Stomata.—The Relation of Stomata to the Environ- ment.—The Lenticels—The Intercellular Spaces.— Diosmosis of Gases into and from Living Cells.—Motive Power in the Distribution of Gases throughout Plants.— Illustrative Studies... . be G04 ‘ 123-137 CHAPTER IX CONSTRUCTION OF THE PLANT’sS Foop The Source and Uses of Food.—Food Building Apparatus.— The Chloroplasts—The Sun’s Energy.—The Palisade Cell the Chief Photosynthetic Unit—Relation of Leaf as a Whole to Photosynthesis.—Conditions Affecting Photosynthesis.—Photosynthesis in the Lower Plants.— Synthesis of Food without Light.—Illustrative Studies . 138-156 CHAPTER X TRANSPORT OF FOODS THROUGHOUT THE PLANTS Need of Circulatory Tissues.—Evidence that the Phloem Carries the Food.—Evidence that the Tracheal Tissues Assist the Phloem in the Upward Transmission of Food. —Relation of Phloem Elements to Other Tissues.— The Course of Food Distribution—Annual Additions to the Food-conducting Tissues.—Relation of One Year’s Phloem Elements to those of the Next.— Character of Food while in Transport.—The Pro- pelling Power in Food Transport.—Illustrative Studies. 157-174 XIV CONTENTS CHAPTER XI STORAGE OF FooD AND WATER Need of Food Storage-—The Kinds of Stored Food.—The Process of Storage.—Location and Extent of Food Storage Tissues.—Fluctuations in the Solubility and Insolubility of Stored Food.—Digestion of Stored Food. —Assimilation of Food.—Relation of Stored Food to Energy Supply.—The Storage of Water.—Character- istics of Water Storage Tissues.—Illustrative Studies. CHAPTER XII SECRETION AND EXCRETION Nature of Secretions and Excretions.—Secreting Cells and Glands in General.—Laticiferous Vessels or Milk Tubes. —Tannin Cells—Special Enzyme-secreting Cells.— Secretion and Excretion of Minerals——The Process of Secretion.—The Excretion of Liquid Water.—Illustra- tive Studies : sh ee ee CHAPTER XIII REPRODUCTION Development of Fern Sporangium.—Division of Spore Grandmother Cells.—Germination of the Spores.— Fertilization of the Egg.—Interpretation of the Processes of Nuclear Division—Two Generations in the Life- cycle.-—Spore Formation in Spermatophytes.—Forma- tion of the Microspores.—Details of Nuclear Division.— Formation of the Megaspores.—Details in Division of Megaspore Grandmother Cell.—Germination of the Megaspore.—Fertilization and Germination of the Egg. —The Triple-fusion Nucleus.—Behavior of Pedigree Hybrids.—Interpretation of Mendel’s Results.—Pater- nal and Maternal Chromosomes.—Bearers of Hereditary PAGE 175-198 £OQ-—212 CONTENTS . xv PAGE Characters—Theory of Pangeneic Interchange.— Necessity of Pedigree Cultures—Mosaic Character of Offspring of Hybrids——Mendel’s Laws.—Practical Applications.—Exceptions to the Rules.—Significance of Sexuality —TIllustrative Studies... 3. . . . . . 213-250 CHAPTER XIV THE PREPARATION OF SECTIONS Cutting Sections Free-hand.—Cutting Sections with.a Micro- tome.—Care of the Section Knife.—Cytological Methods —The Fixing Process——The Hardening Process.— The Process of Imbedding in Paraffin.—Sectioning Material Imbedded in Paraffin.—Mounting Paraffin Sections.—Staining the Sections.—Imbedding in Cel- loidin.—Staining Celloidin Sections —Making Perma- nent Mounts in Glycerine or Glycerine Jelly . «251-270 CHAPTER XV THE USE OF THE MICROSCOPE Adjusting the Microscope.—Drawing to Scale from the Microscope.—Use of the Polariscope-——The Use of Reagents on Microscopic Preparations : Lo... 271-284 CHAPTER XVI REAGENTS AND PROCESSES... . . . oe + . 285-329 CHAPTER XVII MIcCROCHEMISTRY OF PLANT PRODUCTS. . : . « 330-367 CHAPTER XVIII DETECTION OF ADULTERATIONS IN Foops AND Drucs . . . 368-374 INDEX. ...... : ar Pan ain Nine i 375-379 PLANT ANATOMY CHAPTER I THE PLANT CELL The plant body is composed of structural units termed cells. These are minute boxes often barely visible and usually en- tirely indistinguishable to the naked eye. .The walls of the boxes are composed of cellulose, wood, or cork, or a substance allied to cork called cutin. As a rule, the walls are without apparent perforations. The term cell has also come to include the living body which is always present in young cells and very frequently in old ones. The discovery that the plant body is composed of the cell units was made by Robert Hooke, who, about 1660, with a com- pound microscope improved by himself, saw the cellular struc- ture of cork. The cells of cork have the appearance of the cells of honeycomb, and this similarity led to his use of the word “cell” for the structural units of the cork, and ultimately to the. extension of the usage to plants in general. The term is not an unfortunate one for cells considered merely as boxes; but its application to the living parts inclosed within the cell-walls, or to the living part where no wall is present is inapt. The term protoplast (Gr. protos, first, and plastos, formed, the thing first formed) is now in general use to designate the living part of the cell as a morphological unit; and the term protoplasm or plasma is applied to the substance composing the protoplast, just as a ‘brick would be a morphological unit of a brick house, and burnt clay the substance composing it. Following the general usage the word cell will here be applied to the box and its living content I 2 THE PLANT CELL taken collectively, or to the box alone when the living parts have disappeared, and the word protoplast will be used to designate ‘the living part alone. The box or cell-wall is manufactured by the protoplast for its own stability and protection, and the protoplast must, there- fore, exist before the wall which encloses it. Since the pro- toplast is the living structural and physiological unit of the plant body, and since everything that the plant performs is really the work of its individual protoplasts,-it necessarily follows that a satisfactory comprehension of plant anatomy and physiology is impossible without a knowledge of the nature of the proto- plast itself. The Protoplast.—If we study under high magnification sections of an onion root tip that has been fixed, imbedded, sec- tioned, and put up in permanent, stained mounts as directed in the chapter on The Preparation of Sections, we shall find near the root apex young cells that have not yet secreted their entire cell-walls, and we shall often find some degree of plasmolysis or shrinkage of the protoplasts away from their cell-walls so that their contour can precisely be made out. Fig. 1, A, shows us such a protoplast. In the root all of the parts here shown were living, excepting possibly the nucleolus 6. The cytoplasm ¢ constitutes the bulk of the protoplast; the nucleus a is imbedded in the cytoplasm; the plasma membrane d is a specialized outer portion of the cytoplasm; the plastids e are relatively very min- ute parts of the protoplast, but have a special work to do, as will be learned later on. In older cells farther back from the root tip we find that the protoplasts have secreted a wall, as shown in Fig. 1, B. Comparing A and B of Fig. 1, we see that in the younger protoplast the cytoplasm looks something like a sponge with very fine meshes, while in the older protoplast the cytoplasm does not so completely fill out the space inclosed by the plas- matic membrane, some of the meshes having widened into rela- tively large rifts. It seems that as the protoplast grows older the cytoplasm does not keep pace with the general increase in PLASMA MEMBRANE 3 size and its network in consequence becomes broken in places, allowing the small meshes to coalesce into larger ones known as vacuoles. Both the meshes and the vacuoles are filled with cell-sap. The cell-sap is largely water containing in solution salts that have come up from the soil, and food substances together with various other compounds manufactured within the plant. After this general view of the several parts of the protoplast we are ready to examine into the character and uses of each more thoroughly. In doing this let us not lose sight of the fact that the protoplasts taken collectively constitute the living body of the plant and whatever the plant does as a living organism is accomplished by them. They put to use various forces of the external world; they compound the plant’s food and a multitude of other products, which are of various uses to plants themselves as well as to man; they make the cell-wall framework of the plant; they multiply and increase in size so that the plant as a whole is made to grow; they differentiate the various tissues, each suited to perform a particular service; they are sentient to gravity and light, moisture and temperature, and to the general state of the whole organism of which each protoplast forms a part, and they are capable of responding to these things in a definite and useful way. With this knowledge we might anticipate, and it is not surprising to find that each protoplast is a complex thing with visibly distinct parts, and that each part has its own physio- logical significance. We shall now take up the parts of the protoplast in the following order: plasma membrane, general cytoplasm, nucleus, plastids. Plasma Membrane or Ectoplasm.—At the exterior of every protoplast is a very thin, hyaline membrane, which, as has been said, is morphologically a part of the cytoplasm, for when a protoplast is torn or cut in two a membrane is produced from the cytoplasm over the wounded surface. This membrane is known as the plasma membrane or ectoplasm (Fig. 1, A,d). Itis approximately .ooo3 mm. thick, or about za0 of the thickness of this page. After the cell-wall has been formed it is very difficult to distinguish the plasma membrane because of its extreme thin- 4 THE PLANT CELL ness and close contact with the wall; but the greater part of this difficulty vanishes when the protoplast is made to shrink away from the wall. This we can do to advantage with the epidermal Fic. 1.—A, embryonic cells from onion root tip; d, plasmatic membrane; ¢, cyto- plasm; a, nuclear membrane enclosing the thread-like nuclear reticulum; b, nucleolus; e, plastids (black dots scattered about). B, older cells farther back from the root tip. The cytoplasm is becoming vacuolate; f, vacuole. C, a cell from the epidermis of the midrib of Tradescantia zebrina, in its natural condition on the right, and plasmolyzed by a salt solution on the left; g, space left by the trecedence of the cytoplasm from the wall; the plasma membrane can now be seen as a delicate membrane bounding the shrunken protoplast. All highly magnified. cells of Tradescantia zebrina containing a colored cell-sap. We strip a bit of epidermis from the under side of 4 midrib of a leaf and mount it under a coverglass in a drop of water. We bring some of the colored cells under the objective and run a 5 per cent. NaCl solution under the coverglass. The salt solution draws water out of the cells by osmosis and the protoplast soon shrinks away from the walls because it is elastic and had been stretched by the water within the cell. We can now make out the thin plasma membrane at the surface of the shrunken pro- toplast (Fig. 1, C). If we replace the salt solution under the coverglass with fresh water the protoplast quickly swells up and presses against the cell-wall all around as before. In this experiment we can see that while the plasma mem- brane allows the water to be drawn from the protoplast by the salt solution it does not permit the coloring mater or the osmotic substances in solution in the cell-sap to escape, for the color does not at all diminish in the protoplast as it shrinks, and the GENERAL CYTOPLASM 5. protoplast could not again swell up.on the replacement of the salt solution by water if its osmotic substances had been lost. This guardianship of the exchange of materials between the the cells or between the cells and the external world is one of the well-recognized functions of the plasma membrane. Since the plasma membrane lies at the surface of the protoplast it must receive and transmit to the other parts the stimuli that come from without. Until the cell-wall is built the plasma membrane doubtless affords some rigidity and protection to the parts within; and when the time for the building of the cell- wall arrives it seems that thé plasma membrane constructs it by the chemical transformation of its own substance into the substance of the wall. General Cytoplasm.—The cytoplasm is the living matrix in which the nucleus and plastids are imbedded. In very young cells it fills out all of the space not occupied by the nucleus and plastids (Fig. 1, A), but in old cells it becomes a very thin film, hardly greater than .ooo6 mm. in thickness lining the cell-wall. In cells that have been killed, fixed and stained in the usual ways (see chapter on The Preparation of Sections) the cytoplasm has a spongy or netted appearance (Fig. 1, A). It is uncertain whether the cytoplasm is really sponge-like with irregular and intercommunicating canals or alveolar with each cavity a closed sac. Whatever the exact character of the cavities may be, they are -filled with cell-sap or, in many in- stances, with insoluble reserve food, such as starch, proteids, and oils, and excretions, such as crystals of calcium oxalate. As has been stated, as the cell grows older some of the cavi- ties in the cytoplasm enlarge and coalesce, and are then known as vacuoles. A plasma membrane is formed about the vacu- oles similar to the exterior plasma membrane already described, and it exercises a selective function over the passage of mate- rials to and from the vacuole just as does the exterior mem- brane to and from the protoplast as a whole. The spongy, or alveolar, condition of the cytoplasm persists until the divi- sion of the nucleus preparatory to cell division sets in, when a 6 THE PLANT CELL part becomes thread-like and seems to assist in the translocation of the chromosomes (the definite parts into which the nuclear substance becomes segmented during nuclear and cell division) to the opposite pole:of the cell; this process may, therefore, be classed as one of the functions of the cytoplasm (see Fig. 3). Throughout the life of the cell the cytoplasm has many things to do of a chemical nature, but it is very improbable that it often works independently of the nucleus. Where storage of pro- teids and oils is taking place, we find them stowed away in the meshes of the cytoplasm, and this is good circumstantial evidence that the cytoplasm has manufactured them where we find them. This is particularly true of the insoluble proteids. The cyto- plasm probably secretes the ferments by means of which the stored materials are digested when they are wanted for food. Many other of the ceaseless activities of the cell are doubtless accomplished with its assistance. The Nucleus.—In young cells the mucleus is spherical in form and lies at the center imbedded in the cytoplasm and occu- pies from .5 to .8 the diameter of the cell. It consists of the nuclear membrane, nuclear reticulum and nuclear sap, and usually contains one or more nucleoli (Fig. 1, A, 0). The nuclear membrane appears to be really a part of the cytoplasm, similar to the plasmatic membranes lining the exterior of the cell and of the vacuoles. The reticulum is the essential living part of the nucleus. The nuclear sap appears to.be a fluid which fur- nishes to the reticulum water and food and in other ways serves it; while the nucleolus seems to be reserve food of a peculiar kind needed to help in the processes of nuclear and cell division. The nucleus is often spoken of as the center of life of the cell. While the statement is vague, it conveys a meaning not entirely misleading. Some of the facts at the foundation of this conception are these: (2) When a protoplast is segmented into two parts by plasmolysis or other artificial means the part containing the nucleus has the power to construct a wall about itself, while the enucleated part has not. Although the plas- matic membrane is the immediate agent in the construction of THE NUCLEUS i the wall, it cannot do its work without the influence of the nucleus. (6) We have good reasons for the belief that the nucleus is the cause of oxidation in the living cell, and, without it, the tearing down and building up processes of the cell that depend upon oxidations cannot go on. (c) After the nucleus has been removed the remainder of the protoplast soon dies. While, on these grounds, the nucleus may be spoken of as the center of life of the cell, the fact must not be left obscured that a nucleus dissociated from the rest of the protoplast cannot long maintain its existence, and the codperation of the other parts is necessary to the success of the functions of the nucleus. The nucleus is also frequently spoken of as the bearer of the inheritable characters and qualities. This, too, is not with- out foundation, and if true the nucleus stands forth as the architect and master of the form, character, and activities of the cell and entire plant body. The grounds for the assump- tion are: (a) In the embryonic cells of the growing apex or cambium the nucleus is relatively large, having an average diameter 0.6 that of the entire cell. These cells are to undergo profound changes in size, form, and general character as the differentiation of tissues proceeds, and why should the nucleus be allotted so much space within the cells unless it plays a domi- nant part in the hereditary differentiations that are to follow? The evidence here is enough to raise the question, but it does not go far toward solving it. (b) During nuclear and cell -divi- sion the nuclear reticulum, which is the essential part of the nucleus, divides into many pieces with great precision, and these pieces are equally distributed between the nuclei of the two resulting cells. What but the vehicle of hereditary transmission should require such exactness of distribution? Here again the facts afford us hardly more than the suggestion of the ques- tion. (c) In fertilization, when the sperm cell from the pollen grain is fusing with the egg cell within the ovule, the sperm cell consists almost entirely of nucleus, and the egg cell is relatively rich in cytoplasm; yet the offspring may partake as much of the peculiarities of the male as of the female parent. The inference 8 THE PLANT CELL is that, since the male contributed little more than a nucleus and the female a relatively large amount of cytoplasm in addi- tion to the nucleus, it must be that to the nucleus have been given the inheritable qualities to transmit from generation to generation; for if the cytoplasm bears these qualities equally with the nucleus the impress of the female might be expected to preponderate in the offspring. (d) In the nuclear and cell divisions resulting in the production of pollen spores and em- bryo-sac spores the number of chromosomes (see Fig. 3) in each nucleus is reduced one-half. The sperm cell and egg cell arising from the germination of pollen spore and embryo-sac spore each contain the reduced number of chromosomes, but when they fuse in the act of fertilization the original number is restored in the fertilized egg and so continues throughout the body of the resulting offspring until pollen spores and embryo-sac spores are again produced by it with the reduced number. What the significance of this halving and doubling may be is told in Chapter XIII, where it is seen unmistakably to relate.to the transmission and coéperation of paternal. and maternal characters. Since the chromosomes are segments of the nuclear reticulum, the phenomenon here described may be classed as a part of the evi- dence that the nucleus is the bearer of the inheritable qualities. (e) A new line of evidence is at present being opened up in the study of reproduction, and especially in the reproduction of hybrids where the segregation and recombination of parental characters is in harmony with the behavior of the chromosomes during these processes, as told in detail in Chapter XIII. The Plastids.—The plastids are distinct and usually rela- tively small parts of the protoplast. They vary in size from barely discernible points with the highest magnifications to discs and bands that traverse the entire length of the cell. In form they may be orbicular, ellipsoidal, disc, or ribband shaped. They are classed under three names according to their color: the Jeucoplasts are colorless; the chloroplasts contain the green coloring matter chlorophyll; the chromoplasts are yellow, orange, or red. They are, however, really the same thing under different THE PLASTIDS 9 guises and performing different functions, for the leucoplasts may become chloroplasts and the chloroplasts chromoplasts. This fact is well made out in the tomato, for instance, where the very young pistil in the bud contains no other plastids than the leucoplasts and is colorless in consequence; later when the corolla drops away and the pistil emerges into the light the leucoplasts produce chlorophyll and the young fruit is green; but when the fruit begins to ripen the chlorophyll is gradually replaced by red and orange coloring matters, and the chloroplasts become Bic. 2.—A, cell from the epidermis of the upper side of the calyx of Tropzolum majus with crystalline chromoplasts; B, cells from the petal of Lupinus luteus with yellow chro- moplasts; C, cell showing numerous chloroplasts scattered through the cytoplasm. (A, after Strasburger; B, after Frank.) chromoplasts. The leucoplasts are therefore the progenitors of the other plastids, but they have their own functions to per- form as leucoplasts: they take carbohydrates out of solution in the cell-sap and store these within themselves in the form of insoluble starch grains. This is well seen in those cells and tissues, as in the potato and the endosperm of seeds, where reserve food is stored away. The chloroplasts secrete two color- ing matters known as chlorophyll-green and carotin or chlorophyll- yellow. The chloroplasts employ these pigments in arresting the sun’s energy, by means of which they make the food of the plant. Io THE PLANT CELL In Chapter IX the chloroplasts will be more fully discussed in connection with the photosynthetic system. The chromoplasts impart red, orange, and yellow colors to flowers and fruits, where, within the chromoplast body, the reds occur as crystal- line carotin and the yellows as amorphous xanthin. ‘These may be found separately or together in the same chromoplast. The presence of both produces an orange color (Fig. 2). The reddish and bluish pigments occurring in solution in the cell- sap are known as anthocyanin, and it does not appear that the chromoplasts are necessary to their production. Cell Division.—In the growing apices of root and shoot and in the cambium cell division may go on indefinitely. The process of cell division begins in the nucleus and terminates by the formation of a dividing cell-wall, as will be seen in Fig. 3, where the different stages may be followed. The nuclear reticulum (in a) becomes transformed into a thick winding thread (in 6), which in successfully stained sections is seen to consist of colored discs or granules termed chromatin, im- bedded in a colorless matrix called linin. The thread splits longitudinally throughout its length (in c), and then breaks into rod-shaped pieces, each of which consists of two longi- tudinal halves arising. from the longitudinal division of the thread (in d). These rods are known as chromosomes, and their number varies with the species. Next the nuclear wall disappears and threads arise in the cytoplasm and converge to a point at two opposite poles, forming what is known as the nuclear spindle (in ¢). Some of the threads extend uninter- ruptedly from pole to pole, while others become fastened to the chromosomes. The chromosomes in some unknown way line up in an equatorial plane half-way between the poles, and then one-half of each chromosome is drawn to one pole and the re- maining half to the other when they form at each pole a nuclear thread (e, f, and g). This spins itself out into a nuclear reticulum around which a nuclear membrane is soon organized (in h and i). The connecting fibers extending between the poles bulge out at the equator more and more and new ones evidently are formed CELL DIVISION II until the whole equatorial zone is traversed by them (in g and h). The fibers thicken in the equatorial area and the thickenings fuse together, forming a closed membrane known as the cell-plate (progressive stages in g, h, and 7). This is a plasmatic mem- SER , & > he a mar) o a ov o 2B qoueisd mets eqn} 8AeTs ca eqny [Reqoes4” we tnuuy ~9909 Teeqse13 Teutds Wag sarrery ray x x 4 = t = r aqetd esoTs maaullary, “BY pmAyousied yoTosoT sed eudyousiedc aposotsed Cambium ‘Bark Fic. 14.—Diagram to show the topography and character of the tissues that are evolved Cambial activity has not yet begun. from the primary meristems. 32 DIFFERENTIATION OF THE TISSUES walls, is to give strength at a time when the bast and wood fibers have not yet made their appearance or arrived at sufh- cient maturity to be effective. It may occur as a continuous zone or in separate strands. In producing the collenchyma the ground meristem cells divide transversely and vertically, the daughter cells enlarge and elongate vertically; the walls gradually thicken at the angles, but the cellulose composing them does not appear to become essentially altered or replaced. The collen- chyma cells usually contain chloroplasts, and they remain living until cut off from the general circulation by cork cells in the formation of borke (see page 55), as frequently happens in woody plants. Thin-walled Parenchyma lies next to the collenchyma internally, and as a rule constitutes most of the primary cortex. It has been evolved from the ground meristem by transverse and vertical division of the cells of the latter, and the growth of these cells about equally in all dimensions, or with a slight excess of elonga- tion in the vertical or radial direction (Fig. 14). The cell-walls of this tissue remain thin, and their original cellulose is practically: unchanged. The cells frequently contain chloroplasts and remain alive unless involved in the formation of borke, as stated for collenchyma. In'virtue of its chloroplasts this tissue, as well as the collenchyma, is able to manufacture food, and it is much used also in the slow conduction and storage of foods that have come to it from the leaves. Short Sclerenchyma Cells or Stone Cells are frequently found, singly or in groups, distributed amongst the thin-walled paren- chyma (Fig. 14). These, as a rule, are transformed thin-walled parenchyma cells whose walls have become greatly thickened and more or less lignified. The tubular and often branched pits characteristic of the walls of these cells are thin places left as the thickening of the walls progresses. These cells soon die after the completion of their walls and seem chiefly to be used in giving strength and protection. Long Sclerenchyma Cells or Bast Fibers sometimes occur in the primary cortex or in the pericycle (Fig. 14). They take their THE PRIMARY PERMANENT TISSUES 33 origin from strands similar in appearance to procambium strands that have arisen from vertical division of the cells of the ground meristem or protoderm. In the latter instance they would be classified with the epidermis from the standpoint of their origin; Fic. 1 5.—Diagram showing stages in the development of collenchyma, stone cells, and bast fibers. A, collenchyma; 1 and 2, cross and longitudinal section of a collenchyma cell in its primary meristem condition; 3 and 4, longitudinal and cross-sections of the same cell at a later stage, the walls in 4 have commenced to thicken at the angles; 5 and 6, longi- tudinal and cross-sections of a mature cell. The arrows indicate the planes through which the longitudinal sections were cut. The stippling inside the walls indicates the protoplasts. B, stone cells; 1, in the primary meristem condition; 2, the cells have enlarged and the walls have begun to thicken and become pitted; 3, the walls are completed. The primary wall is black, cellulose additions white, and the lignified walls in 3 are stippled. Notice that the protoplasts have disappeared in 3, and the pits in some instances are branched; C, bast fibers: 1 and 2, cross and longitudinal sections of primary meristem cells that are to become bast fibers; 3 and 4, the same at a later stage; 5, longitudinal section of completed bast fibers. In 5 the stippling of the wall indicates lignification. Note that the walls have become pitted and the protoplasts have disappeared from the fibers. but on account of their position they will here be included with the primary cortex. In their procambium-like state the bast fibers are thin- and cellulose-walled and vertically elongated; proceed- 3 ‘ 34 DIFFERENTIATION OF THE TISSUES ing toward maturity the procambium-like cells enlarge, and princi- pally in the vertical direction, so that the ends shove past each other and become pointed; the walls thicken and become lignified as a rule (Fig. 15), and the protoplasts finally disappear, leaving the fibers dead. This is the mode of origin of bast fibers wherever they occur. The bast fibers seem to serve chiefly, if not solely, for giving strength, for which purpose they are fitted, whether they occur in the primary cortex or in the pericycle, by their vertical elongation, thick and lignified walls, and dove-tailing or in- terlacement of the ends. In roots the primary cortex usually consists of thin- walled parenchyma alone (Fig. 16). The innermost layer of the primary cortex is the starch sheath or endodermis Fis. 16.—Portion of « cross. (Fig, 14). This in stems is, as its section of a root-of Allium . < 7 7 . ascalonicum. h, large central name implies, unusually rich in its tescheal tube; % xylem. and 1. starch content, and other than this, in phloem portion of the vascular bundle; , cortex cells; j, endo- stems, it oftentimes has no striking dermis with thin-walled cell at oes k to admit passage of materials, Characteristics. In recent years good ee pericambium. evidence has been brought forward, notably by Haberlandt, to show that the starch grains in this tissue act like the otoliths in the ear, and by falling always to the lower side of the cell as the position of the part to which it belongs is shifted, they furnish by their impact the stimulus for perceiving the direction of the gravity pull. In other instances, and particularly in roots, the endodermal cells become differentiated from the rest of the primary cortex by elongating somewhat in the vertical direction, suberizing their radial walls, and by partially or completely thickening their walls (Fig. 16). The intercellular spaces that can be found in other tissues of the primary cortex are lacking in the endodermis. These charac- teristics have led to the conception that the endodermis possessing THE PRIMARY PERMANENT TISSUES 35 them is intended to reduce permeability between primary cortex and stele; and this conception is strengthened by the occurrence, in such an endodermis, of thin-walled cells just in front of the xylem portion of roots where water absorbed from the soil has need of access to the water tubes (Fig. 16). In old portions of roots it often happens that the outer parts of the primary cortex slough away, leaving the endodermis to protect the stele. : The Pericycle.—The pericycle lies between the starch sheath or endodermis and the outer rim of the phloem part of the vas- cular bundles (Fig. 14). In stems we commonly find it composed of two kinds of tissues, thin-walled parenchyma and bast fibers, the origin of which from the ground meristem is as stated for the corresponding tissues in the primary cortex. The bast fibers @ 6 C80 og) @ @® @? A B Cc Fic. 17.—Diagram to show different plans in the distribution of bast fibers. A, basta continuous cylinder in the pericycle; B, isolated strands of bast in the cortex and in the pericycle in front of each vascular bundle; C, a combination of A and B. (After Green.) may form a continuous zone all around the stem, or they may occur as isolated groups, either associated with, and seemingly a part of the phloem of the bundles, or dissociated from the phloem (Fig. 17). In the stems of most dicotyledonous plants the bast fibers are restricted to the pericycle. ‘They serve, of course, for giving strength; and unless, or until, the cambium later adds a substantial amount of wood fibers or fiber tracheids they remain the chief reliance in this respect. The thin-walled parenchyma cells of the.pericycle, like those of the primary cortex, often contain chloroplasts, and they serve for the slow conduction and storage of reserve foods, particularly of the non-nitrogenous class. In roots the pericycle occurs usually as a single layer of 36 DIFFERENTIATION OF THE TISSUES thin-walled cells (Fig. 16), and its chief significance here is that from the division of its cells the lateral roots take their origin. The Primary Vascular Bundle.—The typical vascular bundle of dicotyledonous stems consists, as already stated, of three parts, an outer or phloem, an inner or xylem, and a median or cam- bium. These, in primary bundles, are all the product of the differentiation of the procambium, and the progress of their evo- lution will now be followed. A typical phloem consists of three elements, the sieve tubes, the companion cells, and the sieve or phloem parenchyma (Fig. 14). A sieve tube consists of a vertical row of cells each of which is vertically elongated and separated from its neighbor above and below by a thickened partition wall that is perforated by many openings. These partition walls somewhat resemble a sieve and have therefore suggested the name for the tube (Fig. 14). In the evolution of a sieve tube a vertical row of procambium cells divides longitudinally, producing a double vertical.row of cells. The cells of one row enlarge transversely and vertically; their transverse or end walls thicken, leaving thin places or pits, which finally become complete openings from one cell cavity to another by absorption of the wall at the bottom of the pits, and the vertical row of cells in this way becomes a continuous tube (Fig. 18.) The vertical walls of the sieve tubes are usually comparatively thin, but they are sometimes markedly thickened. The cells in the row companion to the sieve tubes, known as the companion cells, enlarge in all dimensions somewhat, but remain much smaller than the cells of the sieve tubes. The cells of the sieve tubes remain alive, at least throughout the first year of their for- mation, but they strangely lose their nuclei. This is an anomaly, for cells that have been deprived of their nuclei artificially soon die. It is thought in this instance that the nuclei of the com- panion cells extend their influence to those of the sieve tubes and so keep up there the oxidative and other processes that depend upon nuclear activity. The walls of both sieve tubes and companion cells remain cellulose. THE PRIMARY PERMANENT TISSUES 37 The contents of the sieve tubes are found to be rich in pro- teids, amido-acids, and soluble carbohydrates, and minute starch grains may sometimes be present in abundance. Even proteids that are in solution do not pass readily through cell-walls, and in the sieve tubes the perforations allow them to pass in an unob- structed stream from one cell or sieve tube member to the other. That the sieve tubes are for the vertical flow of proteids and allied substances is shown by direct ob- servation under the microscope while using suitable reagents for the demonstration of proteids; and further by girdling and constric- tion experiments described in Chapter X. The sieve parenchyma cells in differentiating from the procam- bium elongate vertically more or less and increase in their cross diameters (Fig. 18), but they do not become, as a rule, so large in any dimension as the cells of the sieve tubes. Their walls remain cellulose and commonly thicken but little. They appear to serve chiefly in the translocation of car- bohydrates and as storage places Fic. 18.—Stages in the development of sieve tubes, companion cells, and phloem parenchyma. A, a and b, two rows of procambial cells; in c and d, a has divided longitudinally and c is to become companion cells; d, a sieve tube, and b, phloem paren- chyma; B, c, companion cells, and d, a beginning sieve tube from c and d, respectively in A. The cross-walls in d are pitted; b, phloem parenchyma grown larger than in A; C, the same as B with the pits in the cross-walls of the sieve tubes become perforations, and the nuclei gone from the cells composing the tube. for proteids which they are in position to take from the sieve tubes when a surplus is at hand, and they assist in delivering over to the medullary rays materials from the sieve tubes to be stored by the rays or transported inward for storage in the cells of the wood or xylem parenchyma. The sieve tubes, companion cells, and sieve parenchyma cells seem to remain alive and functional throughout the first year of 38 DIFFERENTIATION OF THE TISSUES their origin, and in some instances certainly for several years, but it may be stated as a rule that they soon become crushed by the pressure of surrounding tissues, and their effectiveness is thereby reduced, and after a time altogether destroyed (Fig. 24). The three kinds of cells taking part in the formation of the phloem as described above do not always occur together; in Monocotyledons, for example, the parenchymatous elements are absent, and in Gymnosperms and Pteridophytes the companion cells are lacking. . The primary xylem (namely, that portion of the xylem that has differentiated from the procambium and exclusive of that which is added later by the cambium) may consist of three classes of elements, the tracheal or water tubes, tracheids, xylem paren- chyma, and wood fibers. These elements do not, however, com- monly all occur together. The wood fibers are usually absent, and tracheids are not common in the primary xylem of Angio- sperms; while in Gymnosperms true tracheal tubes do not occur, with few exceptions. . A tracheal tube is produced by the absorption of the end or transverse walls in a vertical row of cells, and at the same time the enlargement in all dimensions of the cells composing the tube, and the subsequent unequal thickening, and lignification of the vertical walls. The thick places in the walls are for strengthening the tube, while the thin places are for the easy passage in and out of water and materials in solution. In the tubes first formed the thick places are in the form of rings or a spiral coil. To realize the use of these, imagine barrel hoops or a flexible spiral coil of wood sewed inside a bag to make it stand open. This type of tracheal tube is differentiated from the procambium not far from the growing apex in internodes that have not yet ceased elongating, and it will be seen that the corresponding growth in length which these tubes must undergo will be but little resisted by the kind of thickenings which their walls possess. In older internodes where elongation has nearly or quite ceased stronger tracheal tubes are laid down having thin places of more restricted area in the form of pits or elongated meshes. THE PRIMARY PERMANENT TISSUES 39 While a tracheal tube consists of a chain of fused cells a tra- cheid is a single cell only with thin places in its walls in the form of pits with overhanging border for greater strength. These are known as bordered pits (Fig. 19). Or the thickenings may be of the spiral and reticulate types. In becoming a tracheid the procambium cell elongates and tapers at the end to a greater or less degree, thickens its wall unequally, and finally lignifies its wall (Fig. 19). The tracheal tubes are primarily for carrying water from roots to and throughout the leaves; and the tracheids have the same function, but they may assume the character of wood fibers and be depended on for strength as well as for conducting water, as in the case of pine wood. The cells of the xylem parenchyma are, as a rule, relatively thin-walled, and the walls are sometimes, and in woody plants commonly, lignified, and they may or may not be pitted. To form the xylem parenchyma the procambium cells divide trans- versely, and the young parenchyma cells enlarge in all dimen- sions, becoming more or less elongated vertically, with end walls at right angles to the vertical or, as a rule, only slightly inclined (Fig. 19). They differ from the tracheids by their less elongation, blunter ends, commonly thinner walls and unbordered pits. The proportion of xylem parenchyma to the other elements of the primary xylem varies greatly in different examples, from occupying the bulk of the xylem to entire absence. Its function is to store reserve water and foods, and possibly to assist in lifting the water to the leaves. The wood fibers are characterized by being much elongated and taper-pointed at the ends, and by thick and lignified walls and small unbordered pits. The steps in the evolution of a wood fiber from a procambium cell are evident. The cell elon- gates, and in doing this the ends of contiguous cells shove past each other; the walls gradually thicken, and finally become, more or less'completely lignified. The wood fibers are chiefly to give strength, and they are assisted in this by their interlacing and dove-tailing together, as well by their thick and lignified walls. 40 DIFFERENTIATION OF THE TISSUES The cells composing the tracheal tubes soon die, so that the ‘tube is not long alive after it comes to maturity. The wood fibers may live but one or only a few years, while the tracheids in respect of length of life seem to vary between the tracheal tubes 4 D Fic. 19.—Stages in the development of the elements of the xylem. A, progressive steps in the development of a tracheal tube. 1, row of procambial or cambial cells that are to take part in the formation of a tube; 2, the same at a later stage enlarged’in all dimensions; 3, the cells in 2 have grown larger, their cross-walls have been dissolved out, and the wall has become thickened and pitted; 4, the walls in 3 have become more thickened, THE PRIMARY PERMANENT TISSUES 41 and the wood fibers. The parenchyma cells are, as a rule, the longest lived of all xylem elements. They have been found still carrying on the vital function of starch stotage in rings of growth nearly a century old. After the elements of the primary xylem and phloem have been completed it is found that a layer of undifferentiated pro- cambium cells remains between them in Dicotyledons, while in Monocotyledons and Pteridophytes the whole of the procambium enters into the composition of phloem and xylem. The layer of undifferentiated cells in Dicotyledons is known as the cambium, and its most important characteristic is that it retains the power of cell division for a longer or shorter time, even indefinitely in the case of woody perennials. - The divisions of the cambium cells may take place trans- versely, or vertically in radial and tangential planes; but the tangential vertical division is by far the most frequent and gives rise to cells known as the tissue mother cells, which by one or more cell divisions produce other cells that differentiate into the elements of the xylem on the one hand and those of the phloem on the other. These new additions to the primary vascular bundle constitute the secondary xylem and phloem. : The medulla or pith is formed by transverse and vertical divisions of the ground meristem, and the subsequent enlarge- ment in all dimensions of the daughter cells. The pith cells are not apt to be much elongated in any one dimension, and their walls, as a rule, remain relatively thin and unchanged the pits have an overhanging border, the walls have become lignified as indicated by the stippling, and finally the protoplasts have disappeared, and the tube is mature and dead; B, stages in the formation of tracheids from procambial or cambial cells. The steps are the same as in A, excepting that the cross-walls remain and become pitted. C, steps in the development of wood fibers from cambial cells; 1, cambial cells; 2, the same grown larger in all dimensions with cells shoving past each other as they elongate; 3, a later stage with cells longer and more pointed and walls becoming thickened and pitted; 4, complete wood fibers with walls more thickened than in the previous stage and lignified, as shown by the stippling. The protoplasts in this last stage have disappeared and the fibers are dead. D, steps in the formation of wood parenchyma from cambial or pro- ,cambial cells. 1, group of cambial or’procambial cells; 2, the same enlarged in all dimen- sions; 3, the same with walls thickened and pitted; 4 and 5 show the same stages as 2 and 3, but here the cells have enlarged radially or tangentially more than they have vertically. The walls of these cells are apt to become lignified, but the cells are longer lived than the wood fibers. 42 DIFFERENTIATION OF THE TISSUES from their orginal cellulose composition; but they are some- times decidedly thickened and lignified. They are short lived; in woody plants the surrounding bundles crowd in and crush them, and in herbaceous plants they often break down and leave a hollow space; in other cases still they may persist for a long time as dead elements. The pith cells are for some time of use in the storage and slow conduction of water and some- times they are employed in the storage of food, even after they are several years old, but they are evidently not commonly depended on long for any essential function. The ground meristem lying between the vascular bundles undergoes cell division vertically and transversely and the cells thus formed enlarge and frequently become elongated in the radial direction. In this way the primary medullary rays are formed. The medullary rays have two distinct functions: they carry water and substances in solution radially, inward and outward as needed, and they store water and reserve foods. Since the primary rays extend usually only a few millimeters vertically they are not suited for transporting materials in that direction. A study of cross- sections needs to be supplemented by an ex- amination of vertical tangential sections to make the extent of the medullary rays clear. a aes In these vertical sections it is seen that the showing how the primary vascular bundles do not maintain an vascular bundles . : anastomose around independent and isolated course through the the medullary rays. stem, but anastomose with each other across The gaps represent the rays. the upper and lower borders of the rays, as seen in Fig. 20. In the foregoing a type of vascular bundle has been chosen known as the collateral type; where one phloem strand stands radially in front of the xylem strand. While this is the prevailing type, there are others that must not be passed unnoticed here. It sometimes happens that a second phloem strand stands THE PRIMARY PERMANENT TISSUES 43 radially within, or on the pith side of the xylem, forming what is known as the bicollateral bundle; in other instances the phloem surrounds the xylem, or vice versa, making a concentric bundle; while in roots it is the rule that the phloem and xylem of the primary bundle are in strands alternating with each other, Fic. 21.—Different types of vascular bundles. A, the concentric type, with xylem, k, surrounding the phloem, h. JB, the collateral type, with phloem, hk, standing in front of the xylem, k. C, a portion of the radial type, shown complete in D, where the part outlined at a, corresponds to C. Corresponding parts are lettered the same in both figures; c, xylem; b, phloem; f, cambium ring; e, pericycle; d, endodermis. C and D are from the tap root of Vicia faba. (After Haberlandt.) neither standing radially in front of the other, thus making what is called a radial bundle. (See Fig. 21 for these types.) In leaves the primordial meristem becomes differentiated into protoderm, ground meristem and procambium strands; and, just as in stems, the protoderm gives rise to the epidermis, the procambium strands to the vascular bundles which make up the greater part of the veins, and the ground meristem to 44 DIFFERENTIATION OF THE TISSUES the mesophyll cells, which correspond to the primary cortex of stems. Near the bases of leaves, below where the vascular bundles have become split up to form the smaller veins, there is often a sheath of cells surrounding the bundles which corre- sponds to the parenchyma cells of the pericycle of stems. Where the vascular bundles first enter the leaves they have essentially the same constitution as in stems, with the excep- tion of a functional cambium, but they become smaller as they proceed and ramify throughout the leaf and have correspond- ingly fewer parts. The rule is that the sieve tubes gradually give place to elongated but otherwise undifferentiated paren- chyma cells; and in the ultimate ramifications only spirally and reticularly thickened tracheids are left to represent the bundle, and these are surrounded by a sheath of parenchyma cells belonging to the mesophyll or primary cortex, and known as the parenchyma sheath. It is the function of these meso- phyll cells to collect and carry toward the base of the leaf the foods manufactured by the rest of the mesophyll (see Fig. go). ILLUSTRATIVE STUDIES 1. Prepare cross and longitudinal sections of the growing apex of stems of Aristolochia sipho by imbedding the material in paraffin and making permanent stained mounts as described under Cytological Methods in Chapter XIV. Use erythrosin and iodine green for the stains or safranin and hematoxylin. Prepare in the same way sections from several successive internodes back from the apex. The object is to follow the progressive development of the tissues from the primordial condition at the apex down to where the primary permanent tissues appear. Find all of the tissues described in the chapter. Draw a few cells from each tissue, using the eyepiece scale (page 279) to get all details to scale. Determine the actual sizes of the cells and thicknesses of the walls. The erythrosin and iodine green will stain cellulose walls pink, and lignified and cutinized walls green, and in this way ILLUSTRATIVE STUDIES 45 it can be determined how far back from the apex the original cellulose walls first become modified for specific purposes. Notice where the first elements of the vascular bundle appear in the procambium and how in older segments of stem these have become longitudinally stretched. 2. Make cross and longitudinal sections of Aristolochia stem where the stem is older than where the sections in the above. studies were taken, but where the cambium has not yet begun to add to the thickness of the stem. The stem in this region will probably be too hard for the paraffin process and the sec- tions may be cut free-hand or on a sliding microtome as de- scribed on page 262. Double stain the sections in erythrosin and jodine green and make permanent mounts in balsam (page 263). Note the changes which each tissue has undergone since the earlier stages and make drawings to scale to show these changes. Think over carefully what you have seen and embody your results in your permanent note-book. If it is thought best not to make the permanent mounts as above suggested the sections can be examined in chloroiodide of zinc (page 292) in which cellulose walls will be blue and all others yellow. One objection to this reagent is that it swells the walls more or less. In its place aniline sulphate might be used (page 287) and then the lignified walls would be yellow and all others would be uncolored. Cutinized walls could then be demonstrated by leaving sections for several hours in alcanna tincture (page 285) when these walls would be pink. 3. Note the functions that in this chapter are attributed to each tissue, and see in what ways the tissues are adapted to them. Enter your observations in your permanent note-book. 4. Make a cross section of a leaf through one of the lateral veins and identify there the epidermis and the vascular bundle of the vein. The rest of the tissues belong to the fundamental or ground tissue, called in the leaf the mesophyll. Since the leaf is to be studied in detail in another place it will suffice here to enter in the note-book a simple diagrammatic drawing showing the relative positions and amounts of these different tissues. CHAPTER III SECONDARY INCREASE IN THICKNESS DICOTYLEDONS AND GYMNOSPERMS If we follow the history of any particular region of stem or root we find that its growth in thickness up to the time when its growth in length ceases is due to the enlargement of the cells that arise from the division of the cells of the primordial and primary meristems. In other words, increase in thickness is at first due to the enlargement of the cells of the epidermis, primary cortex, pericycle, phloem, xylem, medullary ray, pith. Such increase, known as primary increase in thickness, soon ceases, and subsequent growth in thickness is due to the differentiation of additional tissues following the production of new cells by the division of the cambium, or cork cambium in the bark. Growth of the Vascular Bundles.—It will be remembered from the preceding chapter that the cambium ring is composed of two parts: the fascicular cambium consisting of procambium cells lying between the phloem and xylem which remain prac- tically unchanged in form and retain their power of division, and the interfascicular cambium which is formed by the tan- gential division of primary medullary ray cells that lie in a line connecting the fascicular cambium of contiguous bundles. The cambium cells begin active cell division immediately follow- ing the differentiation of the primary tissues told about in the last chapter, and by the differentiation of these new -cells the fascicular cambium adds tissues to the xylem toward the inside, and to the phloem toward the outside, and the interfascicular cambium makes additions in like manner to the primary medul- lary rays. ‘ It is found on comparing the rate of growth of phloem and xylem that the latter increases much more rapidly than the 46 GROWTH OF VASCULAR BUNDLES 47 P) Drocaaat the g U | Sr : | EHH BT BW ET aH ES HIS AAS : ci BH eH EH His H PAT ES ALH| Wel LE TEL LS L 3 2 U1 Wo : Be : HH] : z Hie. LF | HIE: i HELE 1 PCr La GL IN aT HI a | : ; i Wf: LI H CAT aN UIT THEE 1 HL ! HL posuere i te mee Wane # \ if sb es Fic. 22,—Diagram showing additions to the primary tissues through the activity of the cambium and phellogen or cork cambium. Compare this with Fig.14. In this diagram stone cells have been omitted. 48 SECONDARY INCREASE IN THICKNESS former. This is due to the fact that when a cambium cell divides, by the formation of a tangential wall, which it usually does, the daughter cell facing the xylem much more frequently differ- entiates into the permanent condition than the one facing the phloem, the latter continuing as a cambium cell; but sometimes the daughter cell facing the phloem grows to be one of the phloem elements, while the one facing the xylem remains in the cambium condition. The kinds of tissues which the cambium adds to the xylem vary in different plants. In many Gymnosperms wood paren- chyma cells are formed, but, except in a single genus, neither tracheal tubes nor wood fibers, their place being usurped by tracheids which perform’ alike the strengthening and water-conducting func- tions. In Angiosperms are produced tracheal tubes of the pitted type, tracheids, and_transi- tional forms between these two, xylem Fic. 23.—Photomicrograph of cross section of stem of Aristolochia sipho, where cambial activity is just parenchyma and wood beginning. wu, epidermis; b, collenchyma; c, thin-walled fibers, and transitional parenchyma of the cortex, the innermost cell layer ‘of which is the starch sheath or endodermis; d, scleren- forms between these chyma ring of the pericycle; ¢, thin-walled parenchyma yi of the pericycle; f, primary medullary ray; g, phloem; h also (Fig. 22) . On the xylem; 7, interfascicular cambium; j, medulla or pith. phloem side the cam- mee bium adds sieve tubes, and, varying with the kind of plant, companion cells or phloem parenchyma, or both of these, and, in many instances bast fibers. While, by its tangential divisions, the cambium is thus adding to the radial diameters of the phloem and xylem, it is also, but at a slower rate, increasing their tangential diameters by its GROWTH OF VASCULAR BUNDLES 49 radial divisions; so that in cross sections the vascular bundle has the form of a wedge with its apex pointing toward the center (Figs. 22, 23 and 24). As this wedge broadens new or secondary medullary rays are from time to time begun by the, fascicular cambium (Fig. 24). These rays average less than half a milli- meter in vertical extent, although in a few instances they run 100 to 200 millimeters from node to node; and in their tangential diameter they are seldom more than five hundredths of a milli- meter, while radially they keep pace in growth with the phloem and xylem, and so always extend from the place of their origin in the xylem out between the phloem strands. The stimulus to form more medullary rays seems to come from the need of more radial highways as the diameter of stem and root and absorbing surfaces of new roots and food-building tissues of new leaves increase. The details of this will be discussed in Chapters VII and X. The daughter cells of the fascicular cambium, in becoming secondary medullary ray cells, enlarge chiefly in their radial and tangential diameters, becoming elongated radially, as a rule, in the xylem, but often vertically in the phloem. While the vascular bundle is thus enlarging and secondary medullary rays are being laid down, the interfascicular cam- bium is adding new cells to the primary medullary rays and thus causing them to keep pace in radial growth with the vas- cular bundles. Not infrequently, however, the interfascicular cambium forms new vascular bundles which, in cross section, appear to cut the primary ray into narrow strips. In the xylem portion of both primary and secondary medul- lary rays the tangential walls remain relatively thin or become pitted, and the radial walls are thin or pitted where they come into contact with tracheal tubes, tracheids, or xylem paren- chyma; while the transverse walls are apt to be much thickened and lignified. In the phloem portion of the rays the walls remain thin and unlignified. Fig. 24 shows that the cambium adds much more to the xylem than it does to the phloem. The growth of the xylem 4 50 SECONDARY INCREASE IN THICKNESS continues, as a rule, to the middle or end of August, but the growth of the phloem continues after this even until frost. \ mo. Oe QA me Fic. 24.—Portion of cross section of four-year-old stem of Aristolochia sipho, as shown by the rings of growth in the wood. The letters are the same as in Fig. 23, but new tissues have been added by the activity of the cam- bium; and a cork cambium has arisen from the outermost collenchyma cells and given rise to cork. The new tissues are: 1, cork cambium; &, cork; g, secondary phloem from the cambium, and just outside this is older crushed phloem; n, secondary xylem produced by the cambium; m, second- ary medullary ray made by the cambium (notice that this does not extend to the pith). Half of the pith is shown. Notice how it has been crushed almost out of existence. Compare Figs. 23 and 24, tissue for tissue, to find out what changes the primary tissues undergo with age, and to what extent new tissues are added. Photomicrograph X20. The kinds and relative amounts of the tissues in second- ary xylem and phloem vary a great deal in different families, genera, and species; and this fact is often very useful to the anatomist and pharmacognosist in characterizing and identifying materials. Thus, the xylem parenchyma may vary from abun- dance to entire ab- sence; tracheids may prevail or be lack- ing; tracheal tubes may vary greatly in size and number, or in their contact with one another or com- plete isolation; wood fibers may be present or absent, numerous or infrequent. The character of the phloem may vary in like degree; bast fibers may or may not occur; and so with the companion cells and phloem parenchyma. One of the most wonderful things about plants is that the daughter cells of the cambium may become such various things. How is it that a daughter cell facing outward is directed to GROWTH OF VASCULAR BUNDLES 51 take part in the formation of a sieve tube, while one facing inward undergoes quite other transformations to form a tracheal tube? And how can the daughter cells facing inward become any one of the xylem elements, apparently quite at will? They all have the same parentage, and probably the same potentialities, but behave differently, possibly because they are responding to stimuli of different natures. It may be that these stimuli arise in present need or necessity; but the response is modified by the peculiar potentialities of the particular species. Thus, the daugh- ter cells of the cambium in the oak, feeling the need of more water-transporting tissues, would transform themselves into tracheal tubes, while in the pine, under like circumstances, tracheids with large cavities would be formed. The so-called ring of growth throws some light on this question and will now be considered. In the stems and roots of trees and shrubs it is found that the addition made to the xylem during each growing season consists of two more or less well-defined parts, namely, an early growth, in which the tracheal tubes are relatively more abundant and possess larger cavities (Fig. 24), or where, as in conifers, the tracheids have relatively large cavities and thin walls; and a late growth, in which the tracheal tubes are rela- tively fewer and smaller, and the tracheids have smaller cavities and thicker walls (Fig. 52). In the early growth the water conduction elements may be said to preponderate, and in the late growth the strengthening elements; and this is as it should be, for the plant first feels the need of water in the spring, and later the need of greater strength. The first manifestation of growth in the spring is the unfolding of the leaves, and in Dicoty- ledons and Gymnosperms there are more of these than appeared the previous year, for the crown of branches grows larger every year; and even if the old tracheal tubes or tracheids were in direct communication with the new leaves they would not suffice. But the old water channels in the stem do not extend into this year’s leaves and new ones must be formed which will be contin- uous with those in the leaves (see Fig. 25). Later, when most 52 SECONDARY INCREASE IN THICKNESS or all of the leaves have been formed, and tracheal tubes com- municating with these have been laid down in the stem, the oa Growing : Qn apex j > a o C ot S oo 3 S Ee F : oh E wo A 8 55 ‘ ° i oO 3 er) ey a Ee qe s oh * 12 : “a de gs Ad Hy oe a 12 C Oo, b h Ni oe ba} 3 o, i os Bw Hy ho oo ilo A Sd SE ne o3 og i” le on ow A 4O an 39 Hs 6B i He ff ry & S Hi > 2 ys | Ao | Gh y= As I A Wo ff Min We || HS ed Sd B a: & Coal Bus {h ao Pf Shs § ie # wo f | = od a own Sh B afihN 9° “| r of terminal bud Q Scale scars of terminal bud Broken leaf trace or vascular bundle- of year before last Fic. 25.—Diagram showing the relation of this year’s leaves to the wood of the current year. cambium can devote itself more exclusively to the pro- duction of strengthening tissues, the need for which has been caused by the in- creased size and weight of the crown. The two differ- ent regions of a ring of annual growth are, there~ fore, seen to be an ana- tomical expression of vary- ing physiological needs. Sometimes it happens that trees are stripped of their leaves by insects, and later become rehabilitated by the growth of buds that would under normal con- ditions, have lain dormant until the succeeding spring. It is then found that the new crop of leaves stimu- lates the production of a new ring of growth in the stem, much as would have happened the follow- ing spring if things had been left to their wonted course. In those tropical regions where there is no pronounced dry season and the leaves do not all fall off at once, but just a few at a time with gradual renewal, there is no ring of growth formed; but where in the tropics a decided dry period provides plants with too little water, the leaves drop off just as they do outside GROWTH OF VASCULAR BUNDLES 53 the tropics on the approach of winter, and when these plants again clothe themselves With leaves a ring of growth is formed as already described. Such facts as these strengthen the conception that the formation of the ring of growth is at first stimulated by the demand for water on the part of the leaves. Secondary increase in thickness in roots does not differ essentially from that of stems, and the slight differ- ence that occurs is due to the pecu- liar arrangement of the phloem and xylem in the root bundle. It will be remembered that the phloem and xylem strands in roots stand side by side and not in radial line as in stems. (Compare diagrams in Fig. 21.) When secondary increase in thickness begins, the cambium flank- ing the phloem on the inside or toward the center lays down xylem elements, so that, with the already existing phloem, a collateral bundle, such as is typical in stems, is pro- duced. At the same time the cam- bium in front of the original or primary xylem commonly forms a medullary ray (Fig. 26), but it some- times makes phloem elements and thus completes a collateral bundle here also. The cambium then con- tinues to add new phloem and new xylem in both cases, and secondary medullary rays as the dimensions Fic. young root of Phaseolus multi- 26.—Cross section of a florus. m toiFls mips y eqng. =e eqn} eaeTts merece ele he 0 Ny Ear a Fic. 103.—Diagram to show food from the leaves descending through the sieve tubes and being stored in the medullary ray cells and xylem parenchyma, in A, and the digestion ‘of the stored food and its ascent through the tracheal tubes when growth is resumed in the spring, at B. In both diagrams the black bodies indicate stored food; that at the points of the arrows is being stored, and that at the base of the arrows is being digested and carried away. insoluble, or the plasma membrane imposed its interdiction (see page 92). It may well be that the plasma membranes of the ray cells play an important part in this way; but if they do they alter their behavior when growth is resumed in the spring, for they then allow the digested food to pass freely into the tracheal tubes (see Fig. 103). Igo STORAGE OF FOOD AND WATER The phloem part of the medullary rays passes most of the non- nitrogenous foods that come to it over to the xylem part or to the pericycle and cortex for storage, and reserves the bulk of the nitrogenous forms for itself. This fact stands sharply out when a cross section of stem taken in autumn or late summer is placed in a drop of iodine solution. The part of the rays between the phloem strands is then colored yellowish or brown because of its proteid content, and the xylem part is blue or almost black, due to the abundance of its starch. The wood parenchyma, which is the predominant tissue in the xylem of many herbaceous plants, and occurs in greater or less abundance in the wood of trees and shrubs, assists the medul- lary rays in the storage of non-nitrogenous foods, and almost the whole of fleshy roots and stems is composed of these two tissues, which there maintain the storage function. So long as the medullary rays and wood parenchyma remain living they retain the power and habit of storing food. In trees that have heartwood and sapwood all tissues are dead in the heartwood. In other kinds of trees where heartwood is not formed the medullary rays and wood parenchyma may be alive from bark to pith, even in trees that are fifty or more years old. The rule is, however, that most of the stored food occurs in the younger parts of the wood. The phloem parenchyma, like the phloem part of the medul- lary rays, stores up nitrogenous food reserves, and apparently for this purpose it is longer-lived than the other tissues of the phloem, living on sometimes for ten years or more when the sieve tubes and companion cells produced at the same time have long since died. The thin-walled parenchyma cells of the cortex and _ peri- cycle store up both nitrogenous and non-nitrogenous foods, and with them in this the collenchyma is often associated, and altogether they constitute a very significant part of the storage system. Fluctuations in the Solubility and Insolubility of Stored Food.—When the leaves of woody perennials have finished DIGESTION OF STORED FOOD. Ig!i their work and shrubs and trees stand bare and apparently inactive it might be conjectured that their store of food would wait unaltered for the return of spring; but this is by no means the case, for part of the food is rendered soluble and appar- ently is used in respiration throughout the dormant period, and the greater part may be changed from insoluble to soluble and back again as the outside temperature falls and rises. The maximum amount of starch is found in the fall, for a large per- centage of starch in the bark is changed to sugar or oil during the winter, and in softwood trees and shrubs the same thing happens in the wood. In hardwood trees the change is not so great in the wood. A rise of temperature during the winter or early spring incites a change back to starch again. Digestion of Stored Food.—The forms in which foods are stored are suited as a rule to their safekeeping, but not to their distribution and use. Most foods are stored in an insol- uble form such as starch, oil, and the majority of proteids, and only a few in their storage form are capable both of dif- fusion and assimilation, as glucose and saccharose. The chemi- cal processes by which stored foods are made soluble, diffusible, and assimilable are called digestion. In carrying: on digestion the protoplast usually employs to do this work a proteid body known as an enzyme or ferment which it has made apparently by a process of self-decomposi- tion that we call secretion; and of these enzymes the proto- plasts may possibly make as many kinds as there are varieties of food to be digested; and it is also possible that the proto- plasts sometimes incite digestion without the intervention of an enzyme. Free oxygen is necessaty to the formation of enzymes, and these work best at warm temperatures ranging from 20° C. to 60° C. according to the variety; they are also more effective in the dark than in the light, since light, particularly of the violet end of the spectrum, tends to destroy them. It is not known just how the enzymes act in digestion. They incite the necessary chemical changes, but hold themselves Ig2 STORAGE OF FOOD AND WATER apart so that they are not themselves destroyed in the process. The result of this is that a small amount of enzyme can digest a relatively very large quantity of food, even up to 100,000 or more times its own volume. Some of the many kinds of ferments produced by plants have been classified and named. Thus diastase converts starch into maltose (malt sugar). Maltase converts maltose into glu- cose (grape sugar). Jnulase converts inulin into fructose (fruit sugar). Invertase splits saccharose (cane sugar) into glucose and fructose. Cytase changes cellulose to glucose. Pectase changes pectic substances in the cell-wall to vegetable jelly. Emulsin and myrosin are representative of enzymes acting on glucosides and breaking them up into glucose and other substances. A group of enzymes known as lipases or steapsins split up fats and oils into fatty acids and glycerine. The enzymes, called proteolytic enzymes, that digest proteids are similar to the pepsins and trypsins of the stomach and pancreas of animals. The pepsins change proteids to the soluble peptones, and trypsins convert peptones: or proteids directly into amido-acids. The trypsins Fic. 104.—Enzyme-secreting cells of date @Te the more common of coving #5 aed of Sinden of Indo” the proteolytic jenzymes in plants. ae These enzymes occur in every cell where the food ‘that they are fitted to digest is stored even transiently. In fact, dias- tatic, inverting, and tryptic enzymes are so common that they seem to be a part of every protoplast. There are, however, ° in special cases, cells or groups of cells devoted to the secretion of enzymes, as in the root of the horse-radish; and in the whole family of grasses the epidermis of the cotyledon secretes enzymes ASSIMILATION OF FOOD 193 which help to digest the reserve food in the endosperm, and the same thing occurs in the date palm (Fig. 104). Glands devoted to the secretion of enzymes occur in Dro- sera, Dionea and Nepenthes (Fig. 105), but in these instances the enzymes do not act upon food stored within the plant, but upon insects, and the glands are comparable to those in the alimentary canal of animals. It seems that the enzymes do not reach the maximum amount in the cells until the process of digestion is well started, and it is for this reason that more diastase can be extracted from sprouted than from unsprouted barley. The mere presence of the enzymes is, how- ever, not enough to start digestion going; rather the impulse to grow and the actual inception of growth creating a demand for 3. food seems to supply the stimulus that puts —_-Fic._ 105.—Longitu- a . A o dinal section through digestion in motion. Such procedure would 4 digestive gland of of course be a vital one initiated by the living Beas oa protoplast. — Assimilation of Food.—We must keep in mind the fact that the proteids, oils, starches, sugars, and other kinds of food _ have two distinct uses: to furnish chemical elements for the construction of the protoplasts and cell-wall, and other special and useful products, such as nectar, aromatic oils, and enzymes, | and to supply energy in the form and place needed to keep the vital machinery in motion. That food which is taken on by the protoplast and made a part of its own body is assimilated. We say that the ‘food is lifeless but the protoplast is living. Neces- sarily the food passes from the lifeless to the living condition by something that the protoplast does with it. In this process the food loses its identity. Sugar, oil, soluble proteids, etc., enter into new combinations resulting in the formation of protoplasm— the living substance. An attempt to explain how this most wonderful of transformations takes place would be the merest speculation. We know nothing profoundly about it. To 13 194 STORAGE OF FOOD AND WATER begin with, we know very little about the sequence of steps in the formation of even lifeless proteids from simpler substances, and protoplasm is supposed to be a complex aggregation of pro- teids, water, and other possible compounds. When we speak of life we have in mind a kind of energy manifested by the pro- toplasm alone. We may conceive of this manifestation as being due to a combination of certain rates, amplitudes, and paths of vibrations of the protoplasmic molecules or structural units. When new structural units are built from the food they would be suited to the same mode of motion as the others and they would presumably assume this mode because they are under the same conditions as the others. If this conception were true so far as it goes it offers nothing to clear up the great mystery of the creation of living from lifeless matter. That, however, the protoplasm has power to build its own substance by combina- tions of different kinds of food and that the application of this power is self-regulating, resulting sometimes in growth, at other times in merely maintaining a balance between destruction and construction, is satisfactorily established. Relation of Stored Food to Energy Supply.—The energy which plants draw upon to keep the vital activities going comes ‘to them from the sun through the food. It seems that the facts are about as follows: The sun’s energy is used by the -chloroplasts to build carbohydrates. A part of this is decom- posed, yielding energy for the construction of proteids from carbohydrates and other substances. Other parts of the car- bohydrates and some of the proteids, are oxidized, or broken down in other ways, yielding energy for the formation of proto- plasm from proteids, etc. Some carbohydrates, proteids, and a part of the protoplasm itself are broken down and energy ‘is thus set free for doing whatever the protoplast as a living agent has to do. This line of activities traces the source of its energy directly back to the sun. The food, as well as the protoplasm, has sun’s energy stored up in it ready to be set free for doing work and subject to the direction of the protoplasts. The construc- STORAGE OF WATER 195 tion of food is in a manner similar to the winding of a weight, and when the food or the protoplast is decomposed the resulting readjustment of the elements keeps the vital activities going, just as the falling of a weight may set and keep in motion a train of wheels, etc. More than go per cent. of the energy released in respiration appears as heat, and the remaining energy probably manifests itself for the most part in chemical reactions attendant on growth, repair, secretion, and other constructive processes. The Storage of Water.—The need of plants for special water-storage tissues is not so general as their need for tissues in which to store food. Under ‘ ne at) ordinary conditions water can be Al a) freely taken in from the soil which Cea CMe ‘ serves as the water reservoir for plants. But plants of desert regions or growing anywhere under xero- phytic conditions (conditions making water hard to get, as when it is _ actually scarce or difficult to absorb because of low temperature in the substratum, or because there are ee substances in solution in amounts showing large part of the leaf . devoted to the storage of water. a, large enough to act asa poison or to water-storage cells; 6, chlorophyll- retard the osmotic inflow into the Prerr crllsi < cevetal ef calcium ; : oxalate. (After Schimper.) roots) to which this: reservoir is denied or more or less inaccessible, have hit upon various com- pensating devices. One of these is the water-storage tissue. This is seen in its fullest development in succulent stems and leaves. In Mesembryanthemum Forskalii of the Egyptian desert approximately one-half of each succulent leaf is made up of water-storage tissue (Fig. 106); and in epiphytic species of Codonanthe growing on a dry substratum nearly three-fourths of the fleshy leaf is occupied by cells devoted to the storage of water (Fig. 81). The fleshy stems of cacti and some Euphor- biacee are largely composed of the same kind of tissue. In the leaves of Ficus elastica the protoderm of the upper surface cry a . 196 STORAGE OF FOOD AND WATER divides tangentially and gives rise to several cell-layers constitut- ing a typical water-storage tissue (Fig. 79). Water-storage tracheids sometimes occur as terminals of the finer branches of leaf veins, as in Euphorbia splendens and Townsendia cespitosa (Fig. 107); and occasionally the meso- phyll cells have the characteristic wall thickenings of tracheids and apparently serve in water storage. The tubers of the potato and other fleshy underground parts serve for the stor- age of water as well as of food. Fic. 107.—Water-storage tracheids in the leaf of Euphorbia splendens. 6, b, water- - storage tracheids; d, mesophyll cells; c, branch from a milk tube. (After Haberlandt.) Frequently, and especially in xerophytes, cells or groups of cells contain mucilage as a real cell content or as much thick- ened cell-walls, the inner layers of which have become transformed from cellulose to mucilage. Mucilage has a great affinity for water, imbibing it with power and holding it with great tenacity. When the amount of mucilage is considerable, as is frequently the case in desert plants, such as the Aloes, cacti, certain species of Astragalus, and many others, it plays an important part in the water-storage ‘function. Characteristics of Water-storage Tissues.—The water- storage cells are characterized by having thin cellulose walls, or walls, if thickened, having many pits or thin places. These cells readily imbibe water when plenty is at hand, and when the soil water is scarce they deliver their stores gradually to those tissues, such as the photosynthetic and meristematic, in ILLUSTRATIVE STUDIES 197 which the scarcity of water would be most harmful to the well- being of the plant; and the water-storage tissues keep on dis- tributing their stores until they themselves are wilted while the tissues to which they are tributory are maintained in a fresh and turgid condition. ILLUSTRATIVE STUDIES 1. Cut free-hand sections of potato that has lain for some time in 95 percent. alcohol to harden it. Mount the sections in a drop of water and after studying them with low and high powers let a drop of iodine solution diffuse under the cover- glass. Starch grains will be colored blue to black and_pro- teids yellow. The cell-contents can be seen to best advantage around the thinnest edge of the section, but there they will have dropped out to some extent and study of a thicker part of the section may be necessary to find out how densely packed are the cells with reserve food. Draw a few cells with their contents. Measure the starch grains. 2. Study in a similar manner thin sections of soaked lima bean. Notice how the striations of the starch-grains differ from those of potato starch. Notice the very distinct granules of proteid stained yellow to brownish by the iodine. Draw a few cells with their contents, and measure the grains of reserve food. 3. Cut with a dry razor free-hand sections of the endosperm of castor bean and transfer them to a watch glass containing 95 per cent. alcohol 2 parts and castor oil 1 part and enough eosin to make a light red solution. After a few hours mount the sections in ricinus oil-alcohol without the eosin. This treat- ment will bring the aleurone grains out clearly and reveal their several parts (page 182); and it will show the grains to be im- bedded in the meshes of the cytoplasm. Draw a few cells to show this, and on a larger scale a single aleurone grain in all its details. Put other sections for several hours in alcanna tincture (page 198 STORAGE OF FOOD AND WATER 285) arid mount them in a drop of dilute glycerine. The oil will be stained pink. Notice how it gathers in droplets of various sizes around the edges of the section, and the abundance of it in the uninjured cells. 4. Test the endosperm of germinating and ungerminated castor bean for glucose (see under Copper Acetate and Fehling’s solu- tion in Chapter XVI). How do you interpret the results? 5. Examine with a high power in a drop of water starch from ungerminated and germinating barley. What evidence do you find that starch is digested during germination ? Test both germinating and ungerminated barley for glucose by crushing the grains and boiling them in Fehling’s solution. 6. Cut free-hand or on a sliding microtome cross and longi- tudinal radial and tangential sections of grape stem. Mount the sections in glycerine-iodine (see under Glycerine and Iodine in Chapter XVI). Note the extent of the storage of starch and proteids in phloem, medullary rays, and wood parenchyma. Other woody stems will do, but the grapevine is especially fine for showing this. Make drawings from each region of a few cells with contents. 7. Make thin cross sections of rubber leaf and mount them in dilute glycerine. Note the clear tissue between the epider- mis and palisade cells. This is for the storage of water. Make a diagrammatic drawing of the leaf expressing the proportion of leaf devoted o water storage. 8. Cut tangential sections (see Chapter X, page 173, par. 2) of a sunflower leaf and mount them in dilute glycerine. The large clear cells are water-storage cells. Draw a few of these cells with the surrounding mesophyll tissue. ’ CHAPTER XII SECRETION AND EXCRETION Nature of Secretions and Excretions.—Secretions and excretions are distinguished from the reserve food told about in the last chapter by their evident uselessness in supplying materials and energy for growth, repair, etc. They there- fore, for the most part, remain practically unchanged in special cells or tissues, or are eliminated from the cells by excretion at the surface of the plant or into intercellular spaces. Some- times they have a biological function, as in the case of nectar, and sometimes a physiological function, as in the case of enzymes, and organic acids excreted by the roots. By far the larger number of plant secretions belong to the class of ethereal oils and resins. These occur together, the resins dissolved in the oils and forming oleo-resins. When the oil evaporates, as it normally does in time, and can quickly be made to do on heating, the resins are left behind as a solid residue. The amount of resin present varies greatly, from the merest traces in many epidermal glands to more than 7o per cent. in some of the Conifere. The fragrance of these secretions is due to volatile substances entering into the composition of, and even forming a very large part of, the oils, as, for instance, eugenol in clove oil, safrol in oil of sassafras, and cinnamon aldehyde in oils of cinnamon and cassia. Turpentine, produced chiefly by the Conifere, is the most. abundant of the oleo-resin secretions. On distillation this yields oil of turpentine of commerce and resins varying in char- acter with the different genera producing them. No sharp line can be drawn between secretions and excre- tions as these words have come to be used in physiological litera- 199 200 SECRETION AND EXCRETION ture. So long as the substance is in the cell that formed it we are apt to think of it as a secretion, but as an excretion when it is eliminated from the cell into intercellular spaces or at the exterior. The significance of these terms will be better appre- hended by means of their particular application as we proceed. Secreting Cells and Glands in General.—Probably all living cells secrete digestive and oxidative enzymes, and all cells secrete their cell-walls; but in certain numerous families of plants we find single cells or groups of cells called glands that carry on secretion as their special function. The secret- ing cells are sometimes descended from the protoderm, and therefore belong morphologically to the epidermis, and some- times they are descendants of the fundamental meristem and may occur in the cortex, pericycle, medullary rays and pith. Fic. 108.—Glandular hair from the petiole of Pelargonium zonale. ¢, secretion from the globular gland on which it rests; B, portion of across section through a nectariferous bract of Vicia sepium; n, nectar-secreting cells. (After Haberlandt.) Less frequently we find them descended from the procambium and belonging to the phloem or xylem parenchyma, as in the case of some of the resin-secreting cells of Conifers. There are three kinds of glands in regard to their location and form, namely, the superficial type which, descended from the protoderm, is borne at the outer surface and may rise above it in the form of hairs or scales; the interior globular type con- SECRETING CELLS IN GENERAL sisting of a more or less globular group of cells; and the interior tubular type in the form of a tube or canal. Glands belonging to the first type, commonly known as glandular hairs, arise by the tangential division of a protoderm cell producing a multicellular hair, the apical cell of which enlarges and becomes the secreting cell (Fig. 108, A), or a group of secreting cells may compose the gland at the apex. Nectaries are usually of protodermal origin and their cells are frequently elongated radially in the form of papille (Fig. 108, B). The interior globular glands arise by the division of a cell or group of cells, usually of the ground meristem, and where these glands lie near the surface the protoderm may by cell-division con- 201 Fic. 109.—Formation of an interior, globular, ly- sigenous gland of the leaf of Dictamnus fraxinella. A, g, g and c, mother cells of the gland; c, from the protoderm, and g, g, from the fundamental tissue. B, older stage where the cells have begun to form the secretion. The last stage is shown in Fig. 112. (After Sachs.) Fic. 110.—Cross-section through a portion of orange peel showing the cavity of an interior, globular gland at g; crystals of hesperidin at h; calcium oxalate crystals at k. (After Tschirch and Oesterle.) tribute cells to their formation (Fig. 109); or sometimes the protoderm alone gives rise to the gland. Glands of the globular type are found in the clove, rind of orange and lemon, etc. (Fig. 110). An intercellular cavity into which the secretions of the glandular cells are excreted is formed in one of two ways: The secreting cells may split apart at the center of the group and then draw or grow away from the line of separation, leaving an intercellular cavity (Fig. IIz), or the secreting cells may break down altogether, leaving their secretions in the cavity 202 SECRETION AND EXCRETION formed by their disintegration (Fig. 112). The first method of forming the intercellular space is schizogenous, and the second, /ysigenous. Sometimes the two methods are combined by the space beginning schizogenously and then being enlarged Fic. 112.—Lysigenous gland in the leaf of Dictamnus fraxinella. B, Fic. 111.—Schizogenous resin duct in the young gland, with cells beginning to young stem of ivy (Hedera helix), as seen in secrete oil; C, mature gland where cross section. A, early, and B, later stage the secreting cells have broken down in the formation of the duct. g, the mature and left their secretion within the duct; c, cambium; wb, phloem; b, bast fibers. cavity thus formed; o, large drop of (After Sachs.) secreted oil. (After Sachs.) lysigenously, and to designate this method the term schizoly- sigenous is compounded. Interior glands of protodermal origin solely are found in Amorpha, Myrtus, Eugenia, Asarum, Croton, Crotonopsis, and some species of the Moracex, Urticacee, Acanthacez, Saxifragacee, Crassulacee, Geraniacee, and other families. By far the larger number of interior glands, however, are formed by the ground or fundamental meristem. LATICIFEROUS VESSELS 203 Interior tubular or canalicular glands are formed in essen- tially the same manner as the globular. A circular group of cells as seen in cross section, and a long vertical row as seen in longitudinal section, forms an intercellular space schizogen- ously or lysigneously at its center throughout its length. If formed schizogenously the secreting cells form a sheath around the intercellular canal as seen in Fig. 113, and into this canal Fic. 113.—Resin duct in leaf of Pinus silvestris, in cross section at A, and in longi- tudinal section at B; h, cavity surrounded by the secreting cells; f, f, sclerenchyma fibers surrounding and protecting the duct. (After Haberlandt.) are excreted the secretions of the sheath cells. Fine examples of tubular canals are found in the needles and stems of pines. In the few instances where the tubular glands are differen- tiated from procambium strands they have the same method of formation as have those from the ground meristem. The tubular glands often branch and anastomose and thus form a complex glandular system. Laticiferous Vessels or Milk Tubes.—The milk tubes occurring in many families of plants form a much-branched, richly anastomosing system extending practically throughout the whole plant (Fig. 114). They have two methods of origin. In the Lobeliacee, Cichoriacee, Papaveracee, Campanulacee, Papayacee, and a few Euphorbiacee, and many Musacee and Aroidez they are formed by cell fusicns which take place early in the primary meristematic condition by digestion of separating walls. The anastomoses which unite the tubes into practically 204 SECRETION AND EXCRETION one system are formed also by cell fusions, or sometimes by outgrowth of branches from the tubes which push their way through intervening tis- sues and fuse with other tubes or branches. In the Urticacez, Asclepiadacee, Mora- cee, most Euphorbia- cee, and Apocynacee each tube arises from a single meristematic cell which elongates and branches, keeping pace with the growth of the plant, and fusing its branches with those from other tubes and thus forming an intercom- municating system, so that when a wound is made the milk pours forth abundantly. The milk tubes re- main living for a long time and probably take an active part in the production of the very complex latex or milk, Fic. 114.—Laticiferous vessels from the cortex which may contain of root of Scorozonora hispanica. A, as seen under pl astic or food sub- low power, and B, a smaller portion under high power, (After Sache) stances such as sugar, oil, starch, proteid, and aplastic or non-food substances such as tannins, alkaloids, some varieties of gum, caoutchouc, resins, and salts of calcium and magnesium. Some of these may be mere excretions of useless substances from other tissues, and some of them may be TANNIN CELLS 205 products of the tube itself destined for the useful purpose of healing wounds and giving immunity from parasitic attacks, while others are clearly foods which find in the tubes an efficient means of distribution. Tannin Cells.—Tannin cells are found in various families of plants. They occur as single isolated cells or in small groups. The cells are approximately isodiametric or in various degrees of elongation. ‘The longest known occur in the genus Sambucus, where they become twenty or more millimeters in length and sometimes extend through an entire internode. Tannin cells are found in the epidermis, primary cortex, peri- cycle, phloem, medullary rays, and in the mesophyll of leaves. They occur in greatest abundance in the cortex and in the tissues of galls. Tannins seem to be by-products set aside in the tannin cells from the general circulation. It is uncertain whether the tannins are ever used to an appreciable extent in nutrition. They seem to be of service, however, in warding off parasites by their aseptic qualities and astringent taste. Special Enzyme-Secreting Cells.—In the Crucifere, Cap- paridacee, and a few other families are found special cells de- voted to the secretion of enzymes, such as myrosin. The pun- gency of these plants is due to allylic mustard oil, produced, it is said, at the moment of injury to the plant by the action of my- rosin on the glucoside potassium myronate which is associated with the ferment. Glucose and potassium sulphate are other products of this reaction. The digestive glands of insectivorous plants are unique in that their secretions digest animal tissues and are stimulated to activity by the presence of the captive. On the upper side of the leaves of Pinguicula are two kinds of glandular hairs, a long-stalked form secreting a sticky mucilage which holds fast the prey, and a short form, hardly appearing above the epi- dermis, which, when an insect is captured, secretes and pours forth a digestive enzyme (Fig. 115). The short glandular hairs on the leaves of Dionea muscipula behave like those of Pinguicula. 206 SECRETION AND EXCRETION In the pitchers of the genus Nepenthes unstalked digestive glands occur on the inside near the bottom. These pour forth an abundance.of a mucilaginous digestive fluid the water for Sheps B Fic. 115.—Glands from Pinguicula. A, upper surface of leaf showing long-stalked gland at m, and short-stalked gland at 2. B, cross section through a short-stalked gland. (A, after Kerner, and B, after Haberlandt.) which is supplied to the gland by a bundle of tracheids extending close up to the base of the gland. The most highly differentiated glands are found on the leaves of Drosera rotundifolia (Fig. 105). Here the gland proper, which is borne at the apex of a slender stalk, is composed of a bundle of tracheids surrounded by three layers of cells. The outer two layers seem to be especially concerned in producing the secretion. The cuticle is permeable, and ordi- Fic. 116.—Different forms of crystals of calcium oxalate. ; manicata. (After Frank.) D A, from the petiole of Begonia narily an acid, sticky secretion is excreted at the surface in which the feet of insects alighting on the surface become entangled. The capture of an insect stimulates the glands to secrete and SECRETION AND EXCRETION OF MINERALS 207 pour forth more acid, and an enzyme similar to animal pepsin, by means of which the insect is digested. Secretion and Excretion of Minerals.—In some plants single cells, and strands or layers of cells forming a more or less extensive tissue, are devoted to the secretion and excretion of calcium oxalate crystals—excretion in the sense that while the crystals remain within the body of the plant and are con- tained within cells they have been set apart by themselves where, as a rule, their isolation continues throughout the life of the plant. The calcium oxalate crystals occur singly or in groups in a single cell, and either as simple or compound crystals, as shown in Fig. 116. The forms and association of the crystals may be influenced by the strength of the solution in the cell-sap; but evidently the protoplast has a very important influence, for in certain cells and tissues only one kind of crystal may occur, as in the case of the acicular crystals in Tradescantia, Pistia, Ariseema, etc. A secretion of’ relatively rare occurrence is that of calcium carbonate in the cystolith cells of some Mora- Fic. 117.—Cystoliths from the leaf of « es = Ficus carica. A, complete cystolith; B, Cos Urticacez, and Acantha cystolith from which the calcium carbonate cee, occurring commonly but has been removed for use in other parts of the * plant. B is from a leaf that had fallen off in not solely in the leaves. autumn. (After Haberlandt.) Following the development of a cystolith it is found that the outer wall of an epidermal cell, for instance, grows down into the cell cavity, swells out at the end and there becomes warty at the surface (Fig. 117). When a cystolith is treated with hydrochloric acid it quickly diminishes in size with the evolution of bubbles of CO,, show- ing that calcium carbonate is being decomposed. At the com- pletion of the reaction a skeleton of cellulose remains. The cystolith is therefore a cellulose outgrowth of the wall infil- trated and encrusted with calcium carbonate. In nature the calcium carbonate of the cystolith comes and goes, and its secre- 208 SECRETION AND EXCRETION tion seems to be a method of accumulating it for use at some future time. The Process of Secretion.—Secretion is evidently a vital process, that is, it is carried on by the living protoplasm. The substances may be formed directly from foods, or from the disintegration of the cell-wall, or from the decomposition of the protoplasm itself. It is possible that sometimes all of these methods are employed by a single cell or gland. The distinctive behavior of the cytoplasm of some secreting cells is evidence that it produces the secretion by self-decomposition; for, pre- paratory to secretion, the cytoplasm becomes relatively very dense and granular, and as secretion sets in this density dimin- ishes, until, when secretion stops, the cytoplasm has become very much depleted. In some Rutacee the cytoplasm and nucleus of secreting cells disappear altogether. In some instances the evidence is very clear that the secretion has been formed from the substance of the cell-wall, where the secretion appears just beneath the cuticle, and accumulating, pushes the cuticle off from the wall, as seen in Fig. 118. Fic. 118.—Glands from the leaf of Ribes nigrum. Secretions of mucilage A, young stage in the development of the glandwhere and of ethereal oils and the cuticle is already being pushed up by the secre- é i ‘ tion, 7. B, complete gland;k, secreting cells;h, cavity TeSINS take place in this between the secreting cells and cuticle occupied by the secretion. (After Haberlandt.) Ways Of course the secretions do not neces- sarily come entirely from the substance of the wall, for it is possible that the protoplast makes a part of the secretion directly without first repairing the wall preparatory to decomposing it. The Excretion of Liquid Water.—On summer nights “dew” hangs in droplets at the tips and along the edges of leaves of grass and many other kinds of plants. It was long supposed that in all cases these droplets were real dew formed from condensation from the atmosphere, but that this is by no means always the case is shown by chemical analysis of the EXCRETION OF LIQUID WATER 209 drops, by the anatomy of the leaves where the drops occur, and by physiological experiment, as will now be set forth. Droplets from the leaves of Indian corn on evaporation leave behind .o5 per cent. of solid residue, from leaves of Brassica cretica 1 per cent., and so on for different plants; and on incinera- tion 15 to 50 per cent. of this residue turns out to be ash. Real dew being distilled water could not be expected to give such results. Sections through a leaf where the droplets occur are found under a microscope to have specialized cells or groups of cells having the appear- ance of glands. These have been named hydatodes. One of the simplest of these is found on the upper and under surfaces of the leaves of Gonocaryum pyriforme, where each hydatode consists of a single epi- dermal cell differing from the rest in several details, as shown in Fig. 119. A portion of the outer wall grows out and forms a slender projection trav- ersed longitudinally by a canal extend- ing’ from the mucilaginous apex to the cell cavity. The cell cavity is broad Coe and funnel-shaped in its upper part, in cross section at A, and from and again broadened below the neck ca He Ae or of the funnel. As is the rule with secreting cells, the cytoplasm is unusually dense and the nucleus relatively large. In this instance water is absorbed by the hydatode from surrounding epidermal and subepidermal cells and excreted through the narrow canal in the exterior projection. On the under side of the leaves of Phaseolus multiflorus are curved hairs, as shown in Fig. 120, with outer walls thin and but little cutinized through which water filtrates and is excreted at the surface. 14 210 SECRETION AND EXCRETION In another class of hydatodes bundles of tracheids from the terminations of the vascular bundles supply water to water- excreting parenchyma cells or themselves excrete water directly Fic. 120.—Hydatode from the leaf of Phaseolus multiflorus. (After Haberlandt.) into intercellular spaces. Hydatodes of this sort are not uncommon in ferns where they occur chiefly along the leaf margins. This type reaches its highest development where water- stomata are present in the epidermis through which water excreted into the intercellular spaces is expelled. This is illustrated by Primula sinensis where the hydatodes occur in the teeth of the leaf margins (Fig. 121). Physiological experiments show that many hydatodes excrete: water through the activity of their living cells, since excretion stops after they have been killed by brushing over with a solution of cor- rosive sublimate, and they cannot then be forced to excrete water even when the pressure in the tracheal tubes is made very great by connection with a U-tube with its long arm filled with mercury. The majority of hydatodes equipped with stomata will still excrete water, however, after they have been poisoned, and by artificially increasing the pressure in the tracheal system ex- cretion of water is hastened. The amount of water excreted by hydatodes is often remarkably large. A young leaf of Colocasia nymphefolia gave out 48 to 97 cubic centimeters of water in one night, and a mature leaf of C. antiquorum excreted on the centimeters nightly. Fic. 121.—Radial dinal section through a hyda- tode from the leaf margin of longitu- Primula sinensis. 7, upper, and j, lower epidermis; h, palisade cell; ¢, thin-walled parenchyma, called epithem; g, intercellular space; f, guard cell of a water stoma; k, tracheal elements (After Haberlandt.) average 9 to 12 cubic The use of hydatodes seems to be that when transpiration ILLUSTRATIVE STUDIES 2If is checked, as by the going down of the sun, while the absorp- tive activity of the roots is as yet undiminished, they may by the excretion of water prevent its filtration into the general intercellular system which should be kept open for the storage and circulation of the necessary oxygen and carbon dioxide. Hydatodes may take on other functions in special cases, such as the secretion of nectar or digestive enzymes. ILLUSTRATIVE STUDIES 1. Put small segments of pine branch into a saturated aque- ous solution of copper acetate, and after several weeks cut sections free-hand or on a sliding microtome and study them in a drop of dilute glycerine. Resin will be found stained an emerald green. Study longitudinal sections in the same way and draw the tubular glands from both points of view. 2. Put a small handful of cloves into a widemouth bottle and pour water over them. Stopper the bottle with a per- forated cork and insert a U-tube flush with the lower surface of the cork. Let the long arm of the U-tube extend deep into another bottle. Set the bottle containing the cloves into a basin of water kept boiling, and the other bottle into a basin of cold water kept cold by running water or with bits of ice added from time to time; this will condense the steam and oil that distils over. In this way the presence of volatile oil in the cloves can be demonstrated. 3. Soften cloves in water, cut thin sections and mount them in a drop of strong KOH solution. Numerous oil glands will be found, and possibly needle-shaped crystals of potassium caryophyllate formed by the reaction between the KOH and the clove oil. 4. Make thin sections of lemon peel hardened in 95 per cent. alcohol and inclosed for sectioning in elder pith. Note the globular glands in different stages of formation. How close to the surface do they come? Do any of them show an opening at the exterior? Draw one of the glands with the surrounding tissue. : 212 SECRETION AND EXCRETION 5. With a-sharp knife cut germinating date seeds cross- wise into slices about 2 mm. thick and put them through the process of fixing, hardening, and imbedding in paraffin, and sectioning, described in Chapter XV. When it comes to staining the sections place the slide on which they are mounted, after the paraffin has been dissolved away in xylene and the xylene rinsed off with alcohol, in a dish of safranin (page 326) for a few hours, and then rinse out the surplus safranin in water, dehydrate quickly in 95 per cent. alcohol, rinse in xylene and mount in balsam. Study the epidermis of the enlarged coty- ledon. These cells secrete enzymes for the digestion of the endosperm exterior to them. Draw a few of the secreting cells together with the adjoining cells of the cotyledon and endosperm, and by stippling indicate how the secreting cells differ in appearance from the others. 6. Soak grains of Indian corn over night and cut the thin- nest possible free-hand sections across the embryo and endo- sperm. Mount the sections in dilute glycerine. Compare the appearance of the epidermis of the cotyledon with that of the date. These cells of the corn are also enzyme-secreting. 7. Scrape hairs from the surface of Pelargonium zonale or other plant bearing glandular hairs, mount them in a drop of dilute glycerine and study them under high magnification. Draw some of the glandular hairs. 8. Study longitudinal sections of the stem of some milk- weed or of the greenhouse Euphorbia splendens for laticifer- ous vessels. Make a drawing showing the branching and anastomosing habit of these vessels. Treat the section with iodine. Are there indications of starch and proteids? g. Cut cross-sections through the cotyledons of acorns; examine them in water and allow a dilute solution of chloride of iron to run under the coverglass (see under Tannins in Chap- ter XVII). Note the indication of tannin in some of the cells. 10. Cut cross-sections of the seed of Strychnos nux-vomica and test them for alkaloids as described under Alkaloids in Chapter XVII. : CHAPTER XIII REPRODUCTION In previous chapters we have dealt with the structural pro- visions for those functions that have to do with the nutrition and maintenance of the individual. We shall now find that the organization of the individual is not related to its own needs alone, but that it provides also for the continuance and pro- gressive evolution of its race. This is accomplished by the sporogenous cells and tissues occurring in the ovules and anthers of the spermatophytes and in their homologues among the lower forms, by spores themselves, by gametes (sperm cells and egg cells), and by nurse cells having for their chief or only function the nutrition of the new generation, such as tapetal cells in sporangia, and antipodal cells within the embryo sac. These tissues are often meristematic it is true, but their purpose is so different from that of the vegetative meristems (see page 24) that they cannot be classed together. Those tissues which have reproduction for their chief or only function should be classed as Reproductive Tissues. For our introduction to tissues of this sort we cannot do better than to trace the develop- ment of a fern sporangium. Development of Fern Sporangium.—Figure 122 has been made from camera lucida drawings of a longitudinal section through the apex of a sporophyll of Aneimia phyllitidis. At a is the primordial meristem of the growing apex, and at 8, c, d, e, f, are successive stages in the formation of sporangia pro- ceeding from this meristem. It appears that a sporangium has its beginning in a group of primordial cells such as is seen at 7. These evidently divide parallel to the surface (periclinal division) and again perpendicular to the surface (anticlinal division), and the growth of the daughter-cells causes the enlargement which we recognize as the beginning of the sporangium, b. At 213 214 REPRODUCTION a very early period of its develogment, d, we find the sporangium composed of a protoderm and a central cell. The protoderm proceeds to the formation of an epidermis or sporangium wall, sporangium wall Mature tapetum of Tapetal plasmodium formed by fusionof tapetal protoplasts Lge Mother cells have divided to form four spores only three shown Grandmother-—cell has divided producing two mother-cells Fic, 122.—Sporangium and spore formation of a fern, Aneimia Phyllitidis. uw, growing apex; 7, group of three protodermal cells which are to give rise to a sporangium:; b, c, d, e, Ff, g, h, successive stages in sporangium and spore formation, Partly diagrammatic. Further description in the text. DIVISION OF SPORE GRANDMOTHER CELLS 215 while the central cell gives rise to two distinct products, namely, an exterior tissue or fapetwm averaging two cell-layers in thick- ness when mature, e and f, and surrounded by this an arch- esporial cell, e, which is soon to give rise by division to a group of cells known as the sporogenous cells or grandmother cells of the spores, f and g. It is clear that the tapetal and sporogenous tissues must be classified as reproductive because they serve the purpose of reproduction and no other, the sporogenous cells giving rise to the spores, and the tapetal cells acting as nurse cells to these through all the stages of their development. Let us now follow these two groups of cells throughout their career. Cell-division ceases for a time in the sporogenous cells, as at f, and they enter into a state of preparation for a remark- able series of processes that, as we shall soon see, are apparently of fundamental importance to heredity. Meanwhile the walls of the tapetal cells break down, and their protoplasts fuse, form- ing a plasmodium that is free to circulate throughout the spor- angial cavity, g. The sporogenous, or grandmother cells of the spores, now become separated, and the tapetal plasmodium moves in between them, where, completely surrounding them, it is in position to assist in their nutrition to the best possible advantage, g; and in the capacity of nurse the plasmodium con- tinues to function up to the time of the maturity of the spores. Division of Spore Grandmother Cells.—Now the grand- mother cells begin to initiate nuclear division. The processes in this have been followed in detail by Yamanouchi in Osmunda cinnamomea. There at a somewhat later stage than is shown in g, Fig. 122, the nuclear network resolves itself into a thread lying in two parallel strands in close proximity, Fig. 123, A, a and 6, and traversing the nuclear cavity in various directions. Later the threads become drawn to one side of the cavity into a compact mass where the parallel members become more closely associated. After this the threads go back to the looser ar- rangement which they had at the beginning. They now shorten and thicken and break by transverse fision into twenty-two 210 REPRODUCTION double pieces, or pairs of chromosomes (Fig. 123, B, ¢ and d). These become thicker and dispose themselves at the equator of the nucleus; the nuclear membrane disappears and proto- plasmic threads radiating from the poles of the cell fasten upon the members of the pairs of chromosomes in such a way as to draw them apart toward opposite poles, twenty-two chromo- somes to each pole (Fig. 123, C,eandf). As they move toward the poles it is seen that they have become divided longitudinally Cc Fic. 123.—Formation and separation of chromosomes in the spore grandmother cells of Osmunda cinnamomea. A, a, early prophase showing approximation of nuclear thread in parallel strands; b, detail more highly magnified; B, later prophase showing transverse division of double thread, forming pairs of chromosomes; d, two pairs of chromosomes from ¢ more highly magnified; C, e, metaphase showing separation of the members of the chromosome pairs; f, late anaphase showing that each chromosome is longitudinally split. Further description in the text. (After Yamanouchi.) through the middle (Fig. 123, C, f), and that the halves spread apart at their equatorial ends while remaining together at their polar ends. At the poles these longitudinally-split chromosomes branch out more or less into a reticulum and a nuclear membrane is formed; but soon the second division is begun, the split chromo- somes become again recognizable, and give evidence by this fact that they had preserved their identity through the resting period. The nuclear membrane now disappears, the chromo- somes become lined up at the equator, and strands of cytoplasm pressing in from opposite poles become attached to the longit- udinal halves and draw them one to one pole and one to the GERMINATION OF THE SPORES 217 other, where they fuse and form at each pole a resting spore nucleus. Let us now recapitulate these processes in somewhat briefer terms. (a) The chromosomes that appear in the division of the nucleus of the spore grandmother cell are closely associated in pairs. (b) A pair is not formed by the longitudinal division of a preéxisting chromosome, but by the approximation of two parts of the nuclear thread before its transverse segregation into chromosomes. (c) The members of a pair now separate and are drawn to opposite poles. (d) Traveling poleward each chromosome shows that it has become split longitudinally. (e) At the poles the split chromosomes form a nuclear reticulum which becomes enclosed in a nuclear membrane, thus forming Fic. 124.—A, B, C, D, Successive stages of growth of prothallium from the spore in Osmunda cinnamomea; E, growth of fern plant from fertilized egg within the prothallium of Osmunda Claytoniana. (After Campbell.) the resting nucleus of the spore mother cell. (f) When this mother-cell nucleus begins division the split chromosomes appear again as such, and in the anaphase their halves are sundered and removed to opposite poles where the spores are now formed, two for each mother cell, four for each grandmother cell. Germination of the Spores.—When the ripe spores of Osmunda have fallen in a favorable situation they immediately germinate (Fig. 124, A, B, C, D), and produce a small, pros- trate, chloroplast-bearing body called the prothallium, D, which 218 REPRODUCTION is anchored to the soil by hair-like rhizoids, and bears on its under side two sorts of sacs, one the antheridium (Fig. 125, A), containing sperm cells or male gametes, and the other the arche- gonium, B, bearing an egg cell or female gamete. Some pro- thallia, however, bear antheridia only. Since the prothallium bears the gametes we call it the gameto- phyte. Fertilization of the Egg.—The sperm cells swim by means of their cilia to the archegonia, through the water that gathers under the prothallia (Fig. 125, B), being attracted by a substance, B Fic. 125.—A, Antheridium containing sperm cells; B, archegonium containing an egg cell which has been found by five sperm cells. All from Osmunda cinnamomea. (After Campbell.) probably malic acid, diffusing through the water from its place of secretion in the archegonium. The sperm cell now fuses with the egg. Following this the fertilized egg cell begins a series of nuclear and cell-divisions leading up to the full-grown fern (Fig. 124, E); and this produces sporangia and spores essen- tially as already described for Aneimia. Because of its bearing spores the fern plant is called the sporophyte. Interpretation of Processes of Nuclear Division.—The interpretation that is now being put on the behavior of the nuclear substance during division as previously outlined is suggested not only by what we see under the microscope but also by what we observe in carrying on pedigree cultures of hybrids, as will soon appear. We can best lead up to the interpretation by INTERPRETATION OF PROCESSES OF NUCLEAR DIVISION 219 beginning with the fertilized egg. When the sperm nucleus fuses with the egg nucleus the number of chromosomes borne by the sperm is added to the number borne by the egg, and the fertilized egg, therefore, bears twice as many chromosomes as does the sperm cell or the unfertilized egg cell. Now while these chromosomes lie closely associated in the fertilized egg they do not commingle and lose their identity; there is, rather, ©) [Ol [2 A B D (®N (|O) OOOO Es Fic. 126.—Showing method of association of paternal and maternal chromosomes, at A in all vegetative nuclei, and B, C, D, their manner of separation in all vegetative nuclear divisions. Showing also manner of association of paternal and maternal chromo- somes at £ in early prophase of spore grandmother cells and the manner of their separation during the division of the grandmother-cell nucleus at FandG. Showing also the longi-. tudinal division of the chromosomes in the spore mother cells in H, and their separation to form the spore nuclei in I, two of the four spores being purely paternal and two purely maternal. evidence for the conception that they become joined by end-to- end contact, as shown in Fig. 126, A, where the black segment represents a paternal and the white a maternal chromosome. For simplicity only one chromosome from each parent is shown. When division of the fertilized egg takes place the chromosomes break apart where they are joined end to end, divide longitudi- nally and line up at the equator, as shown at B; and so longi- tudinal halves of both paternal and maternal chromosomes arrive at either pole, C and D. Throughout all subsequent cell-divisions leading to the mature plant body this method of division is repeated, and each of the myriad cells is thus sup- 220 REPRODUCTION plied equally with the characters borne by the chromosomes of both parents. When the grandmother cells of the spores are formed their nuclei are constituted as in Fig. 126, A, like the other cells of the body. But now when the grandmother cells begin division a paternal and its homologous maternal chromosome become as- sociated in a pair (Fig. 126, E and F), and so for all of them, and the members of a pair are sent to opposite poles as shown in Fig. 126, G. It would now follow that when the mother cells divide and form the spores, two of the four spores would have only maternal and two only paternal chromosomes (Fig. 126, H and I) descended from any single pair in the prophases of the grandmother cell (Figs. 126, E and 123, B). It can now be seen that when the paternal and maternal chromosomes become segregated in the division of the grand- mother cells the process is the reverse of what happens when the . egg is fertilized; in the latter process the number of chromo- somes is doubled, and in the former it is halved, or, what is the same thing, the original number is restored. But it will be seen that the question is not simply one of the number of chromosomes, it is also a question of their kind, for even though the two parents belong to the same species, or even to the same variety, they are certain to differ in some manner or degree. Two Generations in the Life-cycle.—It is now evident that there are two generations in the life-cycle of such an organism as we have been studying, namely, the x generation and the 2x generation, x signifying a chromosome number. The 2x generation begins when x chromosomes in the sperm cell fuse with x chromosomes in the egg cell, thus forming the fertilized egg which grows to be the mature fern plant or sporophyte, or, as we may now call it, the 2x generation. The x generation begins when in the division of the grandmother cell of the spores as many chromosomes (x) as are supplied by the sperm cell are sent to one pole and as many (x) as are contributed by the egg cell are sent to the other to form the nuclei of the spore mother cells. A mother cell of the spores is therefore the one-celled FORMATION OF THE MICROSPORES 221 stage of the x generation just as the fertilized egg is the one-celled stage of the 2x generation. Prothallium, gametophyte, x genera- tion are different names for the same thing. So, too, fern plant, sporophyte, 2x generation are names referring to the same thing. It will be observed that in Osmunda cinnamomea the value of x is twenty-two. Spore-formation in Spermatophytes.—Let us now see what are the visible processes in the formation of the spores of Sperma- tophytes, or-so-called flowering plants. In these the spores are of two kinds, the microspores or pollen grains, and the mega- spores, which are usually the same thing as the embryo-sac cells. Formation of the Microspores.—When the stamens first appear as minute outgrowths from the receptacle, and for some time thereafter, they consist solely of meristematic cells. These proceed to construct an anther which early gives indications of a four-lobed outline. The anther itself is at first but a mass of meristematic cells, but soon the outer layer becomes differen- tiated into an epidermis. Cross sections of such anthers at different stages in their early development show in each lobe a hypodermal cell (Fig. 127, A, a), or sometimes group of cells, of larger size, richer protoplasmic content, and with larger nuclei than the others. A longitudinal section shows these to be really a line, or sometimes plate of cells (Fig. 127, F, a) extending nearly the length of the anther. This line of cells is called the archesporium. The archesporium divides throughout its length by periclinal walls, producing an outer primary parietal, B, ppr, and an inner primary sporogenous layer, B, ps. By anticlinal divisions the parietal layer is extended part way around the sporogenous cells, and then by periclinal divisions the former gives rise to an outer and an inner layer, C, oand i. By periclinal divisions the outer layer now produces two layers, D and E, 0 0, which constitute a part of the sporangium wall, while the inner layer proceeds to function as tapetum or group of nurse cells, D and E, #, destined to take active part in the nutrition of the spores, as we saw to be the case in Aneimia. The tapetum is extended around the 222 REPRODUCTION Fic. 127.—Showing stages in the formation of anthers and pollen grains or micro- spores of Silphium. A, a, archesporium; B, ps, primary sporogenous cell, pp7, primary parietal layer; C, z, inner layer, 0, outer layer formed from primary parietal layer; s, sporog- enous cells formed from division of primary sporogenous cell; D, s, sporogenous cells; #, tapetum; oo, two parietal layers; E, cross section of one lobe of mature anther; g, spore grandmother cells, 00, and t as in D; F, portion of longitudinal section of young anther showing a, archesporium; G, showing longitudinal section of same stage as D, parts lettered the same as D; H, longitudinal section of about the same stage as E, with parts lettered the same; J and J, pollen grains beginning to germinate of two species of Sil- phium; m, male or sperm nuclei; K, division of mother cell nuclei leading to microspores in L. (After Merrell.) DETAILS OF NUCLEAR DIVISION 223 inner side of the sporogenous layer by the participation of cells next the connective. While this is going on the sporogenous cells are undergoing longitudinal division, giving rise to the mass of cells shown in E and H, g. These are proven by their subsequent behavior to be the grandmother cells of the micro- spores or pollen grains. Details of Nuclear Division.—For the details in the division of microspore grandmother and mother cells we must now turn to the lilies, where these processes have been very critically studied by various investigators. In these plants, as in all others, where sexual reproduction takes place, the nucleus of the microspore grandmother cell has half of its structure from the paternal and half from the maternal side, a constitution that descended to it from the fertilized egg, where paternal chromosomes joined with an equal number of maternal chromo- somes in the act of fertilization, as we saw to be the case with Osmunda. We shall now see that there is a remarkable simi- larity between the ferns and flowering plants in the details of nuclear division in spore grandmother and mother cells. In a lily, as the grandmother nuclei begin the first steps in division one finds the nuclear reticulum resolving itself into parallel strands (Fig. 128, 2); then the synaptic stage ensues where the strands become indistinguishable (Fig. 128, 3). Soon the mass loosens and the parallel strands again come into view (Fig. 128, 4, and Fig. 129, A). In favorably stained prepara- tions the strands appear built of alternating colored and colorless bodies, as in Fig. 129, A, B, etc., the former called chromatin, and the latter limin. The chromatin bodies are also called ids. In the parallel strands the ids are seen to stand opposite each other. The parallel strands now unite and appear as one (Fig. 128, 5, and Fig. 129, B and C), but soon they separate more widely than before (Fig. 128, 6, and Fig. 129, D). The double strands now become split up transversely into several segments (Fig. 128, 8, and Fig. 130, A), and if we count the pair in one segment as one, then there are half as many of these as there are chromosomes appearing in the dividing nuclei of the vege- 224 REPRODUCTION TSA Eero os 5 COT ART TRI BY QOS a = > ZL trae Fic. 128.—Stages in the division of a grandmother cell of microspores or pollen grains of a lily, somewhat dagrammatic. 1, resting stage of grandmother cell; 2, nuclear thread becoming arranged into parallel threads; 3, synaptic stage; 4, parallel threads uniting; 5, double threads so united as to appear as one; 6, threads again separating; 7, thread trans- versely segmented into double chromosomes; 8, diakinesis, that is, chromosomes dispersed about the nuclear membrane; 9, multipolar spindle stage; 10, bipolar spindle with double chromosomes aligned at the equator; 11, reduction division, the double chromosomes separating and at the same time showing longitudinal division, with the longitudinal halves widely apart at their equatorial ends; 12, formation of daughter nuclei, which are really the nuclei of the mother cells of the spores; 13, division of mother-cell nuclei, the longitudinally split chromosomes seen in 11 again appearing; 14, split chromosomes of 13 aligned at the equator ready for the longitudinal halves of each to be drawn apart to opposite poles; 15, separation of the longitudinal halves; 16, formation of the granddaughter nuclei, or nuclei of the spores. (After Strasburger.) DETAILS OF NUCLEAR DIVISION 225 tative cells of root and stem tips, cambium ring, etc.; but if we count each member of the pair a separate chromosome, as we assumed to be the case in Osmunda, and as the earlier stages seem to justify us in doing, one member perhaps of paternal and the other of maternal origin, then there are just as many chromosomes here as in the vegetative divisions. Each member of the pair now undergoes a longitudinal division (Fig. 129, E), but this is soon lost sight of, due to the shortening and thickening E Fic. 129.—Processes of fusion of double nuclear thread followed by their separation, and longitudinal splitting of each. A, double threads before their union, showing their component ids at 1; B, double threads in process of uniting, fusion completed in the two upper figures, where pairs of ids have fused as at 2; C, completion of fusion with the pairs of ids united as at i in B; D, » and z, two pieces such as the one shown in C, with their fused threads again separating; at 1, a and b, we see the two parts that were fused in C; E, a and b correspond to a and 6 of 1 in D;a and b are much thicker in E than in D, and a and bin E have each split longitudinally; a will be drawn to one pole and b to the other, and as they start their longitudinal halves will spread to form Vs as shown in 11 of Fig. 328, (After C. E. Allen.) of the pair (Fig. 128, 9). In this condition the several pairs become lined up at the equator (Fig. 128, 10). In the metaphase the members of each pair begin to separate, the one from the other (Fig. 128, 10, and Fig. 130, B,r and s), and before they are drawn toward opposite poles in the anaphase the longitudinal division in each member again becomes ap- parent by the actual separation of the halves at their equatorial ends (Fig. 128, 11, and Fig. 130, B). It will appear later on that there are good reasons for the as- sumption we have been making, that when one of these longitudi- nally split chromosomes now traveling to one pole is of paternal origin, its former mate now traveling to the opposite pole is of of maternal origin. At the poles the chromosomes form the nuclei of the two mother cells of the microspores (Fig. 128, 12), and if we count each 15 226 REPRODUCTION split chromosome as one, there are only half as many here as appeared in the prophases of the grandmother cell. The divi- sion of the mother-cell nuclei: which now ensues consists es- sentially in the separation of the longitudinal halves of the split chromosomes and their distribution to opposite poles, where they form the nuclei of the microspores, four descended from each grandmother cell (Fig. 128, 13, 14,15 and 16). The micro- spores have just half as many chromosomes as did the grand- A 1 Fic. 130.—From nuclej of grandmother cells of pollen grains of Funkia Sieboldiana A, in diakinesis showing pairs of chromosomes of different sizes; B, metaphase, the members of each pair separating as shown by widely diveiging ends of r and s; 7 and s each show longitudinal division, with the halves beginning to separate where attached to the spindle fibers. (After Kiichi Miyake.) mother cells, as we found to be the case with the spores of Os- munda, and we must therefore class fern spores and pollen grains as homologues. According to the theory of the segregation of homologous pa- ternal and maternal chromosomes, two of the four microspores would possess paternal and two maternal characters from any pair in the grandmother cell. We will defer further discussion of this until the origin of the megaspores has been traced. We must now note that competent observers have found in many instances that in the prophases of the dividing grand- mother cells of microspores and megaspores the nuclear thread does not take the position of two parallel strands, and segment into various side-by-side pairs of paternal and maternal chromo- FORMATION OF THE MEGASPORES 227 somes as just described, but that the thread remains single and segments into end-to-end pairs of chromosomes, the maternal member of each going to one pole and the paternal member to the other, the final result being the same in both cases. Formation of the Megaspores.—The embryology of the flower of Silphium has been worked out by W. D. Merrell, and his work will be followed in tracing the production of the mega- spore, as was done in our study of the microspore. Early in the development of the ray flowers, which alone are fertile in Silphium, when the ovule has reached the stage shown in Fig. 131, A, 0, the archesporium appears at its apex as a single hypodermal cell, D, a. As the archesporium elongates, a single layer of cells surrounding it and constituting the nucellus, grows out with it (B and Ca, and E). Then the upper end of the ovule becomes inverted, Ca and Cé, and tissue around the base of the nucellus grows forward and forms the integument, Ca and Co. When the archesporium enters upon its first nuclear division (Fig. 131, F) the number of chromosomes sent to each pole is 8, which is one-half the number in the ordinary vegetative divisions. The reduction in the number of the chromosomes here points to the archesporium as the homologue of the grand- mother cells of the microspores. Each daughter cell of the archesporium divides once, yielding a row of four cells or mega- spores within the nucellus (Fig. 131, G). Only the uppermost megaspore, m, proceeds to grow. As it elongates it crowds the three disintegrating megaspores toward the micropylar end (Fig. 131, H) where they are soon lost sight of. Details in Division of Megaspore Grandmother Cell.—The details in the division of the megaspore grandmother cell are es- sentially like those in the microspore grandmother cell, and we assume that here also in respect of a group of characters borne by a single chromosome two of the four megaspores descended from the grandmother cell would be of paternal and two of maternal origin, as shown in I, Fig. 126. 228 . REPRODUCTION Fic. 131.—Stages in the formation of the megaspore, its germination, fertilization of the egg and endosperm nuclei, and germination of fertilized egg and endosperm cells. .4, beginning of ovule at o; B, nu, nucellus; Ca, nu, nucellus, a, archesporium; in, forward growth of tissue to form the integument; Cb, nu, nucellus; in, integument; D, more highly magnified drawing of o in A showing archesporial cell at a; E, a, archesporial cell enlarged and ready for division; nu, tissue of the nucellus; F, a, archesporial cell dividing, nu, nucellus; G, row of four megaspores descended from the archesporial cell, only the upper one, m, functional, the others soon to disintegrate; H, the megaspore, m, enlarging, the others disappearing; I, nucleus of the megaspore divided into two, md; J, continued nuclear division has resulted in eight, namely, three antipodal nuclei, an, at the upper end of the GERMINATION OF THE MEGASPORE 229 Germination of the Megaspore.—The megaspore now germinates, its nucleus dividing as shown in Fig. 131, I; the daughter nuclei move to opposite poles and divide, and the youngest nuclei in turn divide giving rise to eight nuclei sus- pended in the cytoplasm of the megaspore, which we may now call the embryo-sac. Two nuclei, called the polar nuclei, one from each of the opposite ends of the embryo sac, move to the center and fuse,- forming the primary endosperm nucleus (Fig. 131, J, pl). This now moves into close proximity to the three nuclei remaining at the micropylar end, one of which is shown by its subsequent history to be the egg cell, and the other two seeming to assist in fertilization are called synergids (Fig. 131, J, eg and ss). All three are termed the egg apparatus. The three nuclei at the other end of the embryo sac, each asso- ciated with a part of the general cytoplasm, become surrounded by cell-walls, and thus are formed the antipodal cells, J and L, an. Fertilization and Germination of the Egg.—The egg cell is now ready for fertilization, and we shall see the relation of the microspore to this process. When the microspores are first formed each is essentially a protoplast with one nucleus. In the germination.of the microspore the nucleus divides once, and one of the daughter nuclei divides once again, giving two that are known as the male or sperm nuclei (Fig. 127, m in I and J). The other daughter nucleus which did not divide is called the vegetative nucleus (Fig. 127, 1, J). These divisions may take place before or after the microspore has become lodged on the stigma. The two male nuclei descend within the pollen tube as it makes its way down the style and into the ovular cavity, and are discharged into the embryo sac, where one fuses with the egg.cell and the other with the primary endosperm nucleus (Fig. 131, K, mn,, and mn,). megaspore cavity, two polar nuclei, ~/, in process of fusion, egg cell, eg, two synergids, 5, s; K, Showing only the lower half of the megaspore or embryo sac cavity, with pollen tube, pt, and male nucleus, mn,, fusing with egg cell, eg, and second male nucleus, mn,, uniting with primary endosperm nucleus, pe’ s, synergid; L, later stage in the embryo sac, an, antipodal cells, em, beginning of the embryo by division of fertilized egg ceil; five dividing nuclei descended from primary endosperm nucleus. (K after W. J. G. Land, all others after W. D. Merrell.) 230 REPRODUCTION The fertilized egg now begins a series of divisions leading to the formation of the embryo, while the primary endosperm nucleus gives rise to an endosperm or food-storage tissue of greater or less magnitude, sometimes constituting the bulk of the seed, as in castor bean, or dividing the seed about equally with the embryo, as in Indian corn, or holding itself in abeyance and only assisting the embryo to take up the food about as fast as it comes for storage, as in the Lima bean and other Leguminose. The division of the endosperm nucleus usually precedes that of the egg, as illustrated in Fig, 131, L, where the embryo has reached the two-celled stage, while five endosperm nuclei are in process of division. The Triple-fusion Nucleus.—The triple-fusion nucleus formed by the fusion of the two polar nuclei to form the primary endosperm nucleus, and the union of this with one of the male nu- clei, has still no assured interpretation. One of the polar nuclei is sister to the egg-cell nucleus, and it might be expected that the fusion of a male nucleus with this would produce a second em- bryo. It has been suggested that the presence of the second polar nucleus is a disturbing element in this; but in some instances only one polar nucleus fuses with the male nucleus, and the result is the same as when both polar nuclei take part. Again it may be that the fusion of these nuclei is not perfect and not of the same nature as the fusion of the sperm and egg cell. This, . if true, would class the endosperm with the gametophyte gener- ation (see page 218), where the general judgment still places it. That the male nucleus may affect the character of the endo- sperm is most clearly shown in Indian corn. The wrinkling of sweet corn is due to the fact that sugar is contained in solution in the cell sap, instead of starch that fills the cells of dent and flint corn, and therefore on drying sweet corn shrinks while field corn remains plump. Now when sugar corn is fertilized by field corn the grains turn out plump and starchy instead of sugary, the second male nucleus from the microspore having trans- BEHAVIOR OF PEDIGREE HYBRIDS 231 mitted this character. Color residing in outer endosperm cell- layers is similarly transmitted. Behavior of Pedigree Hybrids.—Let us now leave this line of our discussion for the present and turn to the behavior of pedigree hybrids, for in them we have the strongest evidence that paternal and maternal chromosomes become segregated in microspores and megaspores. It was Gregor Mendel, teacher of natural sciences in the Realschule in Briinn, Austria, between 1853 and 1868, who, as a result of a long series of experiments in hybridization, in- spired by Darwin’s then recently published Origin of Species, discovered certain laws in the behavior of his hybrids, which: have recently risen to fame under the name of Mendel’s Laws. We will now relate some examples to illustrate the nature of his results. Mendel crossed a variety of pea having lateral flowers with one bearing terminal flowers, and found that the hybrid progeny bore lateral flowers only (Fig. 132, A, B,C, D. Here F,, F,, F, signifies 1st, 2d, 3d filial generation), no matter which variety was used as the mother or the father, and he therefore concluded that, since both characters were in the blood of the hybrid, the lateral character was dominant and the terminal character was recessive. When these hybrids began to blossom all pos- sibility of cross-pollination was excluded, and each was caused .to fertilize itself; and on planting the resulting seeds it was found that each plant’s offspring were lateral-flowered and terminal- flowered in the ratio of three bearing lateral (Fig. 132, a, b, c under F,) to one bearing terminal flowers (d). Self-fertilization was continued in this generation, and the seeds of each plant were planted separately, with the result that the terminal flowering plants gave only terminal flowering offspring (Fig. 132, 4)—one- third of the lateral flowering plants gave only lateral flowering offspring (Fig. 132, 1), while the remaining two-thirds gave both lateral and terminal flowering individuals in the ratio of three lateral to one terminal (Fig. 132, 2 and 3). In thus carrying on pedigree cultures where the seeds of each 232 REPRODUCTION i we we . Dominant Being self fertilized &11 come true to seed, and so for their progeny to any number of ; generations — A | Is proven by its progeny to be pure Dominant VE geny Being self fertilized yields pure Dominant likea inF, These two being self-fertilized are proven hybrids by their yielding hybrid offspring like 6 inF, Hybrid N A Se < TE, SEES SELES SESE EE SS to be Being self fertilized yields pure Recessive ‘like din F, Is proven by its pro Being self fertilized yields pure Dominant like g@ inF, These two being self-fertilized are proven hybrids by their - yielding hybrid offspring like b inF, w x to be Hybrid Being self fertilized yields pure Recessive like dinF, Is proven by its progeny — (— Recessive Being self fertilized all come true to seed, Qa progeny to be pure Recessive _ A and so for their progeny to any number of generations Lb | “7 Fic. 132.—Diagram to show results of hybridization, with reference to dominant and recessive characters to the F, generation. Further description in text. (After data by Gregor Menield A Is proven by its t INTERPRETATION OF MENDEL’S RESULTS 233 plant were planted by themselves, Mendel was able to judge the character of each plant by its progeny, and so demonstrate to be in the blood characters that did not come to view in the parents. Interpretation of Mendel’s Results.—In seeking an explana- tion of his numerical results Mendel came to the conclusion that when those special cell-divisions are begun that are to result in microspores giving rise to sperm cells, and megaspores pro- ducing egg cells, the inheritance bearers received from the parents of the hybrid become segregated, so that two of the four micro- Q- nN ® I >< —> || 2QQC: SO) > &) | Fic. 133.—Diagram showing possible combination of two contrasting characters during self-fertilization of a hybrid. A, row of sperm cells, B, row of egg cells, C, row of fertilized egg cells. Further description in text. (After data by Gregor Mendel.) spores and megaspores from a grandmother cell obtain a partic- ular character from one parent while two receive its mate from the other parent (Fig. 126, I). Applying this theory to the hybrid pea with tendency toward terminal flowers from one parent and toward lateral flowers from the other, each flower of this hybrid would produce two kinds of pollen or microspores and two kinds of emh-yo sacs or megaspores, resulting, of course, in two kinds of sperm and egg cells. Now in pollination according to chance, which is the way 234 REPRODUCTION it actually happens, four different combinations would result as suggested by the pointing arrows in Fig. 133, where at Aisa row of sperm cells, at B a row of egg cells, and at C a row of fertilized egg cells. From this it would follow that, on the average, in every four plants of the second generation after the first cross (in F,), one would possess only maternal inheritance for any specific character, one only paternal, and two would be hybrids like all the plants of the first generation. Since lateral flowers are dominant over terminal, fertilized egg cells 2 and 3 would produce lateral-flowered plants as well as would 4 which has only lateral flowers in its constitution. Mendel’s explanation, it will be seen, agrees perfectly with the results obtained from his experiments (see Fig. 132). Paternal and Maternal Chromosomes.—Let us now turn back to Fig. 126, E to I, where attempt is made to show what happens to the chromosomes during the divisions of grandmother and mother cells of microspores and megaspores. Under the microscope it can be seen that chromosomes divide and become distributed as shown in the figure, but it is impossible, at least in most instances, to tell from their appearance that half of them are paternal and half maternal. That they are this we conclude from Mendel’s experiments, that is, the results of these are pre- cisely what would follow if the chromosomes become segmented into paternal and maternal kinds as represented in the figure. Therefore, when it is said that half of the microspores and mega- spores are paternal and half are maternal for a definite character, let it be understood that the conception is based on direct ob- servation through the microscope of processes in the formation of these spores, and on the kinds of progeny resulting from a union of their gametes (sperm and egg cells). We must now take note of a fact observable in Fig. 132. The crossing of the lateral- and terminal-flowered peas seems to have given rise to nothing new, the progeny are in appearance exactly like one parent or the other; the only evidence that a cross has taken place is found in the fact that half of the individuals (b and c under F,) show their hybrid origin by producing two kinds of BEARERS OF HEREDITARY CHARACTERS 235 progeny. Then hybridization produces nothing new? Mendel, and plant breeders before and after him have obtained results that are quite otherwise. Hybridization does in fact give rise to more new varieties than have been obtained in any other way. In our consideration of hybrid peas, for the sake of keeping the main point in view, we ignored the fact that plants are a complex. of many characters, and that any two varieties taking part in a cross would be apt to have more than one pair of characters contrasting. In fact Lawrence Balls calls attention to twenty- three pairs of such characters in cotton visible to the naked eye, and beyond question there are many others of this kind besides those that are obscure and invisible. . In the two varieties of peas, flowers terminal and flowers lateral made a pair of contrasting characters, and if one of our plants had long leaflets and the other had short, this would give another contrasting pair. Under such conditions Mendel’s experiments showed that all possible combinations of characters occurred in the offspring of the hybrid, provided these were numerous enough to give all combinations a chance of appearing. It was found that the number of these combinations might be estimated by the formula x= 2", where x stands for the number of combinations sought, and n stands for the number of pairs of contrasting characters. In our example above, the value of n would be 2, and the number of possible combinations would therefore be 4, and of the following character; terminal flowers with short leaflets, terminal flowers with long leaflets, lateral flowers with short leaflets, lateral flowers with long leaflets. Bearers of Hereditary Characters.—When we seek for an explanation of these results in processes going on within the heredity bearers or chromosomes we find ourselves on hypothet- ical ground, but where, nevertheless, a survey of possibilities may reveal to us the right conclusion. We may start with the assumption that each chromosome is the bearer of more than one hereditary character, because plants certainly possess many more characters than chromosomes, and, furthermore, there is clearly a greater diversity in chromosome numbers than in 236 REPRODUCTION numbers of characters. Thus, Canna indica has six chromo- somes while Lilium martagon has twenty-four; but there is no evidence that lilies have four times as many characters as have cannas. A still better confirmation of our assumption is found in the Droseras, where of two species closely allied one has twenty and the other has forty chromosomes. We can, there- fore, confidently proceed with the conviction that each chromo- some bears an indefinite group of characters. Can we with the highest powers of the microscope discover organized units composing a chromosome, each of which might be supposed to bear a single character? The utmost we can see under the microscope of the finer structure of the chromo- somes is best observable in the early prophases of nuclear divi- sion. Here with the most favorable subjects it can be seen in preparations properly stained that the nuclear thread is made up of deeply stained granular masses or chromatin, alternating with unstained portions called linen. The chromatin masses, as has been said, have been termed ids (Fig. 129, i in A and B). It has been proposed that an id is composed of various single character bearers or pangenes and that an id therefore rep- resents a group of characters. With this partly theoretical, partly observational ground to build on a very plausible hy- pothesis has been erected to explain the occurrence of all pos- sible combinations of characters in the offspring of hybrids, and the possibility of fixing these combinations so that they will come true to seed. Briefly stated it is this: In a hybrid, during the prophases of nuclear division of microspore and megaspore grandmother cells it is possible that an interchange of pangenes may take place between homologous paternal and maternal chromosomes, one chromosome being considered ho- mologous to another when its pangenes are so matched with the pangenes of the other as to make pairs of contrasting characters. This hypothesis is in harmony with the results of hybridization, and it does not conflict with what can be-seen under the microscope. Theory of Pangeneic Interchange.—Let us turn to the THEORY OF PANGENEIC INTERCHANGE 237 diagrams of Fig. 134 to illustrate what is meant by the above theory of pangeneic migrations. We will suppose that the parents of a hybrid have four pairs of contrasting characters, namely, flowers terminal, flowers lateral, flowers blue, flowers yellow, stems smooth, stems hairy, leaflets short, leaflets long, and that the hybrid offspring all have lateral, yellow flowers, hairy stems and short leaflets, these characters being dominant and those contrasting with them recessive. Let us suppose that when the cross-fertilization took place, the sperm cell con- tributed but one chromosome and the egg cell but one; then in the hybrid when the grandmother cells of the microspores and megaspores enter upon the prophases of their first division the paternal and the maternal chromosome would be found in juxtaposition, either side by side as shown at E, Fig. 134, or end to end. In this position there would be a chance of an inter- change of pangenes (the paternal chromosome is shown black and the maternal white). Then the chromosomes would be drawn to opposite poles, and there each would split longitu- dinally into two equal pieces, and these in turn would be drawn to opposite poles (Figs. F, G, and H) to form the nucleus of the spores, namely, two paternal and two maternal spores. Let us now consider more in detail the interchange of pangenes that might take place in the prophase at E. At I is shown dia- grammatically the two chromosomes of E, each chromosome represented as consisting of two ids, and each id as composed of two pangenes. The stippled chromosome is paternal and the white one is maternal. Let us suppose that at the time of pangeneic interchange the pangenes bearing flower color changed places as indicated in I, and then that the chromosomes separated and went through the steps of spore formation as shown in F, G, and H, and, supposing that the same interchange takes place in the mother cell of megaspores as in that of microspores, the result H would be reached for megaspores also. Now, since the characters possessed by the microspores and megaspores are handed down to their sperm cells and egg cells respectively, sperm cells A, and A, would be formed from microspores 1 and 2 238 REPRODUCTION | parent Maternal » parent Hybrid © offspring Row of sperm cells Row of egg cells Row of fertilized egg cells ff fe Fic. 134.—Diagram to show dominance, segregation, and recombination of characters in the offspring of hybrids. Discussion of figure in the text. (After data from Mendel, Weismann, de Vries, Strasburger, Lotsy, Bateson, Spillman.) THEORY OF PANGENEIC INTERCHANGE 239 in H, and similarly egg cells B, and B, would be formed from corresponding megaspores. When it came to pollination it would happen in the long run that pollen grains 1 and 2 would become so distributed by insects or wind, etc., that sperm cell A, would chance to fertilize egg cell B, resulting in fertilized egg cell C,; and sperm cell A, would fertilize egg cell B,, resulting in fertilized egg cell C,. These fertilized egg cells would grow into embryos which finally would become mature plants D, and D,. It will now be noticed that D, and D, are unlike the hybrid from which they sprang and unlike either of the parents of the hybrid; and, furthermore, it can be seen from the constitution of the fertilized egg cells C, and C, that the offspring of D, and D, produced by self-fertilization are bound to come true to the parental characters, because D, has nothing but lateral, blue flowers, hairy stems and long leaflets in its blood, and likewise D, has nothing but terminal, yellow flowers, smooth stems and short leaflets. Such organisms are said to be homozygote. Given a sufficient number of instances, all possible varieties of interchange of pangenes would take place in E. Let us there- fore now suppose that the interchange of leaflet characters shown in J occurs in the formation of microspores, resulting in sperm cells A, and A,, and that these by chance came to fertilize the same variety of egg cells shown in B, and B,, now represented by B, and B,. Fertilized egg cells C, and C,, and mature plants D, and D, would result. It will be seen in fertilized egg cell C, that yellow and blue color of flowers, and short and long leaflets are in the blood of D,; but, as will be seen in the original hybrid, the yellow flower and short leaflet characters are domi- nant. Likewise D, has yellow and blue flowers, and long and short leaflets in its blood. Such organisms as these are called heterozygote. Now some interesting comparisons can be made. D, and D, will come true to seed when self-fertilized, while D, and D, will not do this. Under self-fertilization the offspring of D, will all have hairy stems and lateral flowers, because they are homo- zygote to these characters, but they will show the following 240 REPRODUCTION variation in leaf and flower combinations: yellow flowers and short leaflets, yellow flowers and long leaflets, blue flowers and short leaflets, blue flowers and long leaflets. The offspring of D, will all have smooth stems and terminal flowers, but they also will show variations with respect to flower color and form of leaflets, as in the case of Dj. Necessity of Pedigree Cultures.—Our diagrams will illus- trate for us how it is that pedigree cultures are a necessity in arriving as soon as possible at a strain of any particular type capable of coming true to seed. Suppose that one in looking over his field finds plants of the types shown in the D row of our figure, and wishes to propagate the sort shown in D, and D,. To be certain that no intermixture comes in from the other varieties, he ties bags over the flowers of his chosen plants so that all foreign pollen is excluded. Self-pollination having taken place, he saves the seed from all his chosen plants and puts them together in one package. The following year on planting these seeds, all from plants seemingly alike and self-pollinated, he is surprised to find the four types K, L, M, and N appearing in his seedbed, the impurity having come, as we can see, from D, plants, which, so far as he could know at the time, were the same as the D, plants.. But now, if in harvesting the seeds he had put those from each plant into a package by themselves, and had planted them in separate plots, the pure and the impure strains would have made themselves at once manifest. Since the parents of the hybrid we are now discussing had four pairs of contrasting characters, we would expect, in accordance with the formula previously given, sixteen varieties of offspring of the hybrid, each showing a different combination of char- acters from any of the others. These are shown in Fig. 135. A great number of seeds would have to be produced to give these various chance combinations opportunity to be made. More- over, in the long run all of these varieties would be produced cap- able of coming true to seed. This would happen when each was the product of a homozygote fertilized egg cell, as is the case in D, and D, of Fig. 134. This possibility is relied on by Burbank MOSAIC CHARACTER OF OFFSPRING OF HYBRIDS 241 and other plant breeders in their practice of combining by hy- bridization the various good qualities of different varieties while eliminating those that are undesirable. It must be remembered, however, that they arrive at their results only after searching over hundreds, and even thousands, of the offspring of hybrids. We may refer to Fig. 134 to illustrate another fact of impor- tance. K, L, M, and N are, as stated, the different possible offspring of D, under self-fertilization. Now how can we deter- mine the degree of purity of these varieties? We can, of course, test their purity by the character of their offspring produced by self-fertilization, and this would be an adequate and final test; but there is another way of arriving at a partial answer to our question. We know that when the offspring of a hybrid shows recessive characters it is pure to those characters, for if the con- trasting dominant were present it would be expressed and the recessive would not appear. The offspring of K, for instance, produced by self-fertilization, would all have smooth stems and terminal flowers, but they might vary in color of flower and form of leaf. The offspring of L would all have terminal flowers, smooth stems, and long leaflets, but might vary in color of flower. M would always give terminal blue flowers and smooth stems, while possibly varying in form of leaflets. N would have none but pure offspring, since, being recessive in all of its characters, it must be homozygote to all of them. Mosaic Character of Offspring of Hybrids.—It would appear from all that has now been said that the offspring of a hybrid are mosaics of the characters of the original parents of the hybrid, and that different combinations of these characters may appear in the different individuals. Since in spore production the characters may separate and recombine independently, like the blocks with which children build different forms of houses, etc., according to their fancy, or like the bits of glass in a kaleido- scope which fall apart and reassemble in various patterns, they have been called unit characters. We may conceive of a unit character as the expression of the hereditary power of a pangene; or we may think of it as this power itself. 16 242 REPRODUCTION Mendel’s Laws.—Applying this conception of unit char- acters to the results of Mendel’s work we may make the following summary: (a) In a hybrid where the two parents have contrib- uted contrasting unit characters, one of these (the so-called dominant) expresses itself, while the other (the recessive) is suppressed. This is the dominance of one character over another. (b) The homologous chromosomes of the parents of a hybrid, namely, the chromosomes that bear corresponding groups of unit characters, which became associated in the fertilized egg, and so continued in every cell of the body sprung from the fertilized egg, become again dissociated, after a possible inter- change of unit characters, during the formation of microspores and megaspores, so that half of these as to any character are maternal and half are paternal. This is the segregation of unit characters. (c) Paternal and maternal chromosomes, modified to a certain extent by the previous exchange of pangenes, again become associated by the fertilization of the egg, the different varieties of modified homologous chromosomes coming together according to chance, and so giving rise in the long run to all possible combinations of paternal and maternal unit characters. This we call the recombination of unit characters. In the F, generation (see Fig. 132) dominance alone in opera- tive; in the division of the grandmother and mother cells of the spores of the F, generation segregation occurs; when sperm cells and egg cells of the F, generation unite in fertilization, giving rise to embryos of the F, generation, recombination of unit characters takes place, as shown in Fig. 134. Practical Applications.—Knowledge of the nature and behavior of hybrids has a very important bearing on the prob- lems of plant breeding. In the first place it shows us that we should not expect to realize the possibilities of hybridization in the F, generation. Many a culture has been thrown away at this stage because the hoped-for combinations did not appear, when, if it had been carried to the F, generation the ideal might have been realized, together with many other unlooked-for and valuable combinations. PRACTICAL APPLICATIONS In the second place, since all possible combinations of © parental characters may be looked for in the F, genera- tion, it is evident that the more seeds we plant of the F, generation (these seeds would, of course, contain the embryos of the F, generation) the greater would be the chance that all possible combinations would come to expression. In the third place, in order that the possible combinations of the different parents may be worked out without dis- turbance, _ self-pollination must be assured in the F, and succeeding generations. Finally, when a_ desired combination of characters ap- pears it may at once be multiplied vegetatively by cuttings, - budding, grafting, etc.; but if propagation is to take place by seeds the indi- vidual bearing the combina- tion must be tested to see if it comes true, that is, whether it is homozygote for each character in the combination. To make this test the selected individual is self-pollinated, Fic. 135.—Illustration of all possible combinations of characters appearing in the offspring of the hybrid shown in. Fig. 134. 244 REPRODUCTION and its resulting seeds, as many as possible, are planted by themselves, and if the offspring show the same combination the homozygote condition is demonstrated, but if they split into new combinations we know that for some characters they were heterozygote. By selecting for seed production only those offspring that show the original combination, and planting the seeds of each of these separately, the homozygote condition will be found sooner of later, and by self-pollination it may be multiplied indefinitely true to its character. Infer- tility with self-pollen, and serious loss of vigor resulting from self-fertilization are sometimes disturbing factors. Exceptions to the Rules.—That we might keep to the main path in our discussion, we have left to the end mention of excep- tions to the rules that have been formulated for us by Mendel and other experimenters in hybridization. Dominance of one contrasting character over another does not always occur. On the contrary the two characters sometimes blend, or express themselves independently side by side, as when a cross between white and red flowers gives pink flowers, or striped red and white. It seems, too, that segregation does not always take place during spore-formation of the F, and succeeding generations, that is, sometimes propagation by seed can straightway take place true to the type that appears in the F, generation, but this is of much rarer occurrence than is failure in dominance. Sometimes, again, entirely new or apparently new qualities made their appearance in hybrids, as when a cross between two smooth varieties gives hairy offspring (Matthiola or ten-week stocks), or when red flowers crossed with yellow gives white (sweet peas and stocks), or when a cross between two white flowered varieties gives red flowers (sweet peas), or when red flowers crossed with white flowers gives yellow (Mirabilis jalapa). These surprising results are not necessarily outside the domain of Mendelian principles. An analysis of the causes of different colors in flowers will sometimes lead to a clear explanation of anomalous results. Let us examine the case of the white- EXCEPTIONS TO THE RULES 245 flowered stocks from the red and yellow cross. The colors of flowers are due to two causes (see pages 10 and 341), pigments in solution in the cell sap and coloring matters held in the body of plastids. Now the red-flowered stocks have in the petals red-cell sap and colorless plastids (A, Fig. 136), and the yellow flowers have yellow plastids and colorless sap (B). Colorless plastids and red sap are dominant, and therefore, when the red and yellow sorts are intercrossed, in F, the offspring all have OME Fic. 136.—Showing how the flower color is produced in the offspring of a hybrid pro- duced by crossing red flowered and yellow flowered ten-week stocks. (After data by Bateson.) red flowers (C). When, however, segregation of characters takes place in the formation of the spores of the F; generation, and recombinations occur in self-fertilization, the following sorts result: red sap plus colorless plastids, giving red flowers (C); red sap plus yellow plastids, giving yellowish-red or orange-colo- red flowers (D); yellow plastids plus colorless sap, giving yellow flowers (B); and colorless plastids plus colorless sap, giving white flowers (E). In many instances the appearance of what seems to be a new character is but the recurrence of a lost one that belonged, it may be, to a far distant ancestor. This would be a case of atavism. The hairy ten-week stocks from smooth parents would be an example of this. There are purple stocks and red stocks 246 REPRODUCTION whose colors are due to pigment in solution in the cellsap. ‘There are also stocks with cream-colored flowers due to yellowish plastids; and there are white-flowered stocks with colorless sap and plastids. All of these are smooth. When the red and purple varieties are intercrossed the offspring are always smooth, but when the cream or white varieties are crossed with the reds or purples the offspring are hairy, and when the cream and white varieties are crossed the offspring are both purple and hairy. It might be deduced from these facts that both the cream and white varieties possess the factor for hairiness, and that the purple and red varieties do not contain it. It also ap- pears that the factor for hairiness cannot do its work without the codperation in some way of the factor for red or purple sap. Furthermore, it appears that the cream and the white varieties possess factors which acting together can produce purple, while separated they are ineffective. Hairiness and purple color are both reversions to an old parental form. Loss of purple color and loss of hairs seems to have been caused by segregation of characters which only by codperation could produce them. When by crossing these characters were recombined the lost parental characteristics reappeared. Significance of Sexuality.—In whatever manner and through whatever influences sexuality may have arisen, it seems clear that any device so wasteful in the countless pollen grains and ovules that come to nought must in the long run bring some com- pensating good to the race; and evidence of this has not been wanting. In his “Animals and Plants Under Domestication”’ Darwin gives a convincing array of evidence to show that cross- fertilization as compared with self-fertilization .results in very marked increase in vigor. There are, as is well known, many exceptions to this rule; but it has, nevertheless, such extensive application as to warrant the statement that sex-differentiation has in some way been the cause of a marked stimulation of vari- ous vital functions. : A very striking illustration of this we find in the behavior of Indian corn under self- and cross-fertilization. Shull has iso- SIGNIFICANCE OF SEXUALITY 247 lated two strains of corn and propagated them by self-fertiliza- tion and selection until they appeared almost if not quite homo- zygous. In this condition they were weak and stunted, and their ears had degenerated to nubbins (Fig. 137, A and D). He then crossed these two strains reciprocally, that is, the strain that was used as the mother in one cross was employed as the : Fic. 137.—A and D, Ears from two nearly homozygous strains of Indian corn: B. offspring of strain A fertilized by strain D; C, offspring of strain D fertilized by strain A, (After Shull.) j father in the next, and vice versa. Immediately the offspring from these crosses mounted in vigor, even above that of the cross-fertilized race from which the strains had been derived; yielding ears as shown in Fig. 137, Band C. Here A pollinated by D produced B, and D pollinated by A produced C. These results led to the notable suggestion by Shull that-since 248 REPRODUCTION corn is richly heterozygous as ordinarily grown, various strains might be derived by self-fertilization and continued selection that are approximately homozygous, now with one combination of characters, and now with another; and that by crossing all possible pairs of these strains the best combination of parents could be determined for each particular purpose for which corn is used, and for the different environments in which it is grown. Sexuality seems to have possible significance in another very important way, namely, in the evolution of organisms—in the derivation of new varieties and species from others preéxisting. The possibility of thus obtaining new species has been much in dispute. Theoretically it might be confidently expected, for a new combination of unit characters in hybrids might stimulate the expression of entirely new qualities, or bring secretions into new chemical combinations that might cause alterations in various characters. If now such a hybrid should prove to be stable, a new species would have resulted without the aid of artificial self-pollination and continued selection, to bring it to the constant homozygous condition. When we turn to the records, which is much more to the point, we find Focke’s list of more than forty wild species that have been proven of hybrid origin by their duplication through artificial hybridization; we find de Vries’s hybrid primrose produced by crossing Oenothera muricata and O. biennis, and Burbank’s primus and phenomenal berries, both produced by intercrossing a blackberry and raspberry. This suggests to us that in one thing, at least, there is no dis- pute, namely, that the fact of sexuality is of tremendous impor- tance to the work of the plant breeder, for the primary reason that its attendant segregations and recombinations of characters make it possible for him to combine in an improved strain the good qualities of related varieties, while at the same time eliminating those that are undesirable. ILLUSTRATIVE STUDIES 1. Cut small pieces bearing only a few sporangia from sporo- phylls of Aneimia, Osmunda, or Botrychium, taking care to ILLUSTRATIVE STUDIES 249 include sporangia in all stages of development, and treat them as described under Cytological Methods, page 257. Stain with Flemming’s triple stain. Study with #5 or 4 oil immersion objective (see page 277 for method of use). Search for all phases of nuclear division in grandmother and mother cells of the spores, and compare with Figs. 123 and 128. Make drawings to show different stages of sporangial development. These should follow the differentiation of sporangium wall, tapetum, and sporogenous tissue, and the segregation of spor- ogenous tissue into single cells or groups of cells, and the forma- tion and migration of the tapetal plasmodium. The fate of the plasmodium as the spores come to maturity should likewise be followed. 2. Make cultures of fern spores as described on page 323. Make drawings to show spores in process of germination as shown under high powers. 3. Mount mature prothallia under a coverglass in a drop of water. Draw as seen under low power, and study antheridia and archegonia with a high power. Now cut out portions of prothallia bearing antheridia and archegonia, put them ina drop of water on a glass slip, and tease them out under adis- secting lens, so as to separate as nearly as possible the antheridia and archegonia from the rest of the tissue. Look for sperm and egg cells im situ under the oil immersion objective. A more satisfactory study of the gametes can be made from sections of prothallia prepared as described under Cytological Methods. Make drawings. 4. Make preparations by cytological methods of anthers of lilies, Tradescantia, Podophyllum, etc., in different stages of development. Stain with Flemming’s triple stain. If tips of the anthers are cut off before fixation the fixative will penetrate more surely and quickly. Care must be taken to have the series begin with anthers so young that division of the grandmother cells has not yet taken place. Preliminary tests can be made by teasing out anthers in gentian violet (p. 304) and soon replacing the stain with water or dilute glycerine. 250 REPRODUCTION 5. Prepare ovules of the flowers from which the anthers are taken, beginning with the youngest stages. Several ovules may be removed together from the ovary by cutting out a thin strip of the placenta to which they are attached. Prepared in this way they are much easier to orient as desired on the microtome than where a single ovule is handled by itself. Hunt for the stages shown in Fig. 131, and make drawings. 6. Treat as above ovules taken at different periods after polli- nation, and make drawings to show processes of fertilization. 7. In connection with the laboratory work read from the following books. Morphology of Angiosperms, by Coulter and Chamberlain. D. Appleton & Co., N. Y. Fecundation in Plants, by D. M. Mottier. Carnegie Institution, Washington, D.C. Mendel’s Principles of Heredity, by W. Bateson. G. P. Putnam’s Sons, N. Y. Die Stofflichen Grundlagen der Verer- bung, by Eduard Strasburger. Gustav Fischer, Jena. Vorlesun- gen iiber Descendenztheorien, by J. P. Lotsy. Gustav Fischer, Jena. . Proceedings American Breeders’ Association, Vols. IV and V, Washington, D. C. Plant Breeding, by L. H. Bailey. The Macmillan Co., N. Y. Plant Breeding, by Hugo de Vries. The Open Court Publishing Co., Chicago. CHAPTER XIV THE PREPARATION OF SECTIONS a The preparation of thin sections of plant tissues is an abso- lute necessity in the study of plant histology, not only that cell structure may be clearly seen but that the association of cells into tissues and the mutual relationship of the different tissue systems may be brought to light. Whether sections can be cut forthwith without special preparation of the subject to be sectioned depends upon the nature of the material and the partic- ular question regarding it that is to be solved. The method of procedure to fit different cases will now be given. Cutting Sections Free-hand.—Good histological work can be done with some materials, such as the mature parts of “XI, Fic. 138.—Manner of holding the razor and object in cutting sections free-hand. 9 stems, roots, and leaves, by holding them between the thumb and forefinger of one hand while the section razor is wielded by the other (see Fig. 138). The forefinger is held hori- zontal and the razor rests upon it, being pushed from point to heel in cutting the section. There is never danger of cutting too thin sections by this method; rather, most of the sections are too thick, and skill comes only with much practice. Sup- pose a cross-section of a stem is being cut, it is not necessary that the section be complete, and the small but thin bits which one is sure to get in his efforts to secure thin sections are the most satisfactory under high powers. A very small segment 251 252 PREPARATION OF SECTIONS of the stem usually suffices, provided it extends from the sur- face to the pith. Tender, flexible parts, such as the blades of leaves, will need to be inclosed in elder pith before sectioning, and a good stock of pith should be kept on hand for this pur- pose. A piece of pith about an inch long is laid on the table and while held firmly between the thumb and fingers to keep it from cracking it is halved longitudinally with a sharp knife. If a leaf section is desired a strip of the leaf is held between’ the halves of pith while the section is cut through pith and all. Sections of delicate stems and roots and of buds and flowers may be made in the same way, only a groove should be made in the pith, of a size to hold the parts firmly enough while not crushing them. It is surprising how much really good work can be done with simple appliances of this sort. To get sections of the stone-cell tissue of nuts saw off as thin slices as possible with a hack saw and rub these down to the requisite thinness between two water hones kept wet. This is a slow process but it yields fine sections. A simpler way is to whittle off fine shavings with a very sharp knife. These shavings roll up and must be forcibly straightened out. They will break when this is done but the small bits will do. (See p. 310.) A sharp razor is a necessity to successful section cutting; and it is not sharp enough until it will clip a hair held so it is free to bend before the razor. A razor half hollow-ground on both sides ‘is a good one for this purpose. The dealers offer razors ground flat on one side, but it is impossible to keep them sharp by the usual methods. A good shaving razor, so. only the blade is not ground too thin, makes a suitable sec- tion razor. While cutting sections keep the razor blade wet with about 60 per cent. alcohol, and slide the sections into a dish of water before they have time to become dry. Never let sections be- come dry at any time, else they will shrivel and their cells will become filled with air which will prove a nuisance under the microscope. In studying stem and root structure three sections, each CUTTING SECTIONS WITH MICROTOME 253 from a different point of view, are necessary to an understand- ing of the character and extent of the different tissues; these are a cross-section, a longitudinal section parallel to a medul- lary ray, known as a longitudinal radial section, and a longi- tudinal section at right angles to a medullary ray, called a longitudinal tangential section (Fig. 139). Good longitudinal sections are more difficult to get than cross-sections, but much of the difficulty is avoided if most of the surface is pared down so that only a small elevation is left to be sectioned, as shown in Fig. 140. It is a good plan to keep material that is to be sectioned in equal parts of alcohol, glycerine, and water; in this it may remain indefinitely, but only after several weeks will its best effects in softening the harder tissues and toughening the weaker be produced. Cutting Sections with a Microtome.— A simple form of microtome that can be clamped to the laboratory table is often of great advantage in cutting sections with a razor (Fig. 141). If the material is hard enough to bear the strain it may be clamped directly in the jaws of the object holder by — ric. 139.—Showing the means of the thumb-screw S; or it may first Planes in which sections é . 7 ‘ are cut, A, transversely; be inclosed in elder pith, in velvet cork, 2B, longitudinal radially; or even in soft wood, before clamping in. feo ra The object is fed up a very little at a time by turning the milled-head M of the micrometer feed-screw. The section razor is laid flat on the plate glass ways PP and pushed across the object with a long sliding motion from point to heel of the razor as shown in Fig. 142. In doing this the razor must be held firmly against the glass ways. After several sections have accumulated on the razor, which is kept wet with dilute alcohol, they may be swept with the finger into a dish of 254 PREPARATION OF SECTIONS water. If it is desired to keep the sections in the serial order in which they were cut they may be transferred one by one into small phials, a single section to a phial. More elaborate microtomes have devices for carrying the section : knife, or for holding the knife stationary oe Go while the object is made to vibrate back Zo s and forth against it, and in this way sec- : tions can be cut with increased rapidity |{ and accuracy. In some forms there is an _——| automatic feed which can be set to any tik duahepina iow desired thickness of section. One of the to trim a block for cutting simpler forms of microtomes with a knife longitudina] sections. : & ‘ . carrier is seen in Fig. 143, where the principle of its operation will quickly be recognized. As there shown the knife should be set at an angle to make a long sliding cut in all cases excepting where material imbedded in paraffin is to be sectioned. In the latter event the knife is to be set square across at right angles to the direction of its motion, so that the sections are chopped instead of whittled off. In this 44s Fic, 141.—Simplé microtome for clamping to table. P, P, plate glass ways for the section knife; S, milled-head for clamping the object; M, micrometer milled-head for turning the screw that raises the object as the sections are cut; C, milled-head for clamping the microtome to the table. CARE OF SECTION KNIFE 255 way the edges of the sections adhere as they are cut and form a ribbon which preserves the order of the series perfectly. In cutting paraffin sections with the sliding microtome of the type shown in Fig. t43 the knife needs to move through only a short distance each way, so the elbow may rest upon the table and the knife may be operated with a wrist movement merely. Fic. 142.—-Showing the manner of holding the knife blade on the glass ways, and, by the arrow, the direction of sliding the knife while cutting the sections. Care of the Section Knife.—As has been said, and it will bear repeating, the section knife or razor must be kept sharp— what we call perfectly sharp, or as sharp as one can make it. The test is that it should clip a hair at a slight touch. If it will not do this it may need honing on a stone and then strop- ping on leather, or the stropping may be all that it needs. To tell what to do moisten the ball of the thumb and draw it lightly over the edge of the knife from tip to heel; if the edge gives the 256 PREPARATION OF SECTIONS sensation of taking hold of the skin throughout its length only stropping is needed, but if not, the knife must be honed. ii Li Lb a i Hl Before honing or stropping a microtome knife a steel back should be slipped on it so as to tip the edge to the proper angle, Mee Me Me Se . CYTOLOGICAL METHODS 257 but an ordinary razor will not need this back. While honing hold the knife in the position shown in Fig. 144, keeping the back as well as edge against the stone, and while pushing the knife edge foremost slide it at the same time from point to heel as shown by the arrow. Then turn the other face of the knife to the stone and repeat the stroke from point to heel toward the other end of the stone, and so on until the thumb test above de- Fic. 144.—Showing the manner of honing the section knife or razor. t scribed is satisfactory. Do not allow the stone to gum up; keep plenty of oil upon it if it is an oil stone, or if a water stone keep it well lathered with soap and water, and wipe the stone clean after honing. Then strop the knife, drawing it over the leather back foremost from heel to point (Fig. 145), reversing the face for the back stroke, and keep this up until the knife readily clips a hair. Cytological Methods.—Within comparatively recent times methods have been worked out whereby the anatomy of cells 7 258 PREPARATION OF SECTIONS and tissues can be laid bare in their finest details. These methods are intended first of all to preserve the structure of the proto- plasts in its normal form, and then to cut a single cell into several sections while keeping these in their natural sequence, and finally to stain the sections so that different structures will take on different colors. The preservation of the structure of the protoplasts is ac- complished by plunging the material into a solution, known Fic. 145.—Illustrating how the section knife or razor is drawn across the strop. as the fixative, which instantly kills the-protoplasts so that de- composition incident to slow dying is prevented, and then harden- ing the protoplasts by transferring the material to alcohol, be- ginning with weak alcohol and gradually increasing its strength until absolute alcohol is reached, so as to avoid undue shrinkage. The material is next imbedded in paraffin, and sections ad- hering in ribbons are cut usually .oo5 mm. to .oro mm. thick, and these after mounting on a slide and being freed from paraffin are stained with two or three different stains and then sealed in balsam in the form of permanent mounts. The processes thus briefly outlined will now be given in detail. The Fixing Process.—For the study of the finer structures THE FIXING PROCESS 259 of the protoplast Flemming’s fixative has given on the whole the best results. The formula for this is: One per cent. chromic acid....... 16 parts, Two per cent. osmic acid ...... ..+. 3 parts, Glacial acetic acid .. .4 2.05. eae oe OP part. Make the 1 per cent. chromic acid solution by dissolving 1 gram of chromic acid crystals in 99 c.c. of distilled water, and dissolve 1 gram of osmic acid in 49 c.c. of distilled water to make the 2 percent. solution. Then mix together 16 c.c. of the chromic acid solution, 3 c.c. of the osmic acid solution, and 1 c.c. of glacial acetic acid. Of course more of the fixative can be made, so only the ingredients are kept in this proportion. The osmic acid solution must be made with extreme care to avoid all contamination with organic substances, which are sure to spoil it, as shown by the formation after a time of a black precipitate. For this solution procure a glass-stoppered bottle, wash it thoroughly with soap and water, rinse it many times, pour into it a saturated solution of bichromate of pot- ash in strong sulphuric acid, stopper the bottle and shake it vigorously, let it stand for a while and shake again, then pour out the solution and rinse the bottle again and again with dis- tilled water. Clean the stopper as thoroughly as the bottle. The osmic acid comes sealed in glass tubes and it is best to obtain it with one gram to the tube. Clean the outside of such a tube in the manner described for the bottle, stirring it about with a glass rod in the bichromate of potash solution and sub- sequent rinsings, and keeping the fingers off of it; then guide it with the rod into the clean bottle; pour into the bottle 10 c.c. of distilled water, stopper the bottle and strike it against the palm of the hand until the tube of osmic acid is broken; then pour in the remaining 39 c.c. of distilled water necessary to make the 2 per cent. solution. If the whole 49 c.c. of water were poured in at first it would have been more difficult to break the tube. Of course the distilled water must have been kept in receptacles free from organic matter. The fumes of osmic 260 PREPARATION OF SECTIONS ‘acid are hard on the eyes, nose, mouth, and lungs, and the face should be kept away from them. About twenty times as much Flemming’s fixative should be used as of material to be fixed, and the material should be cut into pieces not greater than 2 mm. in any dimension, so that the fixative may penetrate quickly to all parts. It is conveni- ent to do the fixing in small phials, and if the material has a tendency to float it may be pushed under with a piece of filter paper that tightly fits the phial. Material should be fixed at once after it is gathered and if it grows at any distance from the laboratory the fixative should be taken along. Keep the material in the fixative for forty-eight hours and then remove it and pin it in little cheese-cloth bags which one can quickly make himself of the size wanted, and put these in running water for about six hours, or over-night. If running water cannot be had then place the material in a bucket of water which is to be changed several times. A simpler and cheaper fixative which gives good results, but not quite equal to the above for dividing nuclei, is the chrom- acetic fixative. This is made by dissolving 1 gram of chromic acid in 99 c.c. of distilled water and adding 0.5 gram of glacial acetic acid. Use as described for the above fixative. The Hardening Process.—The material still kept in the bags is, after washing, placed in 20 per cent. alcohol for two hours, and then it is carried through a series of alcohols, each of the series 10 per cent. stronger than the one before it, remain- ing in each grade of alcohol for two hours until absolute alcohol is reached. If the material is not to be imbedded in paraffin at once it may be left in the 70 per cent. alcohol until needed, and then it may be carried on into the higher grades as if no interruption had occurred. The process of hardening may be considered complete when the 90 per cent. grade of alcohol has been reached, and the sojourn in absolute alcohol is intended to complete the dehy- dration of the material preparatory to its imbedding in paraffin or celloidin. In order to make dehydration more certain it is PROCESS OF IMBEDDING IN PARAFFIN 261. a good plan to have two bottles of absolute alcohol in each of which the material remains for two hours before it is transferred to the solvent of paraffin or celloidin. The Process of Imbedding in Paraffin.—Transfer the material from the absolute alcohol to a phial containing equal parts of absolute alcohol and chloroform, and after two hours place it in a phial of pure chloroform, and again after two hours transfer it to another phial of chloroform, and in these instances enough chloroform to keep the material submerged is all that is needed. Chloroform is a solvent of paraffin and the ob- ject now is to infiltrate the material with paraffin very gradually. Accordingly after two hours put a small shaving of paraffin into the last phial of chloroform where the material is, and shortly after this has dissolved add another shaving, and so on until the chloroform is saturated with paraffin at the temperature of the laboratory. All this while the material may have been left in the little bag of cheese-cloth for conveni- ence in handling, but now it should be taken out of the bag and laid back loose in the phial of dissolved paraffin. Place this phial on the top of a paraffin oven heated to the melting point of the paraffin, which should be about 52° C. Remove the cork from the phial and let the chloroform evaporate. Add more paraffin a little at a time if needed to keep the ma- terial submerged. Keep the phial on the paraffin oven until the paraffin no longer has a sweetish taste, indicat- ing that all of the chloroform has evaporated. Make a small paper tray by turning up the edges of a piece of paper all around to the height of a centimeter and half fill this with melted paraffin heated hardly above its melting point, and into this pour the contents of the phial—paraffin and material. It is best to have the paper tray on something cold so that a crust of solid paraffin will quickly form at the bottom, and then with heated dissecting needles the material can be disposed in or- derly fashion over this crust, and when the paraffin has entirely hardened each piece of the material can be cut out with a good border of paraffin all around it. When the material has been 262 PREPARATION OF SECTIONS arranged over the bottom crust blow upon the surface of the paraffin to harden it the more quickly, and plunge the tray into cold water as soon as the surface crust will bear this. The more quickly the paraffin is cooled the more firmly it sets about the material. The material may be left thus imbedded in paraffin until it is needed for sectioning. Sectioning Material Imbedded in Paraffin.—Tear off the paper tray and with a knife score deeply around the piece of desired material on both top and bottom surfaces, and then break the piece out. This will be called the paraffin block. Melt a piece of paraffin on the surface of the object carrier of the microtome. In the microtome shown in Fig. 143 the object carrier may be simply a piece of pine wood about a centi- meter in cross-section. which is to be clamped firmly in the jaws of the microtome. Before the melted paraffin on the object carrier has time to harden press into it the paraffin block, setting it up in the position to give sections in the desired direc- tion; then pass a hot needle around the base of the block so as to fuse it thoroughly with the paraffin bed and make a firm union. Pare the sides of the paraffin block so that the opposing faces are parallel, and adjust the object carrier on the microtome so that the knife, standing at right angles to its line of motion, will have its cutting edge parallel with the face of the block turned toward it. Now the sections may be cut and they should adhere and form a ribbon. In cutting paraffin sections the knife does not need to be wet with alco- hol or anything else as in other cases. If the paraffin breaks away from the material as the sections are cut the infiltration may not have been successful, or the temperature of the room may be too low. If the sections crumple up as they are cut the room is probably too warm. The ribbons ought to be straight, and if the front and back faces of the paraffin block are trimmed parallel they are pretty sure to be straight. Sec- tions seldom need to be cut thinner than .oo5 mm., and .oro mm. is a suitable thickness for most purposes. In micrometry MOUNTING PARAFFIN SECTIONS 26 3 the term mikron is applied to .ocor mm. and the above thick- ness would be called 5 and 10 mikrons. Mounting Paraffin Sections.—The paraffin sections are made to adhere to the glass slips by means of albumin water. The stock solution of this is made as follows: Shake together equal parts of white of egg and distilled water and add to this a pinch of salicylate of soda to keep it from spoiling. For use add one drop of the stock solution to one ounce of distilled water. This dilute solution will be referred to as albumin water. Rinse thoroughly and wipe dry a glass slide that has been kept in a saturated solution of bichromate of potash in strong sulphuric acid. Place at the center of the slide a drop of albu- min water and ‘with a dissecting needle drag the drop out in a thin film covering the space that is to be occupied with the sections. The albumin water should stay just where you put it.without creeping away in the least. If it does creep the slip is not clean enough and it should be rinsed off and rubbed with a cloth moistened with alcohol. Cut the paraffin ribbon up into the desired lengths and lay these on the film of albumin water, keeping the glossy side of the ribbon down, namely the side that was down on the knife after cutting, for this side adheres better to the slide than the other. When as many sections have been put on as will fill out under the coverglass, or a less number if so desired, warm the slip over a flame until the ribbons lie perfectly flat and then draw away the albumin water with filter paper; at the same time keep the pieces of ribbon close together and properly lined up, and then place the preparation where it can dry for an hour or so at a tem- perature a little below the melting point of the paraffin. After this stand the slip on end in a dish of xylene to dissolve away the paraffin, and then in a dish of 95 per cent. alcohol to rinse out the xylene, and after this the sections will be ready for staining. If the sections have been at all blackened by the osmic acid, as often happens, they should be bleached .before staining. To do this place the slide for a few minutes in a 264 PREPARATION OF SECTIONS dish containing one part of hydrogen peroxide to twenty parts of 60 per cent. alcohol. Staining the Sections.—The finest results in staining are obtained with Flemming’s triple stain, safranin, gentian violet and orange G, made by Griibler. Prepare the stains separately as follows: Make a saturated solution of safranin in 95 per cent. alcohol and dilute it with an equal amount of distilled water. Make a saturated solution of gentian violet in distilled water, to be used without dilution. Make a saturated solu- tion of orange G in distilled water and dilute it with five times its bulk of distilled water. Put the safranin and gentian violet into Stender dishes or tightly covered tumblers, and the orange G into a drop bottle. In addition to the stains have conveniently at hand: A drop bottle containing absolute alcohol. A drop bottle containing clove oil. A Stender dish or tumbler of xylene. A Stender dish or tumbler of 95 per cent. alcohol acidulated with a drop of concentrated hydrochloric acid. Proceed with the staining as follows: 1. Stand the slide on end in the safranin for a few hours or over night. 2. Remove the slide from the safranin, drain it, rinse it quickly in water, and set it on end in the dish of acidulated alcohol until the safranin stops coming off in clouds and the sections seem almost or quite decolorized. 3. Rinse the slide quickly in water and set it on end in the dish of gentian violet for ten minutes. 4. Remove the slide from the gentian violet, rinse it in water, hold it horizontally, and flood the sections with orange G from a drop bottle for four seconds. 5. Rinse off the orange G in water, drain the slide, and while holding it slightly slanting downward thoroughly dehy- drate the sections by having absolute alcohol flow over them from the drop bottle. 6. Set the slide horizontally and flood the sections with STAINING THE SECTIONS 265 clove oil from the drop bottle. This will gradually extract the gentian violet, and the preparation should be watched under the lower power of the microscope so that this action may be stopped as soon as the gentian stain has lost its too great in- tensity and become transparent while yet distinct. Then drain off the clove oil and set the slide in the dish of xylene to thor- oughly rinse away the clove oil. 7- Remove the slide from the xylene, drain it, place a drop of Canada balsam toward one end of the group of sections and lower a coverglass over it, beginning at the end where the drop of balsam is, by bringing the coverglass into contact with the slide first at that end and gradually lowering it toward the opposite side so as to drive forward any air bub- bles that may become entangled with the balsam. Then set the slide where the balsam can dry for several days at about sor Ce With this three-color stain the cytoplasm should be gray or brownish, the nucleus violet, the nucleolus red, cellulose walls uncolored or grayish, lignified, cutinized, and suberized walls red. In dividing cells the chromosomes should be red, the spindle fibers violet, and the rest of the cytoplasm gray or brownish. Where a fine differentiation of the parts of the protoplasts is not so much sought after as a differentiation of the tissues, other simpler methods of staining may be used to good advan- tage. Double staining with cyanin and erythrosin gives ex- cellent results. For this are needed a saturated solution of cyanin in 95 per cent. alcohol, in a Stender dish or covered tumbler, and a saturated solution of erythrosin in clove oil, in a drop bottle. Set the slide on end in the cyanin for about ten hours, then rinse it in 95 per cent. alcohol until the cyanin no longer comes away in clouds, and this should require only a few moments; then flood the sections for about four seconds with the erythrosin solution, drain and rinse thoroughly with xylene and seal in balsam. If the clove oil is not completely rinsed out in xylene the stains will fade out after a time. The 266 PREPARATION OF SECTIONS time ratios in the stains will need to be varied for different materials. Jodine green may be used in place of the cyanin. It is cheaper than cyanin and is easier to work with. Cyanin and erythrosin can be used for sections cut free- hand or in any other way, and loose sections may be stained in watch glasses. A beautiful differentiation of the protoplast in shades of gray is obtained by iron alum-hematoxylin. Place the sections for two hours in a 3 per cent. aqueous solu- tion of ammonia sulphate of iron, then wash in water for half an hour and place for about ten hours in a 0.5 per cent. aqueous solution of hematoxylin that has ripened in a bottle plugged with cotton, to let in the air, for two months. Remove from the hematoxylin, rinse in water five minutes, and place again in the iron alum to reduce the too infense stain. Keep watch of the bleaching process under the microscope until the parts of the protoplast appear no longer muddy, but still well defined. Now wash in water for an hour or more, and pass through alcohol and xylene, and mount in balsam. After the last wash- ing in water the sections may, if desired, be very lightly counter- stained in a weak aqueous solution of fuchsin or orange G, and then, after again rinsing, be carried through alcohol and xylene for mounting in balsam. It must be borne in mind that always when sections in water or aqueous stains are to be mounted in balsam they must pass from the water into 95 per cent. or absolute alcohol for dehydration, and then into xylene which is a solvent of balsam. If it is found that the sections look milky or opaque when taken from the alcohol to xylene it is a sign that dehydration was not com- plete, the alcohol was not strong enough or the sections were left in it for too short a time. Opaque sections of this kind will clear up more or less after long standing in xylene. Imbedding in Celloidin.—Sometimes material that is not suitable for sectioning free-hand will also not give good results when imbedded in paraffin, on account of its size, hardness, or brittleness. In such cases we may get help in celloidin or collodion (gun cotton) for imbedding. The process of obtain- IMBEDDING IN CELLOIDIN 267 ing sections in this way is a slow one, and it is difficult to get sections as thin as ten mikrons. Therefore cellodin is to be looked upon as a last resort in a difficult situation. Material to be imbedded in celloidin is to be prepared in all respects as when paraffin is the imbedding material up to the go per cent. alcohol in the dehydrating process. From this alcohol it is put into equal parts of ether and g5 per cent. alcohol (which we call ether-alcohol) for several hours and then into a 2 per cent. solution of celloidin in ether-alcohol, where it should remain for several days and then be transferred to a 5 per cent. solution of celloidin in ether-alcohol, whence after a few days it is to go into a 12 per cent. solution of celloidin, and after it has remained here a few days longer it is ready to be mounted on a pine block preparatory to being sectioned. Prepare a pine block large enough in cross-section to sup- port the material and with other dimensions adaptable to its being clamped in the object carrier of the microtome. Leave one end of the block rough and soak this end in ether-alcohol for a while and then dip it for a moment in the 2 per cent. cel- loidin solution. Remove the material from the thick celloi- din and set it in right position on the prepared end of the block. Let the celloidin on the block stiffen for a moment only and then dip the celloidin end into the thick solution, remove it and hold it upright so that the new coating of celloidin may spread out somewhat over the end of the block and make a solid union, and as soon as the celloidin has hardened a little at the surface drop the preparation into a dish of chloroform. After the celloidin has hardened in the chloroform for a day put the preparation into equal parts of glycerine and 95 per cent. alcohol where it is to remain until wanted for sectioning. If it is more convenient to obtain ordinary collodion or gun cotton in place of celloidin it will do just as well as the latter. When wood tissue is to be imbedded in celloidin it has been found helpful first to boil it thoroughly to drive out the air, then to soak it for about two weeks in equal parts of commer- cial hydrofluoric acid and water, or in stronger solutions up to the 268 PREPARATION OF SECTIONS pure acid with very hard woods, to remove silicious deposits that add much to the difficulty of sectioning. After this wash out the acid completely and put the material for several days into equal parts of 30 per cent. alcohol and glycerine preparatory to starting in go per cent. alcohol on the way through celloidin, as above described. The hydrofluoric acid solutions must, of course, be used in a dish coated with paraffin. Sectioning Celloidin Material.—Clamp the block in right position in the object carrier of a sliding microtome (Fig. 143), set the knife slanting so that a long gliding cut will be made, wet the knife with the alcohol-glycerine mixture, and proceed to cut the sections as thin as ‘they can be made; but hope not for success if the knife is dull. With a camel’s-hair brush sweep the sections from the knife into a dish of 70 per cent. alcohol. Staining Celloidin Sections.—Transfer the sections from the 70 per cent. alcohol to the safranin solution described under the three-color method (page 264), and after ten hours or longer rinse them in 70 per cent. alcohol containing one drop of con- centrated hydrochloric acid for every fifty cubic centimeters, until the safranin is very faint in the celloidin but still of sufh- cient intensity in the subject itself. Then pass the sections quickly through 95 per cent. alcohol and transfer them to a mixture of equal parts of bergamot oil, cedar oil, and carbolic acid where they will be further dehydrated and cleared, and then they are ready for mounting in Canada balsam. Safranin and Delafield’s haematoxylin combine well for double staining celloidin sections. To make the hematoxylin solution dissolve one gram of hematoxylin in 6 c.c. of abso- lute alcohol and add this drop by drop to 100 c.c. of a satu- rated aqueous solution of ammonia alum. Leave this exposed to light and air for one week, then filter it and add 25 c.c. each of glycerine and methyl alcohol and after five hours filter again. Let this ripen about two months before using. To double stain the celloidin sections place them in safranin for a day, rinse them in 50 per cent. alcohol, put them into MAKING PERMANENT MOUNTS : 269 hematoxylin for about ten minutes, rinse them thoroughly in water and then in 35 per cent. alcohol and again in 50 per cent. alcohol; pass them quickly through acid alcohol (one drop hydrochloric acid in 50 c.c. of 70 per cent. alcohol), and then put them through 70, 85, and 95 per cent. alcohols, leaving them about two minutes in each grade. Now clear the sections for about two minutes in the mixture of equal parts of bergamot oil, cedar oil, and carbolic acid, and mount them in Canada balsam. Here, as in all staining, the time ratios for the different reagents will need to be determined for different materials. Making Permanent Mounts in Glycerine or Glycerine Jelly.—Filamentous alge and fungi are pretty certain to shrink and become plasmolyzed when put through the process of mount- \ NUitin Oa ine Mirror Bar____. ° sw eceeePttlarn, SPENCER’ L! ele a Pea eo Fic. 147.—Illustrating the parts of a compound microscope. 271 272 USE OF THE MICROSCOPE it by the pillar below the stage, never by the arm or fine adjust- ment pillar, since the fine adjustment bearings might be injured in that way. Take a position near a window where the light from the sky will be unobstructed, but where direct sunlight will not fall upon the microscope. A north window is prefer- able where the too bright light from the sun cannot interfere with good vision. Place the microscope on the table, as shown iv Fic. 148.—Showing correct position at the compound microscope. po : — 2 Pe Ss4 a Cat ay | Hat qu i (e in Fig. 148, so that with the microscope erect you can look into the eyepiece without putting yourself in a constrained position. The better microscopes have an inclination joint, so that the microscope can be inclined to suit the height of the observer; but since in histological work fluid reagents and mounting media are to be used it is better to keep the microscope upright ADJUSTING THE MICROSCOPE 273 and adjust yourself to it by adjusting the height of the table or chair. If the microscope is to be inclined pull back on the fine adjustment pillar and not on the body tube. ‘In nearly all histological work the object is seen by trans- mitted light; that is, by light that shines through the object, and this is accomplished by reflecting the light from below by means of the mirror, and where high powers are employed the light should pass through a condenser. The mirror has a plane and a concave surface, and where there is a condenser the plane surface only should be used, and without a condenser the concave mirror can in a measure fill its place, but it does not give so good results with high powers as does the conden- ser. The condenser should have an iris diaphragm by means of which the light can be adjusted to the object used, as will appear later on. For histological work there should be a medium and a high power objective, such asa #anda¢in. or a 16 anda4mm., mounted on a revolving double nosepiece, as shown in Fig. 147. Assuming that the microscope is thus equipped revolve the lower power, that is, the % in. or the 16 mm. objective into position (in the figure the higher power is shown in position) and get ready some object for examination. To begin with there is nothing better than starch from the potato. To pre- pare this for examination a glass slip and coverglass obtain- able from dealers in microscope supplies will be necessary. A good supply of these should be at hand. Clean a glass slip thoroughly so that it is crystal clear when you look through it; lay it flat and put a small drop of water on it near the middle. Cut open a potato.and with the point of a pocket knife scrape up a very small portion of the pulp not more than the size of two pin heads. Better take too little than too much. Put this in the drop of water-on the slip. Clean a coverglass thoroughly and lay it over the drop. The coverglass must be put on with some care else air bubbles will become entangled in the prepara- tion, and they are a nuisance, particularly to a beginner who may mistake them for some part of the thing he is studying. To 18 274 USE OF THE MICROSCOPE avoid the air bubbles place one edge of the coverglass on the glass slip first and then gradually lower the coverglass while holding it between thumb and finger pressed against opposing -edges, and support it with a dissecting needle held under the upper edge as the coverglass begins to flatten out the drop of water. If now air bubbles are being entangled in the water the coverglass may be rocked gently up and down with the dissecting needles until the bubbles are broken and driven out. When the coverglass is in position the water should fill out under it to the edges, but no more. If it runs out over the slip the drop was too big and the surplus should be wiped off with a piece of filter paper or clean cloth. Take note in this first prepara- tion whether the drop was too large or too small so as to avoid the mistake in the future. The potato in the preparation should now appear as a very thin film. If it is in a lump it may be flattened out by pressing on the coverglass with the dissecting needle. Now place the slip on the stage under the clips (see the figure for the parts of the microscope as they are mentioned) and. bring the preparation over the center.of the condenser or to the center of the opening in the stage if there is no condenser. With the plane mirror, if there is a condenser, reflect light from the sky into the condenser and adjust the mirror so that the prep- aration is seen to be illuminated while looking at it directly and not through the ocular. Then with the rack and pinion coarse adjustment run the body tube down until the lower power object- ive, which has been revolved into position, is within a quarter inch of the coverglass. Look in through the ocular and the field of view should appear circular and brightly illuminated; if it is not, adjust the mirror slightly until it is so. Now, still look- ing through the ocular, slowly rack the tube back until the ob- ject comes into view, and with the micrometer head of the fine adjustment, turning to right or left as seems to be necessary, bring the object into sharp focus. The torn and crushed tissue of the potato will be seen and many very minute grains of starch which need to be seen with the higher power objective. Ac- cordingly draw the body tube up with the coarse adjustment ADJUSTING THE MICROSCOPE 275 and swing the high power objective into position, and then while watching outside the microscope run the body tube down until the objective all but touches the coverglass. It must be very close indeed, and you must hold your eye on a level with the stage to be certain about it and see that the objective does not ‘press against the coverglass. Now look through the ocular and slowly draw the body tube up by turning the micrometer head of the fine adjustment contra-clockwise, until the preparation comes sharply into view. Starch grains of the potato have circular, excentric striations, and if you can see these clearly with the high power it is an indication that the microscope is adjusted to give a good image. If the striations cannot be seen you may be sure something is the matter. See that the mirror is adjusted to give plenty of light and that the opening in the iris diaphragm is hardly more than } in. in diameter, and see that the lenses of the ocular and the front lens of. the objective are perfectly clean. These are the chief things to look to in securing a good image. If the lenses need cleaning breathe upon them and quickly wipe them off with a clean soft cloth; or dip the cloth into water and wash the lenses and then dry them with gentle pressure of a dry part of the cloth. A good tentative rule about the dia- phragm opening is to have it about the size of the front lens of the objective in use. If there is then not enough light the open- ing may be made somewhat larger, but it will be seen that the image becomes less sharp the larger the diaphragm opening is made to be. Make it a rule from the beginning never to put up with a poor image. There are some details about the working of a microscope that need special mention. The tube length should be ad- justed to 160 mm. by sliding the draw tube in or out (see Fig. 147). When a revolving nosepiece is used the tube length should be measured from the eyelens to the lower edge of the nosepiece. The coverglasses should be No. 2, excepting when oil immersion objectives are employed with a working distance so short as to require the thinner No. I coverglasses. It is to be noted that the micrometer fine adjustment works through only a 276 USE OF THE MICROSCOPE short distance, and therefore the coarse adjustment should always be used excepting for the finishing touches in getting a sharp image. If at any time the fine adjustment has been run to the extreme end of its range one way or the other the body tube should be run up with the coarse adjustment and the micrometer head of the fine adjustment should then be turned in the requisite direction to bring the fine adjustment to about the middle of its range. In some microscopes the condenser is provided with a focus- ing screw, by means of which the condenser can be raised or lowered to give the best results. Where there is a fixed mount- ing for the condenser the latter should be kept with its front lens flush with the upper surface of the stage. It is important that the lenses of the condenser should be kept as crystal clear as those of the ocular and objectives. For nearly all work the mirror bar should be kept parallel with the long axis of the microscope, so that the light will be reflected along the axis of the entire system of lenses. The clips (Fig. 147) are intended to press gently upon the glass slide so that it can be moved about over the stage steadily with gentle pressure of the fingers, but this cannot be done well unless the stage is kept clean. It has already been said that where there is a condenser the plane mirror should be used. The reason for this is that the condenser is made to bring parallel rays of light to a focus in the plane of the object, and if the concave mirror is used con- vergent rays would enter the condenser and be brought to a focus within the condenser system and the object would not be so well illuminated. Keep the fingers off from the surfaces of the lenses, for they are sure to leave a film that will need to be washed off. Most of the trouble which beginners experience in the use of the microscope comes from dirty lenses due to contact. with the fingers or reagents, and to water or other fluids in which the object is mounted running out from under the coverglass and coming between the latter and the objective. These troubles ADJUSTING THE MICROSCOPE 277 are easily avoided with a little thoughtful attention. One can soon get into the habit of not touching the lenses with the fingers, and reagents will not run out from under the coverglass unless more than is necessary has been used. One should soon learn how large a drop of reagent is needed for a given size of coverglass. For bacteriological and cytological work an oil immersion objective will be necessary. A vz in. or a 2 mm. objective is best for general work in these lines. If such an objective is to be used it would be best to have a triple revolving nose- piece to carry the medium, the high, and the immersion object- ive all at once. The use of the oil immersion objective is very simple. Put a small drop of cedar immersion oil on the coverglass directly over the object and run the body tube down with the coarse adjustment until the front lens of the immersion objective enters the drop and comes almost into contact with the coverglass. This is to be done while watching the objective outside the microscope. Then while looking through the ocular draw the objective up with the fine adjustment until the object comes into focus. The higher the power of the objective the smaller the field of view, and when it comes to the oil immersions one sees but a very small area at once, and it is therefore much more difh- cult to find a small object with them than with the lower powers. For this reason it is a good plan to find the object with a lower power and bring it to the center of the field, and then it will probably be in view when the immersion objective is used, but if it is not it cannot be far out of the way and a very slight movement of the preparation one way and another should suffice. With the oil immersion, when the object has been stained, the opening of the diaphragm may be made as wide as is necessary to let in plenty of light. Indeed a good image can under these conditions be secured with a wide open diaphragm. When through with the oil immersion objective wipe it off with a clean soft cloth or with a piece of Japanése lens paper, and wipe off the coverglass in the same way. t | y i 278 _ USE OF THE MICROSCOPE A very good artificial light for use with the microscope can be obtained with a Welsbach gas mantle and a balloon flask filled with a light solution of ammoniacal copper sulphate (Fig. 149). Dissolve a very small crystal of copper sulphate in enough water to fill the flask and add ammonia a little at a time until the solution loses all opalescence and becomes perfectly clear blue. Adjust the light and the blue condenser in front of the microscope until an image of the mantle, about natural size, falls on the mirror. Looking through the micro- Fic. 149.—Method of illuminating compound microscope with gaslamp. C, Balloon flask filled with ammonio-sulphate of copper; G, Welsbach mantle. scope the light should appear white; if yellow, add more cop- per sulphate; if blue, dilute the. solution. This light cannot be excelled and makes one independent of the weather condi- tions or the time of day. Drawing to Scale from the Microscope.—There are two ways of drawing from the microscope to a scale that can be accurately determined; one is by the use of an eyepiece mi- crometer, that is, an eyepiece containing a glass disc with a fine scale etched on it. For purposes of drawing, a disc ruled off in very small squares is preferable (Fig. 150). In using this we need to know how large with a given objective an object would be that just fills out the space across one of these squares DRAWING TO SCALE FROM MICROSCOPE 279 or divisions of the scale. This we can determine with a stage micrometer scale, which is simply a glass slip with a scale etched on it divided into tenths and hundredths of a millimeter. Put this in position on the stage and focus with the medium power and find how many of its smallest divisions extend across one of the squares or divisions on the eyepiece scale. Suppose it takes fifteen of them to do this, then we know that any object that is found with the same objective to extend across one of the divisions on the eyepiece scale is exactly .15 mm. in diameter. Try the high power in the same way, and of course the scale on the stage will be magnified —_—Fis. 150.—Glass disc x he os. ruled into squares to more and a less number of its divisions than serve as a micrometer before will now cover a division in the eye- ee Mean eros piece scale. If three of them now do this _ is to be placed in an we know that any object under the high ren nes power extending across one division of the eyepiece scale has an actual diameter of .o3 mm. An arbitrary but accurately determinable scale can now be fixed upon for drawing from the microscope. We may decide to draw 5 mm. long any- thing that covers one eyepiece scale under the medium power, in which case the magnification of the drawing would be 1s= 33-3. This will serve to illustrate the method. When the eyepiece micrometer is ruled. in the form of squares it can be conveniently used in determining the number of any particular structures in a given area, as in a square millimeter. For in- stance, using the medium power with which an object .15 mm. in diameter would extend across the diameter of the square of the eyepiece scale, suppose we can count 9 stomata in ‘the under epidermis of a leaf within one of these squares and we want to determine how many there would be in a square millimeter. That portion of an object which fills one of the squares would have an area equal to .15X.15 mm.=.0225 sq. mm. How many times would this area have to be taken to make up one square millimeter? Of course the answer would be found by 280 USE OF THE MICROSCOPE dividing 1 by .0225, which would give 44.4. Then the number of stomata in one square millimeter would be 9 times 44.4= 399.6. The other method of drawing to scale is carried out with a camera lucida, the most convenient form of which is shown in Fig. 151. The main structural details of this instrument are, behind the opening P a prism silvered on one of its surfaces excepting for a narrow circular area at the center; and the plane Fic. 151.—-Camera lucida. M, Mirror; P, opening to reflecting prism; K, knobs for regu- lating diaphragms that govern illumination from object and drawing paper. mirror at M. Through the unsilvered part of the prism one can look and see the object; the mirror reflects the drawing paper placed on the table below it to the silvered surface of the prism and this reflects it into the eye. In this way the object and the drawing paper and the pencil held over the drawing paper are all seen at once superimposed; the object appears spread out over the paper and with the pencil its outlines can easily be traced. To use the camera lucida successfully it is necessary to have some means of illuminating the object and drawing paper with equal intensity, for if one appears brighter than the other they cannot both be seen with equal clearness. If the object, for instance, is too bright the point of the pencil cannot be accurately followed, and if the paper has the stronger illumination the ee TO SCALE FROM MICROSCOPE 281 pencil can be seen distinctly enough but the outline of the object becomes too dim. In the camera lucida of Fig. 151 there are two revolving diaphragms with handles at K, with a series of = | fe Fic. 152.—Convenient drawing board for use with camera lucida. openings, all but one of which in each series are covered with a graduated series of different intensities of smoked glass. With this provision one can decrease the light entering the eye from the drawing paper or from the object until the object and the point 282 USE OF THE MICROSCOPE of the drawing pencil can be seen with equal clearness. It is best to begin the adjustment with the free opening in both dia- phragms in position, that is, with no smoked glass intervening between drawing paper or object, and then if the object, for instance, should be too bright the diaphragm relating to it can be revolved until this is corrected, or if the drawing paper is too bright its diaphragm is to be revolved until the right degree of smoked glass is in position. . In drawing from the camera lucida it is a convenience to have a drawing board (Fig. 152) adjusted to the same height as the microscope stage, and if the microscope is used tilted the drawing board should be set to the same angle as the stage. The mirror should be adjusted to bring the center of the drawing vertically below the middle part of the mirror, for the projection of the image off to one side of the mirror causes its distortion. The determination of the magnification of a drawing done with a camera lucida is made by projecting the scale of a stage micrometer upon the drawing paper by means of the camera lucida and drawing it there and measuring the drawing with a millimeter scale. Then the magnification is obtained by dividing the value of the magnified drawing by the actual value of the scale. For example, if one of the finest divisions of the micrometer scale (.or mm.) measures 5 mm. in the drawing the magnification would be .zy=500. Of course the magnification would have to be determined for each objective used and the tube length must be kept the same for the micrometer scale as for the object, and the distance of the drawing paper below the mirror must be kept the same. Use of the Polariscope.—The polariscope is very uel in detecting the presence of minute starch grains and crystals and in bringing out sharply fragments of sclerenchyma tissues in powdered drugs, etc. The polariscope consists of the polarizer which is placed beneath the stage, and the analyzer surmounting the eyepiece. Proceed with the polarizer as follows: Adjust the mirror so as to reflect light into the microscope, and looking into the eyepiece rotate the analyzer. It will be seen that during this USE OF REAGENTS 283 rotation through 360° the illumination of the field changes from brightness to blackness and back to brightness again. Turn the analyzer so that the field is at its brightest and place on the stage some potato starch mounted in a drop of water or dilute glycerine; these will then appear when looking through the eyepiece as though no polarizing apparatus were employed. Now turn the analyzer through 180° or until the field is black and each starch grain will be seen to be traversed with a bright cross. It will be found that when thin sections of plant tissues containing crystals of calcium oxalate are treated in the same way some portions of them shine brightly out in the dark field, and the same thing is true of many sclerenchyma cells and fibers. It will be seen from this that the use of the polarizing apparatus may be of great service in identifying the parts of powdered foods and drugs where the fragmentary condition of tissues, cells, and cell contents increases the difficulty of finding out what the differ- ent parts are. The Use of Reagents on Microscopic Preparations.— When an object has been mounted in a drop of water under a coverglass the water can be replaced with other fluid reagents without removing the coverglass or removing the preparation from its position under the objective. Suppose we wish to treat with a solution of iodine, starch from the potato that we have already examined in water: put a small drop of the iodine upon the glass slip close to but not touching the coverglass, and then with a dissecting needle or broomstraw drag the drop into contact with the coverglass, when it will diffuse under and give to the starch the characteristic blue color which iodine is known to impart to it. The drop of reagent is not put at first in contact with the coverglass because there would then be danger of its running over the top of the latter, wetting the front lens of the objective and preventing a good image. If this accident should ever happen in the use of a reagent there is nothing to do but to remove and wash the coverglass, and wash and wipe dry the objective lens. Sometimes it is desirable to hasten the replace- ment of the water under the coverglass by the reagent, and to do 284 USE OF THE MICROSCOPE this it is only necessary to put a strip of filfer paper in contact with the edge of the coverglass opposite the drop of reagent, when the water will run into the paper and the reagent will be drawn under the coverglass by capillarity. This process is called irrigation. It must be remembered, however, that very minute objects such as starch grains are pretty certain to be swept along in the currents, so that the slower process of diffusion should be depended on wherever practicable. Some reagents should not be diluted with water at all. Such, for instance, is chloroiodide of zinc. If a preparation already mounted in water is to be treated with such reagents the cover-- glass should be removed and all of the water drawn off with filter paper; then the reagént should be put on before the preparation has time to become dry. If acid reagents are to be used especial care must be taken not to allow them to get upon any parts of the microscope; if this should happen they must be washed off at once with plenty of water. The use of such acids as hydro- chloric and sulphuric-‘must be quickly over with, since their fumes are injurious-to the eyes as well as to the microscope. When through with the microscope see that it is clean in all its parts and put it away under cover, where it will be free from dust. CHAPTER XVI REAGENTS AND PROCESSES The different kinds of cell-walls and cell-contents may be demonstrated by the use of reagents which, in some cases, im- part characteristic colors to walls and contents; in other cases act as selective solvents, dissolving some of the walls and con- tents, leaving others undissolved; or the reagents may produce precipitates the nature of which furnishes good evidence regard- ing the character of the substance which has united with the reagent to produce the precipitate. These reagents together with their uses, will now be given in alphabetical order. Acetic Acid dissolves most ethereal oils, while most fatty oils are insoluble in it; dissolves calcium carbonate with evolu- tion of CO,, while calcium oxalate is unaffected by it, and it therefore serves to distinguish between these two salts of cal- cium; solvent of crystals of hesperidin which have been depos- ited from the cell-sap of oranges, etc., when these have lain for some time in alcohol; when various lichens are treated with it, crystals of calycin in acicular form are deposited after the lichens thus treated have been powdered and dried; 1 per cent. solution dissolves globoids in aleurone grains, while any crystals of calcium oxalate present are unaffected by it; when pieces of potatoes, carrots, etc., are macerated in it, the separate cells become isolated. Used in the preparation of various fixatives. Albumen.—The white of egg is used with an equal amount of glycerine and a trace of salicylate of soda for fixing micro- tome sections to the glass slide, the sodium salicylate acting partly as an antiseptic. (Page 263). Alcannin.—This is a coloring matter, obtained from the roots of Alcanna tinctoria. A tincture of alcannin to be used 285 286 REAGENTS AND PROCESSES as a reagent is prepared by placing alcanna root in 95 per cent. alcohol for about ten hours, or until a deep red solution is ob- tained, and then filtering off the solution and diluting it with an equal bulk of water. (1) Suberized and cutinized walls, when treated with a solu- tion of alcannin for some hours, take on a pink color. (2) Alcannin tincture mixed with 1 per cent. glacial acetic or formic acid is used to fix and stain sections of elaioplasts from fresh material. (3) When sections containing fatty oils are treated with tincture of alcannin, the oil is colored pink. Sections containing ethereal oils and resins behave in the same manner. Alcohol.—The commercial alcohol obtained in this country is about 95 per cent. alcohol. In making alcohols from this of different strengths it answers all practical purposes to pro- ceed as if the commerical 95 per cent. alcohol were absolute— that is, very nearly 100 per cent. Thus, if 50 per cent. alcohol is desired, 50 c.c. commercial alcohol and 50 c.c. distilled water will give sufficiently accurate results for all histological work. If absolute alcohol is desired, it may be prepared by pouring the commercial alcohol over unslacked lime, and then distilling from this. Or better still, drive off the water of crystalliza- tion from copper sulphate by heating in an iron vessel. Put the powder into a bottle and pour 95 per cent. alcohol over it. Keep the bottle tightly stoppered. Water will be extracted from the alcohol and absolute alcohol results. Ammonium Molybdate.—A concentrated solution of am- monium molybdate in a saturated solution of ammonium chlo- ride. This gives a yellow precipitate in sections containing tannins. _ Ammonium Vanadate.—This is used as a test for solanin. The sections are treated with a solution prepared by dissolving 1 part of ammonium vanadate in 1000 parts of a mixture of 98 parts of concentrated sulphuric acid and 36 parts of water. If solanin is present, a yellow color appears, which merges into orange, then different shades of red, and finally into violet, and then all color disappears. ANILINE OIL—BERLIN BLUE 287 Aniline Oil.—Excellent for dehydrating sections, since it will dissolve about 4 per cent. of water and may be kept dehy- drated by a small piece of solid KOH which is insoluble in it. The sections may be transferred from the aniline immediately into Canada balsam. Aniline Sulphate.—Make a saturated aqueous solution. As a test for lignified membranes mount the sections in the solu- tion and add a drop of sulphuric acid, and a yellow color is given to the lignified membranes. Or pour sulphuric acid slowly into aniline oil until a pre- cipitate is produced throughout and then add water until the precipitate is dissolved. This will not require the addition of sulphuric acid to the sections. Balsam.—Canada balsam dissolved in xylol is, on the whole, the best medium for making permanent mounts of sections under a coverglass. For the method of doing this see page 265. Balsam in xylol can be obtained ready prepared of the dealers. Barium Chloride.—This is sometimes used to distinguish calcium oxalate from calcium sulphate. When barium chlo- ride is run under the coverglass, calcium oxalate, if present, is left unchanged, while a fine granular layer of barium sul- phate comes to incrust any crystals of calcium sulphate. (2) To determine the presence of tartaric acid, barium chloride and antimonic oxide in hydrochloric acid is run under the cover- glass, producing, with tartaric acid. rhombic crystals of anti- monium-barium-tartrate, whose obtuse angles measure 128°. Benzol.—Used in detecting caffeine, thus: Sections are heated on the slide in a drop of distilled water until bubbles arise, © then the water is allowed to evaporate, and the residue is dis- solved with a drop of benzol. The benzol is then allowed to evaporate and the caffeine is deposited on the edge of the drop in the form of colorless needle-crystals. Berlin Blue.—Useful in the study of the growth in thickness of the cell-membranes. In the study of marine alge—notably, Caulerpa prolifera—it is used in the following manner: A vigorous 288 REAGENTS AND PROCESSES alga is submerged for a few seconds in a mixture of one part of sea-water with two parts of fresh water in which has been dis- solved sufficient ferrocyanide of potassium to give it the specific gravity of sea-water. The alga is then rapidly rinsed in sea- water and placed for about two seconds in a mixture of two parts of sea-water and one part of fresh water, to which has been added a few drops of freshly prepared ferric chloride. This produces in the membranes of the alga a precipitate of Berlin blue. The alga is then transferred to sea-water for further growth. In case new lamellz are added to the membranes, the new portions will appear colorless, while the older portions will appear blue because of the Berlin blue which was precipitated in them. Bismarck Brown.—This is preéminently a nuclear stain. The powder is soluble with difficulty in water. Jt is a good plan to treat with boiling water and after a day or two to filter. Ora saturated solution may be made in 70 per cent. alcohol. Al- though Bismarck brown stains rapidly, it does not overstain. It may be used for staining im toto or for staining sections on the slide. Boracic Acid.—Used as a mounting medium for sections containing mucilaginous membranes. The sections are cut from dry material and placed in a ro per cent. solution of neutral lead acetate to harden the mucilaginous layers. Then the sec- tions are stained in a solution of methyl blue, washed in water, and mounted under a coverglass in a 2 per cent. solution of boracic acid. The coverglass should be sealed down with a mixture of paraffin and vaseline, which is applied with a brush while melted. Borax-carmine.—A 4 per cent. solution of borax in water is made and to it is added 3 per cent. of carmine; an equal bulk of 70 per cent. alcohol is then added to this. The mixture is left standing for a day or so and then filtered. Sections should lie in the stain for about twenty-four hours, and should then be transferred without previous washing to acidulated alcohol, made by adding four drops of hydrochloric acid to 100 c.c. of alcohol. Here they should remain until they become bright BORDEAUX RED—BROWN DISCOLORATION 289 and transparent. This is a useful stain for, aleurone grains, for differentiating cell-contents from cell-walls when the sections are subsequently stained with methyl green, and much used also in the differentiation of the cell-contents of filamentous alge. Bordeaux Red.—Used in conjunction with hematoxylin in staining nuclear figures, particularly where Heidenhain’s pla- tinic chloride fixative has been used. The sections are placed in a weak aqueous solution of the Bordeaux until they are in- tensely stained; they are then rinsed and placed in a 2 to 5 per cent. solution of ferric-ammonium sulphate for three hours. If the sections are mounted on a slide, they should be placed upright in this solution, so that any precipitate may not gather on the slide. Then the sections are carefully washed in an abundance of water, and placed for twenty-four hours in a solu- tion of hematoxylin prepared as follows: 1 gm. of hematoxylin is dissolved in 10 gm. of alcohol and go gm. of water. This is allowed to stand for about four weeks and then an equal bulk of distilled water is added. The stain is then ready for use. When the sections are taken from the hematoxylin, they will be found overstained; they are, therefore, rinsed and placed in a 2.5 per cent. solution of ferric-ammonium sulphate, where they remain until examination of the sections under the microscope shows the desired intensity of color. Then rinse in water 15 minutes, dehydrate in alcohol, and pass through xylol for mount- ing in Canada balsam. Borodin’s Method.—To determine the nature of a precipi- tate Borodin treats it with a saturated solution of the same sub- stance as the precipitate is supposed to be. Thus, if the pre- cipitate is supposed to be asparagin, it is treated with a saturated solution of asparagin. If the precipitate dissolves by this treat- ment, it is then some other substance than asparagin. This method is not very reliable for substances which are very readily soluble, such as potassium nitrate. Care must be taken that the solution used for the test is entirely saturated. Brown Discoloration of Material in Alcohol.—Some plants, such as Monotropa, are apt to become quite brown in 19 290 REAGENTS: AND PROCESSES . t alcohol. This can be prevented by placing the fresh material in alcohol which is acidulated by vapor of sulphuric acid in the following manner: For each 100 c.c. of alcohol several cubic centimeters of concentrated sulphuric acid are ‘poured over 4gm. of sodium sulphite, and the vapors arising are conducted into the alcohol. This operation need require hardly more than a minute. After twenty-four hours the material should be transferred from the acid alcohol to neutral alcohol. There- after the material will not discolor and will take stains very well when used for histological purposes. Calcium Nitrate.—(z). Used to differentiate more clearly the lamelle of starch grains. Potato starch, for instance, is placed in a rather strong aqueous-solution of methyl violet. After the grains have become deeply colored, they are treated with a weak solution of calcium nitrate, when the methyl violet becomes pre- cipitated, particularly. in the less dense lamellae of the starch grains. (2) Calcium oxalate is precipitated in the form of crystals when sections containing oxalic acid are treated with a solution of calcium nitrate. The calcium nitrate is thus a test for the presence of oxalic acid. * Canarin.—This is often used as a stain for tissues which have been cleared in caustic potash. Canarin is not affected by this reagent. Carbolic Acid (Phenol).—Used as a clearing agent. If leaves which have been hardened and bleached in alcohol are placed in three parts of turpentine and one part of carbolic acid, or in pure carbolic acid, the leaves will become so trans- parent that their cellular structure may be made out from one surface to the other. Pollen grains may be made transparent in the same manner.: . Carmalum, Mayer’s.—Carminic acid 1 gm., alum ro gm.; ‘dissolve in 200 c.c. of hot distilled water; filter and add a few crystals of thymol, or o.1 per cent. of salicylic acid, or 0.5 per cent. of sodium’ salicylate. This stains material well in bulk, with little danger of overstaining. If this happens, it may be corrected by washing with a 0.1 per cent. solution of hydrochloric CEDAR OIL—-CHLORAL HYDRATE 291 acid. Material which has been stained in bulk with carmalum may be sectioned, and the sections may then be double-stained with some aniline stain, such as blue de Lyon. See Borax- carmine for another carmine stain. Very fine double staining may be achieved by placing sections first in an aqueous solution of iodine green and then for a somewhat longer time in carmalum. By this treatment lignified membranes are stained by the iodine green, while the unlignified membranes are stained by the carmalum. ' Cedar Oil.—Sections which are to be mounted in balsam may first be examined in cedar oil to determine their fitness for permanent mounts; if they are satisfactory, the cedar oil may be drained off and the balsam immediately added to the slide. Cedar oil has a clearing effect on sections which are treated with it. Thicker cedar oil with a refractive index of about 1.515 is used as an immersion fluid for homogeneous immersion lenses. Cedar oil is often used as an intermediary between alcohol and paraffin in paraffin-imbedding, but for plant tissues chloro- form is rather to be recommended. Chloral Carmine.—This is useful in clearing pollen grains and staining their nuclei at the same time. It is prepared as follows: Carmine 0.5 gm. and 30 drops of officinal hydrochloric acid (specific gravity, 1.13 or 17° B.) are added to 30 c.c. of alcohol, and this is heated for about thirty minutes on the water-bath; then, after cooling, 25 gm. of chloral hydrate are added, and the solution is filtered until clear. . Chloral Hydrate.—Dissolve five parts of chloral hydrate in two parts of water. The chloral hydrate may be taken in, grams and the water in cubic centimeters. This is one of the best clearing agents. Whole leaves, when boiled in this solu- tion, clear quickly to such an extent that they may be studied by transmitted light throughout all of the cell-layers. Crys- tals in leaves may be plainly demonstrated in this way. This reagent is also very useful in clearing pollen grains, and em- bryos within the ovules. 292 REAGENTS AND PROCESSES Chloral Hydrate-iodine.—Dissolve five parts of chloral hydrate in two parts of water and add enough finely powdered iodine to leave an excess undissolved after long standing. Shake before using. This is the best reagent for demonstrating the presence of starch in chlorophyll corpuscles and in pyrenoids, or in any situation where the starch is surrounded and obscured by other substances. Chloroform.—Used as a solvent for fatty oils and of caro- tin. Used as a solvent for paraffin in the process of imbedding in paraffin. See page 261. Chloroiodide of Zinc.—Dissolve 30 gm. of chloride of zinc, 5 gm. of potassium iodide and 0.89 gm. of iodine in 14 c.c. of distilled water. Chloroiodide of zinc solutions should be kept in the dark. This reagent is one of the most | generally useful in determining the character of plant mem- branes. By it cellulose walls are colored violet, lignified mem- branes a yellowish-brown, cutinized and suberized membranes from yellow to yellowish-brown. When sections containing sieve tubes are treated with chloroiodide of zinc and a rather weak solution of potassium iodide-iodine, the walls of the sieve tubes appear violet, while the pits in the sieve plates are a reddish-brown, due to the strands of protoplasm which ‘penetrate them; the callose plates are stained a reddish-brown. Mucilaginous walls are colored violet by this reagent. Chloro- iodide of zinc stains protoplasmic cell-contents from yellow to brown, and starch from purple to almost black. Chlorophyll Solution.—A freshly prepared strong solution of chlorophyll in alcohol is used to demonstrate suberized and .cutinized membranes. When sections are kept in the chloro- phyll solution for an hour or so in the dark, cutinized and sub- erized membranes are stained green, while lignified and cellu- lose membranes remain unstained. The chlorophyll solution will not keep, and should be freshly prepared whenever needed. Chromic Acid.—Solutions of 1 per cent. and 0.5 per cent. have been much used for fixing plant tissues. The material to be fixed should lie in the chromic acid for a day or more, ‘. CLEARING 293 according to, the size of the pieces of material to be fixed. The material should then be thoroughly washed out in water and dehydrated by slow degrees in ascending grades of alcohol (see page 260). A concentrated aqueous solution of chromic acid may be used as a macerating fluid to cause the separation of tissues into their separate cells. To this end rather thin bits of the tissue to be macerated should be placed in the chromic acid for about half a minute, and then carefully washed in water. This operation may be carried on with sections under the cover- glass.. Silicious skeletons of diatoms, incrustations on the epidermis of Equisetum, etc., may be prepared by allowing the material to lie in concentrated sulphuric acid until it becomes black, and then, after transferring to a 20 per cent, solution of chromi¢ acid for some minutes, washing thoroughly in water. In the case of Equisetum and the like the tissues should be scraped away from the inside down to the epidermis before treat- ment with the acids. Chromic acid is useful in the recognition of tannins, since sections containing tannins, when treated with a I per cent. solution of chromic acid, yield a brownish precipitate. Clearing.—For clearing media see Carbolic Acid, Cedar Oil, Chloral Hydrate, Canada Balsam, Clove Oil, Eau de Javelle, Glycerine, Origanum Oil, Turpentine, Xylol. A very successful method of clearing whole leaves is to boil them, if fresh, in 95 per cent. alcohol to extract the chlorophyll, place them in 5 per cent. hydrochloric acid for about ten hours, and then leave them until quite transparent in a saturated solution of chloral hydrate. In the case of leaves that have been dried, or preserved in formalin or alcohol, and have in consequence become discolored and difficult to clear, place them in a 1o per cent. solution of hydro- chloric acid overnight and then keep them for three to seven days in a saturated aqueous solution of chloral hydrate. From this © they are to be taken and thoroughly washed in water and brought into a 5 per cent. solution of potassium hydrate, and there kept until they are clear and transparent; then they are to be thoroughly washed and transferred to dilute glycerine, or, if they are to be 294. REAGENTS AND PROCESSES stained, to saturated chloral hydrate. To stain the'leaves transfer them to a deep red solution of safranin in saturated chloral hydrate for a few hours or overnight; then rinse in water and place them in saturated chloral hydrate till all but the tracheal elements of the veins is clear of the stain. Now the leaves are to be put into dilute glycerine and, after this has concentrated by evaporation in a place free from dust, the leaves may be per- manently mounted in glycerine gelatine (see page 269). This clearing and staining method may be used to advantage with sections of leaves, stems, etc., and for these a double staining with safranin and Bismarck brown is often desirable. If this is to be done, after clearing out the safranin stain from all but the lignified walls in chloral hydrate, rinse the sections in water and stain for five to fifty seconds in a saturated aqueous solution of Bismarck brown, then rinse quickly in water and transfer the sections to dilute glycerine, from which they can be brought into glycerine gelatine as above suggested. If the brown stain is too intense at first, it must be used for a shorter time. Experi- ence only will show for the specific subject in hand what the time length for this stain should be. Clove Oil.—This is an excellent clearing medium, but it has the power of extracting certain stains, and so cannot be used in all cases; it is, however, for this very reason of great advantage in the safranin-gentian violet-orange method of staining. See ‘under this head. Collodion.—Used as an imbedding medium (see page 267). Congo-red.—This stain is particularly useful in studying the growth of membranes. Old membranes are, as a rule, left unstained, by it, while the newly formed membranes are colored red. In a o.o1 per cent. solution—that is, 1 part of the stain to 10,000 of water—alge may continue to live and grow, and they are, therefore, well adapted to the study of the growth of membranes with the employment of this stain. Copper Acetate.—Used in the determination of tannins. Small bits of the plant to be tested are placed in a saturated solution of copper acetate, where they remain for eight or ten CORALLIN—CYANIN 295 days; the sections are then placed on a slide in'a drop of a 0.5 per cent. solution of ferrous sulphate; after a. few minutes the sections are washed in water, then in alcohol, and are finally treated with a drop of glycerine and examined under a cover- glass. This gives an insoluble brown precipitate with tannins. An alcoholic solution of copper acetate, to which has been added a small amount of acetic acid and glycerine, is used to demonstrate glucose in position within the cells where it occurs. The sections are laid in a mixture of the above solution, and an equal volume of sodium hydrate in alcohol, and the whole is brought to boiling on the water-bath. Since glucose is insoluble in alcohol, the cuprous oxide which indicates the presence of glucose in this reaction is found to be deposited within the cells which contain the sugar. For other tests for sugar with a salt of copper see Fehling’s Solution. See under Resin in next chapter. Corallin.—This stain is to be dissolved in a 30 per cent. or a saturated solution of sodium carbonate. It is particularly useful in staining the callose of sieve tubes. It is best to over- stain the sections and then to reduce the intensity of the color by immersing the sections in a 4 per cent. solution of sodium carbonate. Corrosive Sublimate.—See Fixatives. Cuprammonia.—This should be freshly prepared as needed in the following manner: Put copper filings into a bottle or flask, which is provided with a ground-glass stopper. Pour concentrated ammonia upon the filings and rock back and forth. Only sufficient ammonia should be used to cover the filings. When the solution will dissolve cotton, it is ready for use. This reagent is a solvent of cellulose. When sections are placed in it for some time and are then rinsed with ammonia and finally with distilled water, crystals of cellulose are precipi- tated within the cells which are stained blue with chloroiodide of zinc and red with Congo-red. The crystals are again dissolved on the addition of cuprammonia. " Cyanin.—This stain is almost insoluble in water, and should be dissolved in 50 per cent. alcohol. This is a useful stain for 296 REAGENTS AND PROCESSES fats and all ethereal oils. Sections of fresh material, or mate- rial fixed in an aqueous fixative, such as an aqueous solution of corrosive sublimate or picric acid, will be sufficiently stained when left in the cyanin solution for about half an hour. Over- staining may be reduced with glycerine. The alcoholic solution of cyanin, to which has been added an equal bulk of glycerine, is a good stain for suberized membranes, particularly after the sections have been treated with eau de Javelle, which destroys the tannins that prevent the membranes from taking the stain. When sections are placed in a dilute solution of cyanin,—say 20 drops of a concentrated alcoholic solution of cyanin in 100 c.c. of water,—for ten hours or longer, and are then washed in alcohol and placed in oil of cloves containing eosin, the lignified and suberized walls will be stained blue, while cellulose walls will be red. The sections may then be mounted in Canada balsam. When sections are placed for a quarter of an hour in a concentrated alcoholic solution of cyanin, and are then washed in alcohol and transferred for a quarter of an hour to a 5 per cent. ammoniacal solution of Congo-red, the lignified mem- branes will appear blue, while the unlignified membranes will appear red. After washing in alcohol and afterward in xylene, such sections may be mounted in Canada balsam. See also page 265. Dahlia.—An aqueous solution of from o.oor per cent. to 0.002 per cent. is used for staining live nuclei. The dividing nuclei of Tradescantia Virginica, for instance, when kept in this stain for a few hours, become weakly stained. The struc- ture of pyrenoids is well demonstrated by fixing them in equal parts of a 10 per cent. solution of potassium ferricyanide and a 55 per cent. solution of glacial acetic acid and then staining with dahlia, and finally swelling the pyrenoids somewhat in a weak solution of potassium hydrate. Decalcification.—Three per cent. of nitric acid in 70 per cent. alcohol is a good decalcifying reagent. The material should be left in the solution for several days. Chromic acid has a decalcifying action; a 1 per cent. to 2 per cent. solution DECOLORIZING—DESILICIFICATION 207 should be used, and the material should be left in this until decalcification is found to be complete. Decolorizing.—Material which has become brown in alco- hol may be decolorized in the following solution: To each 100 c.c. of alcohol is added from 0.2 to 0.5 c.c. of concentrated sulphuric acid and as much potassium chlorate as can be trans- ferred on the point of a knife. The material is to lie in this solution for eight or ten days, and is then to be transferred to alcohol or to equal parts of alcohol, glycerine and water for preservation. See also under Brown Discoloration and under Clearing. Dehydration.—This is best accomplished by cutting the material into as small pieces as is practicable, and then placing it in 20 per cent. alcohol, and then into ascending grades of alcohol of 10 per cent. increase at intervals of about two hours. Microtome sections mounted on the slide may be transferred. to strong alcohol without injury. In passing from water or aqueous stains to Canada balsam, the material should first come into strong alcohol, and then into xylol to insure complete dehydration, and to infiltrate the material with a solvent. of balsam—namely, xylol. Aniline is also a good dehydrating agent. The preparations may pass directly from water into the aniline and from the aniline into the balsam. A stick of potas- sium hydrate placed in the aniline will keep the latter dehy- drated. Potassium hydrate is not soluble in aniline. Very. thin microtome sections which are found not to be injured by drying may be allowed to dry, and then may be placed in xylene and thereafter transferred to balsam. See page 260 for further description of the process of dehydrating. Desilicification.—This is accomplished by hydrofluoric acid. A glass vessel is coated on the inside with melted paraffin to prevent the action of the acid on the glass. Alcohol is then poured into the vessel and the material is immersed in the alcohol; then the hydrofluoric acid is added, drop by drop. The process should be completed in a few minutes. Care must 2098 REAGENTS AND PROCESSES be taken not to breathe the fumes of the acid, since they attack the mucous membranes. Diastase.—This may be prepared as follows: Germinate barley in the incubator between pieces of blotting-paper until the plumule has reached a length of about 2 mm.; then dry the barley on the water-bath and grind to a fine powder. When a diastatic solution is desired, pour over 10 gm. of the powdered barley 1 liter of water containing 2 c.c. of chloroform; let stand for ten hours at about 15° C. and filter. The water filtered off will contain the diastase in solution. Add a little chloroform and preserve in a dark place. Starch grains may be mounted in this solution under a coverglass and kept from drying in a moist incubator, and the effect of the diastase on the starch may be studied from time to time under the microscope; or a I per cent. starch paste may be made to which about an equal amount of the diastatic solution may be added, and then at intervals samples from the mixture of starch and diastase. may be tested with a solution of iodine. The starch will, after a time, be changed into dextrines and grape-sugar and will no longer give a blue color when tested with a solution of iodine. Digestive Fluids.—To remove from sections aleurone grains which are so numerous as to obscure the nucleus, the sections should be treated for twenty-four hours with a digestive fluid prepared by mixing 1 part of pepsin-glycerine with 1 part of pancreatin-glycerine, and 20 parts of a 0.3 per cent. solution of hydrochloric acid. Differences in the character of the proto- plasmic cell-contents, and particularly in the dividing nucleus, may be demonstrated by treating sections of fixed material with a digestive fluid made by mixing 1 part of pepsin-glycerine with 3 parts of water acidified with 0.2 per cent. of chemically pure hydrochloric acid. Diphenylamine.—This is a test for nitrates in plant tissues. Five centigrams of diphenylamine are dissolved in 10 c.c. of pure sulphuric acid. The presence of nitrates is to be assumed when sections treated with this reagent take on a blue color. It EAU DE JAVELLE 299 seems, however, that in the presence of lignified tissues the reaction may fail, even when nitrates are present in abundance. Diphenylamine is also used to distinguish between crystals of asparagin and potassium nitrate. Asparagin dissolves without color in this reagent, while potassium nitrate assumes a deep blue color on dissolving in it. Eau de Javelle.—Prepared by adding to an aqueous solution of chloride of lime a solution of potassium oxalate so long as a precipitate is formed. The solution is then filtered and diluted somewhat with water before using. Or 20 parts of a 20 per cent. solution of calcium chloride is diluted with 100 parts of water, and after this has stood for some time, a solution of 15 parts of pure potassium carbonate in 100 parts of water is added. If a film should form on the surface of this on exposure to the air, a few drops of the solution of potassium carbonate should be added and the precipitate filtered away. ‘Lignin is extracted from sections of woody tissues which have lain in the eau de Javelle solution for some time, and there- after, on treating with chloroiodide of zinc, the membranes show only a cellulose reaction, staining only purple with the chloroiodide of zinc. Starch grains included in chloroplasts may be demonstrated by first treating sections, or even whole leaves, with eau de Javelle until the chloroplasts are dissolved (this may take from one to twenty-four hours), and then treating the material with asolution of potassium iodide-iodine. The starch grains will take on a blue or violet color. In some cases, however, the starch grains themselves are dissolved with the eau de Javelle. In such cases, and indeed in most cases, chloral hydrate and iodine is to be preferred for demonstrating starch inclusions in chloroplasts (see under this head). When the forms of the cells simply are to be studied, eau de Javelle is very useful in clearing the sections by dissolving the cell-contents. If the sections become too clear in the eau de Javelle, this defect may be corrected by treating the sections with alcohol or with a solution of alum. See under Cyanin for 300 REAGENTS AND PROCESSES use of eau de Javelle in differentiating cutinized and suberized membranes. Eosin.—An aqueous solution of eosin is an excellent stain for protoplasmic cell-contents and cellulose walls. The solution should be quite dilute. For the use of eosin in double staining see under Cyanin and Gram’s Method. See also in the next chapter under Aleurone Grains. Fehling’s Solution.—Prepare three separate solutions: (1) 17.5 gm. of copper sulphate in 500 c.c. of water; (2) 86.5 gm. of sodium-potassium tartrate in 500 c.c. water; (3) 60 gm. of caustic soda in 500 c.c. of water. To prepare for use, mix 1 volume of each of these with 2 volumes of water. The solutions keep well separately, but the mixture becomes changed after a time, and for this reason the solutions should not be mixed until needed. Sections may be treated with this solution on the glass slip. Two small drops of distilled water are placed on the slip with 1 small drop of each of the three solutions; then sections, not too thin, of the material which is to be tested for glucose are placed in the mixture on the slide. It is best to cut the sections without wetting the razor, and the sections should not be placed in water, but should be transferred directly to the mixture on the slide. The sections should be covered with a coverglass and the slide carefully heated over the flame of an‘alcohol lamp, or a very small flame from a Bunsen burner, until bubbles rise in the solution. If glucose is present, the sections will appear reddish from very small crystals of cuprous oxide which have been reduced from: the solution. If it is not desired to observe the crystals of cuprous oxide within the cells, but simply to demonstrate the presence of grape-sugar, small pieces of the tissues to be tested may be placed in a test-tube containing a few cubic centimeters of the solution, which is then heated to boiling; if grape-sugar is present in considerable quantity, a copious precipitate will after a time settle to the bottom of the tube. See under Copper Acetate. Thisis particularly suitable for demonstrating the presence of grape- sugar in those cells which contained it in the uninjured tissues. FERRIC CHLORIDE—FUCHSIN 301 Ferric Chloride.—An aqueous solution is used as a test for tannin. When sections containing tannin are placed in this solution on the slide, a color is produced which may vary from dark blue to green. Ferricyanide of Potassium.—Used in demonstrating the structure of pyrenoids. Alge containing pyrenoids are placed in a mixture of equal parts of a ro per cent. solution of ferri- cyanide of potassium and a 55 per cent. solution of acetic acid, and then treated as described under Dahlia. Fixatives and Fixation.—See page 258. Fischer’s Method of Demonstrating Cilia.—The following method is highly recommended for demonstrating cilia of cer- tain bacteria: An exceedingly small amount of the culture con- taining the bacteria is spread out as thinly as possible on the cover- glass. After the film has dried on the coverglass: the latter is passed through the flame of ‘an alcohol lamp or Bunsen burner (care being taken to avoid a too excessive heat), and then a few drops of-a mordant are put on the film on the coverglass. The mordant is prepared by dissolving 2 gm. of tannin in 20 c.c. of water. The coverglass is then passed back and forth over a small flame until vapor arises from the mordant. The mordant is now washed off by means ef water from a wash-bottle, and then one edge of the coverglass is held in contact with a piece of filter paper to draw away the surplus water. Next, a concen- trated aqueous solution of fuchsin is spread over the film on the coverglass, and the coverglass is held over a flame until the fuch- sin solution begins to boil; the coverglass is then washed off, and is allowed to dry. At any time thereafter the coverglass, with the film side down, may be cemented to the slide with balsam.. In successful preparations made by this method cilia, when pres- ent, will stand out quite sharply. Fuchsin.—Dissolve 1 gm. of fuchsin in 100 c.c. of absolute alcohol and roo c.c. of water. An excellent single stain. Espe- cially to be recommended for preparations that are to be photomicrographed. It stains different tissues different tints of red. 302 REAGENTS AND PROCESSES An excellent double stain with fuchsin and methyl blue is obtained as follows. Leave sections in the above fuchsin solution overnight or for several hours. Wash the sections thoroughly in water and rinse in 95 per cent. alcohol quickly and remove them quickly to water while the stain is still coming off in clouds and transfer them to a saturated solution of methyl blue diluted with an equal bulk of water. Leave the sections in this for a few minutes, then rinse them in water and again in 95 per cent. alcohol; transfer them to xylene and mount them in Canada balsam. The time ratios for the two stains will vary with different materials, and the ratios are to be changed as experience teaches. Fuchsin, Acid.—Excellent for staining crystalloids. The material containing the crystalloids should be fixed in a concen- trated alcoholic solution of corrosive sublimate. Then the sec- tions should be immersed for twenty-four hours in a 0.2 per cent. solution of acid fuchsin, to which a little camphor has been added. To demonstrate crystalloids in plastids the sections should be treated as follows: The sections are placed in a solution of 20 per cent. acid fuchsin in 100 gm. of aniline-water. This solution is heated somewhat while the sections remain in it from two to five minutes; they are then rinsed in a solution of 1 part of a concenrated solution of picric acid in alcohol and 2 parts of water. This solution should be warmed to about 40° C., and the sections should be rinsed in it until they cease giving off color to it. Thereafter they are dehydrated in strong alcohol, passed into xylene, and mounted in Canada balsam. Acid fuchsin is an excellent stain for leucoplasts and plas- tids in general. The material is fixed in a concentrated alco- holic solution of corrosive sublintate in absolute alcohol, where the material remains for twenty-four hours; then the fixative is washed out in alcohol containing iodine. Sections from this material are placed in a 0.2 per cent. solution of acid fuchsin in distilled water. After remaining twenty-four hours they are taken out, washed in running water for a time, and then are examined in glycerine or are allowed to dry, after which they GELATINE 303 are mounted in Canada balsam. The sections cannot be dehy- drated in alcohol, because this will extract the stain from the plastids. The following method may also be used: The mate- rial is fixed in a solution of 5 gm. of corrosive sublimate in 100 gm. of absolute alcohol, which is acidulated with 10 drops of hydrochloric acid. Then the fixative is removed by placing the material in pure alcohol, which is several times replaced. Sections from this material should be stained by immersion for about twenty minutes in a solution of 20 gm. of acid fuchsin in 200 c.c. of distilled water and 3 c.c. of aniline oil. They are then washed in a mixture of 50 c.c. of a saturated alcoholic solu- tion of picric acid and 100 c.c. of water until color ceases to be given off from the sections. ‘Then the picric acid is washed from the sections in pure alcohol. The sections are next placed in chloroform for ten minutes and are then ready to be mounted in Canada balsam. When desired, sections cut from fresh material may be fixed and stained as above. Or the material may be fixed and im- bedded, and after microtome sections have been cut-and mounted on the slide they may be stained as above directed. A beautiful double stain for nuclei is prepared from acid fuchsin and methyl blue as follows: The microtome sections mounted on the slide are immersed for half an hour in a 0.001 per cent. aqueous solution of acid fuchsin, then quickly washed in water, and immersed for about one minute in a 0.002 per cent. aqueous solution of methyl blue. The surplus stain is then washed off in alcohol and the preparation is allowed to dry; then the sections are immersed in olive oil from six to twenty- four hours, after which they are washed in absolute alcohol or in a mixture of absolute alcohol and xylol until the stains are quite clear, and the preparation is ready to be mounted in Canada balsam. Gelatine.—Motile swarm spores and the like are some- times mounted for observation in a solution of gelatine, which renders their movements less rapid, and in this way facilitates the study of these bodies. About 1 gm. of gelatine is dissolved 304 REAGENTS AND PROCESSES in roo c.c. of water; a drop of this is placed upon a slide which has been somewhat warmed, and then a drop of the fluid con- taining the motile bodies is added to the drop of gelatine solu- tion and mixed with it by stirring, after which the coverglassis put on. See also under Nutrient Media. Gentian Violet.—A 1 or 2 per cent. solution of acetic acid, to which gentian violet is added until the solution appears of a deep violet color, is effective in instantaneously fixing and staining the nuclei of fresh tissues. Anthers and sporangia need only to be teased out with a needle in this fluid or crushed under the coverglass, when the nuclei of the pollen grains and spores or the mother cells of these will be fixed and stained for immediate examination. See also page 264 and Gram’s Method. Glycerine.—This is frequently used as a mounting medium, but since objects are apt to become very transparent in it, only those sections which have been stained should be mounted in it. Sections, such as of wood, which are not apt to shrink easily may be mounted in glycerine directly from water, but delicate tissues should first go from water into a mixture of 10 parts of water and 1 part of glycerine; this should then be allowed to concentrate by the evaporation of the water, when the sections may be mounted on the slide in a drop of pure glycerine. The coverglass should be quite clean and the glycerine should not be allowed to run back over it. After putting on the cover- glass the surplus glycerine should be taken up with a bit of filter paper and the slide about the edge of the coverglass should be made quite clean with a cloth moistened in water and then wiped dry with a dry cloth; then the slide may be put in position on the turntable, where a ring of Brunswick black, or of shellac to each ounce of which 20 drops of castor oil have been added, may be spun around the edge of the coverglass. This process should be repeated several times, allowing each coat to harden before putting on the next, until a strong ring of the cement has been formed. When certain stains are used, such as hematoxylin, the glycerine must be entirely free from acids; but with other GLYCERINE-GELATINE 305 stains, such as the carmine stains, an acidulation with 1 per cent. of acetic acid is of advantage. Dilute glycerine, in which sufficient chrome-alum has been dissolved to give a clear blue color, is recommended as a mount- ing medium for the Schizophycee and Floridez, since the natural colors of these plants are retained in this medium. Sections containing mucilaginous membranes may. be mounted in a drop of pure glycerine in which the membranes will not swell, and then, by irrigating the mount with water the process of the slow swelling of the membrane may be observed. Glycerine-gelatine.—This is for most subjects a better mounting medium than glycerine alone. It is prepared as follows: One part by weight of the best gelatine is soaked for about 2 hours in 6 parts by weight of distilled water. Then 7 parts by weight of chemically pure glycerine are added, and finally, to each 100 gm. of this mixture 1 gm. of concentrated carbolic acid. ‘The mixture is warmed for about 15 minutes, and at the same time constantly stirred until it becomes clear; then, by means of a hot-water funnel, or while kept warm in an incubator, the mixture is filtered through glass-wool or filter paper which has been washed with distilled water after being placed in the funnel. To mount sections in glycerine-gelatine the glass slip is warmed and a small bit of the gelatine is placed upon it. If the slip is not warm enough to melt the gelatine, it should be passed back and forth above the flame of an alcohol lamp. If the sections are of a character not liable to shrink, they may be transferred directly from water to the melted gelatine; if, however, there is danger of shrinking, the sections should first be placed in a to per cent. solution of glycerine, which is then allowed to con- centrate by evaporation of the water, and then, from the concen- trated glycerine the sections may be transferred to the drop of melted glycerine-gelatine. To avoid air-bubbles the cover- glass should be put on with the precautions given on page 265 for putting on the coverglass when Cariada balsam is the mount- ing medium. If several sections are being mounted under 20 306 REAGENTS AND ‘PROCESSES one coverglass, and: these should come to lie over each other in putting on the coverglass, they may be properly arranged without attempting to remove the coverglass (which usually makes the matter worse) by heating the slide until the gelatine becomes quite soft, and then drawing a hair under the coverglass, with which the sections may be manipulated. It is sometimes a difficult matter to put just the right amount of the gelatine on the slip. To overcome this difficulty, heat the gelatine and pour it out in a thin film over a clean glass plate. When it has become cool, strip it from the glass; then cut off small squares of different size, melt them separately on glass slips, and cover’ with the coverglasses of the size to be used with subsequent preparations. The film of gelatine should then be cut into wafers of the size found to exactly fill out the space under the coverglass. These wafers should be kept from drying too much and free from dust in tightly-stoppered bottles. Glycerine Gum.—Dissolve 10 gm. of powdered gum arabic in 10 c.c. of water and add about 4o drops of glycerine. This is useful for imbedding hard seeds, pollen grains, etc., prepara- tory to sectioning them. The wood tissues of hard seeds may be softened as described on page 267. Put a drop of the glyc- erine gum on a suitable pine block and submerge the material in it, and leave the preparation in the air to dry. Sections may then be cut free-hand with a razor or on a microtome. Wash out the gum from the sections in water. Glycerine-iodine.—See under Iodine and Glycerine. Gram’s Method.—This method is specially recommended for staining bacteria, either in coverglass preparations or in -sections. The sections are stained in a mixture of 100 c.c. of aniline water (prepared by combining about 5 c.c. of aniline with 95 c.c. of distilled water), and 11 c.c. of a concentrated alcoholic solution of gentian violet or, better, methyl violet. This is filtered, and ro c.c. of absolute alcohol are added to it. The preparation is taken from the stain, rinsed in alcohol, and transferred to a solution of 2 parts of potassium iodide, and 1 part of iodine in 300 parts of distilled water, where it remains GUM ARABIC—HAMATOXYLIN, DELAFIELD’S 307 from 1 to 3 minutes. Then it is rinsed in alcohol, transferred to clove oil, and thence mounted in Canada balsam. A good double stain is obtained if the clove oil has some eosin dissolved in it. Gunther’s modification of the Gram method is as follows: The preparation is stained and passed through the potassium iodide-iodine solution as above. Then it is placed for 1 to 2 minutes in alcohol, next for 10 seconds in a 3 per cent. solution of hydrochloric acid in alcohol, then again for several minutes in pure alcohol, until no more color comes away, and then it is passed on into xylol, and finally is mounted in Canada balsam. Gum Arabic.—The study of the spermatozoids of ferns, etc., is facilitated by adding a 10 per cent. solution of gum arabic to the drop of water containing the spermatozoids, which are then unable to move so rapidly in the thicker fluid. - Hematein.—Dissolve with heat 1 gm. of hematein in 50 c.c. of go per cent. alcohol, and add to this a solution of 50 gm. of alum in 1 liter of distilled water. After cooling, filter if neces- sary, and add a crystal of thymol to prevent the growth of fungi. The solution is ready for use at once. Sections stained in this solution should be washed in water and transferred to glycerine- gelatine for mounting, or may be dehydrated and mounted in Canada balsam. The stain may be reduced in overstained sec- tions by allowing the preparation to stand for some time in a 1 per cent. solution of alum. A sediment is apt to settle from this solution, but this is not an indication that the stain is spoiled. The sediment can be partly prevented by adding to the solution about 2 per cent. of glacial acetic acid, which, on the whole, increases the effectiveness of the stain. The acid should be ‘entirely washed from the sections with water before permanent mounts are made. Hematoxylin, Delafield’s.—Prepared by mixing 4 c.c. of a saturated solution of hematoxylin crystals in absolute alco- hol with 150 c.c. of a saturated solution of crystals of ammo- nium alum in water. After standing for a week exposed to the light and air, this should be filtered and mixed with 22 c.c. 308 REAGENTS AND PROCESSES of glycerine and 25 c.c. of methyl alcohol. Before using this it should be allowed to stand until all precipitates have settled. Sections are transferred from water into the stain, where they remain for several minutes; they are then placed in a dish of 70 per cent. alcohol, acidulated with a drop of HCl, and after a minute they are rinsed in 95 per cent. alcohol, then in xylene, and mounted in balsam. Hematoxylin and Safranin.—Sections stained in safranin and washed in water may be placed for a few minutes’ in Dela- field’s hematoxylin, where they are treated as described under this stain. By this treatment lignified and suberized walls are stained red and cellulose walls violet. Hanging-drop Culture.—A hanging-drop culture is useful in the study of various microérganisms. Spin a ring of melted paraffin on the slide the size of the coverglass to be used. Wash the coverglass with soap and water, rinse it and rub it bright with alcohol on a clean cloth, and sterilize it by baking in an oven. Handle it thereafter with sterilized forceps. By means of a sterilized glass rod or pipette transfer a drop of nutrient solution (see under Nutrient Media) to the middle of the coverglass, and to this drop transfer with a sterilized needle a very minute por- tion of the culture or material to be studied. Invert the culture over the paraffin ring and press it down firmly so that a tight union is made. This is necessary to keep the drop from evap- orating. If the paraffin is too hard to make a close union, put a thin layer of vaseline over the ring before putting on the cover- glass. The drop should of course hang free in the cell and not touch the slide. Sometimes it is desirable to draw the drop out a little over the coverglass with a sterilized needle, or even to flatten it out entirely by placing over it a coverglass enough smaller than the first not to touch the paraffin ring. The dealers furnish hollow-ground slides that are excellent for hanging drop cultures. With these no paraffin ring is needed. Cultures prepared in.this way can be studied at any time, even with high powers, without disturbing them. Hardening Processes.—The hardening of tissues is accom- HYDROCHLORIC ACID—-HYDROGEN PEROXIDE 309 plished by the withdrawal of water from them. This is in most cases best accomplished by means of successively higher grades of alcohol, as described on page 260. A quick method of hardening fresh tissues, and at the same time preparing them for immediate sectioning, is to freeze them by the evaporation of ether or the expansion of liquid carbonic- acid gas. This process requires the use of special apparatus, for a description of which the student is referred to the catalogue of Bausch & Lomb, Rochester, N. Y. For an imbedding mass, either a drop of the white of egg or a thick solution of dextrin in a solution of carbolic acid 1 part, water 4o parts, may be placed about the object before freezing. If the dex- trine solution is used, it would be better to pump the air from the object while immersed in the solution; then place the object on the object-holder, pour a small amount of the solution about it, and freeze. This method will answer very well in some cases when it is desired to prepare a large number of sections quickly for class use, but it can by no means take the place of fixing the material in an appropriate fixative, hardening slowly in alcohol, and imbedding in paraffin or collodion. The mucilaginous layer of certain seed coats ‘may be har- dened with a 1o per cent. solution of neutral acetate of lead. The sections are cut from dry seeds, hardened in the lead acetate, and stained with methyl blue. They are then washed in water and mounted in a 2 per cent. solution of boracic acid. Hydrochloric Acid.—This reagent has such manifold appli- cation in histology that its uses are best learned in the specific cases of its application. See in the next chapter under Amy- lose, Berberin, Caffeine, Calcium Oxalate, Calcium Sulphate, Ethereal Oils, Magnesium Sulphate, Middle Lamella, Myro- sine, Pectic Substances, Phloroglucin. Theobromine, Vanillin. See also in this chapter under Maceration. Hydrogen Peroxide.—One part of hydrogen peroxide mixed with 20 parts of 60 per cent. alcohol will, in a few minutes, remove from sections the dark discoloration due to osmic acid which has been used as a fixative (see page 263). ! 310 REAGENTS AND PROCESSES India Ink.—The gelatinous sheath of the conjugate may be demonstrated by placing the alga under investigation in water in which India ink has been rubbed up until the water has a dark gray color. In this the gelatinous sheath becomes sharply demarked. Infiltration.—For infiltration with glycerine gum see page 306, with paraffin, page 261, with celloidin, page 266. The stony tissues of seeds, etc., which are too hard and brittle to ‘be sectioned with a knife, and must, therefore, be ground to the requisite thinness on a stone or by means of emery powder, may be protected against breaking during this process if fairly thin sections are first cut with a fine saw and then placed in a rather thin solution of Canada balsam or copal in chloro- form, which is then allowed to evaporate to the’ thickness of syrup; the sections are allowed to dry and are then cemented by means of a thick solution of gum arabic to a glass plate preparatory to grinding. Only a thin layer of gum arabic should be used, and this should be quite dry before the grind- ing is begun. The sections may now be ground thin on a clean, dry Arkansas or Wichita stone. Before the section has been brought to the desired thinness, the surface should be polished by rubbing it on a piece of soft leather which has been dressed with tripoli. The stone on which the sections are ground may be cleaned of the balsam from time to time by means of a cloth dipped in xylol or turpentine. When one side has been polished, the section may be freed from the glass plate by soaking in water, and then the polished side should be cemented to the glass plate and the reverse side ground and polished as before. The sec- tions should be examined from time to time with the microscope, so that the process of grinding may be stopped as soon as the desired transparency has been obtained. They may then be washed from the glass plate with water, and after drying should be stained with fuchsin and mounted in Canada balsam. Iodine.—The fumes from heated crystals of iodine serve well in many cases as a fixative. Small objects in drop cultures may be readily fixed by pouring over them the fumes arising IODINE AND ALCOHOL. 311 from iodine heated. in a test-tube. Algze may be fixed by placing a few crystals of iodine in the bottom of a test-tube, cautiously. inclining the tube slightly with the. mouth downward, then placing the algee in the test-tube near the mouth directly from the water in which they were growing, and thereafter heating the crystals so that the fumes from them pour down over the alge. The iodine may afterward be expelled by warming the fixed material to 30° or 40° C., and the material will then need no further washing out. Iodine has a wide application in plant histology and micro- chemistry. See under Aconitine, Atropine, Carotin, Cellulose, Colchicine, Gums, Gram’s Method, Lipochromes, Lignin, Nicotine, Proteids, Starch, Suberin. Iodine and Alcohol.—A good fixative for very small organ; isms is a solution of 3 parts of iodine in 100 parts of 70 per cent. alcohol. This at the same time permits the staining effect of iodine on the cell-wall and cell-contents. Iodine and Glycerine.—A mixture of potassium iodide- iodine with glycerine in equal parts gives good results when the action of iodine is to be observed. The glycerine keeps the preparation from drying and at the same time has a clear- ing effect. Iodine and Phosphoric Acid.—Used as a test for cellulose, which it colors violet. Prepared by dissolving with heat 0.5 gm. of potassium iodide and a few crystals of iodine in 25 c.c. of concentrated aqueous solution of phosphoric acid. Iodine and Potassium Iodide.—This: solution is prepared by dissolving 0.5 gm. of potassium iodide and 1 gm. of iodine in a small amount of water, and then diluting this with roo c.c. of water. The solution is left standing over any iodine which may crystallize out. This formula is recommended by Arthur Meyer in his work on “Starkekorner” as best adapted to the study of starch grains. A rough-and-ready method of preparing an iodine solution is to dissolve a small amount of potassium iodide in distilled water and then dissolve crystals of iodine in this until a brown color is obtained. This can be 312 REAGENTS AND PROCESSES diluted with water as is found necessary. A rather pale solution of iodine is sufficient to color starch blue. To stain modified cell-walls the solution needs to be stronger. Iodine Green.—See page 266 for the use of iodine green in double staining. A 2 per cent. solution of glacial acetic acid with iodine green dissolved in it serves well in the instant fixing and staining of the nuclei of fresh material. Iron Acetate.—Used in the detection of tannins, which see in the next chapter. Lactic Acid.—Dried alge and fungi may be prepared for study with the microscope by soaking them first in water and then in concentrated lactic acid, in which they are heated until small bubbles are formed; they may then be studied in the lactic acid. A 1o per cent. solution of lactic acid is recommended for fixing bacteria. This fixative is said not to interfere in any way with the subsequent processes of staining with alcoholic solutions of aniline dyes. Lead Acetate.—A 10 per cent. solution of neutral lead acetate is used to harden the mucilaginous layers of seed coats. For subsequent treatment see under Boracic Acid. Lithium Carbonate.—Useful in removing from material picric acid, which has been used as a fixative. A few drops of a cold, saturated, aqueous solution of lithium carbonate are added to the alcohol, used to wash out the fixative. Maceration.—In studying the forms of cells it is sometimes desirable and even necessary to isolate them by the process of maceration. Where cells with lignified walls are to be isolated Schultze’s maceration process is best employed. Put a small amount of concentrated nitric acid into a test-tube and add a small crystal of potassium chlorate. Heat this to boiling and drop into it sections containing the tissues under investi- gation. The sections will soon turn quite white. When this occurs, and before the sections have time to dissolve altogether, pour the contents of the test-tube into a large dish of water. Select a section and tease it out in a drop of water on a glass slide with dissecting needles, and examine the preparation METHYLENE-BLUE 313 under a coverglass.. Bast and wood fibers and stone cells can be isolated by this process, but thereafter they give the reaction for cellulose instead of for lignin. Another process, known as the Mangin process, gives good results with sections. Place the sections for forty-eight hours in a mixture of four volumes of alcohol and one volume of hydrochloric acid. Wash the sections in water and put them into io per cent. ammonia for fifteen minutes, then mount a section in a drop of water under a coverglass and press upon the coverglass until the cells are forced apart. Chromic acid also is used for maceration. Place sections in a concentrated solution for a minute or so, then rinse them in water, mount them in a drop of water under a coverglass and press upon the coverglass. If the cells do not then come apart they should be put for a longer time in the acid. Methyl-blue.—An aqueous solution is an excellent stain for cellulose membranes. It. may be used as a double stain with safranin as follows: Stain in safranin over night (see under Safranin) and then rinse in water and acidulated alcohol; place in a concentrated aqueous solution of methyl-blue for fifteen minutes, rinse in strong alcohol and xylene and mount in balsam. See also under Fuchsin. Methylene-blue.—A good nuclear stain. For cells filled with protein granules it is particularly good in differentiating the nucleus. Methylene-blue is useful in differentiating pectin compounds. The protoplast and lignified walls are stained a bright blue, while pectin compounds are stained a violet blue. Cells containing tannin will accumulate methylene-blue from very dilute solutions. The sections of living tissues are placed in a solution of 1 part of the stain in 500,000 parts of filtered rain-water. The cells containing tannin soon take on a distinct blue color, and, later, a deep blue precipitate is formed in them. The gelatinous sheaths of live conjugate may be stained by dilute aqueous solutions of methylene-blue without injury to the living organism. A o.oo1 per cent. solution of methylene- blue in water will stain the living nuclei of diatoms and other 314 REAGENTS AND PROCESSES simple organisms. The central body of the Cyanophycee may be stained by the above dilute solution if, after twenty-four hours’ treatment, the stain is strengthened to a o.1 per cent. solution. Methylene-blue and carmine form a good differential stain for bacteria occurring in sections of tissues. Methylene-blue and Carbol-fuchsin.—This double stain- ing method is used in the differentiation of Bacillus tubercu- losis. The material first coughed up from the lungs by the patient on waking in the morning should be expectorated into a wide-mouthed bottle or covered jar. The person who is to make the examination should afterward pour this out into a shallow glass dish. This should be placed on a dead-black background, and one of the small, yellowish, lenticular bodies which usually occur in tuberculous sputum should be removed and placed on a coverglass. A second coverglass should be placed over this; then press the coverglasses gently between the thumb and forefinger, and rub to and fro until the material is spread out in a thin film on the coverglasses. Then slide the coverglasses apart, and allow them to dry in the open air. When dry, hold them with a pair of forceps and pass them three times through the flame of the Bunsen burner or alcohol lamp. (The film should not be allowed to turn brown, else the preparation will be ruined.) Next pour over them carbol- fuchsin prepared by rubbing 1 gm. of fuchsin with roo c.c. of a 5 per cent. aqueous solution of carbolic acid, with the gradual addition of 10 c.c. of alcohol. Hold the coverglasses over a flame with forceps until vapor begins to arise from the surface of the stain. Then hold away from the flame, except in inter- vals of gentle heating, by which they are kept warm for a minute or two. They are next washed in water and decolorized by being moved about in a 25 per cent. solution of nitric or sulphuric acid. When the previously deep red color has changed to a * greenish tint, the preparation is washed in 60 per cent. alcohol to remove the color set free by the acid. If any red color still remains, the preparation should be rinsed in water and again treated with the acid-bath. By the above process the fuchsin METHYL-VIOLET—-NUTRIENT MEDIA 315 has been removed from everything but the tubercle bacilli. The double staining is accomplished by now pouring over the preparation a mixture of 3 parts of water with 1 part of a concen- trated alcoholic solution of methylene-blue. After a few minutes the methylene-blue is washed off with water, and the prepara- tion is allowed to dry; when dry, it may be mounted in Canada balsam. Other bacteria than the tubercle bacilli are decolor- ized by the acid-bath, and are subsequently stained blue by the methylene-blue. Methyl-violet.—Starch grains may be stained by treatment with a dark aqueous solution of methyl-violet. If the starch grains after staining are treated with a very dilute solution of calcium nitrate, the stain becomes deposited particularly in the less dense layers of the grains. Useful as a stain for elaioplasts. See under this head in the next chapter. See also in this chapter under Staining Intra Vitam. Millon’s Reagent.—This should be prepared fresh, as needed, by dissolving mercury in an equal weight of nitric acid and then diluting this solution with an equal weight of distilled water. Proteids are colored a brick-red with this ° reagent. Sections to be tested are to be mounted in a drop of the reagent on a glass. slip. Warming the slip hastens the reaction. Nutrient Media.—Nutrient media must be sterilized by heat to keep them from spoiling and to make it possible to grow in them pure cultures—that is, cultures of organisms of any desired sirigle species. Sterilization may be accomplished by steaming the medium for about twenty minutes each day on three days in succession, after having poured it into test- tubes or flasks which have previously been tightly plugged with cotton rolled into the form of a stopper of the proper size and baked in an oven until the cotton is slightly scorched. The tubes and cotton plugs should be baked together. Or, if an autoclav is available in which steam can be generated under pressure, and accordingly at a higher temperature than that of boiling water at ordinary atmospheric pressure, the cotton 316 REAGENTS AND PROCESSES plugs and tubes, or flasks, will not need to be baked, but may be sterilized, together with the nutrient medium already poured into them, by subjecting them for fifteen minutes to a tempera- ture of 115° C. in the autoclav. At this temperature a single sterilization suffices. A good artificial nutrient medium for yeasts is made by adding 0.05 per cent. of tartaric acid to a 10 per cent. solution of cane- sugar. A filtered aqueous extract of malted barley also gives good results. To prepare this, barley is germinated until the plumule just begins to protrude; the barley is then dried and ground up, and water is poured over it until there is about twice as much water by volume as of the powdered malt. The water should stand over the malt, with occasional stirring, for about an hour, when it may be filtered off and sterilized. Steril- ized grape juice is also ari excellent nutrient medium for yeasts. Cultures of yeasts grown in the above media may be made to produce spores in about twenty-four hours if some of the culture is transferred to the surface of sterilized bits of flower- pot which are half submerged in water and kept covered by a bell-jar. Cohn’s normal solution for the culture of bacteria is pre- pared as follows: Dissolve in 200 gm. of distilled water 1 gm. of acid potassium phosphate, 1 gm. of magnesium sulphate, 2 gm. of neutral ammonium tartrate, and o.r gm. of calcium chloride. An infusion of meat for the culture of bacteria is prepared by covering finely chopped lean beef with water and allowing it to stand for twenty-four hours in an ice-chest, after which it is to be filtered through a muslin bag, using pressure of the hands to make the filtration more complete. The filtrate is then cooked and again filtered, and neutralized by the gradual addition of a solution of carbonate of soda. The solution should be tested with litmus paper, and the addition of carbonate of soda should cease as soon as neutralization is accomplished. To this solution is added 0.5 per cent. of common salt. Ten grams of peptone may be added to a liter of the infusion. NUTRIENT MEDIA 317 In place of the meat infusion as prepared above, meat extract may be used in the ratio of 4 to 5 gm. per liter of water. Bouillon is prepared by adding 1 liter of water to 1 pound of chopped lean beef. This is cooked for half an hour, then filtered and neutralized with carbonate of soda, then again boiled for an hour to precipitate albuminoids. After a final filtering the bouillon is poured into flasks or test-tubes and sterilized. Infusions of hay and dried fruits may also be used for nutrient media. A hay infusion for the growth of Bacillus subtilis may be prepared as follows: Chopped hay is placed in a beaker and barely covered with well-water; this is kept in an incubator at a temperature of 36° C. for four hours, after which time the extract is poured off and diluted, if necessary, to a specific gravity of about 1.004. The extract is now poured into a flask which, having been closed with a cotton plug, is placed in a steam sterilizer and subjected to a gentle evolution of steam for about an hour. The flask is then placed in an incubator at 36° C. for a day or two, after which time a film produced by colonies of Bacillus subtilis will have formed over the surface of the extract. The spores of this bacterium are particularly resistant to heat, and for this reason while the spores of other bacteria are killed by the process of steaming, those of Bacillus subtilis still retain their vitality. Solid culture media may be prepared by adding to any of the fluid culture media a sufficient amount of a gelatinous substance to keep the mixture from liquefying at the tempera- ture of the laboratory, or, if desired, at the higher temperature of an incubator. One of the most used of the solid media is prepared by adding to the peptonized infusion of meat, as above described, 10 per cent. of the best French gelatine. The gelatine may be increased up to twice this amount, as the tem- perature may require. One hundred grams of gelatine is allowed to soak in x liter of the meat infusion until the gelatine becomes swollen, and then a gentle heat is applied until the gelatine is completely dissolved. After the gelatine is dissolved the solu- 318 REAGENTS AND PROCESSES tion should again be neutralized, if necessary, with carbonate of soda. When the solution stands at a temperature of about 50° C., an egg stirred up in roo gm. of water is added while the mixture is stirred with a glass rod. The mixture is then kept at the boiling-point for about ten minutes. This coagulates the egg-albumen and clarifies the liquid. The clarified liquid is now filtered by means of a hot-water funnel or while kept warm in an incubator, the high temperature being necessary for the reason that the mixture would become stiff at a low temperature, and so incapable of being filtered. The medium should be distributed while warm in sterilized test-tubes or flasks, which are then stoppered with baked cotton plugs. It should then be subjected to a temperature of 100° in the steam sterilizer for ten minutes at four successive intervals of twenty-four hours. For the reason that gelatine loses its power of solidifying at ordinary temperatures after being subjected to the temperature of boiling water for a long period, the time of each sterilization is necessarily reduced to about ten minutes and the number of sterilizations is increased to four; whereas, with other solidifying substances, such as agar-agar, the length of each sterilization may extend to one hour, and the number of sterilizations need be only two or three. In pouring the filtered medium into the test-tubes care should be taken not to get any of the medium on the upper portion of the tube where the cotton plug would be likely to come in contact with it, else the plug would later be difficult of removal. A solid nutrient medium which will remain solid at a higher temperature than the gelatine medium may be prepared from agar-agar, a substance obtained from certain gelatinous alge, as follows: Two gm. of the agar are broken into small pieces and soaked in cold water for twenty-four hours. Then the water is poured off and the swollen agar is added to 1 liter of the peptonized meat infusion. The mixture is boiled for several hours until the agar is completely dissolved. The solu- tion is then neutralized with a solution of carbonate of soda, filtered, distributed in flasks or test-tubes, and sterilized by NUTRIENT MEDIA 319, steaming for 1 hour at two or three successive intervals of twenty- four hours. , Cooked potatoes afford a solid nutrient medium which is quickly prepared and which is particularly adapted for the culture of chromogenic bacteria. Potatoes free from wounds are selected and scrubbed in water until they are perfectly clean, and the eyes and any unsound spots, if these could not be avoided, are cut out with a knife. Then the potatoes are placed for an hour in a solution of 1 part of mercuric chloride in 500 parts of water to disinfect the surface. They are next steamed for about an hour in a steam sterilizer, and after twenty- four hours the steaming is repeated for about half an hour. The sterilized potatoes are then placed in glass Petri dishes, are cut in halves with a sterilized table-knife, and the cut sur- faces are inoculated. If the source of the inoculation is not a pure culture, an isolation of forms may be approximated by making long scratches over the surface of the potato with a sterilized platinum needle which has been in contact with the source of the inoculation. It will add to the security of the process of sterilization if each potato, before being placed in the bath of mercuric chloride, is wrapped in a piece of tissue _paper, and so protected until it is cut open for inoculation. Another method of preparing potatoes which is, on the whole, more convenient and certain, is to cut out long cylindrical plugs from sound potatoes by means of a cork-borer or any metal tube of the proper size, and then to cut the potato cylin- ders very obliquely in two pieces, each of which is then to be placed in the bottom of a test-tube so that the oblique surface stands uppermost. After plugging the tubes with baked cotton, the potato cylinders are subjected to a temperature of 100° C. in the steam sterilizer for one hour at three successive intervals of twenty-four hours. A sterilized paste made from potatoes or bread serves well for the culture of molds as well as of bacteria. A decoction of horse-dung furnishes a good medium for the culture of Mucor and various other molds. The decoction is prepared by boiling the dung in water, then filtering and sterilizing 320 REAGENTS AND PROCESSES the solution. By placing the dung of different kinds of animals in a moist chamber, as, for instance, in dishes floating on water and covered with a bell-jar, characteristic fungi will after a time appear on it. Single spore cultures of Mucor may be obtained in the following. manner: Glass slides are thoroughly cleaned and sterilized by baking. By means of sterilized forceps a single sporangium of Mucor is picked from a spontaneous growth of this fungus on horse-dung or stale bread kept in a moist chamber. The sporan- gium is placed in a sterilized decoction of horse-dung con- tained in a sterilized watch-glass, which may be placed on an inverted tumbler in a plate of water and then covered with a bell- jar which should dip into the water and form a germ-proof moist chamber. After a few hours the sporangium will have burst open and the spores, which are now distributed through the decoc- tion, will have swollen to several times their original diameter, and can all the more readily be discerned in subsequent manipu- lations. A needle which has been disinfected by heating in a flame is now dipped into the decoction and the point of it drawn along the surface of a glass slide which has been cleaned and sterilized as above directed. By this process the decoction which has adhered to the needle is drawn out in the form of a narrow streak, and if several spores of Mucor are present, they will be separated from each other. A single spore may be located with a medium power of the compound microscope, and all other spores present in the streak may be wiped off with a cloth which has been sterilized by heat. Then a drop of the decoction of sterilized horse-dung should be added to the small amount con- taining the spore on the slide. The slide should be placed in a moist chamber where the spore will soon give rise to a mycelium visible to the naked eye, and from the mycelium numerous spo- rangia will be produced after a time. The slide may be taken from the moist chamber from time to time and the stages in the development of the fungus examined, but as much care as possi- ble should be taken to prevent the contamination of the culture. Knop’s nutrient solution, which is particularly good for the _NUTRIENT MEDIA 321 culture of alge, consists of 4 parts of calcium nitrate, 1 part of magnesium sulphate, 1 part of potassium nitrate, 1 part of potas- sium phosphate. These should be dissolved in sufficient water to make a o.2 per cent. solution of the combined salts. The potassium salts should first be dissolved, then the magnesium salt, and last the salt of calcium should be added after having been-dissolved by itself. By this procedure only a small amount of insoluble calcium phosphate is formed. The zoospores of Vaucheria may be induced to form at almost any time by transferring this alga from the above solution, in which it has been growing exposed to a bright light, to pure water; or cultures in a 0.1 per cent. or 0.2 per cent. nutrient solution which have been exposed to the light need only be placed in a dark place in order to incite the production of zoéspores. A 2 per cent. to 4 per cent. solution of cane-sugar may be used as a nutrient medium for alge. Filaments of Spirogyra may be made to conjugate by transferring them from the water in which they have been growing to a solution of cane-sugar as above, which is then placed in a well-lighted place. The formation of zodspores may be incited in @dogonium by transferring filaments of the alga from water at a low temperature (say at the temperature of the early morning) to a 2 per cent. or 3 per cent. solution of cane-sugar which is kept at a constant tem- perature of about 26° C. Convenient flasks for the preservation of Getilized fluid nutrient media may be made from glass tubing as follows: A piece of glass tubing o.2 inch in diameter, or larger, is held with its lower end in the flame of a blow-pipe, the tube being constantly revolved about its long axis to insure an even heating of the end. of the tube until the end of the tube becomes soft and just begins to draw downward in the form of a large drop. By this time the mouth of the tube has become closed. Then quickly the tube is removed from the flame, and while the melted end of the tube is still held downward, air is blown;in at the upper end of the tube by means of the mouth, so that the molten glass at the lower end of the tube is forced outward in the form of a rounded flask. 2r 322 REAGENTS AND PROCESSES After cooling so that it may be handled, the tube is held in the flame close to the bulb, and by constant turning the tube is heated equally on all sides until it becomes so soft that it may be drawn out. This process is accomplished by taking the tube from the flame and pulling on it gently so that it may be drawn out quite long and narrow. The length of the stem of the bulb should be equal to the depth of the vessel from which the nutrient medium is to be drawn into the bulb. The stem may be severed from the tube by holding it in the flame of the blow-pipe at the proper dis- tance from the bulb, where it will soon become soft enough to be pulled off from the main tube. Then the end of the capillary neck is held in the flame until a bead is formed; in this way the flask is hermetically sealed. To fill the flask with nutrient fluid the neck is sterilized near the end by passing it through a flame, and the head is broken off with sterilized forceps. The bulb is then heated in the flame of an alcohol lamp or Bunsen burner to expand the air. The end of the neck is next quickly dipped into the nutrient fluid, which is forced up the neck into the bulb as the air in this cools. When the bulb is two-thirds full, the neck is withdrawn from the fluid and hermetically sealed in a flame. In filling the bulb the greatest care must be taken to keep the stock of nutrient medium from any source of contamination, if it has once been sterilized. Chemical flasks with narrow necks serve well for a common receptacle. These should be kept stoppered with a cotton plug, and to fill the small flasks the plugs need only to be lifted slightly while the sterilized capillary neck of the small flask is thrust past the plug into the nutrient fluid. If the nutrient fluid is freshly prepared, and has not yet been sterilized, the small flask may be filled, sealed up in the flame, and sterilized in the steam sterilizer or in a vessel of boiling water for an hour each day on three successive days. The nutrient fluid will keep indefinitely in the little flasks, and when a drop is wanted for a drop culture, it is only necessary to sterilize the end of the capil- lary neck in a flame, break off the bead with sterilized forceps, invert the flask, and place the palm of the hand over the bulb. The heat of the hand will expand the air over the fluid and force OSMIC ACID 323 the latter down the neck. With a little practice just the desired amount of fluid can be forced out by the heat of the hand. The hand must not be placed on the bulb until the flask is inverted. If it is desired to make cultures within the little flasks, snip off the end of the capillary neck as before, and thrust a long platinum needle, the end of which has been in contact with the source of inoculation, down the neck into the fluid. Then withdraw the needle and hermetically seal the neck in the flame. When cul- tures are to be made in the flasks, these should be only one-third filled by the nutrient medium; there will then be sufficient air in the flasks for the success of the culture after the flasks have been inoculated and hermetically sealed. Pollen grains may be made to germinate in hanging-drops com- posed of 100 parts of well-water, 3 to 30 parts of cane-sugar, and 1.5 parts of gelatine. This should be made as needed, or it may be sterilized and kept indefinitely in the little flasks just described. The amount of cane-sugar to give the best results varies with the species of pollen, and can only be determined by experiment, but 3 parts will probably answer for most pollen grains. Spores of ferns may be made to germinate on pieces of flower- pot which are kept half submerged in water and are covered by a bell-jar. They should be set before a north window. They should never be exposed to the direct light of the sun, since in such a position the temperature under the bell-jar would become very great. Osmic Acid.—The method of preparing a solution of osmic acid and of its use in Flemming’s fixative is given on page 259. The vapor of osmic acid may be used as a fixative for very small organisms. In order to accomplish this a drop of water con- taining the organisms need only to be inverted over a bottle con- tdining a 2 per cent. solution of the acid. Osmic acid colors ethereal and fatty oils from brown to black, but other organic substances are also darkened by it; and as a test for oils it is not absolutely reliable. Aleurone grains in sections of Ricinus which have been freed from their oil by standing for a time in strong alcohol may be stained brown, and the crystalloid and ground 324 REAGENTS AND PROCESSES substance differentiated by immersing the sections for a short time in a 1 per cent. solution of osmic acid. Paraffin.—The directions for imbedding material in paraffin are given on page 261. Paraffin of about 52° C. melting-point sections to good advantage at a temperature. between 21° and 24° C., or 70° and 75° F. Good cells for hanging-drop cultures may be made by placing glass slides on the turn-table and spin- ning rings on them by means of a camel’s-hair brush dipped in melted paraffin. Pepsin.—One part of pepsin-glycerine and 3 parts of water acidulated with o.2 per cent. of chemically pure hydrochloric acid. When sections containing protoplasts are subjected to this reagent at blood temperature, certain structures of the pro- toplast which are insoluble in the reagent may be isolated from those which are soluble. In the dividing nucleus the kinoplasmic spindle-fibers persist after the chromosomes and nuclear plate have been dissolved by this reagent. By the action of digestive ferments on aleurone grains the ground substance is first dissolved and then the crystalloid more slowly, while the limiting mem- brane of the vacuole occupied by the aleurone grain persists. Digestive ferments are thus found to be excellent reagents for demonstrating the difference in constitution of the finer structures of the protoplast and protoplasmic cell-contents. Phloroglucin.—This furnishes one of the most reliable tests for lignin. Sections are placed in alcohol containing a trace of phloroglucin, transferred to a drop of water.on a slide and covered with a coverglass. A drop of hydrochloric acid is then applied to the edge of the coverglass and, as the acid comes.in contact with the lignified members, these are colored a bright violet-red. Phospho-molybdic Acid.—This is used as a test for pro- teids. Sections are treated for an hour or two with a solution of 1 gm. of sodium-molybdenum phosphate in 90 gm. of distilled water and 5 gm. of concentrated nitric acid. Proteid materials then take on the appearance of yellow granules. Picric Acid.—The structures of aleurone grains are well differentiated by fixing in a concentratéd alcoholic solution: of PICRO-ANILINE BLUE—PICRO-NIGROSIN 325 picric acid and subsequent staining with eosin. The sections are to remain in the alcoholic fixative for several hours. They are then to be washed out in alcohol and stained for a few min- utes in a solution of eosin in absolute alcohol. Then the sec- tions are successively washed in absolute alcohol, transferred to oil of cloves, and mounted in Canada balsam. The ground substance is dark red, the crystalloid yellow, while the globoid remains colorless. The pyrenoids and chloroplasts of alge may be simultaneously fixed and stained by placing the alge for an hour or longer in a concentrated solution of picric acid in 50 per cent. alcohol, to which has been added about 5 drops of a solu- tion of 20 gm. of acid fuchsin in 100 c.c. of aniline water. The aniline water is prepared by shaking up 3.5 gm. of aniline in 96.5 gm. of water. The alge are then washed in alcohol, trans- ferred to xylol, then to a thin solution of balsam in xylol, and are finally mounted in the thicker solution of Canada balsam in xylol. Alcohol is a better solvent of picric acid than water, and accord- ingly it gives quicker results in washing out the acid from the fixed material than water does, but running water may be used to wash out the fixative whether the latter has been dissolved in alcohol or in water. Picro-aniline Blue.—A double stain, which is very rapid in its action, is prepared by adding aniline blue to a saturated solu- tion of picric acid in 50 per cent. alcohol until the solution has a blue-green color. By this treatment the unmodified cell-walls and the cell-contents are stained blue, while the lignified walls are stained by the picric acid. Picro-nigrosin.—A solution of nigrosin in a concentrated solution of picric acid in water or 50 per cent. or 95 per cent. alcohol is a good fixative and stain for algee and leucoplasts, and for double-staining modified and unmodified cell-walls. The solution may, in some cases, need to act for twenty-four hours. The strong alcoholic solution is particularly recommended for material containing chlorophyll, since this will be extracted by the strong alcohol. Nuclei and leucoplasts are stained a steel blue by the nigrosin. 326 REAGENTS AND PROCESSES Potassium Alcohol.—Used for bleaching sections. It may be prepared by mixing a concentrated aqueous solution of potas- ‘sium hydrate with go per cent. alcohol until a sediment is formed. This is allowed to stand for twenty-four hours with frequent violent shaking, and then the clear liquid is poured off and is diluted for use with 2 or 3 parts of water. Potassium Hydrate.—For general use, dissolve 5 gm. of potassium hydrate in 95 c.c. of distilled water. This is used as a clearing agent for sections and small organisms. The process of clearing may require from several hours to several days. After clearing, the potash should be washed out in plenty of water, and then the preparation may be neutralized with acetic acid. This will tend to make the objects more opaque, and if too much is added, the objects may be cleared again by caustic potash or ammonia. A dilute solution of caustic potash, as above, may be used for the maceration of cork, while delicate tissues in gen- eral may be macerated by boiling for a few minutes in a so per cent. solution of potassium hydrate in water; the tissues should then be washed in water and teased out on a slide in a drop of water. Ruthenium Red.—An aqueous solution is an excellent stain for pectic substances and for gums and slimes which have been derived from these. Ruthenium red is not soluble in alcohol, clove oil, or glycerine, and, therefore, preparations stained by it may be dehydrated and mounted in glycerine or balsam. Safranin.—A saturated solution of safranin in alcohol should be made, and this should be diluted with an equal bulk of water, or with an equal bulk of a saturated aqueous solution of saf- ranin. This is an excellent general stain, and gives good differ- entiating effects when used singly. It is one of the few stains which are particularly adapted to the staining of pectic com- pounds. It also gives beautiful results in staining the cell- contents of Spirogyra and other alge. The alge, after fixing in a fixative containing chromic acid, should lie in the alcoholic solution diluted with an equal bulk of water for twelve or twenty- four hours. They should be transferred to 50 per cent. alcohol, SALICYLATE OF SODA—-SILVER NITRATE 327 to which strong alcohol is then added, drop by drop. The color will begin to be extracted in the alcohol, and when the right inten- sity has been reached, the material should be transferred to dilute glycerine, where it is to remain while the glycerine slowly concen- trates in a place protected against dust. ‘Then permanent mounts may be made in glycerine or glycerine-jelly. The stain given by safranin is quite permanent. See also page 264 and the direc- tions there given for the three-color method. Salicylate of Soda.—A clearing reagent which for small objects is not inferior to chloral hydrate is furnished by dis- solving crystals of salicylate of soda in an equal weight of dis- tilled water. With tincture of iodine added this reagent will cause starch to swell, at the same time imparting a blue color to it. Salt.—A 4 per cent. or stronger solution of common salt, or of potassium nitrate, may be used to cause plasmolysis in living cells. This process may be all the more clearly seen by adding eosin to the salt solution. Shellac.—A thick solution of shellac in alcohol, to each ounce ‘of which are added 20 drops of castor oil, makes an excellent sealing medium for preparations mounted in glycerine or glycerine- jelly, or in an aqueous medium. Silver Nitrate.—A solution of silver nitrate is used to bring out more clearly the striations in bast fibers and starch grains. Sections containing striated bast fibers are allowed to dry and are then impregnated with the silver salt. Without previous washing the sections are transferred to a 0.75 per cent. solu- tion of common salt. They are then placed in distilled water and exposed to the light for a considerable time; thereafter they are allowed to dry and may be examined to good advantage in anise oil. Dry starch grains are put to soak in a 5 per cent. solution of silver nitrate. After a time they are allowed to dry super- ficially and are then treated with a 0.75 per cent. solution of common salt, in which they are finally exposed to the direct light of the sun to reduce the chloride of silver which has been formed within the grains. The less dense lamine of the starch 328 REAGENTS AND PROCESSES grains will show a gray color, due to the reduced silver. See page 177 for a description of the structure of starch grains. Staining Intra Vitam.—Living protoplasts may accumu- late certain stains from very dilute solutions without injury to themselves. Dahlia, methyl-violet, mauvein, and methylene- blue are particularly suitable for this purpose. Solutions con- taining o.oo1 per cent. or 0.002 per cent. of any of the first three stains have given good results in staining living nuclei, while I part of methylene-blue in 500,000 parts of filtered rain-water is used for staining living cells containing tannin. A large amount of these very dilute solutions should be employed in order that a sufficient amount of coloring matter may be at hand for accumula- tion by the living cells. Living protoplasts have the power of reducing and accumulating metallic silver from solutions of certain of the salts of silver, while dead protoplasts have not this power. The simplest method of producing this reaction is to place a few filaments of Spirogyra in a liter of a mixture of 1 part of silver nitrate in 100,000 parts of water with 5 c.c. of lime-water. The experiment will be completed in about half an hour if the temperature is approximately 30° C. Tannin and Antimonium-potassium Tartrate.—These are used successively as a mordant for methyl- and gentian- violet, fuchsin, and safranin when sections stained with these are to be mounted in glycerine. The sections before staining are placed in a 20 per cent. solution of powdered tannin in cold water. After washing well in distilled water, they are placed for twenty-four hours in a 2 per cent. solution of antimonium- potassium tartrate. After washing again in distilled water, they are transferred to the stain. From the stain the sections are washed quickly in distilled water and placed in strong alcohol, where the coldr is washed out until the desired degree of intensity is reached. They are now ready for mounting in- glycerine, or, if desired, they may be placed in xylol and then mounted in balsam. If the sections are so deeply stained that they cannot be suffi- ciently: washed out in alcohol, they should be placed for a time in a 2.5 per cent. solution of tannin. TURPENTINE-—-XYLENE 329 Turpentine.—This may be used to dissolve paraffin from sections which have been cut from material imbedded in paraf- fin. See also under Carbolic Acid. Venetian Turpentine.—To prepare a mounting medium _from Venetian turpentine, the productas it comes from the apothe- cary is diluted with an equal volume of strong alcohol, and after the mixture has become clear by long standing or by filtering after being well shaken, it is thickened somewhat on the water- bath. Objects may be’ mounted directly from strong’ alcohol into Venetian turpentine as above prepared. Objects which are found to shrink by this treatment may be transferred from strong alcohol to a mixture of ro parts of the turpentine with too parts of alcohol. The alcohol is then to be withdrawn from this mixture by placing the latter, together with a dish of calcium chloride, under a bell-jar. In order to keep the mixture of tur- pentine and alcohol from mounting the sides of the vessel which contains it, the rim of the vessel should be coated over with hot paraffin. The turpentine hardens quite slowly, and in order to quickly fasten a coverglass to the slide when the turpentine is being used for a permanent mount, a wire which has been heated in a flame should be quickly drawn around the edge of the coverglass. Xylene.—This is used as a solvent for paraffin, either in re- moving paraffin from sections or in preparing a dilute solution of paraffin to be used in the gradual infiltration of tissues with this substance. Used also as a solvent of Canada balsam. Xylol is the trade name for xylene CHAPTER XVII MICROCHEMISTRY OF PLANT PRODUCTS Aconitine, C,,H,,NO,,.—An alkaloid occurring in especial abundance in the rootstocks of Aconitum Napellus. To demon- strate aconitine treat sections with potassium iodide-iodine, or with a solution of potassium permanganate. The first re- agent produces a carmine-red coloration in the presence of aconitine and the second gives a red precipitate of aconitin permanganate. Aleurone.—See page 181 for a description of the nature of aleurone grains. The protein nature of aleurone is dem- onstrated by its dissolving with a red color in Millon’s reagent and by its being colored yellow or brown with iodine reagents. Aleurone grains should be studied in a mixture of equal parts of castor oil and 95 per cent. alcohol slightly colored with eosin. In water they are in danger of going more or less into solution. Permanent preparations of the aleurone of Ricinus may be made by placing small bits of the endosperm in a saturated alcoholic solution of picric acid, rinsing in alcohol, imbedding in paraffin (see page 261), sectioning on the microtome (see page 262), staining in an alcoholic solution of eosin, rinsing in oil of cloves, and then in xylene, and mounting in balsam. By this process the ground substance should be red, the crystalloid yellow, and the globoids colorless. For reaction of aleurone to other reagents see in the last chapter under Borax-Carmine, Digestive Fluids, Pepsin. Alkaloids.—Sections to be tested for alkaloids should be thick enough to leave one cell layer intact. In order to make the determination of the alkaloid more certain, sections for con- trol should be soaked for a day or so in a solvent of alkaloids prepared by dissolving 1 part of tartaric acid in 20 parts of alcohol, 33° ALLYL SULPHIDE—AMYLODEXTRINE 331 and rinsing in water for a day to wash out the acid. Mount sections thus treated under a coverglass with untreated sections and apply reagents for detecting alkaloids. The following reagents give with alkaloids amorphous or crystalline precipitates: potassium iodide-iodine, potassium bismuthiodide, chloroiodide of zinc, potassium-mercuriciodide, chloride of gold, ammonium- molybdate, potassium permanganate. See under Aconitine, Atropine, Berberin, Brucine. Caffeine, Corydalin, Curarin, Cytisin, Morphine, Narceine, Narcotine, Nicotine, Piperine, Sinapine, Strychnine, Theobromine, Veratrine. Allyl Sulphide or Garlic Oil, (C,H,),S.—This may be demonstrated by treating sections of species of Allium with palladous nitrate which produces a kermes-brown precipitate; or sections may be treated with a solution of silver nitrate, when sulphide of silver will be formed. Amygdalin, C,,H,,NO,,—This nitrogenous glucoside is particularly abundant in bitter almonds and in the bark, leaves, and flowers of Prunus padus. It can be extracted in boiling water, and on addition of alcohol it crystallizes out in the form of pearly scales. It is split into prussic acid, oil of bitter almonds and sugar by the enzyme emulsin which occurs associated with the glucoside. Amylodextrine.—This carbohydrate occurs in those starch grains which take on a reddish color with iodine, and it is formed by the action of diastase and acids from the amylose of those starch grains which are colored blue with iodine. By the action of diastase on the starch of germinating seeds the amylose of the starch is converted first into amylodextrine, and this in turn into dextrine and isomaltose. The microchemical behavior of amylo- dextrine is given by Arthur Meyer as follows: Water at 70° C. dissolves crystals of amylodextrine slowly, while at 100° the crystals are dissolved at once. A solution of 10 gm. of pure calcium nitrate in 14 gm. of water dissolves crystals under the coverglass very slowly. After some hours, if a solution of iodine is added, the calcium nitrate solution is colored brown, which indicates that the crystals of amylodextrine have at least been 332 MICROCHEMISTRY OF PLANT PRODUCTS partially dissolved. A solution of 2 gm. of purest potassium hydrate in 100 gm. of water dissolves small crystals within two hours, while the solution of larger crystals requires a longer time. A solution of iodine, prepared as directed on page 311, colors the crystals dark brown. A 25 per cent. solution of hydrochloric acid dissolves large and small crystals immediately. When this solution is diluted with 4 parts of water, it takes on a brownish- red color with the iodine solution. When 1 drop of malt extract is added to 5 drops of a neutral solution of amylodextrine this becomes inverted within 1o minutes, so that it no longer is colored by the iodine solution. To prepare the malt extract treat 1 part of malt with 3 parts of water and filter the solution. The solution of crystals of amylodextrine by the malt extract requires several days. At a temperature of 40° C. saliva dissolves the amylodextrine crystals within forty-eight hours. To prepare the saliva, mix human saliva with a drop of chloroform, filter, and preserve over a few drops of chloroform. Amyloid.—This carbohydrate occurs as reserve material in the seeds of Tropeolum majus, Impatiens balsamina, Peonia officinalis, and in many other plants. It is colored blue by dilute solution of iodine, but with a concentrated solution it is colored a brownish-orange. It is soluble in cuprammonia only after a day. Treated with a 30 per cent. solution of nitric acid it swells strongly, and finally dissolves. This is different from the amyloid produced by the action of acids and certain chlorides on cellulose. Amylose.—Starch grains which are colored blue by iodine —that is, most starch grains—are, according to Meyer, com- posed of crystals of two kinds of amylose, named by Meyer a-amylose and §-amylose. The a-amylose has been isolated ‘ in crystalline form, but the 8-amylose has not been isolated, and its microchemical behavior has only been determined by experiments with starch grains. The microchemical behavior of the a-amylose is as follows, the reagents being prepared as directed under amylodextrine: Water at from 60° to 100° C. does not soon dissolve the crystals of this amylose. Treat- ANTHOCHLORIN 333 ment with the calcium nitrate solution for 30 minutes does not appear to affect the crystals. The solution of iodine does ‘not color the crystals at first, but after a longer time it imparts a brownish color. The solution ‘of hydrochloric acid dissolves the crystals at once, and the solution, diluted with four times its bulk of water, is colored deep blue with the iodine reagent; but after the solution has stood for 12 hours it is colored brown- ish or not at all by the iodine. The solution of potassium hydrate at ordinary temperatures affects the crystals so that they are colored blue by the iodine after the solution has heen neutralized with acetic acid. In boiling potassium hydrate the crystals are changed into viscid drops. If the solution is now neutralized with acetic acid and diluted with four times its bulk of water, it takes on a deep blue color with the iodine reagent. If a drop of malt extract is added to the solution formed by boiling crystals of a-amylose with the potassium hydrate solution, and exactly neutralizing with acetic acid, it is found after 5 minutes that the solution takes on a red color, due to the form- ation of amylodextrine by the influence of the malt extract. Saliva and malt extract have very little effect upon a-amylose. After treatment with these reagents for 15 days at.a constant temperature of 40° C., no essential change could be detected. &-Amylose is insoluble in cold water, but at a temperature of 70° C. it forms viscid masses or minute droplets. The solu- ‘tions of calcium nitrate, potassium hydrate, and hydrochloric acid have the same effect as water, excepting that the solution in hydrochloric acid is more complete than in water. The solution of 8-amylose acts precisely as the solution of a-amylose. Undissolved f-amylose, however, is colored blue by the iodine solution. The swelling of starch in hot water is probably due to the 8-amylose which it contains. Meyer considers a-amylose and f-amylose to be the same substance, but that the latter contains water of crystallization, while the former does not. Anthochlorin.—A yellow coloring matter occurring in solution in the cell-sap and differing from the yellow coloring matter xanthin occurring in chromoplasts in that it is not 334 MICROCHEMISTRY OF PLANT PRODUCTS changed to a blue color by the action of concentrated sulphuric acid. , Anthocyanins.—These are coloring matters of flowers, leaves, and other parts of plants which impart red, violet, blue, blue-green, or green colors, the character of the color being dependent on the alkalinity or acidity of the cell-sap. The anthocyanins are soluble in water, alcohol, and ether, and are decolorized in strong alkalies. Anthoxanthin.—This yellow coloring matter in the chro- moplasts of flowers and fruits takes on a blue color with con- centrated sulphuric acid. Since the chromoplasts of flowers and fruits were first of all green, anthoxanthin is probably a derivative of chlorophyll. Anthoxanthin is also called xanthin and xanthophyll. Arabin.—This is the gum derived from species of Acacia and known as gum arabic. Arabin is soluble in hot and cold water, and insoluble in alcohol and ether. The aqueous solu- tion will mix with glycerine, but concentrated glycerine has little effect on the hard gum. Asparagin, C,H,NH,.CONH,.COOH.—This is a_nitrog- enous compound of simpler constitution than proteids. It is formed within plants both analytically by the decomposition of proteid, and synthetically probably by the combination of simpler substances. Asparagin is soluble in water and in the cell-sap, and is one of the most important nitrogenous com- pounds capable of solution and circulation within plants. It combines with non-nitrogenous compounds to form proteids, and is apt to accumulate in those parts of plants where there is not sufficient non-nitrogenous material at hand for the formation of proteids. The accumulation of asparagin is par- ticularly apt to occur in plants which are grown in the dark, so that carbon assimilation does not take place. Thus, Pfeffer found that when seedlings of lupin were grown in the dark, they contained a large amount of asparagin, but when they were brought to the light, the asparagin disappeared. He found that this was not due simply to the influence of the light; ATROPINE—BERBERIN 335 for when the seedlings were exposed to the light in an atmos- phere destitute of carbon dioxide, the asparagin persisted in the seedlings. For the ready demonstration of asparagin, tubers of Dahlia may be employed. Rather thick sections are cut from a tuber while the razor is kept dry and transferred to a few drops of alcohol on a glass slide and covered with a coverglass. On the evaporation of the alcohol crystals of aspara- gin in the form of rhombic plates are deposited on the coverglass and slide. To determine whether the crystals are asparagin, they are treated with a few drops of an entirely saturated solution of asparagin, which must be of the same temperature as the preparation. If the crystals are asparagin, instead of being dissolved they will increase in size, while other substances than asparagin will dissolve in the saturated asparagin solution just as they would in water. It is characteristic of asparagin that if the crystals are heated to 100° C., they lose their water of crystallization and appear like bright droplets of oil. At 200° asparagin becomes decomposed and forms frothy brown drop- lets which are no longer soluble in water. Atropine, C,,H,,NO,.—This alkaloid with its isomers, hyoscyamin, pseudohyoscyamin, and hyoscin, occurs widely distributed in the Solanacee. Sections of roots of Atropa Bella- donna contain atropine and yield a brownish precipitate when treated with potassium iodide-iodine. Bassorin.—Gum tragacanth, obtained from certain cells of the pith and medullary rays of several species of Astragalus. Swells strongly in water, but does not go into complete solution. Is not colored either by iodine or chloroiodide of zinc. - Berberin, C,,H,;NO,+6H,O.—This yellow alkaloid .oc- curs in the young parenchymatous tissue, and in the older xylem portions of Berberis vulgaris, and in representatives of the most various families. With potassium iodide-iodine it _ forms a reddish-brown precipitate which, by treatment with alcoholic potassium iodide-iodine, becomes changed into tubu- lar or hair-like forms having a brownish or iridescent green color. Ammonia and nitric acid impart to berberin a reddish- 336 MICROCHEMISTRY OF PLANT PRODUCTS brown color. A solution of potassium bichromate or potassium iodide in 50 per cent. sulphuric acid produces, with berberin, an intense purplish-red color. One part of nitric acid mixed with too parts of water added ‘to sections containing -berberin will produce clustered acicular crystals of berberin nitrate within the berberin-bearing cells. Betulin, C,,H,,O,+H,O.—This glucoside occurs in the form of fine granules in. the thinner walled cork cells of birch bark. It is accompanied by the enzyme betulase, which splits it into glucose and methylsalicinic ester. In order that it may be studied to good advantage under the microscope, the air should be pumped from sections immersed in water, and then the sections should be examined in water under the microscope. Betulin is insoluble in water, but is soluble in alcohol. It is strongly antiseptic, and protects birch bark against the attacks of lower organisms. Betuloretic Acid, C,,H,,O,.—This is secreted by the glandular hairs on the leaves of Betula alba. It is obtained from the thick, pale yellow secretion by successive solution in boiling alcohol, ether, and an aqueous solution of sodium carbonate: It is colored a beautiful red by concentrated sul- phuric acid. Brucine, C,,H,,N,O,+4H,O.—The alkaloid brucine oc- curs along with strychnine in the seeds of various species of Strychnos. Ammonium vanadate in sulphuric acid gives with brucine a yellowish-red color. When sections containing brucine are treated with a mixture of nitric and hydrochloric acids, the cell-contents are colored a reddish-orange, which merges into yellow. Caffeine, C,H,,N,O,+H,O.—The narcotic alkaloid in many foods and drugs. It occurs in plants of various families; for instance, in Thea, Coffea, Theobroma, Cola, Ilex, Sterculea, Neea. When sections containing caffeine (theine, methyl- theobromine, trimethyl-xanthin) are treated with a drop of concentrated hydrochloric acid, and then after a minute with a drop of a 3 per cent. gold chloride solution, somewhat slender, CALCIUM—CALCIUM PHOSPHATE 337 yellowish, silken crystals of a double chloride of gold and caffeine begin to be formed on the evaporation of the reagent. However, theobromine forms quite similar crystals when treated as above. Another method for the detection of caffeine is to place sections in a few drops of water and heat to boiling; then to allow the water to evaporate slowly and to treat the residue with a drop of benzol. On the evaporation of the benzol, caffeine appears in the form of fine needle-crystals. Calcium.—When the ash of plants is treated with sulphuric acid, this unites with the calcium present to form crystals of gypsum. If calcium sulphate is already present in the ash, its characteristic crystals may be detected when an aqueous solu- tion of the ash is allowed to dry slowly. If calcium is present in sections, it may be deposited in the form of crystals of cal- cium oxalate if the sections are treated with a solution of ammo- nium oxalate. Calcium Carbonate, CaCO,.—This rarely occurs in the crystalline form within the cells. It may, however, be found imbedded in, or incrusted on, the cell-walls. Calcium carbonate dissolves with effervescence when treated with dilute acetic acid. When treated with concentrated hydrochloric acid, it dissolves with the evolution of carbon dioxide gas. The in- growths from the walls of certain cells of .the leaves of Ficus elastica, known as cystoliths, are thickly incrusted with calcium carbonate and afford excellent material for the demonstration of this salt within plant tissues. Calcium Phosphate, Ca,(PO,),.—This salt of calcium occurs usually, if not always, in solution in the cell-sap. It may be deposited in the form of spherocrystals when plant tissues containing it are kept for a long time in strong alcohol. When treated with sulphuric acid, the spherocrystals are dissolved and crystals of calcium sulphate are formed in their stead. When sections containing calcium phosphate are heated on a slide in a drop of ammonium molybdate acidulated with nitric acid, a yellow precipitate is produced. This reaction may be hindered by the presence of certain organic compounds, such 22 338 MICROCHEMISTRY OF PLANT PRODUCTS as potassium tartrate, in which case the sections should be treated with a mixture of 25 volumes of a concentrated aqueous solution of magnesium sulphate with 2 volumes of a concen- trated aqueous solution of ammonium chloride and 15 volumes of water. In this case a crystalline precipitate of ammonio- magnesium phosphate is formed. Calcium Oxalate, CaC,O,.—Crystals of calcium oxalate occur so commonly in plants that it is safe to assume that any crystals observed in fresh tissues are of this substance until the contrary is demonstrated. The crystals may occur singly in the cells, in which case their definite crystalline form can be made out, or in the form of agglomerated star-shaped clusters of crystals, or in bundles of parallel needle-shaped crystals, or they may occur very numerously in cells in the form of very minute crystals. The crystals are insoluble in water and acetic acid, but dissolve without effervescence in hydrochloric acid. When they are treated with sulphuric acid, crystals of calcium sulphate are formed in their place. Calcium oxalate appears to be an excretion formed by the union of salts of calcium, which have been absorbed from the soil, with oxalic acid which is formed by the plant. Calcium Sulphate, CaSO,.—Minute crystals of calcium sulphate occur in many desmids. ‘They are insoluble in con- cenirated sulphuric acid.