ett Halley i {fi} Ha ite Ha Hh su i i i: Hn Hil Hn | . nti Ht i il u Hi i HA ti SHE) nt Hi i a CORNELL UNIVERSITY LIBRARY FROM H.F. Roberts Haining oli Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924031488558 eA ae LABORATORY AND FIELD MANUAL OF BOTANY BY JOSEPH Y. BERGEN, A.M. AUTHOR OF ‘ ELEMENTS OF BOTANY,” ‘‘ FOUNDATIONS OF BOTANY,” ‘PRIMER OF DARWINISM,” ETO. AND BRADLEY M. DAVIS, PxD. RECENTLY ASSISTANT PROFESSOR OF PLANT MORPHOLOGY IN THE UNIVERSITY OF CHICAGO GINN & COMPANY BOSTON - NEW YORK -. CHICAGO - LONDON vw CORNELL UNIVE RELY fof te hse (a ie CoPpYvRIGHT,, 1907, BY ~ JOSEPH Y. BERGEN AND BRADLEY M. DAVIS ALL RIGHTS RESERVED 77.3 The Atheneum Press GINN & COMPANY: PRO- PRIETORS « BOSTON : U.S.A. PREFACE ‘This manual offers material for much more than a year’s laboratory work.’ This is made necessary by the fact that in- structors differ widely in their views as to what matter should be presented in an introductory course under the variety of conditions obtaining where botany is taught. A course must necessarily be framed selectively, and the chief alternatives are discussed in the opening paragraphs of the Introduction. The authors fully recognize the fact that no set of directions of only moderate fullness can tell the student all that he needs to know about choice of material, apparatus, and manipulation. It is assumed that much is left to be explained by the instructor, and constant mention is made of general and special laboratory guides which may be consulted for needed details. The student in the laboratory is not to consider himself as merely the corroborator of facts already ascertained: he is to interrogate mainly not the instructor, not the manual, but the plant itself. The directions here given are, therefore, for the most ‘part suggestions on methods of procedure and indications as to the plants or parts of plants in which to look for desired information. Since the amount of ground that can be covered by labora- tory divisions varies so largely with many circumstances, it has , seemed desirable to designate two courses, a briefer and a fuller one. The matter which may be omitted from the latter to frame the shorter course is printed in smaller type and consists in the main of rather more difficult or detailed studies than those which appear in the larger type. In a general way the order of treat- ment follows that of the authors’ Principles of Botany, but the Ht iv PREFACE shorter course does not cover many more topics than are dealt with in Bergen’s Hlements or Foundations of Botany, and may be used with either of those books. Part I consists mainly of studies on the gross anatomy and the histology of seed plants, together with a set of separately numbered experiments to illustrate some of the main principles of plant physiology. Part II deals with type studies of spore plants, outlining the evolution and classification of the plant kingdom. Here will also be found studies on the gametophyte-phases and the life histories of seed plants to show their relationships to the spore plants. Part IT is introduced by outlines on the plant cell to illustrate the chief principles of growth and reproduction. Part III is concerned with a series of laboratory and field studies which may serve to offer at least an outline for the treatment of ecology as a scientific subject. Profound ecological studies demand far more knowledge of taxonomy, plant phys- iology, meteorology, the physics and chemistry of soils, and kindred subjects than can be required of beginners in botany. However, the authors believe that it is quite possible to illustrate, even to beginners, something of the kind of quantitative discus- sion of variations in environment and the responses of plants to changed conditions, which must distinguish the ecology of the future. Hearty acknowledgments for valuable suggestions are due to A. T. Bell, F. E. Clements, W. N. Clute, W. F. Ganong, B. Gruen- berg, Miss Lillian J. MacRae, G. J. Peirce, and R. B. Wylie, who have wholly or in part’ read the manuscript or the proofs. : J. Y. B. CamBripcGE, March, 1907 B. M. D. CONTENTS PAGE INTRODUCTION . ‘ ‘ © ¥ ‘ 7 1 LABORATORY METHODS AND > EQUIPMENT r ‘ r = V4 PART I—STRUCTURE AND PHYSIOLOGY OF SEED PLANTS Introductory Study of a Seed Plant and its Organs : : 15 The Seed and its Germination F - : ‘ ‘ 4 ‘ = dT Storage of Food in the Seed . . : o S21 Movements, Development, and Manghalegy of the ‘Siete. ‘ 27 Roots. : ‘ : . 29 Some Properties of Cells ane their Functions in the Huot . . 86 Stems. 7 . : : : é : ; ‘ . 37 Structure of the Stem ‘ , : 2 : 3 . 5 ‘ . 89 Work of the Stem . : 5 A ; 7 E ‘ ‘ 45 Buds ‘ - : : ‘ F . 3 . 48 Leaves . . : : 7 ‘ . 61 Leaf Arrangement with Héterenee to Light ‘ ‘ . . 5 . 58 Minute Structure and Functions of Leaves . : : . . 55 The Flower of the Higher Seed Plants . ‘ ‘ a‘ a 64 Pollination and Fertilization . . . ‘ ; ; z : 68 The Fruit : : . : : . ; : : Z 5 . 69 PART II— TYPE STUDIES PRECEDED BY THE STUDY OF THE PLANT CELL The Plant Cell, its Structure and Reproduction ‘ . 75 The Flagellates, or Flagellata : . ; ‘ . : . 88 The Slime Molds, or Myxomycetes P ; 2 - m . . 88 The Blue-Green Algx, or Cyanophyces : i ‘ F 2 . 84 The Green Algz, or Chlorophycee . 3 a . 87 The Brown Algz, or Phzophycez . ‘ : r 5 . 97 The Red Algez, or Rhodophycee . a : : ‘ 2 : . 100 The Bacteria, or Schizomycetes . b, : 7 ‘ : . 102 The Yeasts, or Saccharomycetes . ‘ : 7 z : A ~ 105 The Alga-like Fungi, or Phycomycetes’ . : ‘ ‘: “ : . 107 Vv vi CONTENTS The Sac Fungi, or Ascomycetes. : , The Lichens . . s . ‘ The Basidia Fungi, or ‘Bedidioniyecton 5 E The Liverworts, or Hepatice . . 7 7 . The Mosses, or Musci_. F ‘ ; : : The Ferns, or Filicinee . : : The Horsetails, or Equisetinez The Club Mosses, or Lycopodinez The Gymnosperms, or Gymnosperme The Angiosperms, or Angiosperme PART III— ECOLOGY Parasitic and Carnivorous Plants . : . How Plants protect themselves from poor n Pollination of Flowers . 2 . How Plants are scattered and pronaeatedl Competition and Invasion Plant Successions Ecological Classes . C Plant Formations ; Zonation . z ‘ Study of Types of Seed Plants ‘i - re BOTANICAL MICROTECHNIQUE General Reagents employed in Temporary Preparations Some Special Reagents for Microchemical Tests and Temporary Peep. rations. é ‘ s Killing and Fixing . : 3 The Preservation of Material . General Staining Methods Mounting in Balsam and Glycerin Imbedding in Paraffin Sectioning . Staining on the Slide CULTURE METHODS The Culture of Alge . 5 . The CultureofFungi . . . . The Culture of Liverworts and Mosses . The Culture of Ferns. The Culture of Seed Plants CONTENTS vii MATERIAL, APPARATUS, AND SUPPLIES PAGE Lists of Preparations for the Microscope 2 $ . . 217 Suggestions on Material for the Study of Plant Histology di , . 220 Apparatus for the Laboratory F : . 7 7 : . 222 Chemicals for the Laboratory 7 i ‘ x f ; . 224 Dealers in Material, Apparatus, and Bapaltes: : ‘ ‘ 2 . 225 BIBLIOGRAPHY . ‘ F 5; F me bs : ‘ ‘ . 227 APPENDIX . p : , ‘ ‘ ‘ ‘ ‘ i ‘ . 233 GLOSSARY . . ‘ 3 ; ; , é ‘ P : . 241 INDEX . . . ‘ : : s, ; ‘ ‘ 5 . 255 LIST OF EXPERIMENTS PAGE I. Temperature and germination 7 . a F z . 19 II. Amount of waterinseeds . . ‘ s : : 20 III. Relation of air to germination F : ‘ . : 2 21 IV. Effect of germination on air . ; Z 3 ‘4 . 21 V. Use of the pea cotyledons after germination ‘ ; F + 21 ~ VI. Relation of food in seed to rate of growth . ; . 22 VII. Occurrence of starch in secds . : 2 : : 24 VIII. Oil in flaxseed ‘ 4 : ‘ ‘ ‘ ‘ ; . 25 IX. Proteidsin seeds . 5 . : F 3 - . 26 X. Plant foods in Brazilnuts . - . ‘ : ; . 26 XI. Cause of arch of hypocotyl . , Fi 3 F . 27 XII. Discrimination between root and lynoeatyl z 2 P . 28 XIII. Growing region of root . ‘ : . 3 . 28 XIV. Percentage of water in the plant body : : : , . 33. XV. Waiter cultures : : - . ‘ . 33 XVI. Root absorption with dimindaned resiperauine ‘ : . 34 XVII. Region of bending in the root : : : . 385 XVIII. Pressure of root tip x % : : z : . 385 XIX. Cause of downward ae of sab é : : . 38 XX. Osmosis . i i 7 , i : , . . 36 XXI. Osmosis of Begonia leat. : : F . F : . 37 XXII. Course of water in stems . 45 XXIII. Relation of loss of water to fines of tissues : 46 XXIV. Use ofcork . F : - A F i i : . AT Vili XXV. XXVI. XXVIL XXVIIL XXIX. XXX. XXXL XXXIL XXXII. XXXIV. XXXV. XXXVI XXXVII. XXXVIIL. XXXIX. XL. XLI. XLII. CONTENTS Reserve sugar in onion bulb Proteids in onion bulb F Cause of nocturnal position of leaves . Values of illumination for leaf positions ‘ Adaptation of growing leaves to changed light lations Heliotropic movements of English ivy Oxygen making by plants : Starch in Tropwolum leaves Consumption of starch in Tropwolum ieee Effect of sealing stomata on starch production Effect of darkness on chlorophyll production Transpiration 7 Side of Ficus elastica leat white ‘cananlnes PAGE 48 48 53 53 54 55 57 57 58 59 59 60 61 Relative transpiration of Hydrangea Hortensia and “Pies elastica Passage of water from ieee to leaf Rise of water in leaves P Starch contents of leaves at various seasons Production of pollen tubes . 61 63 63 63 68 LABORATORY AND FIELD MANUAL OF BOTANY INTRODUCTION It is intended that these laboratory outlines shall be found adaptable to several methods of approach in framing a general course in elementary botany. First. By beginning with Part I the student may consider first the more general features of the morphology of the seed plant and the most important of its physiological activities. This may then be followed by studies of a series of spore plants (Part II), to outline the chief steps in plant evolution. Such work as is possible in plant ecology (Part IIT) is thus deferred to the end of the course. Second. By commencing with Part II the student will be introduced at once to the principles of cell structure, growth, and reproduction, and can then trace the evolution of the plant king- dom. By this arrangement selections from Part I will follow the studies of Part II, and Part III will receive attention last. Third. Part I may be followed at once by Part III, and the studies of Part II be used only to illustrate such types and topics concerned with spore plants as may seem desirable. Fourth. It is by no means necessary that the matter of Part I be taken up in the order given. Instead of beginning with the plant as a whole or with the seed, a course may be readily shaped so as to commence with the fruit or with the leaf. The planning of a course depends upon so many factors, such as season, material, equipment, maturity of students, and the time 1 2 INTRODUCTION at the disposal of the class, that it must vary greatly with the different conditions. The authors, recognizing these difficulties, have tried to present a flexible outline in a thoroughly practical manual containing sufficient material to permit of a wide range of choice. In general they believe that the best results will be obtained, when a full year can be devoted to the subject, by taking the matter in the order given in the jirst or second of the alternatives presented above. If only a half year is avail- able, the best course in their judgment is that indicated in the third alternative. For the guidance of any who may care for such suggestions the authors have designated by double asterisks (* *) those experiments and studies which they consider to be the most valuable. A brief discussion of laboratory methods and equipment is presented immediately before the laboratory outlines and experi- ments of Parts I, IJ, and III. It is hoped that the instructor may find some helpful suggestions in this, and certain parts are written expressly to aid the student to an understanding of the spirit of laboratory work, methods of drawing, note taking, and the care of instruments. j The essential methods of botanical microtechnique and the preparation of the material are taken up after the laboratory out- lines. This account has been introduced to assist the instructor and the advanced student in the collection and preservation of material and in the more detailed studies of plant histology and cytology, which demand the preparation of microtome sections and critical staining methods. The discussion does not attempt to give such details covering special studies as may be found in several more exhaustive treatises to which the reader will be referred. It endeavors rather to outline standard methods of killing, fixing, preserving, cutting, and staining plant structures, which cannot fail to give good results, with the reasons why they have been selected. Some simple directions for the culture of alge, fungi, moss protonema, fern prothallia, etc., follow the account of microtechnique. INTRODUCTION 3 A section entitled “ Material, Apparatus, and Supplies” gives lists of preparations for the microscope, favorable material for histological work, apparatus and supplies, with the addresses of dealers who furnish these to the trade. The bibliography has been chosen with the purpose of present- ing a group of books many of which are within the possibilities even of a well-equipped school library, rather than a lengthy list of detailed literature which is usually only handled by the spe- cialist. These works are numbered and the references to them throughout the manual will be by the author’s name and the number. An appendix with suggestions to instructors follows the bibliog- raphy. This contains matter which it is not necessary for the student to read in connection with his laboratory work, although in many cases it may be of interest for him to do so. The ap- pendix is really a collection of practical notes based on the experience of the authors or gathered from conversations and correspondence with many teachers. Indeed, it is a feature which the authors have introduced in the hope that it may bring forth other helpful and practical suggestions from those who use the book, and correspondence upon this subject is cordially invited. A glossary gives a selected list of botanical terms, including the most important of those used in this manual and in the authors’ Principles of Botany. Only a few necessary abbreviations have been used, to econo- mize space. As stated above, books listed in the bibliography are referred to by the author’s name and number in the list. Principles designates the Principles of Botany; App., the ap- pendix; l.p., m.p., and h.p. refer to low power, medium power, and high power of the compound microscope respectively ; lens means either hand lens or dissecting microscope as the case may be; cp. means chemically pure. The usual abbreviations for the units of the metric system are frequently employed. LABORATORY METHODS AND EQUIPMENT THE LABORATORY AND ITS EQUIPMENT The essentials of a laboratory are, of course, good light, con- venient tables, and sufficient apparatus. While north light is preterable, since its quality is more constant, east, west, or south light can be perfectly regulated by translucent shades which may be pulled up to any desired distance, and so temper direct sun- light when necessary. Moreover, it is desirable that some win- dows have the sun for part of the day, since aquaria and glass cases for growing plants require some sunlight and may be placed in such parts of the room. Excellent suggestions on the arrange- ment of laboratory tables, lockers, glass growing case, sink, black- board, ete., are given in Ganong, T, Chapter V, and in Lloyd, 8, Chapter IX, books which should be read by every teacher of botany. The equipment of a laboratory will depend largely upon the nature of the work, whether very elementary or covering a strong full course of a year or more, and also upon the attitude of the instructor, who may emphasize especially either physiology or a more detailed morphology. Physiology requires its own special apparatus, and detailed morphology demands the equipment necessary for imbedding, microtome section cutting, and staining. Much of the work with this apparatus can best be conducted at tables in the center or back of the laboratory, which will not interfere with the tables for the more general class exercises. In the choice of equipment and its storage the instructor is again referred to the admirable discussions of Ganong and Lloyd. Lists of the chemicals, apparatus, and supplies necessary for the work outlined in this manual are given in Secs. 215, 216. The cost of compound microscopes is the item of greatest expense in the equipment of a laboratory, and their selection 4 GROWING PLANTS IN THE LABORATORY 5 ' demands careful thought. The laboratory should have enough microscopes so that every student in a section may have his own instrument. If this is not possible, it is better that the course should be planned along such lines that the microscopic work is largely in the nature of demonstrations by the instructor on such microscopes as are available. Two or three students working together at the same microscope create confusion and secure poor results. There are anumber of medium-priced instru- ments on the market, with varying merits, from which the instructor must choose for himself. 2. Note the toothed sheaths at the joints (nodes). Each tooth represents a leaf. 8. Observe the developing, branched, vegetative shoots. Have they the same jointed structure with toothed sheaths as the spore-bearing shoots ? 4, Trace the spore-bearing shoots and also the develop- ing vegetative shoots to the creeping rootstock (rhizome). What is the structure of the rootstock? Has it joints? sheaths? Where do the roots arise? Note the tuberous bodies, if present. Tllustrate the above features in a habit sketch, with such details of the joints and sheaths as are necessary to make clear the fundamental morphology. In plants collected somewhat later, with well-developed vege- tative shoots, note: 5. That the spore-bearing shoots have died. 6. That the vegetative shoots have developed into tall green, much-branched stalks. How do they feel to the touch ? 144 TYPE STUDIES 7. Study the jointed structure of the branches, the sheaths, the method of branching. What part of the plant per- forms the work of photosynthesis ? Draw a portion of the vegetative shoot to illustrate the above points. B. The cone and its scales, or sporophylis. Study from pre- served material. 1. Draw a cone in detail Gf not drawn under A), showing the arrangement of the hexagonal scales in circles around the stem. Why should their form be six-sided? Com- pare the circles of scales with the nodes or joints of the stem bearing circles of leaves forming sheaths. 2. Cut off several scales, noting the central stalk bearing the six-sided plate and the sac-like sporangia hanging down from the plate. Draw two side views of the scales as seen somewhat obliquely from above and below. The scales are highly specialized spore leaves, or sporophylis. What are some of the reasons why they should be so considered ? C. The sporangium and its spores. Split a sporangium open with the point of a needle. Examine under h.p. Note: 1. The spores, each bearing four filamentous elaters, devel- oped from four segments of the outer spore wall, which separate from one another and the spore except at one point. 2. Allow the spores to become dry and note the position of the elaters. Draw. 3. Breathe gently on the dry spores or moisten them and observe the behavior of the elaters. Draw various spores. 4, Study the structure of the sporangium wall, the cells of which are irregularly thickened. Draw a portion under h.p. How does the sporangium wall rupture? What mechanical forces are at work to make it split open ? D. The cell structure or histology of the stem. Cut sections across the stem between the nodes. Observe under l.p. the LYCOPODIUM 145 air spaces, the distribution of green tissue, the rigid tissue around the outside, the small fibro-vascular bundles. Show these structures in an outline sketch. 1. The detailed structure of the stem is complicated and highly specialized for its work of photosynthesis under severe xerophytic conditions. There are many interesting adaptations to these eco- logical relations, as shown by the position of the stomata protected within the lengthwise grooves, the heavy layers of thick-walled tis- sue outside of the green tissue, etc. The study of these adaptations is very interesting, but rather special, requiring well-cut sections. E. The growing points of stems and roots. These are occupied by remark- able, large apical cells whose structure and activities are best studied by means of microtome sections. They are among the best illustrations of growth from apical cells. F. The gametophytes. These can be obtained only by sowing spores when very fresh. The prothallia are irregularly lobed and the antheridia and archegonia are generally developed on separate plants (see Prin- ciples, Fig. 285). REFERENCES. Campbell, 23; Goebel, 16. Questions. What are the structural characters of the horse- tails especially adaptive to severe conditions of heat and drought (xerophytic conditions)? Where is the work of photosynthesis performed? Have the leaves anything to do with it? Have the leaves any very obvious functions ? Why are they present especially on the underground root- stock? What is the form and structure (morphology) of the cone? What are some of the advantages of the elaters on the spores? What are the advantages in the spores clinging together so that they germinate in groups ? THE CLUB MOSSES, OR LYCOPODINEA 134. Lycopodium, the lycopod, or club moss. Species of Lycopodium with well-differentiated cones, such as L. annotinum, L. complanatum, L. clava- tum, etc., furnish the best material for type studies. Observe when possible the life habits, noting the creeping stems from which arise the upright branches bearing cones, : 146 TYPE STUDIES A. General morphology. Well-mounted herbarium sheets are excellent for this study. Note: 1. The upright stems, with spirally arranged, needle-shaped leaves. 2. The long terminal cones, composed of spirally arranged scales (sporophylis). 8. The creeping stems with leaves similar to those of the upright stems. 4. The rather infrequent roots. Illustrate the above features in a habit sketch. B. The cone and its scales, or sporophylis. Examine preserved material. 1. Draw a cone in detail, showing the spiral arrangement of its scales (sporophylls) if not illustrated under A. 2. Cut off several scales and draw one as seen from the inside, showing the large sporangium at its base. 8. Construct a diagram illustrating the attachment of the scales to the axis of the cone and the way in which they overlap one another. The scales are specialized spore leaves, or sporophylls What are some of the reasons why they should be so considered ? C. The sporangium and its spores. Split a sporangium open and note the immense number of minute spores. Draw a group. What is the sig- nificance of the angles along one side ? Like the spores of the bryo- phytes and pteridophytes generally, they are developed in groups of four, tetrads, from spore mother cells. D. The cell structure, or histology of the stem and leaf. This is a profitable but detailed study. 1. Cut cross sections of the stem. Note the epidermis, the thick corti- cal regions of ground tissue, the vascular strands called leaf traces, leading out to the leaves from the central jfibro-vascular bundle. In the fibro-vascular bundle observe the more or less parallel regions of wood (xylem), composed of large tracheids, and bast (phloém) within the bundle sheath. Draw the details of cell structure in a series of figures from cross and lengthwise sections. 2, Examine the surface of the leaves for stomata. Cross sections of the leaves will show their simple cell structure. E. The germination of spores, and the gametophytes. Gametophytes have not been found in America and the spores have not been germinated beyond the first few cell divisions. The propagation of plants is chiefly by the branching of stems which separate as older parts die away, and in some species by peculiar vegetative buds. Rererence. Campbell, 28. 135. Selaginella. Various species of Selaginella have quite differ- ent habits of growth and arrangements of leaves and branches. SELAGINELLA 147 S. rupestris is generally the most available of our native species, but S. opus is often easily obtained. S. Kraussiana is a delicate form frequently cultivated in conservatories. S. Martensii and other tropical species are large, erect, much-branched, ornamental plants cultivated in greenhouses, and when in good fruit are ex- cellent for type study. A single plant in fruit will supply a large class with material, but the smaller species are also good. A. General morphology. Note: 1. 2. 3. The character of the stems, upright or creeping. Describe their method of branching, generally in one plane. The scale-like leaves. These are spirally arranged in some species (as S. rupestris), but in other forms are distributed in four rows and are of two sizes. What is their arrange- ment in the type studied ? Have the stems an upper and lower side (dorsiventral symmetry) ? What advantage is there in this symmetry in relation to the spreading habits of growth ? Search for a very small triangular structure, called the ligule, at the base of the leaf. Its significance is not known. The spike-like cones composed of crowded scales (sporo- phylls). How are these arranged? How many rows? What is the geometrical form of the cone ? . The fibrous forking roots. In tropical species roots may be developed from the tips of special descending branches (rhizophores). Illustrate the above points in habit sketches, paying special attention to the arrangement of the leaves on the stem. B. The cone and its scales, or sporophylls. 1. 2. Draw a cone in detail, showing the arrangement of the scales, or sporophylls (if not illustrated under A). Note the sporangium attached at the base of each sporo- phyll. Are the upper and lower sporangia of the same cone alike? Cut off the scales and compare them, dis- tinguishing between oval sporangia containing minute spores, microspores, and larger-lobed sporangia containing 148 TYPE STUDIES a few very large spores, megaspores. These two forms of sporangia are termed respectively microsporangia and mega- sporangia, and the scales which bear them are microsporo- phylis and megasporophylls. Draw the microsporophylls and megasporophylls, viewed from the inside, showing the form of the sporangia. 3. Construct a diagram illustrating the attachment of the two forms of scales to the axis of the cone and their distribution above and below. The scales, as stated above, are sporophylls differentiated into two forms. What are the reasons why they should be so consid- ered ? The differentiation of spores into two sizes, microspores and megaspores, which develop respectively male and female gametophytes, is called heterospory. C. The microsporangium and microspores. Split a microspo- rangium open and note the immense number of minute microspores. Draw, under h.p., a portion of the wall of the sporangium, showing the cell structure, and a group of spores. D. The megasporangium and megaspores. Split a megasporan- gium open and count the large megaspores. Is the number the same in all sporangia? Draw a megaspore under h.p., showing the form and markings on the wall. What is the significance of the angles on one side? Like the spores of the bryophytes and pteridophytes generally, and also like the microspores, they are developed in groups of four, tetrads, from spore mother cells. Under the same magnifica- tion draw a microspore by the side of the megaspore to show comparative size. E. The cell structure, or histology of stems and leaves. 1. Cut cross sections of the stem. Note the epidermis and the cortical ground tissue surrounding two or more air spaces crossed by delicate filaments. In the center of each space lies a fibro-vascular bundle consisting of a strand of wood (xylem) surrounded by bast (phloém). The further examination of these elements in cross and lengthwise sections may constitute a detailed study, , SELAGINELLA 149 2. Compare the cell structure of very young leaves with that of older ones. The cells, in the younger leaves, contain a single large chro- raatophore, which becomes divided into a chain of segments in the older cells. . The germination of spores, and the gametophytes. The microspore pro- duces a very small male gametophyte, which remains contained within the ruptured spore wall and develops two-ciliate sperms (see Prin- ciples, Fig. 290, A, B). The megaspore gives rise to a small female gametophyte, which emerges somewhat from the ruptured spore and develops several archegonia (see Principles, Fig. 290, C). The develop- ment of these gametophytes requires several weeks, and their study demands microtome sections, which are difficult to prepare, so that they can hardly be treated in an elementary course. Marsilia (Sec. 182) is a better type for the study of reduced male and female gametophytes associated with microspores and megaspores. G. The development of the sporophyte. This study, like that of gameto- phytes, requires microtome sections of material difficult to obtain and to cut, and is hardly practicable for a general course. Marsilia (Sec. 182) is ' a better type to illustrate the relation of the embryo sporophyte to the female gametophyte in heterosporous pteridophytes. The embryo de- velops in the interior of the gametophyte at the end of a structure called the suspensor. Three regions are differentiated, — the stem with young leaves forming a bud, the root, and the large foot (see Principles, Fig. 290, C). The foot absorbs food from the tissue of the gameto- phyte within the megaspore. In certain species of Selaginella, as S. ru- pestris, the sporophytes are developed while the megaspores are still held mechanically by the sporophylls on the cone (see Principles, Fig. 290, D). This retention of the megaspores on the sporophyte is suggestive of the seed habit (see Principles, pp. 835 and 336). REFERENCE. Campbell, 23. QueEstions. What are the growth habits of the type of Selagi- nella which has been studied? Are there any xerophytic adaptations? Describe any peculiarities in the arrangement of the leaves and suggest reasons therefor. What is the form and structure (morphology) of the cone? Why are its leaves called sporophylls ? What is the relation between the large size of the megaspores and their production relatively few in a megasporangium? What is heterospory ? What are its advantages in giving the sporophyte a better start in 150 “TYPE STUDIES life? Describe the life history, distinguishing between the sexual phases, gametophytes, and the asexual phase, sporo- phyte. Draw and arrange a series of diagrams illustrating the chief stages throughout the life history, using two col- ored pencils to designate the gametophytic and sporophy tic generations respectively (App. 18). Construct a life-history formula that will express this succession (App. 18). 136. Isoetes, quillwort. Study when possible the life habits, noting whether the form is aquatic or terrestrial. A. General morphology. Note: 1. The rush-like leaves arranged around a very short, conical stem, and the cluster of forking roots below. Illustrate in a habit sketch. These leaves, late in the season, produce sporangia at their bases, thus becoming sporophylls. At such times the stem may be com- pared to a cone of sporophylls. 2. Strip the sporophylls from the stem. Those on the outside, and consequently lower on the stem, are likely to be megasporophylis, as shown by the basal sporangium containing megaspores. Those in the interior, and consequently higher up on the stem, are likely to be microsporophylis, as shown by the basal sporangium containing microspores. B. The sporophylls. Examine the base of a megasporophyll, viewing it from the inner side. Show in a figure : 1. The large megasporangium containing megaspores, held in a hol- low at the base of the leaf and partially covered by a membrane, the velum. 2. The ligule, a triangular scale situated on the sporophyll above the sporangium. Diagram the position of the ligule and sporan- gium as they appear in a lengthwise section of the base of the sporophyll. 3. Draw the megaspore under h.p., showing the markings on the thick wall and the angles on one side. What do the angles signify ? 4. Compare the base of a microsporophyll with that of a megaspo- rophyll. ‘5. Draw a group of microspores under h.p. to show their size in com- parison with that of the megaspore. The structure of the leaf. Section the leaf across and lengthwise. Note the large air spaces separated by partitions. Show their arrangement in an outline drawing and the small fibro-vascular bundle traversing the interior of the leaf. Are stomata present ? THE PINE 151 D. The gametophytes. The germination of the spores of Isoetes and the development of the gametophytes is even more difficult to follow than that of Selaginella and requires detailed study. REFERENCE. Campbell, 23. THE GYMNOSPERMS, OR GYMNOSPERMA 187. Cycads. Cycas revoluta is a large form frequently cultivated in park conservatories, and may be used to illustrate the general morphology of the cycads, that is, the trunk-like, unbranched stem bearing the crown of com- pound leaves at the top. This cycad occasionally develops sporophylls in the greenhouses, which may then be collected and preserved for study. The carpels are developed more commonly than the stamens. Zamia is a small cycad which grows in Florida and is an admirable type for a study of the cycad cone, together with the development of the ovules and pollen, the structure of the male and female gametophytes, processes of fertilization, and development of the embryo. Cones of Zamia may be readily shipped, and since the ovules retain their vitality for a considerable time they can be studied alive in the North or killed and preserved for micro- tome sections. Zamia may also be readily grown from seed in greenhouses, and will produce cones under cultivation. Its study is recommended wher- ever possible. 138. The pine (App. 21). Several species are available for this study, such as the Scotch pine (Pinus sylvestris), the Austrian pine (P. laricio), P. Strobus, P. palustris, or some of the scrub pines, such as P. Banksiana. Living material for general mor- phological or histological work may be obtained at any time of year. The young cones should be collected and preserved in alco- hol at the time of pollination in May, when the year-old cones and two-year-old cones may also be gathered and preserved for comparison with the first. Dried, open cones and seeds should be collected late in the summer. The pine should also be studied in the field to determine its growth habits and the ecological adap- tations of its foliage to conditions of severe cold and drought. A. General morphology. Observe: 1. The main stem and branches, each ending in a terminal bud ; the arrangement of the branches. 152 FYPE STUDIES 2. The very numerous dwarf branches, each bearing a cluster (fascicle) of two, three, or more needle-like leaves, accord- ing to the species studied. 8. The thin scales on the dwarf branches wrapped around the base of the cluster of needle leaves. Are these scales morphologically leaves? Why? 4. The bases of old scales spirally arranged, and covering the main branches from the axils of which the dwarf branches arise. Younger full-sized scales at the tips of the shoots. What are these scales morphologically ? 5. The bud scales, or leaves, enveloping the terminal bud. Illustrate these features in habit sketches. How many forms of leaves are there on the pine? 6. On a branch two feet or more in length note the regions that indicate the beginnings of one year’s growth and the end of another’s. Draw such a region and explain the peculiar arrangement of the scales upon it. 7. Observe the position of cones on the branches. How many sizes do you find and what are their ages as shown by their positions? 8. Note the branch scars on older portions of the stem and main branches. B. The cell structure, or histology, of the stem. Cut cross sections of a three- or four-year-old branch. These may be stained with advantage (Sec. 212) and mounted in balsam. 1. Observe under l.p.: (a) the restricted region of pith; (b) the layers or rings of wood, or eylem, around the pith (what is the significance of their number ?); (c) a layer of bast or phloém outside of the wood and separated from it by a thin cambium ; (d) the outer bark com- posed of larger cells, in places green, but on the exterior dead and forming scales ; (e) medullary rays appearing as radiating lines running through the wood and bast; (f) resin ducts in the wood and outer bark. Are the medullary rays of the same length? Do any of them penetrate to the pith? Show the position of these tissues in an outline sketch. 2. Study the structure of the wood in the region between the growth of two successive years. Ina detailed figure show the form of the THE PINE 153 empty wood cells and explain differences in size. Also include in the figure a medullary ray, the cells of which have dense protoplas- mic contents, and also a resin duct. Note the cross sections of pits in the wood cells, which will-be better understood after the study- outlined in C. In what faces of the cells are they found ? 3. Study the region of the cambium and show the form of its cells in a detailed figure, together with some of the wood on the inside and the bast on the outside, including a medullary ray. The bast is composed for the most part of sieve tubes. What are the peculiari- ties of the cambium tissue characteristic of a region of growth ? 4, Study the outer bark, showing in figures (a) the form of the old bast cells having a crushed appearance ; (b) the green parenchyma, comparing it with the pith ; (c) the manner in which the medullary rays merge with the cells of the bark. . Cell structure of the wood. Use the cross sections employed above and also radial (lengthwise) sections and tangential (lengthwise) sections, staining if desired (Sec. 212). 1. In radial sections note (a) the long, empty wood cells, tracheids, with walls bearing bordered pits (pits characterized by two circles and peculiar to certain groups of gymnosperms); (b) the medullary rays, like long knife blades, piercing the wood, mostly composed of cells with dense protoplasmic contents but some of them empty and pitted. Show these points in a detailed figure. 2. In tangential sections note (a) the wood cells, tracheids, with cross sections of the bordered pits; (b) the cross sections of the medul- lary rays. Illustrate in a detailed figure. 8. Examine the cross sections again to understand clearly the appear- ance of the pits and medullary rays in the light of your study of radial and tangential sections. Make a new figure if the study out- lined in B, 2, is not satisfactory. 4, Draw a cross section of a pit, showing the delicate membrane, or primitive cell wall (middle lamella), which crosses it, and the second- ary thickening of the cell wall on both sides. 5. Construct a figure of the appearance of a cube of wood under high magnification, several cells wide, as viewed from an angle so as to show cross, radial, and tangential sections (App. 21). Include in this figure also one or more medullary rays and a resin duct. . The cell structure, or histology, of the pine needle. Cut cross sections of a pine needle free-hand (Sec. 194) or use prepared slides (Sec. 212). Observe : 1. The heavy epidermis, with lengthwise grooves, in which are situated the stomata. 154 TYPE STUDIES 2. The layer of rigid tissue (sclerenchyma) beneath the epidermis. 8. The broad layer of green tissue, mesophyll, whose cells have in- folded walls. 4. Resin ducts in the green tissue. 5. A central area containing in most species of pine two fibro-vascular bundles and bounded by a bundle sheath. The fibro-vascular bundles lie in a special region of so-called transfusion tissue, composed in part of empty pitted cells and in part of cells containing protoplasm. Each bundle consists of a region of wood (xylem) and bast (phloém) and contains rather ill-defined medullary rays. 6. The sections of the stomata show epidermal cells on either side of the groove and below them two small guard cells containing chloro- phyll. Each stoma opens into a chamber within the green tissue. Show the position of the tissues of the needle in an outline drawing and then treat the details in separate figures. E. The staminate cones. These are short-lived structures de- veloped in the late spring with the appearance of the new growth from the terminal buds. They are variously clustered in different species of pine. Draw a habit sketch of a group, showing the arrangement of the cones on the new growth, with its developing needles. 1. Observe the position of the staminate cone in the axil of a pointed scale leaf. Draw in side view to show the some- what spiral arrangement of the closely crowded stamens (microsporophylls). 2. Split the cone lengthwise and diagram the attachment of the stamens along its axis. What is the morphology of the staminate cone as indicated by its position on the stem and the nature of the stamens (see F below) ? F. The stamen and pollen. Remove a stamen from a cone which has not yet shed its pollen. 1. Observe its form and structure, —a short stalk, broaden- ing beyond, on the lower face of which are borne long pollen sacs (microsporangia). The tip of the stamen is turned upwards and fits over the pollen sacs of the stamen above. Draw under a hand lens the stamen in end and side views to illustrate these points. ere THE PINE 155 2. Open a pollen sac and mount the pollen grains (micro- spores) in water. Under h.p. note (a) the two wings at- tached to the pollen grain; these are developed from the outer wall of the cell; () within the pollen grain the tube nucleus lying near the center and the generative cell against the wall at the side farthest away from the wings, also occasional remains of a prothallial cell between the gener- ative cell and the wall. Draw a pollen grain showing this structure. The pollen grains are developed in groups of four, tetrads, within pollen mother cells. Their method of formation shows them to be microspores, and the pollen sac is consequently a microsporangium and the stamen a microsporophyll. The nuclear and cell divisions within the pollen grain are stages in the germi- nation of this microspore to form the male gametophyte. G. The carpellate cone at the time of pollination and its scales. These cones appear on the new growth from the terminal buds in the late spring at the same time as the staminate cones. They are borne singly or in groups of two or three at the tips of branches. Draw a habit sketch of the carpellate cones on the new growth. 1. Draw a side view of the carpellate cone, showing the spiral arrangement of the cone scales. Each scale is be- lieved to be a group of fused carpels or megasporophylis. 2. Detach acone scale carefully and draw it viewed from the inner face. Note (a) the two ovules at either side of its base, each with two horn-like appendages ; (6) a point on the cone scale above the ovules and between them. — 3. Draw a side view of the cone scale, noting a small bract in the axil of which the cone scale is borne. ‘The ovule is a megasporangium with a protective envelope, the integument, but the evidence for this conclusion can only be understood after the study of sections of later stages (see J). H. The year-old carpellate cone and its scales. Search for year- old cones, establishing their identity by their position on 156 J. TYPE STUDIES the branches. Compare their size and texture with the car- pellate cones at the time of pollination. Draw a side view of the cone, if time permits, and then detach a scale and draw two views as described in G, 2, 3, noting and comparing the relative position of the structures described there. . The two-year-old carpellate cone and its scales. Search for two-year-old cones, establishing their identity by their posi- tion on the branches. Compare their size and texture with the year-old cones and the cones at the time of pollination. 1. Draw a side view, if time permits. 2. Cut into the cone with a heavy knife, carefully detaching one of the scales. Draw the scale as viewed from the inner face, noting (a) that the ovules are ripening into winged seeds, the wings developing from a tissue that separates from the inner face of the scale; (0) the relative position of the point, above and between the maturing seeds. 3. Draw a side view of the cone scale to show the position of the bract in the axil of which the cone scale is borne and the relation of parts in comparison with the cone scale at the time of pollination. The ovule on the year-old cone. Sections of the ovule on the scales from a year-old cone may be cut free-hand, but stained microtome sections are much more satisfactory (Sec. 212). They should be cut lengthwise of the ovule and per- pendicular to the surface of the scale. Observe: 1. The enveloping integument meeting at-the tip of the ovule where there was formerly an opening, the micropyle, at the time of pollination. 2. Within the integument and below the micropyle the pollen chamber in which germinated pollen grains may be found sending their tudes into the interior of the ovule. 3. A conical structure, the nucellus, into which the pollen tubes have grown, lying within the integument. 4, A large area in the interior of the nucellus, called the embryo sac, which contains a delicate tissue, endosperm, Pitts THE PINE 157 and at the micropylar end several reduced archegonia with very large eggs. Show in an outline drawing the relations of the structures described above, treating such details as are possible in separate figures. The endosperm, with the archegonia and eggs, constitutes the Semale gametophyte, derived from a megaspore which became the embryo sac. The megaspore was one of a group of four cells, tetrad, formed in the interior of the nucellus, which is conse- quently a megasporangium. The integument is a special protec- tive envelope, possibly comparable to an indusium. The pollen tubes are later developments of the male gametophytes formed by the germination of the microspores or pollen grains. The morphology of the cone scale has been and is still a diffi- cult problem. Because of the arrangement of the fibro-vascular bundles in the scale there are reasons for believing it to be a group of fused megasporophylls or carpels, probably two fertile carpels, each bearing an ovule, and possibly a third sterile carpel represented by the point on the scale, situated above and between the ovules. The cone scale is therefore much more complex than the stamen. According to this theory the cone scale is a group of megasporophylls in the axil of a bract, and the carpellate cone is not a simple cluster of sporophylls arranged along a shoot (like the staminate cone), but is a compound structure consisting of groups of sporophylls in the axils of bracts. The staminate cone has the same morphology as the cones of club mosses and some simple flowers of angiosperms, but the carpellate cone is comparable to an inflorescence, or cluster of flowers, each cone scale representing a highly modified flower. K. The gametophytes. A full study of the gametophytes of the pine would require the examination of stages in material covering many months of development, which is impracticable in a general course. In slides of the stage treated in J it will be possible to determine the following structures : 1. In the female gametophyte: (a) the delicate cell structure of the endosperm; (b) the protoplasmic structure of the large egg with its 158 TYPE STUDIES prominent nucleus; (c) frequently the presence of a single canal cell (ventral canal cell) above the egg ; (d) a layer of cells differen- tiated from the endosperm, forming a jacket around the egg ; (e) one or two tiers of cells, four cells in a tier, above the egg, and repre- senting the neck of the much-reduced archegonium (see Principles, Fig. 300, D). 2. In the male gametophyte: should the tips of pollen tubes be found entering the necks of archegonia they may be expected to show two large sperm nuclei, and possibly the remains of the tube nucleus, now degenerating, with that of the stalk ceil also. L. The seed. Take seeds from an open cone. It generally opens at the end of the third summer, when the cone is approxi- mately two years and three months old. Sketch to show the wings. Section such seeds, or, better still, cut open.some of the large edible seeds of the nut pines, pifion, obtainable from fruit dealers. Note: 1. The tough seed coat, or testa, which is the ripened integu- ment, and beneath the testa a membranous seed coat which is largely the remains of the nucellus. 2. The endosperm, filling the seed except for the embryo. The former is a development from the endosperm of the embryo sac and consequently gametophytic in character. 3. The straight embryo,- developed from a fertilized egg, attached to the micropylar end of the seed by a suspensor and imbedded in the endosperm. The embryo consists of a short hypocotyl, bearing above a circle of cotyledons. Construct a diagram of a lengthwise section of a seed to show these structures in relation to one another. M. The germination of the seed. The pine seed germinates rather slowly, but it will be of interest to plant some and watch them as they sprout, comparing them with such seed- lings of the angiosperms (squash, bean, pea, corn, etc.) as may have been studied. REFERENCE. Principles, Secs. 350-356. Quxstions. What are the growth habits of the pine? What are the peculiarities of its foliage ? its adaptation to drought JUNIPER AND ARBOR VITE 159 and cold? Where is the resin and turpentine formed ? Can you suggest any advantage to the plant in its produc- tion? How and when is pollen formed and how abundantly ? How does it reach the ovule? What is the history of the carpellate cone after pollination? When are the seeds tipened ? What is the morphology of the pollen grain ? Describe the male gametophyte, with its habits, after the germination of the pollen grain in the pollen chamber. Describe the structure of the ovule. Describe the female gametophyte and its life habits within the nucellus (mega- sporangium). From what does the embryo arise and how does it obtain the food for its development? How many generations are represented in the tissues of the seed? Describe the life history, distinguishing between the sexual phases, gametophytes, and the asexual phase, sporophyte. Draw and arrange a series of diagrams illustrating the chief stages throughout the life history, using two colored pencils to designate the gametophytic and sporophytic generations respectively (App. 18). Construct a life-history formula that will express this succession (App. 18). 139. The morphology of the juniper and arbor vite. The juniper (Juniperus) and arbor vite (Thuya) are interesting types to study comparatively with the pine: (1) as regards the arrangement and forms of the leaves and conse- quent appearance of the foliage ; (2) as regards the structure of the carpel- late cones, whose scales are opposite instead of being distributed spirally, and present other peculiarities of structure and habits of ripening ; (8) with reference to the special characteristics of the stamens. These genera pre- sent a higher type of gymnosperm evolution in these respects than the pine. Certain cedars (Cupressus) are equally good for this comparative study. THE ANGIOSPERMS, OR ANGIOSPERMA 140. The morphology of the angiosperms. The general morphol- ogy of the angiosperms, including roots, stems, leaves, flowers, and fruits, together with many principles of plant physiology, have been treated in Part I, The Structure and Physiology of 160 TYPE STUDIES. Seed Plants. The outlines presented here will deal entirely with the gametophyte generations and the organs of the sporo- phyte, stamens and pistil (composed of carpels), especially con- cerned with their development. For outlines covering general flower structure see Secs. 44-46. An outline for a general type study of an angiosperm such as the lily is presented in Sec. 162. 141. The lily studied with reference to its gametophytes (App. 22). The lily is a favorite subject for the study of pollen formation and the develop- ment and fertilization of the embryo sac, partly for its clearness and partly for the relative ease with which material may be obtained. Other types of the lily family, such as the trillium, the tulip, the Roman hyacinth, etc., are also good. Satisfactory studies on the gametophytes of the angiosperms require microtome sections of the structures involved. Directions for the preparation of these are outlined in Sec. 212. The wild lilies, such as Lilium philadelphicum, furnish excellent material, but various cultivated lilies are equally good or better. A. The stamen of the lily. Dissect away the perianth of the lily flower to show the stamens and pistil. 1. Observe the arrangement of the stamens around the pistil. Draw a stamen to show the stalk (filament) and the attachment of the anther. 2. Note how the pollen is discharged from the anther. . Draw a pollen grain under h.p. to show the markings on its wall. 4. Section the anthers and observe that the pollen is developed in four locules, or pollen sacs, running lengthwise of the anther. 5. In microtome sections properly stained (Sec. 212) note that the pollen grains will show a large central nucleus, tube nucleus, and at one end the generative nucleus, which gives rise later to two sperm nuclei. -B. The development of pollen. To obtain the stages of pollen formation anthers must be taken from very young unopened buds, the stamens of which when cut across exude a watery fluid from the pollen sacs. Should the fluid be milky or yellowish the stamen is too old and will certainly contain pollen grains. Stamens of the proper age must be fixed, imbedded, and cut lengthwise on the microtome and stained as described in Sec. 199, D. Such preparations will show various stages in development and division of the pollen mother cells to form the pollen grains in groups of four, or tetrads. The following conspicuous stages are likely to be found and should be drawn. 1. The spore mother cells before division, with their nuclei in a resting condition. They constitute a spore-forming tissue (archesporium), oo 6. THE LILY 161 and, increasing in size, gradually round off and separate from one another. . Synapsis, a very common stage in which the chromatin within the nucleus of the spore mother cell will be found collected in a dense mass, generally near the nucleolus at one side of the nucleus. Synapsis is a very important stage, since it appears to be charac- teristic of the time when the sporophyte number of chromosomes is reduced by half to the number of the gametophytes (see Principles, Sec. 334). The gametophyte number of chromosomes in the lily is twelve, and first appears in the nuclear divisions within the pollen mother cell and the embryo sac (D, 3). . The first nuclear division, or mitosis, where a large spindle will be found within the spore mother cell, and the chromosomes will be either at the center, forming the equatorial plate (see Principles, Fig. 302, B), or separated into two groups of daughter chromosomes that pass to the poles of the spindle. . Two daughter nuclei in a resting condition following the first mitosis and the division of the spore mother cell into two daughter cells. . The second nuclear division, or mitosis, in which two spindles are formed simultaneously in the two daughter cells, resulting in four daughter nuclei. The final division of the spore mother cell into four daughter cells, forming a tetrad, each of which becomes a pollen grain. The processes of pollen formation are identical in all essentials with those of spore formation in the bryophytes and pteridophytes, and show that the pollen grains are microspores, —a conclusion sustained by their further de- velopment into reduced male gametophytes. The pollen sac is therefore a microsporangium and the stamen a microsporophyll. Pollen formation in the lily and related types is an attractive subject for the study of nuclear and cell division in the higher plants. C. The pistil of the lily. Draw the pistil in side view, showing : 1. The ovule case, or ovary, below; the style above, bearing the three- lobed stigma, or receptive region, for the pollen. Note that the ovule case is three-angled, and the position of the angles with reference to the lobes of the stigma. . Cut sections of the ovule case both from flowers which have recently opened and from those whose perianth has been withered several days. Under 1.p. note the three lvcules, or chambers, of the ovule case and the ovules within them. Show their position in an outline drawing. The three locules of the ovule case and the three lobes of the stigma present evidence that three carpels are involved in the formation of this 162° TYPE STUDIES pistil. That the carpels are megasporophylls is proved by the structure and development of the ovules (see D). D. The development of the ovule and embryo sac. These can best be stud- ied from microtome cross sections of the ovule cases prepared as described in Sec. 212. The ovule cases from open flowers contain mature embryo sacs. Those collected from three to four days after polli- nation may show stages in fertilization (the time varies in different lilies). Stages in the development of the embryo sac must be sought in unopened buds along with or somewhat later than stages in pollen formation. The following stages are likely to be found in material of graduated ages and should be drawn: 1. Young ovules with the two integuments beginning to develop around the nucellus. The end of the nucellus will be exposed, and at the tip, under a layer of cells, is to be found a large cell with deeply staining protoplasmic contents and conspicuous nucleus. This becomes the embryo sac. 2, Later stages show the inner integument grown beyond the nucellus and forming the micropyle at the tip of the ovule. The outer integument extends around on the outside nearly to the end of the inner integument. 8. The embryo sac, gradually enlarging with the growth of the ovule, is the seat of three nuclear divisions, or mitoses, by which the number of nuclei is increased to eight. The gametophyte number of chromo- somes, twelve, appears in the first of these mitoses as in the pollen mother cell (B, 2). The first two of these mitoses have peculiarities (see Principles, Sec. 860 and footnote) which show. that they are of the same kind as those characteristic of pollen formation and spore formation when tetrads are developed within mother cells. But tetrads are no longer developed in the nucellus of the lily, although present in the ovules of many other angiosperms (see Principles, Fig. 304). The four nuclei resulting from the first two mitoses in the embryo sac of the lily, although comparable to megaspore nuclei, have all become included in the very much reduced female gametophyte that is developed in the embryo sac. 4, The eight nuclei of the mature embryo sac become distributed as follows: (a) three nuclei form the egg apparatus at the micropylar end of the sac, one being the egg nucleus and lying slightly below and between the other two, which are termed synergids; (b) three nuclei form the group of antipodal nuclei at the opposite end of the sac; (c) the other two, called polar nuclei, approach one another in the center of the sac. This is the structure of the mature female gametophyte (see Principles, Fig. 306, B). POLLEN GRAIN OF ELDER 163 The first two mitoses in the embryo sac of the lily show that it is a mega- spore mother cell in this plant (as also in related types), which later contains the female gametophyte. The nucellus is therefore a megasporangium and the carpel a megasporophyll. E. Fertilization and double fertilization. Stages showing the nuclear fusions of fertilization and double fertilization are, of course, not common, but one preparation will serve for a demonstration of the processes. The pollen tube brings two sperm nuclei into the embryo sac. One of these unites with the egg nucleus, fertilizing it, and the other unites with the two polar nuclei, forming a triple fusion which results in the large endosperm nucleus in the center of the sac (see Principles, Fig. 307). F. The development of the. embryo and endosperm. The fertilized egg nucleus with surrounding protoplasm becomes the fertilized egg, and, forming a cell wall about itself, proceeds to develop the embryo sporo- phyte at the micropylar end of the embryo sac. The endosperm nucleus gives rise through successive divisions to a very large number of nuclei, which become distributed in the layer of protoplasm which lines the em- bryo sac (see Principles, Fig. 808). Cell walls are later formed between these nuclei, and the embryo sac becomes filled with a delicate tissue called the endosperm, in which the developing embryo lies imbedded. It is important to note that the endosperm of all the angiosperms is a development following fertilization and therefore not strictly comparable to the endosperm of gymnosperms, as illustrated in the pine, which is formed before fertilization and is therefore strictly gametophytic in charac- ter without the complication of double fertilization on the union of polar nuclei. The group of three antipodal nuclei in the embryo sac of the angiosperms may represent the endosperm of gymnosperms, but this is not fully established. The female gametophyte of the angiosperms is much simpler than that of the gymnosperms, having only eight nuclei, one egg, and no clearly defined archegonium. The male gametophyte of the angiosperms is likewise simpler, containing only three nuclei, two sperm nuclei, and the tube nucleus. 142. The pollen grain, or microspore of the elder. Some pollen grains at maturity contain. the male gametophytes much further advanced than is shown in those of the lily. This is well illustrated in the elder (Sambucus). Unopened stamens should be fixed and preserved in alcohol. The anthers of such may be teased apart in water, allowing the pollen grains to escape. When stained with eosin these grains will be seen to contain three nuclei, a tube nucleus lying freely in the center of the cell, and two sperm nuclei somewhat at one side, surrounded by denser protoplasm, forming two male cells (see Principles, Fig. 305). The nuclear divisions are completed in this male gametophyte at the time the pollen is shed, and the only further 164 TYPE STUDIES development is that of the pollen tube, which carries the sperm nuclei to the embryo sac. Microtome sections of the ripe anthers of the elder (Sec. 212) will give excellent preparations of this interesting condition. The pollen grains of many plants, as the lily, contain but two nuclei at the time of pollination. These are the tube nucleus, above mentioned, and the generative nucleus, which later gives rise to the two sperm nuclei. 148. Capsella, the shepherd’s purse, studied for the development of the flower, ovule, pollen tube, and embryo (App. 23). The shepherd’s purse is in many respects an excellent type for a general study of a dicotyledonous seed plant, although the flower is rather small. It is a particularly good subject for the study of the topics indicated in the above heading. A. The development of the flower. Tease apart in a drop of water, under a dissecting microscope, the extreme tip of a flower cluster (which is a raceme), to obtain the youngest flowers (microscopic) just below the growing point. Older stages may be cleared by adding potash solution if necessary (Sec. 169). Search for the following stages and draw: 1. The young flowers appearing as small protuberances just back of the growing point. 2. A circle of sepals developing at the tip of the young flower. 8. The growth of the sepals over the tip of the flower and the origin of the stamens in a circle within. 4. The development of two carpels, more or less united below, at the tip of the flower. 5. The late appearance of the petals between the sepals and stamens, after all the other parts of the flower are present. 6. The final folding one over the other of the sepals in the young bud, the growth of the petals, the differentiation of the stamens into anthers and stalks (filaments), the union of the carpels above to form the pistil, with the ovule case or ovary below, in which the ovules ap- pear as outgrowths along the surface. 7. Microtome sections cut lengthwise of the tip of the raceme (Sec. 212) give excellent stages in the development of the flowers, and especially the closing together of the carpels to form the pistil and the origin of the ovules along their inner surface within the ovule case. B. The development of the ovule. Pick to pieces some of the youngest flowers that can be easily seen with the unaided eye. Open the ovule ; cases, or ovaries, in a drop of water, with the point of a needle, thus exposing the ovules. A variety of stages will be presented, Search for the following and draw: 1. The young ovule, consisting of the nucellus at the end of a short stalk, with the two integuments just beginning to appear like collars at its base. CAPSELLA 165 2. A stage in which the integuments are further developed, the outer arising somewhat below the inner one. The large embryo sac is gener- ally evident at this time in the center of the nucellus. 8. A stage in which the outer integument has grown over and beyond the inner one, so that only the extreme tip of the nucellus is seen. At this time the ovule begins to bend over at its basal region. 4. Finally the outer and inner integuments grow completely over the nucellus, almost meeting beyond to form the small opening called the micropyle. Meantime the bending of the ovule brings the micro- pyle close to the stalk of the ovule, so that the latter is therefore completely bent on itself (campylotropous). This condition will be found in the ovules of rather young, unopened flowers, and such preparations may be cleared with potash. C. The development of the pollen tubes. Mount the pistil of an open flower in water and examine the stigma under m.p. Note the papille on its surface, and among the papille the germinating pollen grains, which will be found sending tubes into the tissue of the stigma. Clear with potash if too opaque. Draw. D. The development of the embryo. Remove the pistils from flowers, the petals of which have begun to wither. Open the ovule case with a needle and mount the ovules in a potash solution. The embryo may sometimes be clearly seen‘lying in the embryo sac. Press gently on the cover glass and the embryo will be crushed or squeezed out. Note the row of cells forming the suspensor, the lower one of which is much enlarged. Make a number of preparations of younger and older ovules, which are likely to show the following stages, and should be drawn : 1. The suspensor before the formation of the embryo, consisting of a filament attached by a large basal cell near the micropylar end of the embryo sac. 2. The development of the embryo, beginning at the free end of the suspensor, by the formation of walls in three planes, thus differen- tiating a globular structure. 8. The later growth of the embryo, with the appearance of two cotyle- dons, and the development of the root at the point where the embryo is attached to the suspensor. 4, Lengthwise microtome sections of the ovules (Sec. 212) will show the relation of the developing embryo to the enlarged and curved embryo sac with its endosperm, and the integuments of the ovule (see Principles, Fig. 309, H). 166 TYPE STUDIES Part II of this manual presents a series of type studies fur- nishing an outline of the comparative morphology and life histories of plants, upon which are based systems of classifica- tion and various theories of the evolution of the groups. While botanists are in general agreement on the principal lines of plant evolution and in full accord as to the fact that there has been an evolution, the details of the history of development must always remain speculative problems for the reason that evolutionary processes have been in force since very early geo- logical ages and the records of plant life in former periods, pre- served as fossil remains, are relatively scanty. Consequently, while it is often of great interest to draw or diagram lines of plant evolution indicating relationships of groups, such outlines should generally be considered as provisional attempts to help forward discussion rather than as expressions of final judgment. Such a study of comparative morphology as may be framed from the matter presented in Parts I and II forms an excellent foundation for detailed work in plant physiology and the funda- mentals of ecology. Indeed, it may, be said to be essential to extended work in these subjects, for a knowledge of structure must always precede a study of functions and life activities. When a course in botany is planned to begin with type studies such as may be chosen from Part IJ, the usual procedure would be to supplement or end the course with the more special exam- ination of the seed plants. These latter studies may be not only morphological and physiological but also ecological, and would involve a selection of such topics and experiments from Parts I and III as seem best suited to the conditions under which the work must be given. Part III ECOLOGY PARASITIC AND CARNIVOROUS PLANTS 144. Field study of parasites. A. Study out of doors any parasitic seed plants that you can find. In most parts of the country the dodders are of much more fre- quent occurrence than any other parasites among the higher plants. Frequently several species of dodder can be found. Note and collect the host plants on which the parasite grows and observe the mode of attachment between parasite and host. Find out whether the host is at all injured by the parasite. B. If possible transplant thriving specimens of the parasite upon new kinds of host and see whether they will grow there. Collect seeds of any parasitic plants found, germinate the seeds, and make studies of the behavior of their seedlings. Are the seedlings green at first ? Rererence. Kerner-Oliver, 2. 145. Field study of carnivorous plants. A. Examine any carnivorous plant which you can find growing sponta- neously (various species of Drosera and Sarracenia are the kinds most widely distributed). Make notes on the total number of insects cap- tured by a single plant and by a single leaf. B. In the case of Drosera study and draw various leaves, some expanded and some closed over insects. C. Put into 50 per cent alcohol or 2-4 per cent formalin solution all the insects obtained from any one kind of carnivorous plant and bring them to the laboratory for determination of the groups. 146. Laboratory study of carnivorous plants. A. Put living flies in the pitchers of Sarracenia containing their usual amount of liquid, and note whether any of the flies escape, or what finally becomes of them. B, Feed leaves of Drosera with bits of raw meat, particles of cheese, very small insects, bits of sand, or broken glass. Place the objects very 167 168 ECOLOGY carefully on the tips of the glandular hairs, note what movements, if any, occur, make sketches of the leaf in various positions, and keep a complete daily record of the behavior of the leaf until it returns to its ordinary form. Rererences for Secs. 145, 146. Darwin, 64; Darwin and Acton, 11. HOW PLANTS PROTECT THEMSELVES FROM ANIMALS 147. Field study. If possible, visit pastures grazed by horses, cattle, or sheep, and large barnyards where weeds are abundant. Make a sketch map of a small part of the pasture-or barnyard, showing the clumps of weeds that have been left uneaten ; number the clumps, and at the bottom of the map indicate what plants make up each group. Study the characteristics of some plants that are not usually eaten, and state the most obvious means of protection of each plant. 148. Laboratory study. Examine in detail any of the plants of your region which are. left unharmed by grazing animals, and make out a tabular list of the protective equipment of each plant. Use the microscope to study and make sketches of cutting edges of grasses and sedges and the rough or stinging hairs or spines of such plants as mulleins, nettles, thistles, and so on. Do not taste plants suspected of being poisonous, but try those which are known not to be so. If some plants are attacked by insects, though not by grazing quadrupeds, make a note of it. Record the notes in a table like the one on the following page.! ‘ POLLINATION OF FLOWERS 149. Studies in insect pollination.** The student cannot gather more than a very imperfect knowledge of the details of cross pollination in flowers with- out actually watching some of them as they grow and observing their insect visitors. If the latter are caught and dropped into a wide-mouthed, stop- pered bottle containing a bit of cotton saturated with chloroform, most of them may be identified by any one who is familiar with our common insects. The insects may be observed and classified in a general way into butterflies, moths, bees, flies, wasps, and beetles, without being captured or molested. Whether these out-of-door studies are made or not, several flowers should be carefully examined and described as regards their arrangements for attracting and utilizing insect or bird visitors. 1 It will probably be necessary for the instructor to determine for the student many of the species studied. ADAPTATIONS FOR POLLINATION 169 Means or Prorection oF CERTAIN PLANTS ® ; b = » eo Q a B c eo so £4 g 3 3s an a) =| 5} os B |o % S jo (Sy a2 2 /BS/8e/ 2 | 2 seize] & [38 6 | &el/S Gl] a - | SAS H ° a] a a Qs S| oO ® 2) og ga B ® |eala S| 6 a5 " ~~ al cae Es ele fe | 2 |Fe tt Jimson weed (Datura) | x 2 x | x REFERENCES. Kerner-Oliver, 2; Ludwig, 51. OuTLINE FoR Stupy or ADAPTATIONS FOR POLLINATION! [ 1, greenish, nectarless, inconspicuous perianth ? I. Is the flower 2, appearing before the leaves ? characterized by | 3, dry, dusty pollen? U4, feathery stigmas ? If several of these characteristics are found, it is probably wind-pollinated. IL. Is the fower 5, curving stamens which bring the anthers into con- pineaeeioed y tact with the stigma? If so, it is self-pollinated m (see Principles, Fig. 380).? 1 Pollination by water is not discussed here, as the cases are rather few and material is not usually available for study. 2 Many self-pollinated flowers are not easily distinguished as such. 170 ECOLOGY ( 6, odor ? 7, color (not green) ? 8, nectar ? 9, sticky pollen ? 10, opening during only part of the day ? 11, bilateral symmetry ? 12, facilities for insect visitors? (See Principles, III. Is the flower Figs, 324, 325.) 1 characterized by | 18, mechanism for holding visitors imprisoned until covered with pollen ? 14, mechanism for pollinating visitors? (See Prin- ciples, Figs. 331, 332.) 15, two or three lengths of stamens and pistils (dimorphism or trimorphism) ? 16, unequal maturing of stamens and pistil (dichog- amy) ? ff-any of these characteristics (III) are found, the flower is pollinated by insects, birds, or other animals. _ Make a list of all the attractions displayed by the flower examined, and if possible find out what visitors it receives and how their visits are utilized. (17, a sticky stem or flower stalk ? 18, water reservoirs along the stem ? 19, a slippery flower stalk ? 20, a sticky-hairy or slippery nodding calyx ? 21, a corolla with closed throat ? 22, stamens or pistils covering the nectaries ? 23, a long calyx or corolla tube ? 24, long spurs, with the nectar stored at the bottom ? Is the flower under examination pro- tected from unde- ¢ sirable visitors by means of XN * Make a list of these protections, and if possible study their operation. The following list includes a considerable number of the most accessible flowers of spring and early summer, about which it is easy to get informa- tion from books. List oF InsEct-PoLLINATED FLOWERs ? I 1. Flax. . .. . . . Linumiusitatissimum . . . . . . Knuth 2. Missouri currant. . . Ribesaureum. . . . . . . . . Knuth 1 For very many other devices for pollination see Knuth-Davis, 62, and Kerner-Oliver, 2 2 The plants in this list are arranged somewhat in the order of the complexity of their adaptations for insect pollination, the simplest first. It would be well for # ADAPTATIONS FOR POLLINATION 171 3. Snowberry . . . . Symphoricarpus racemosus . . . . Knuth 4. Lilac . . é Syringa persica. . . . . . . Knuth 5. Periwinkle - - Vinca minor . : Knuth 6. Mignonette . . . . Resedaodorata . . : Knuth 7. Lily of the valley . . Convallaria majalis : . . Knuth 8. Dead nettle . . Lamium album... . . Lubbock 9. Bleeding heart . Dicentra (Diclytra) spectabilis . Knuth 10. Columbine . . . . Aguilegia vulgaris . . . . Knuth 11. Monkshood . . . Aconitum Napellus. . . . . Knuth II 12. Larkspur. . . . . Delphinium elatum, D. consolida . . Knuth 18. Herb Robert. . . Geranium robertianum . . . . Knuth 14. Pink . . . . .,. Dianthus (various species). . Knuth 15. Fireweed. . . . Epilobium angustifolium . : Gray 16. ‘Nasturtium’. . . Tropeolum majus . . . . Newell, Lubbock 17. Pansy. . . . Violatricolor. . . . 1. we, Knuth 18. Heal-all . . . . Brunella (Prunella) vulgaris. . . . Knuth 19. Groundivy . . . Nepeta Glechoma . . Knuth, Newell 20. Lousewort . . Pedicularis canadensis . . Knuth, Newell 21, Snapdragon. . . . Antirrhinum majus . : . . . Knuth 22. Iris. . . . . . . Trisversicolor . . . . . . . Newell 23. Bellflower . . . . Campanula rapunculoides , . Knuth 24, Horse-chestnut . . . Aisculus Hippocastanum. . - Newell III 25. Yarrow . . . . . Achillea millefolium . . . . . . Knuth 26. Oxeye daisy . Chrysanthemum Leucanthemum . . Knuth 27. Dandelion . . . . Taraxacum officinale . . - Knuth, Newell IV 28. Barberry. . . . . Berberisvulgaris . . . . . . Lubbock 29. Mountain laurel . . Kalmialatifolia. . . . 1. . + Gray each student to take up the study of the arrangements for the utilization of insect visitors in several of the groups above, numbered with Roman numerals. Ex- planations of the adaptations can be found in the works cited by abbreviations at the right. Knuth stands for Knuth-Davis’s Handbook of Flower Pollination, 62; Lubbock, for British Wild Flowers, considered in Relation to Insects ; Gray, for Gray’s Structural Botany ; and Newell, for Miss Newell’s Outlines of Lessons in Botany, Part II. Consult also Weed’s Ten New England Blossoms, and Kerner- Oliver, 2. The instructor may find it necessary to identify most of the species. 172 30. 31. 32. 33. 34, 35. 36. 37. 38. 39. 40. 41. 42. ECOLOGY v White clover Trifolium repens +. Knuth Red clover Trifolium pratense . Knuth Locust. , Robinia Pseudacacia Gray Wistaria . Wistaria sinensis Gray Vetch . Vicia Cracca . Knuth Pea. Pisum sativum Knuth Bean Phaseolus vulgaris Gray Groundnut Apios tuberosa Gray VI Partridge berry . Mitchella repens . A Gray Primrose . Primula grandiflora, P. officinalis . Lubbock Loosestrife Lythrum Salicaria . Gray VII Milkweed . Asclepias Syriaca Knuth, Newell Vill Lady’s slipper Cypripedium acaule . Newell HOW PLANTS ARE SCATTERED AND PROPAGATED 150. Field study of vegetative propagation. A. Collect, by digging them up, sketch, and describe any underground stems of use in multiplying plants. There are many rootstock-produ- cing species, such as June grass, quick grass, Bermuda grass, Canada thistle, common sorrel (Rumex Acetosella), wild iris, wild ginger, sweet flag, various sunflowers, and some mints. If possible dig away the earth from one side of a hill of potatoes, and sketch some of the roots, subterranean branches, and tubers. Bulbs are borne by a large pro- portion of the members of the lily family. B. Make sketches to illustrate the spread of plants by rooting branches, as in the raspberry, strawberry, and cinquefoils. C. In any cleared or partially cleared bit of woodland note the way in which some trees produce a crop of sprouts from the stump. D, Examine the neighborhood of black locust trees (Robinia), silver- leaved poplars, or balm of Gilead poplars, to find out how far these trees ‘spread by the root.””? Dig up a young sprout, with a piece of the parent root, and sketch it. Rererence. Beal, 63. DISSEMINATION OF SEEDS 173 151. Laboratory study of vegetative propagation. A. In moist earth or sand plant pieces of potato tubers, each containing one or more “eyes,” and others without eyes. Note results and sketch any plants that are produced. B. Plant bulbs and bulblets of onion and note the compara- tive growth of the plants produced. C. In sand which is kept moist plant cuttings of any of the following plants: “geranium,” Tradescantia, willow, cotton- wood currant, raspberry, blackberry, and grapevine. When the cuttings have rooted, sketch some of them, and decide whether the roots spring indifferently from any part of the stem. D. If obtainable, put some vigorous Bryophyllum leaves on moist sand, cover with a bell glass, and sketch the leaf with young plants, if any appear. 152. Field study of dissemination of seeds. A, Examine the region about any tree or shrub which has no near neighbors of its own kind, and try to trace the distance to which its seeds have been carried. In case enough seedlings are found to war- rant it, make a map to show their distribution with reference to the parent tree. B. Discuss the means by which the seeds have been carried. C. Watch such trees as elms, maples, lindens, willows, sycamores, and cottonwoods, when the fruits or seeds are fully ripe, and find out how far they travel. What trees hold many of their seeds after they are fully ripe ? What are the advantages of this ? D. Look for as many contrivances for seed dispersal as possible, and classify the plants studied into those with fruits or seeds dispersed : (1) by wind. (2) by water. (8) by animals. (4) by some contrivance for shooting or slinging the seeds. REFERENCE. Kerner-Oliver, 2. 153. Laboratory study of dissemination of seeds. A. Find out which of the wind-carried fruits and seeds fall most slowly from the laboratory ceiling to the floor. 174 ECOLOGY B. Test some of the following water-tarried fruits and seeds to see which will float longest: aquatic grasses, rushes, and sedges, polygonums, water dock, bur reed, arrowhead, water plantain, pickerel weed, alder, buttonbush, water parsnip (Sium), water hemlock (Cicuta), water pennywort. (Hydro- cotyle), lotus (Nelumbo). C. Sketch and describe in detail the fruit or seed which appears to be best adapted to each of the four modes of dis- persal above mentioned (Sec. 152, D). COMPETITION AND INVASION 154. Field study of competition. ; A. Find a spot in which many weed seedlings have sprung up, stake off one or two square feet, count the plants, and then watch their growth for as long a period as possible. Stake off a similar plot, pull up all but one or two of the weeds, and compare the growth of these plants with that of the crowded ones. B. Beginning as soon as weed seedlings start in the spring, stake off a square foot of very weedy ground, pull up and count all the seedlings which grow on the plot, continue the count as others spring up, and make a list of the kinds obtained 1 and the total number of each kind. C. Allow any large, vigorous weeds to grow up among lettuce, radish, carrot, or other seedlings, and notice which set of plants prevails. Give as many reasons as possible for this result. REFERENCES. Clements, 59; Principles, Chapter XXXIV. 155. Field study of invasion.* * A. Look for places in which pastures or mowing fields are beginning to **run out,’’ and daisies (Chrysanthemum), sorrel (Rumez), cone flowers, and similar weeds are taking the place of the grass. Look for a lawn that is too much shaded and becoming filled with chickweed or other weeds. B. Find a pond in process of drying up and notice what changes are taking place in the character of the vegetation. C. Study an abandoned strawberry bed. D. Examine a clearing in which young saplings have begun to grow to a height of 15 or 20 feet. In every case make a list of the older inhabit- ants of the territory 1 and of the newcomers! and give as many reasons 1 Identified by the instructor. of ECOLOGICAL CLASSES 175, as possible for the prevalence of the latter. State what would prob- ably be the condition of the piece of ground examined if left unmo- lested for ten years or more. Rererencus. Clements, 59; Principles, Chapters XXXIV, XXXV. PLANT SUCCESSIONS 156. Field study of successions. A. Examine a field of wheat, rye, oats, or barley, when the grain is not more than a foot high, again when it is ready for reaping, and finally as long as possible after reaping, before fall plowing or frost destroys the plants upon it. Make a list of all the plants that can be recog- nized at each period, and note which ones are in blossom or in fruit. B. Study wood lots with full-grown trees upon them, others from which the trees have recently been cut off, others still which have been cleared for many years. Make a general statement of the kinds and relative numbers of seed plants of all sorts found in each case. C. If possible, study the changes in the vegetation of old fields allowed to grow up to weeds and bushes. Draw up a general account of what you have found to be the order of succession of plants in grain fields, in cleared woodland, and in abandoned fields. Try to give some reasons why the plants succeed one another in the order actually observed. Rererences. Clements, 59; Schimper-Fisher, 56; Warming-Graebner, 57; Principles, Chapter XXXV. ECOLOGICAL CLASSES 157. Field study of ecological classes. * * A. Examine the vegetation of any accessible lake, pond, marsh, or river, of ordinary woods, thickets, and grass lands, and of the driest areas in the region, such as sand hills or dunes, barren knolls or banks, ledges or outlying masses of rock. Select some of the typical inhabitants of each region and make a list of: a) living only in water. @) Hyarophytes{ living either in water or in very wet soil. (2) Mesophytes. (8) Xerophytes. 176 ECOLOGY Describe the characteristics of each group, giving atten- tion to all the vegetative parts of the plant body. B. In woods or thickets make lists of the sun plants and shade plants, and classify the trees roughly as regards their tolerance of shade con- ditions. Measure the relative illumination of some of the plants which live in the deepest shade by the method given in Exp. XXVIII. C. Study the distribution of plants as related to the character of the soil, looking for species characteristic of limestone and of sandy soils. If possible, find assemblages of plants in loose sand, especially of sand dunes, and report on their peculiar form and habits. If there are accessible localities for halophytes, examine the vegetation of salt marshes, of the sea beach, or of ‘‘alkali’’ lands, and describe some of the most noticeable characteristics of these plants. D, Examine and report on any epiphytes that may be found. Rererences. Clements, 59; Warming-Graebner, 57; Pound and Clem- ents, 58. 158. Labotatory study of ecological classes.** Make a sketch of at least one typical member of each of the three principal ecological classes as based on water requirements. Make careful studies of any available material and ‘discuss as many as possible of the following topics : A. Relative importance of the root system in aquatic plants. B. Special provisions for photosynthesis, respiration, and circulation of air in the plant body of aquatic plants. C. Comparison of the structure of the leaves of deciduous trees, broad- leaved evergreens (such as holly, live oak, and some rhododendrons), -and needle-leaved evergreens. In this study pay especial attention to the total area of the three kinds of leaves, to their relative thickness, to the thickness of the cuticle and the epidermis, and to the protection of stomata by their position at the bottom of grooves or pits in the epidermis. D. Appearance of plants in their resting condition (during winter’s cold or summer’s drought) in any available bulbous- or tuberous-rooted species or in fleshy-rooted biennials (e.g. parsnips, beets, or carrots). E. Characteristics of plants slightly, moderately, and decidedly xero- phytic, as illustrated by wild plants of the neighborhood or (if these are not obtainable) by such species as Huphorbia splendens, houseleek, Echeveria, and cactuses. F. Difference in the appearance of a species grown in dry soil, with leaves exposed to warm, dry air, and that grown in damp soil under a PLANT FORMATIONS 177 bell glass (e.g. young plants of any xerophytic grasses, houseleeks, dandelions, shepherd’s purse, gorse !), G. Tolerance of salt, as determined by water cultures, in one to six per cent solutions of seedlings of ordinary garden annuals and seedlings of such halophytes as the salt-marsh grasses (Spartina and others), marsh rosemary (Statice), samphire (Salicornia), and saltwort (Salsola). The plants which live longest with the roots immersed in a solution of any given strength are the most decidedly halophytic. Rererences. Kerner-Oliver, 2 ; Schimper-Fisher, 56 ; Warming-Graebner, 57; Haberlandt, 33. PLANT FORMATIONS; ZONATION 159. Field study of formations.* * Visit any well-defined forma- tions that are readily accessible, such as lake or pond, marsh, river valley (“bottom land ”’), upland woods, hill or cliff side, wet prairie, dry prairie, and other formations. A. Note what are the characteristic plants of each kind of formation and, if necessary, collect specimens of these and bring them to the laboratory to be named. B. Make a detailed study of at least one formation, noting all that is possible of the physical conditions, especially of mois- ture and light, the habits of life, and mutual relations of the plants which compose it. For example, if the formation is a wooded one make lists of : (1) The trees, their grouping, relative height, relative density of shade, comparative number of each species, probable origin (e.g. means by which seeds were planted and source from which they came). (2) The larger shrubs. (3) The undershrubs. (4) Herbaceous plants. _ (5) Parasitic or saprophytic seed plants. If there is any law to account for the way in which the plants of (2), (3), and (4) are unequally distributed over the 1 Ulex. 178 ECOLOGY forest floor, try to establish it, noting especially their relation to the relative moisture of the soil and to light. Rererences. Clements, 59; Principles, Chapter XXXVII ; Warming-Graebner, 57 ; Schimper-Fisher, 56. Fic. 6. Model for grouping of drawings in type studies A, base of a plant of shepherd’s purse (Capsella bursa-pastoris), x 4; 7, the main root; B, upper part of the inflorescence, X 1; C, two leaves, —I from the upper part; II from the base of the plant, x 1; D, a flower, x 3; E, the same, with sepals and petals removed, Xx 3; F, petal; G, sepal; H, stamen, X 10; f, filament; an., anther; J, a fruit with one of the valves removed to show the seeds, X 4; J, longitudinal section of a seed, x 8; K, the embryo removed from the seed, X 8; J, the first leaves (cotyledons) ; s¢., the stem end- ing in the root; Z, cross section of the stem, x 20; /b., fibro-vascular bundle; M, a similar section of the main root, x 15; NW, diagram of the flower. — After Campbell 160. Field study of zonation. A. Select any locality where two or more plant formations meet and determine : 1. The characteristic plants of each formation, TYPE STUDIES, SEED PLANTS 179 2. What plants (if any) are common to two or more for- mations. 3. Some of the causes of the boundaries between formations, and whether these boundaries are fixed or shifting. B. Make a sketch map of the series of zones on the same gen- eral plan as that of Fig. 366 in Principles, and if possible secure one or more photographs of the series, with a large numbered placard set up in a prominent position in each zone. STUDY OF TYPES OF SEED PLANTS * 161. Family Pinacez.! 162. Family Liliacez. Any obtainable genus may be used to represent the family. Lilies, tulips, dogtooth violets (Erythronium), Scilla sibirica, or Roman hyacinths answer excellently. Since it is to be had of florists during the winter months and in gardens for a long time in spring, the lily of the valley (Convallaria) is here taken as a type. As an alternative the study of Erythronium is also outlined. Convallaria majalis, L. A. Sketch the entire plant. B. Does the underground portion all belong to the root system? Give reasons for conclusion. Is it highly specialized for storage of reserve material ? 1. Test a piece of it (collected in winter) for starch, Sec. 12. 2. Cut a cross section of the most vigorous portion, examine with m.p., and decide which of the types of stem studied in Secs. 25, 26, it most resembles. 8. In early spring note the manner in which the plant emerges from the ground. C. In the portion above ground note first the scale leaves, and, higher up, the foliage leaves. Label these in your sketch. 1. Are both leaf surfaces equally or unequally exposed to the light ? Hold a leaf up to the light and study its venation. Describe it. * To THE InstrucToR: As these studies consume much time, it may be found desirable to select only a few of them. One of the longer ones, like Sec. 165 or Se 167, thoroughly worked out, is worth more than the whole series hurriedly one. 1A detailed study of the pine may be found in Sec. 188. It may, if necessary, be simplified to make it homologous with those studies of families of angio- sperms which here follow by omitting some of the histological work and the larger part of the details about the process of reproduction in gymnosperms. 180 ECOLOGY 2. Cut across section of a leaf, examine with m.p., and sketch a por- tion extending from the one epidermis to the other. Describe in a few words the characteristics of the leaf structure as compared with any others you have studied. D. Note the mode of origin of the peduncle (scape). E. Note the position of the flowers. Uses of this. Describe the flower, making a diagram of a longitudinal section and a cross section. ¥. If fruit of the preceding season (in alcohol or formalin) is at hand, describe it. Does it reproduce mainly by seed or by other means? "G. Does this appear to be a sun plant or a shade plant? Reasons for conclusion. Is it a mesophyte or a xerophyte? What is its habitat in a wild state ? Look for insect visitors. What attractions has the flower for these ? Could it be readily pollinated without them ? Erythronium. A. Sketch the entire plant. B. If possible, dig away the earth with a trowel and make a diagram to show how the aérial parts stand above ground and how the un- derground portion is distributed. Describe the bulbs. Of what use are they ? 1. Test one for starch, Sec. 12. 2. Can they be drawn from the ground by pulling the leaves and scape? Advantage? Has the plant a stem ? C. How do the young leaves emerge from the ground? What is their position when full grown with reference to. light? Strip off epidermis from both surfaces and study with m.p. Differences? Explain. D. What is the position of the fully opened flower? Advantage? De- scribe the flower. 1. Make a diagram of the cross section. 2. Make a diagram of the longitudinal section. 8. Look for nectar and nectaries. What insect visitors frequent the flower ? E. Do many seeds ripen? Can the plant reproduce itself otherwise than by seed ? F. Is the species of Erythronium studied a sun plant or a shade plant, a mesophyte or a xerophyte? Do its leaves and blossoms mature earlier or later than those of the larger plants amid which it grows? Advan- tages? What becomes of the leaves during the summer ? Plants of the lily family are readily distinguished from those of the near- est related families. They differ from the rushes on the one hand in having a well-developed, not membranous, perianth, and from the members of the amaryllis family and the iris family on the other by having hypogynous flowers. The Liliacew are divided into about ten subfamilies of very TYPE STUDIES, SEED PLANTS 181 unequal numbers. Some of the most obvious differences between these relate to the bulb or rootstock or the aérial stem. Erythronium belongs to the lily subfamily and Convallaria to the aspara- gus subfamily. Give reasons why the lily, dogtooth violet, tulip, trillium, asparagus, lily of the valley, hyacinth, crown imperial, and onion should be classed as of the same family. 168. Family Ranunculacee. Study any obtainable kind of buttercup, discarding such of the following questions as do not apply to the species in hand. Ranunculus abortivus. A. Sketch the entire plant. B. Describe the root system. C. Cut the stem across and note any peculiarity of its structure. D. Note the three kinds of leaves, — ‘‘ root leaves”? at the base of the stem, ordinary leaves, and involucral leaves. Sketch one of each kind. What is the advantage of having the upper leaves parted into narrow divisions ? of having the root leaves long-petioled ? E. Describe the floral organs. 1. Make a diagram of a longitudinal section of the flower. How much apparent union of parts of the same or of different circles is there ? 2. Do all the anthers mature together? Advantages? 3. Do the stigmas and anthers mature together? Advantage ? 4, Look for the nectar and nectar glands. What insect visitors occur ? Are the flowers self-pollinated ? ; F. Study a head of mature akenes and describe it. Does seed mature abundantly ? Why cannot this species become a troublesome weed like R. bulbosus or R. acris? G. Test the flavor of any species of Ranunculus that you can get, by biting the fresh stem. Explain uses. Does this buttercup seem to be a more or less highly specialized plant than the columbine (Aqui- legia) or the larkspur (Delphinium) of the same family? In what respects ? 164. Family Rosacee. Study any kind of rose except the cultivated varieties with double flowers, or any ‘kind of cherry or plum. Rosa humilis, A. Describe the height and mode of branching of this rose. Are all specimens equally prickly ? Where do prickles occur ? B. Sketch a typical leaf (of five leaflets). How and how much do the leaves vary ? Study a cross section of a leaf with m.p. to find out how much the epidermis protects against excessive transpiration. If con- venient, compare with a greenhouse species, e.g. tea rose. 182 ECOLOGY C. Describe the occurrence of the flower buds. When and for how long do the flowers open? Advantage ? Look for insect visitors. What do these collect ? Are roses probably dependent on insect pollination ? Make a diagram of a longitudinal and of a cross section of the flower. D. Study fruit of the rose from material in alcohol or formalin. Are fresh rose hips edible ? Are the seeds? How long do rose hips remain on the branches? Advantage ? Have youseen birdseating them? At what season ? What becomes of the seeds when birds eat the pulp of the fruit? Are most rosebushes browsed by cattle ? Reasons? E. Is this rose adapted to a moist or a dry habitat ? How ? Prunus serotina, wild black cherry. A. What is the shape of full-grown trees? Size of the largest ones in your vicinity ? B. Sketch and describe the leaves. C. Sketch a flower cluster. Do cherry and plum trees blossom before or after the leaves develop? Are they all alike in this? Advantages of blossoming first ? 1. Make a diagram of a longitudinal and a transversé section of the flower. How many ovules are there? Do all mature? 2. Look for nectar and nectaries. Do the anthersall mature together ? Are there insect visitors ? D. Is the fruit edible? Do birds gather it? What evidence is there of wide distribution of the seeds? Why do cherry trees often grow beside fences ? The rose family (as found in temperate regions) is divided into four sub- families, — Spireoidew, Pomoidew, Rosoidee, and Prunoidee. Familiar rep- resentatives of these are the Spircea, the apple, the rose, and the cherry. The most obvious differences between these subfamilies depend on the development of the receptacle and the way in which the carpels are borne on or within it. In the Spircoidew the receptacle is flattish and the carpels are borne on its surface. In the Pomoidew the flowers are epigynous and the carpels appear to be grown fast to the hollow inner wall of the recep- tacle. In the Rosoidew the carpels are in some genera (as in the rose) mod- erately attached to the interior of a hollow receptacle, and in other genera (as in the raspberry, the blackberry, and the strawberry) they are borne on the outside of a more or less elongated and thickened receptacle. In the Pomoidee there is often but one carpel, which ripens only a single seed, inclosed in a fleshy stone fruit. Is there any general similarity in the size, habit, and degree of woodiness of rosaceous plants ? How could all rosaceous plants be roughly classified as regards their leaves ? TYPE STUDIES, SEED PLANTS 183 What can you say of the economic importance of the family ? Make a list of all the cultivated Rosacee that you know and state for what each is valued. Uses of some wild species ? 165. Family Leguminose. The black locust (Robinia Pseudacacia), one of the vetches (Vicia or Lathyrus), or the common pea (Pisum) are good types for study. Robinia Pseudacacia. A. Sketch a well-grown tree (best before the leaves appear). B. Examine the roots for tubercles. In the locust, as in the Leguminose generally, these serve an important purpose in manufacturing soluble nitrogen compounds for the use of the plant. C. Sketch a twig to show the arrangement of the thorns. 1. 2. What are the thorns? How related to the winter buds ? Why? Are the twigs mature and alive to their tips? How is the growth of the twig continued in the spring? What other trees or shrubs do you know with the same characteristics ? . Cut off a large branch of locust. Is there much distinction between sapwood and heartwood ? For what is the wood most valuable ? What other woods are notable in the same way ? D. Sketch a locust leaf. 1. 2. 3. Is the number of leaflets constant ? Study and report on the positions of leaves on horizontal and vertical twigs. Explain. Study the leaflet position : (a) on outer branches in sunlight ; (6) on inner branches or in dull weather ; (c) at night. . Sketch a leafy twig as seen in positions of (a), (b), and (c). Explain the use of each position. Try to ascertain by studies “of the leaves on the tree what percentage of the noon illumination on a perfectly sunny day is necessary to produce position (a) and what to produce posi- tion (b). (For method of measuring relative intensities of illumina- tion see Exp, XXVIII.) E. Note the time of appearance of the flowers relatively to that of the leaves. Is it the same in all individuals? In southern Italy the flowers usually appear before the leaves. Advantage of this ? 1. 2. 3. . Make a detailed drawing of the longitudinal section of a large flower Sketch a twig with a flower cluster in its natural position. Sketch an entire flower and another dissected. Why is a flower of this shape called papilionaceous ? Make a diagram of the longitudinal section. bud, two to four times natural size. Show plainly the beard of hairs below the stigma. 184 ECOLOGY 5. Remove from a just-opened flower all the floral organs except the pistil and make an enlarged drawing of the pistil, side view. Where is pollen accumulated on it ? 6. Which matures earlier, stigma or anthers ? Watch a bee visiting the flowers and note what happens when she alights on the wings. Why are the wings and keel fastened together ? Imitate the action of the bee by pressing the wings and keel down- ward. . Explain why cross pollination is almost sure to occur. 8. Make a list of all the attractions which this flower has for insects. What insect visitors have you observed ? F. Study the ripe fruit of the locust. What becomes of it in the autumn ? Are the seeds likely to be destroyed by animals? Reasons? Part of the locust seeds of each season grow within a year, but others do not grow until succeeding years. Advantage of this ? G. Write a brief essay on the ecology of the locust, explaining all its adap- tations with reference to utilizing bacterial symbionts, to light supply, to browsing animals, to pollinating insects, and to reproduction by seeds. Lathyrus odoratus, sweet pea. A. Sketch the entire plant. Study the distribution of the hairs on its surface. Of what use may these be? In southern Europe, where this plant is common in a wild state, snails are among the most important enemies of vegetation and they rarely attack hairy plants. B, Examine the roots for tubercles. What kind of a climber is it? How does it climb? Why ? C. Sketch several leaves with tendrils in various stages of development. How does the tendril pull the plant toward any support on which it fastens ? D. Sketch a bit of stem with a flower cluster in its natural position. Make a detailed study of the flower as described under Robinia. E. Study the ripe fruit of the sweet pea. Are the pods likely to be eaten by animals ? Reasons? Can you find out how the seeds are distributed ? Explain how the vetches are equipped to hold their own as dwellers in thickets and in tall grass or among other large herbaceous plants. The family Leguminose is a very large and important one, divided into three subfamilies — Mimosoidew, Ceesalpinioidee, and Papilionatee — whose characteristics are based on the structure of the flowers. The first, the Aca- cia subfamily, is mostly tropical ; the second, the Cassia subfamily, contains three quite familiar North American genera, — Cassia or wild senna, Cercis or redbud, and Gleditschia or honey locust. Most of our familiar Legu- minose, however, belong to the third subfamily, Papilionate, readily recog- nizable by their papilionaceous flowers. Make out a list of those which you know are useful or ornamental and give their uses. = 166. TYPE STUDIES, SEED PLANTS 185 Family Violacee. Study any species of violet; if some of the points suggested in the following outline do not fit the species in hand, omit them. Viola palmata. A. Sketch the entire plant. B. Has it any stem? Explain. C. Sketch one of the first leaves of the season and a later one. D. Sketch a flower in profile in its natural position. 1. 10. 11. What kind of symmetry has it? Does the flower, seen from in front, appear open or closed? How much of the stamens and pistils can be seen ? . Make a diagram of the cross section of the flower. . Remove the petals. Is the spur a part of the calyx or the corolla? It serves as a nectary. . Note the two nectar glands which project from stamens into the spur. . Make a sketch of the magnified pistil, surrounded by the stamens. . Are the filaments united ? the anthers? Do the anthers discharge inwardly or outwardly ? Is the pollen dry or sticky ? . Note that the pollen when shed collects in a sort of cone formed by the united anthers, which is closed at the narrow end by the pistil. When a visiting insect, as a bee seeking nectar, thrusts its tongue into the small end of the cone, what would become of any pollen which the insect brought with it? Would other pollen be carried away? Result ? . Thrust a slender, moistened toothpick gently into the opening of the corolla, and after withdrawing examine it with a lens. Result ? . What means have violets of advertising their supply of nectar ? Compare the attractiveness of several species. Look for insect visitors. Do they all explore the interior of the flower, as shown in Principles, Fig. 325 ? Many violets form most of their seed from apetalous cleistogamous flowers (see Principles, Sec. 408). Is Viola palmata one of these ? Study specimens in late summer or early fall to determine this point. What are some advantages of cleistogamy ? disadvantages? How would a plant with some insect-pollinated flowers and other cleistog- amous ones avoid the disadvantages mentioned ? E. What mechanism have the capsules for distributing seeds ? F. Are any violets moderate xerophytes ? Are any hydrophytes? Does the species studied belong to either class ? The Violacee constitute a small and unimportant family, but the flowers are decidedly interesting from the perfection of their adaptation for cross and self pollination. 186 ECOLOGY 167. Family Composite. Most of the genera of this family found in the United States are summer or autumn flowering. Two common genera which flower in spring are Taraxacum and Erigeron. Taraxacum officinale, common dandelion. : A. Sketch the entire plant. 1. 3. Notice the rosette formed by the leaves, all borne close to the ground or even pulled below the surface by contraction of the tap root. What is the use of this shortening of the tap root? What other plants show it ? . Sketch the plant as seen from above. How much do the leaves overlap and shade each other? How much do they interfere with the growth of grass in a lawn? Advantages ? Taste the root and leaves. Use of this taste? Are the leaves easily injured by frost ? B. 1. Sketch the slender scape with its head of flowers. How do you oo or know that the whole yellow “dandelion” is an inflorescence and not a flower ? Advantage of grouping flowers in a head ? . Changes in length of scape as it grows older? Advantages ? . Describe the involucre. . What is its condition in the bud? ina fully opened head? in a head that is past blooming ? in a head when the fruits (‘‘seeds”’ ) are beginning to disperse ? . Of what use is the involucre ? . Make a longitudinal section through a newly blooming head and note whether all the flowers mature together. . The cushion-like expanded extremity of the scape, from which the flowers spring, is a common receptacle for all the flowers of the head. . Note and describe the change in form of the corolla as the buds open. . Decide from the number of teeth at the tip of the corolla and the number of stamens what is the numerical plan of the flower. . Slit open the corolla of a bud with a needle and scalpel, or two needles, under the magnifying glass, and note the structure of the flower. How many stamens are there? From what part of the flower do the filaments spring? Are the anthers attached to each other ? How do they open? Position of the anthers relative to the style? How many branches has the stigma? What is their form and position (a) in the bud? (b) in the newly opened flower ? (c) in the flower just before withering ? What portion of the stigmas is hairy ? . When the stigmas emerge from among the anthers what do they bring with them? Importance of this? Could the movements of TYPE STUDIES, SEED PLANTS 187 the stigmas cause self-pollination ? At what period in théir develop- ment ? Advantages of this ? 5. Look for nectar. How high does it rise in the corolla tube ? Acces- sibility to small insects ? 6. How many kinds of insect visitors do you find ?1 7. Is the head open on sunny and cloudy days alike? Is it open all night ? Advantages? Mark a head by tying twine loosely about the scape and note how long it remains in blossom, how long it remains closed after blossoming, and how long after reopening the last fruits are dispersed. D. Sketch a fruit somewhat magnified and label the parts. Test the traveling powers of some akenes in a gentle breeze. KE. Study the distribution of dandelion plants in your neighborhood, state where they thrive best, and give reasons. F. Write a brief essay on the ecology of the dandelion, discussing : (1) Relations to other plants. (2) Relations to leaf-eating insects and grazing animals. (8) Relations to pollinating insects. (4) Relations to weather. (5) Distribution of seed. Erigeron philadelphicus, common fleabane (or other species). A. Study the fully opened heads and make out a list of resemblances to and differences from the head of the dandelion. Note that every flower in the dandelion head has a strap-shaped corolla, and is bisexual. 1. Where are strap-shaped flowers of the fleabane ? Are they bisexual? 2. Sketch under the lens a tubular flower. B. Look for insect visitors. C. Discuss the relative equipment of the dandelion and the fleabane for success in life. The family Composite is the largest family of seed plants, comprising about eleven thousand species. It is usually considered to be the highest ‘family. Not many Composite in temperate climates are shrubby or tree- like, but as herbs they show the greatest diversity of form and ecological characteristics. As a rule, they are extremely successful in maturing and distributing seed, and for this and other reasons constitute very formi- dable weeds. Make a list of some of the commonest weeds of this family in your neighborhood. Rererences. Principles, Chapters XXXII, XXXIII; Strasburger, Noll, Schenck, Karsten, 1; Warming-Moébius, 87; Engler, 36; Knuth- Davis, 62 ; Kerner-Oliver, 2. 1 Nearly a hundred species have been noted in a single locality. BOTANICAL MICROTECHNIQUE 168. Introduction. This section will describe the technique of a number of well-known histological and cytological methods involving the preparation of material. Its object is to present simple and clear descriptions of tried methods that can be depended upon to give good results. Detailed treatments may be found in a number of treatises.1 The best general accounts of his- tological methods in English are those in Strasburger-Hillhouse, 6, which is based on Strasburger’s Das botanische Praktikum. The subject-matter of the following brief account will be taken up under the following headings : General reagents employed in temporary preparations. > Some special reagents for microchemical and other tests and temporary preparations. Killing and fixing. The preservation of material. General staining methods. Mounting in balsam and glycerin. Imbedding in paraffin. Sectioning. Staining on the slide. GENERAL REAGENTS EMPLOYED IN TEMPORARY PREPARATIONS 169. General reagents employed in temporary preparations. A. Iodine solutions. Dissolve 5 grams potassium iodide in 100 cc. distilled water, and add 1 gram of iodine. This gives a good strength for most purposes. It may be diluted if desired, and should be used of half this strength, or weaker, when the color of the solution might interfere with the clearness of vision, as when zodspores are stained to show their cilia. Iodine solutions kill protoplasm quickly, staining it a deep brown, especially the nucleus and chromatophores. They furnish the simplest 1 Zimmermann-Humphrey, Botanical Microtechnique, Henry Holt & Co., New York, 1893. Poulsen-Trelease, Botanical Micro-Chemistry, 8. E. Cassino & Co., Boston, 1884. Chamberlain, Methods in Plant Histology, The University of Chicago Press, Chicago, 1905. 188 GENERAL REAGENTS 189 tests for starch (Sec. 12), coloring the grains blue, or a deep brown if the solution be too strong. Cellulose (Sec. 12) is generally stained yellow or brown, which changes to blue if a strong solution of sulphuric acid be applied after the iodine. . Chlorzinc iodine. This is a troublesome reagent to prepare, but the best test for cellulose. Dissolve zinc in pure hydrochloric acid and evapo- rate the solution (with metallic zinc present in it during the process) to the density of sulphuric acid. Add as much potassium iodide as the solution will dissolve, and finally as much metallic iodine as it will take up. The solution will keep better away from the light. Chlorzinc iodine stains pure cellulose a clear blue or violet. It reacts best on preparations in water. . Potash solution. A 5% solution of potassium hydrate, or caustic potash, in water is an excellent clearing and softening agent. A 15% solution is necessary for some subjects, as firm leaf sections. The potash solution may be neutralized by washing the sections in commercial acetic acid and then mounting them in the latter. Potash solutions must be kept tightly stoppered. Rubber stoppers answer well; glass ones should be covered with paraffin, otherwise they are likely shortly to become stuck beyond the power of removal. . Acetic acid. A 1% solution of glacial acetic acid in water will fix and frequently bring out clearly the nucleus and other protoplasmic struc- tures of a cell. Beautiful temporary preparations may then be made by staining with gentian violet (see F) or methyl green (Sec. 186, B). . Eosin. A strong solution of eosin in water is the most useful. Alco- holic solutions may be employed when the preparation is in alcohol. This stain has the peculiar advantage of coloring protoplasm alone, leaving the cell wall unaffected. . Gentian violet. A deep violet solution in 1% acetic acid is a good strength for temporary staining of fresh material, or after fixing with 1%, acetic acid (see D). . Alcohol. Alcohol has its chief value for temporary preparations in driving out air bubbles from material which will not wet easily in water, as, for example, the mycelium of fungi. . Distilled water. Temporary preparations of living plants are mounted in tap water, or that in which they live, if aquatic. Distilled water is used when the preparations are made from preserved material. . Glycerin. A solution one third glycerin and two thirds distilled water is very useful in preserving temporary preparations. A drop or two placed at the edge of the cover glass will prevent the preparation from drying up. This solution, or one considerably stronger, is also used for permanent preparations when inclosed in a cement ring (Sec. 188). 190 BOTANICAL MICROTECHNIQUE SOME SPECIAL REAGENTS FOR MICROCHEMICAL TESTS AND TEMPORARY PREPARATIONS 170. Some special reagents for microchemical tests and temporary preparations. A. Alkanet root tincture. Add enough bits of alkanet root to 95% alcohol to color it deep red. The solution serves as a test for oils and resins (Sec. 12), coloring them red. B. Ammonia. Ordinary commercial ammonia water is used after treat- ment of sections, etc., with nitric acid to give the xanthoproteic reaction (Sec. 12), coloring proteids yellow or orange. C. Chloral hydrate. A solution of eight parts of chloral hydrate in five parts of water by weight forms an excellent clearing reagent for growing points and pollen grains. D. Chloroform. Removes oil from sections of seeds which are to be examined for aleurone grains. E. Fehling’s solution. This reagent may be bought of dealers in chemicals. It is usually made up in the form of two or three solutions, which are to be mixed only at the time of using. The following formula is convenient, and keeps well in a cool place. Dissolve 34.64 grams pure crystallized copper sulphate in 200 cc. of distilled water. Mix the solu- tion with 150 grams of neutral potassium tartrate dissolved in about 500 ce. of a ten per cent solution of sodium hydrate. The whole is then to be diluted with water to 1 liter, and 100 cc. of glycerin added. The solution serves as a test for sugar (Sec. 12). F. Millon’s reagent. Dissolve metallic mercury in its own weight of c.p. concentrated nitric acid and dilute the solution with its own volume of distilled water. This reagent swells cell walls and usually colors pro- teids (Sec. 12) a characteristic brick red. G. Nitric acid. C.p. nitric acid, slightly or not at all diluted, is used as a test for proteids (Sec. 12). It is also used in Schultze’s macerating mixture (see M). H. Olive oil. This is used as a mounting fluid for sections of seeds with aleurone grains. I. Phloroglucin. 1-5% solutions in water or alcohol are used as a test for lignified tissue (Sec. 12). J. Potassium chlorate. This is used as an ingredient of Schultze’s macer- ating mixture (see M). K. Potassium permanganate. A 4% solution of this compound in water is used as a stain to distinguish roots from stems in very young seedlings. L. Safranin. A saturated or sometimes a half-saturated aqueous solution of this stain is valuable for differentiating tissue elements, e.g. in stem SPECIAL REAGENTS 191 sections or leaf sections. There are several good formule for safranin stains (Sec. 184). : M. Schulize’s macerating mixture. This mixture is used to disintegrate tissues (e.g. wood) to obtain individual cells. The material to be treated should be in small bits cut lengthwise. Place the sections in a test tube and pour on just enough strong nitric acid to cover the material. Add a few crystals of potassium chlorate and heat gently until bubbles are given off and the substance treated becomes white. If violent action occurs and abundant reddish fumes are evolved, repeat the operation with fresh bits of the substance to be macerated, using less chlorate. The process must be conducted out of doors or under a hood, as the acid vapors produced are very corrosive and injure microscopes and most metallic apparatus. When the maceration is finished the fibrous material left should be thoroughly rinsed with successive por- tions of water until all traces of acid are removed. It may then be teased apart with needles and preserved in glycerin, and portions mounted for examination as required. N. Sugar. Cane sugar is used in the preparation of solutions for the culture of pollen tubes (Experiment XLII), algz (Sec. 200), and germi- nating spores of mosses and ferns (App. 19 and App. 20). Solutions of the required strengths can be made by weighing out the necessary amounts of granulated sugar and adding to measured or weighed amounts of tap water. One and a half per cent of gelatin may, with advantage, be added to most of the solutions for the culture of pollen tubes. KILLING AND FIXING 171. The principles of killing and fixing. Fixing is the preservation of the structure of protoplasm immediately after death as nearly as possible like that of the living cell. Killing and fixing are generally accomplished by the same fluid. Fixing agents have in their composition elements (as chromium, osmium, platinum, etc.) which living protoplasm normally never or but rarely encounters, or severe combinations of poisons that are utterly foreign to it. The ability of a killing fluid to fix undoubtedly rests on its power to subject protoplasm to a shock so sudden and great that there is little or no time for great structural changes to take place, while the reagent itself must not cause disorganization. Fixed material must later be preserved, a process involving quite different methods and reagents (Secs. 177, 178). 172. Chrom-acetic acid. The combination of chromic and acetic acids in various proportions has proved to be a very satisfactory general fixing agent, and is among the cheapest. Three grades of chrom-acetic acid will be found 192 BOTANICAL MICROTECHNIQUE useful, —a weak, a medium, and a strong. Any shrinkage of the cel con- tents during fixation indicates that the solution is too strong in chromic acid, which has a tendency to contract the protoplast, partially compen- sated by the acetic acid which is employed because of its tendency to swell the cell contents. A. Weak chrom-acetic acid}: 1% chromic acid 25 cc. making .25% chromic acid. 1% acetic acid 10 cc. making .1% acetic acid. distilled water 65 ce. 100 cc. This is generally the most satisfactory strength of chrom-acetic acid for alge and fungi, and the more delicate structures of the liverworts and mosses will be excellently fixed by it, likewise fern prothallia. B. Medium chrom-acetic acid : 1% chromic acid 70 cc. making approximately .7% chromic acid. glacial acetic acid 5 cc. making approximately .5% acetic acid. distilled water 30 ce. 100.5 ce. A good fluid for most work on the histology of the pteridophytes and seed plants and the firmer structures of mosses and liverworts. C. Strong chrom-acetic acid: 1% chromic acid 100 ce. making approximately 1% chromic acid. glacial acetic acid __1cc. making approximately 1% acetic acid. 101 cc. This solution may prove more satisfactory than medium chrom-acetic acid when the tissue is dense or with very heavy cell walls. The determinations of the proper relative strengths of chromic and acetic acids become matters of experience and experiment which must be tested with untried subjects, but those who use this fixing fluid are soon able to judge very accurately the strength and time necessary for good results. Chrom-acetic acid keeps perfectly, and costs so little that it may be made up in large quantities; it is the most useful general fixing agent in the labo- ratory. It should be employed in liberal quantities, perhaps one hundred times the bulk of the material, or in such amounts that the fluid is not noticeably discolored by the material. 1 The formule in this account are generally given in terms of 100 cc. The proportions may be multiplied by tens and hundreds for larger quantities. Chrom-acetic acid is so useful a reagent that a laboratory should always have a stock supply. Another plan is to keep on the same shelf or table a large bottle of 1% chromic acid, another of 1% acetic acid, and a small bottle of glacial acetic acid, together with a large and small graduate and the formule posted on the wall. Solutions may then be made up at any moment. KILLING AND FIXING 193 Material is generally left in chrom-acetic acid for twelve hours or more, but very delicate structures require only an hour or two. Some alge with soft cell walls, such as Polysiphonia, will go all to pieces if left in the weak formula more than five or ten minutes. Solutions employed upon the marine algee must be made up in salt water instead of distilled water, and the fixed material must also be washed in salt water. Tissues with hard or very firm cell walls are improved by being left for longer periods, perhaps several days, in the fluid, for the chromic acid acts on the cell walls, softening them somewhat. Material fixed in chrom-acetic acid must be washed thoroughly before being carried up into alcohol for final preservation (Sec. 178). This may be done most satisfactorily with firm tissues in a gentle stream of tap water circulating through the vessel (a wide-mouthed bottle with a piece of gauze tied over the mouth to hold the material within is convenient). Washing may occupy several hours or, with firm tissues, a much longer time without danger. It is necessary to get all of the chromic acid out of the material, otherwise a precipitate will be formed by the alcohol and the protoplasm will not stain well. : It is not generally known that the chromic acid can be washed out by running the material through the grades of alcohol to 70% (Sec. 178), provided the bottle of material is kept inthe dark. The precipitate referred to in the paragraph above is only formed by alcohol in the presence of light. The 70% alcohol must of course be changed until there is no trace of chromic acid. This method works especially well with small objects and saves much time and the somewhat difficult operation of washing small objects in water. 173. Chrom-osmo-acetic acid (Flemming’s fluid). The most successful of the formule containing chromic, osmic, and acetic acids were developed and perfected by Flemming and are accordingly called Flemming’s fluids. The addition of osmic acid to the chrom-acetic basis gives somewhat better fixation of material than chrom-acetic acid alone. This better fixation appears in the cytological details of nuclear division and in a more brilliant reaction of material to the stains safranin and gentian violet, which with orange G form a group often used together as a triple stain (Sec. 199, D) after this fixing agent. Flemming’s fluids penetrate slowly, and material should be cut up into small pieces or slices, perhaps an eighth of an inch in thick- ness, to obtain the best results. The expense of the osmic acid rather pre- cludes the use of these fluids for general morphological and histological studies where fortunately the cheap chrom-acetic formule are in the main quite satisfactory. Flemming’s fluids do not keep in the light, and it is best to make them up fresh just before fixation. Solutions of osmic acid must be kept in the dark. 194 BOTANICAL MICROTECHNIQUE A. Weak chrom-osmo-acetic acid (Weak Flemming) : 1% chromic acid 25 cc. making .25% chromic acid. 1% acetic acid 10 cc. making .1% acetic acid. 1% osmic acid 10 cc. making .1% osmic acid. distilled water - 55 ee. 100 ce. This well-known formula is used for the alge and fungi and delicate tissues of the higher plants. It has the same strength as weak chrom- acetic acid but with osmic acid added. Half the amount of osmic acid in the above formula gives, according to our experience, better results with many algz and fungi. . B. Strong chrom-osmo-acetic acid (Strong Flemming) : 1% chromic acid 75 cc. making .75%, chromic acid. glacial acetic acid 5 cc. making 5% acetic acid. 2% osmic acid 20 cc. making .4% osmic acid. 100 ce. This formula has a medium strength of chromic acid but an exceptional strength of acetic and osmic acids. It may easily be modified by vary- ing the amounts of its components. Thus Mottier recommends for anthers the following proportions: 1% chromic acid, 80 cc.; glacial acetic acid, 5 cc. ; 2% osmic acid, 15 cc. Strong Flemming naturally finds its use on the same sort of subjects as require the medium or strong formule of chrom-acetic acid, as for example the firmer tissues of the higher plants. Material fixed by Flemming’s fluids must be washed to remove the chrom- acetic acid, as described in the previous section. The osmic acid always blackens the material, but this discoloration is not treated until just be- fore staining (Sec. 198), when the preparations are bleached with hydrogen peroxide. The chromic acid must be dissolved in sea water, when these fluids are used upon marine alge, and the material also washed in sea water. 174. Absolute alcohol. The fixing fluids based on chromic acid penetrate rather slowly, and consequently very dense tissues or structures with heavy hard cell walls are sometimes not at all well fixed by them, the protoplasts appearing shrunken. There is also occasional difficulty in immersing or wetting material in these water solutions. For such material some of the fixing fluids based on alcohol are preferable. The best of these are absolute alcohol and Carnoy’s fluid. Absolute alcohol alone is not an especially good fixing agent except for very small objects, which it can penetrate almost instantly. Material is, of course, ready very quickly for preservation in 85% alcohol, or for the process of imbedding in paraffin. PRESERVATION OF MATERIAL 195 175. Carnoy’s fluid. absolute alcohol 60 cc. chloroform 380 cc. glacial acetic acid 10 ce. 100 cc. This is a strong fixing fluid which penetrates very rapidly, and conse- quently should only be used for a few minutes, —ten to thirty minutes is probably long enough for most subjects. There is always danger of leaving material too long in it. The material is washed in changes of absolute alcohol until there is no odor of acetic acid, and is then best imbedded at once, but may be transferred to 85% alcohol for preservation. The staining of chromosomes after Carnoy’s fluid is sometimes very brilliant, but spindle fibers and other kinoplasmic structures are apparently less perfectly preserved than by the chrom-osmo-acetic formule. If the subject be very resistant to penetration, as for example the mega- spores of the pteridophytes, the proportionate amount of acetic acid may be greatly increased. Thus two parts glacial acetic acid, one part absolute alcohol, and one part chloroform have been recommended as giving good results for the spores of Selaginella. However, even in these cases, long treatment with medium or strong chrom-acetic acid, especially if applied hot, aided by mechanical cutting or pricking of material, to assist penetration, will frequently give better results than Carnoy’s fluid. 176. Concluding suggestions on fixing. It is important to facilitate mechan- ically, in every way possible, the rapid penetration of the fixing fluid. Thus an ovary of a lily should be pared along the angles and then sliced in pieces three eighths of an inch thick or cut lengthwise. Small objects, such as fila- mentous alge, may be examined at various stages in the process of fixation to see if the cell contents are in good condition. Material that must be sec- tioned cannot, however, be so easily observed, and shrinkage may occur, which was not caused in the fixing, but at some later stage in the manipula- tion leading to sectioning or staining. Consider results critically, and when unsatisfactory attack the problem as one of physics and chemistry, and find just where the methods failed. Close attention to these details will soon give a sure command of a few simple methods of fixing which are likely to give satisfaction. THE PRESERVATION OF MATERIAL 177. Alcohol. Material collected for general morphological study may be placed at once in 95% alcohol. It must later be transferred to a lower grade, such as 70%, or it will become very brittle. Alcohol mixed with glycerin, half and half, or one fourth glycerin, will keep material from becoming 196 BOTANICAL MICROTECHNIQUE brittle, and is especially good for firm structures that are to be sectioned free-hand, such as the various parts of seed plants. Alcohol is the best all-round preservative. Other fluids have appeared from time to time as rivals, as for example formalin, but they none of them have supplanted it. It is somewhat uncertain whether denatured alcohol, now on the market, will be just as good as the pure alcohol for preservative purposes, and it should be used with some care until its effects are known. ; 178. Bringing fixed material into alcohol. Botanists are coming to depend more and more upon fixed material even for general morphological studies, since it is very little trouble and expense to fix in chrom-acetic acid, and the superior results are worth the attention required. This is especially true of type material of the thallophytes and bryophytes. Material fixed in chrom-acetic acid or in chrom-osmo-acetic acid (Flem- ming’s fluids) must be washed as described in Sec. 172, and then passed, or ‘run up,” through several grades of alcohol to 70% (or 85% if the material is delicate), where it may rest indefinitely. It is well to begin with 15% alcohol and pass successively through 25%, 35%, 50%, to 70%. Sinall objects such as lily anthers will not require more than an hour in the lower grades. They should remain, however, twice as long in the 35% and 50%. Larger objects must remain from four to eight hours in each grade. The process should be planned so that material is not left for so long a time as over night in a grade of alcohol below 50%, and it should not remain in 50% longer than over night. Generally the entire process can be finished in one day. The grades of alcohol are made from 95%, which for general purposes is regarded as being pure. Material fixed in fluids based on alcohol, as for example Carnoy’s fluid, should be passed directly into a grade of alcohol corresponding to that in the fixing fluid. 179. Formalin. Much was expected of formalin when it appeared a num- ber of years ago. The most important claims have not been fulfilled. It will not preserve the green color of plants in the light, and shades of red, blue, and brown are generally modified after a few months. Unless the tissue is firm it is apt sooner or later to soften or macerate ; this is especially true ot the lower plants. Finally, formalin is intensely disagreeable to work with on account of its effect on the nose and eyes. Formalin, which is about 40% formaldehyde, is added to water to make a 2-5% solution. It is convenient to carry, since a small quantity will make many quarts of the preserving fluid. If material is to be used within a short time, formalin will prove satisfactory. Material may also be transferred from formalin to alcohol, being carried up through the grades. However, its ad- vantages are rather doubtful when chrom-acetic acid and alcohol are at hand, STAINS 197 GENERAL STAINING METHODS 180. Methods of staining. There are two principal methods of staining, — (1) in bulk or loose sections, and (2) on the slide. The second method gener- ally follows the process of sectioning in paraffin, and is given special con- sideration in Secs. 197-199. Staining in bulk is, on the whole, much less precise in its results than the staining of microtome sections. The methods of staining in bulk also apply to sections cut free-hand (Sec. 194), either from preserved or living material. 181. Eosin. Saturated solutions in water or alcohol are used. Material is stained almost at once, and should then be transferred to 1% acetic acid for a minute (which renders the stain less soluble), after which the acid should be thoroughly washed out. Permanent preparations are generally made in glycerin after the method outlined in Sec. 188. If the preparations are to be mounted in balsam (Sec. 187), the staining should be with alcoholic solu- tions, or, better still, with a solution in absolute alcohol from which the material may pass directly into xylol, and there is no need of treatment with acetic acid. Eosin does not stain cell walls and never overstains protoplasm. It is especially useful for the fungi, which are generally mounted in glycerin, and is one of the best of the quick, simple stains. 182. Iron-alum hematoxylin. This method, developed by Heidenhain, gives the most satisfactory results of all the hematoxylin stains in the differ- entiation of protoplasmic structure. Delafield’s hematoxylin (Sec. 188) is a somewhat better stain for tissues, because it colors cell walls sharply. Iron- alum hematoxylin does not color cell walls heavily, and is consequently a very useful general stain for the algee and fungi which are to be stained in bulk and mounted without sectioning. Two separate solutions are used : (1) A 2% aqueous solution of ammonia sulphate of iron (iron alum). (2) A 4% solution of hematoxylin dissolved in hot distilled water. The solution of iron alum acting as a mordant prepares the tissue to take up the hematoxylin. Bring the material from water (running it down through the grades of alcohol, if preserved in the latter) into the iron-alum solution, which for delicate structures may be diluted to 1%. Leave in the iron alum from one to three hours, rinse for a few minutes in water, and place in the hematoxylin solution. If the hematoxylin becomes too muddy, ¥eplace it with fresh. Leave the material in the hematoxylin from three to ‘ten hours (over night does no harm) and then,place in iron alum again. The black stain extracts rapidly, and the material must be examined from time to time under the microscope. When the stain has been extracted to the proper point, place the material in considerable tap water for a half hour, or 198 BOTANICAL MICROTECHNIQUE as much longer as convenient, to remove all trace of the iron alum. The material is now ready to be mounted in balsam (Sec. 187) or glycerin (Sec. 188). If mounted in balsam it must be carried through xylol (never oil of cloves, which fades hematoxylin). The hematoxylin can be extracted to a point where the nucleus is prac- tically the only structure stained, and is consequently one of the best of the nuclear stains. Such material may be counterstained (that is, stained in addition) with safranin (Sec. 184), thus differentiating the nucleus (gray or black) from the rest of the protoplasm (red). Iron-alum hematoxylin is probably on the whole the most satisfactory of all the staining methods for protoplasmic structures. It is subject to great latitude in the time limits, which may be set for the different stages of the process except that of extrac- tion, which must of course be watched carefully ; but these are soon learned with experience. It is perhaps the least uncertain of the stains, and although the process is somewhat long it can always be depended upon to give good results. 183. Delafield’s hematoxylin. This stain reacts very differently from iron- alum hematoxylin. It stains cell walls sharply, but does not differentiate protoplasmic structures as well as the latter. It is one of the best stains for tissues of higher plants, and may be combined very effectively with safranin, as described below and in Sec. 185. Delafield’s hematoxylin is made as follows: a solution of 1 gram hema- toxylin in 6 cc. absolute alcohol is added drop by drop to 100 cc. of a saturated solution of ammonia alum. Filter after exposing for a week to the air and light. Then add 25 cc. of glycerin and 25 cc. of methyl alcohol. Allow the mixture to stand for several hours (4-7), until the color is dark, and then filter. The solution should then remain two months in a tightly stoppered bottle to ‘‘ripen.’’ The prepared stain may be purchased from dealers (Sec. 218). Material is transferred to Delafield’s hematoxylin from water or 25% -alcohol. Staining will take place rather rapidly, requiring from a few min- utes to an hour or more. The stain may be diluted to half or a fourth of the above strength, and the staining, although longer, is frequently better. Wash the material in tap water until a rich purple color develops. If the sections or other subjects are overstained, or if a precipitate is formed when the material is placed in alcohol, rinse in acid alcohol (7, cc. hydrochloric acid in 100 cc. 70% alcohol). The acid alcohol takes out the color, which may thus be extracted until the nucleus alone remains stained. When washed in acid alcohol the material must be placed in tap water until the purple color returns. Then run up in the grades of alcohol through 95%, and absolute alcohol, clear in xylol, and mount in balsam, as described in Sec. 187, or pass from water into glycerin, as outlined in Sec. 188. STAINS 199 Material stained in Delafield’s hematoxylin may be counterstained with alcoholic safranin, but very good results may be obtained with the tissues of higher plants by staining first with safranin, as described in Sec. 185. 184. Safranin. There are various kinds of safranin sold, some of which dissolve more readily in water and some inalcohol. The stain should always be placed in its appropriate solvent. A 1% solution in water is a good strength, and a saturated solution in 95% alcohol mixed with an equal vol- ume of water, making a 50% alcoholic solution, is also good. The alcohol solutions are the most convenient. Anilin safranin is prepared from a satu- rated solution in 95% alcohol mixed with an equal amount of anilin water (made by shaking anilin oil in distilled water, when a small percentage of the oil is taken up by the water). Anilin safranin is considered by some to be the best of the safranin stains. Safranin colors cell walls as well as protoplasm. It is therefore a general stain, but when properly extracted it may be made to differentiate certain nuclear structures sharply (chromosomes and nucleolus), and is much used in staining on the slide, especially in combination with gentian violet and orange G (Sec. 199, D). Material may remain in safranin from one hour or less to twelve hours or more. The stain is extracted in 50% alcohol until the desired coloration is obtained, or, if very much overstained, the material may be placed in acid alcohol (2, cc. hydrochloric acid in 100 cc. 70% alcohol). The acid alcohol, if used, must be thoroughly washed out. 185, Safranin and Delafield’s hematoxylin. Safranin followed by Delafield’s hematoxylin is an excellent stain for the tissues of higher plants, whether in free-hand or microtome sections. Sections cut free-hand from fresh material may be fixed for 10-15 minutes in absolute alcohol or medium chrom-acetic acid (Sec. 194); those from preserved material may be stained at once. They should remain in the safranin several hours (over night). Wash in 50% alcohol (acid alcohol if desired) until the stain is extracted from all parts except lignified cell walls, as in fibro-vascular bundles. Remove the acid alcohol if used. Stain in Delafield’s hematoxylin for. several minutes (1-30). Wash in tap water, or extract the stain if necessary in acid alcohol (as described in Sec. 183), which must be followed by tap water until the stain is purple. Carry through 95% alcohol, then absolute alcohol, clear in xylol, and mount in balsam. Sections cut on the microtome are stained on the slide (Sec. 197) in the same manner as described above. 186. Other anilin stains. There are numerous anilin dyes of great value in special cases, but few of them have such general usefulness as eosin, safranin, and gentian violet. The following, however, are important. A. Acid fuchsin. A 1% solution may be made in water or in 70% alcohol. The stain acts rapidly and is very brilliant. It may be extracted in 95% alcohol from overstained material or sections. 200 BOTANICAL MICROTECHNIQUE B. Methyl green. A saturated solution in 1% acetic acid keeps well, or it may be made up simply in distilled water. Dilute if desired. This is a good stain for living cells, but it is especially valuable in combination with acid fuchsin, forming an effective double stain for the,tissues of higher plants. Sections from preserved material may be stained at once, those from fresh material must be fixed in absolute alcohol or chrom- acetic acid (Sec. 194). Stain first with methyl green for two hours or more and wash in distilled water until the green remains in the ligni- fied cell walls alone. Then stain with acid fuchsin for a few minutes, — not long enough to affect the lignified tissues, — and pass through 95% alcohol to absolute alcohol and into clove oil and balsam (Sec. 187). C. Erythrosin. This stain is similar to eosin and may be used in saturated solutions in water or 70% alcohol. It is a good counterstain following hematoxylin or green and blue anilin dyes. MOUNTING IN BALSAM AND GLYCERIN 187. Mounting in balsam. Canada balsam is the most satisfactory medium for permanent preparations. It should be used whenever possible, but there are some subjects, such as delicate filamentous alge and fungi, which cannot easily be carried into balsam without shrinkage, or which cannot be teased apart when brought into that medium because the clearing agents such as xylol or clove oil render the filaments much less flexible. For such subjects glycerin, glycerin jelly, or Venetian turpentine are better media. Material is carried into balsam from absolute alcohol through a clear- ing agent. It must first be brought up through the grades of alcohol to 95% (Sec. 178), where it is best left several hours (it may remain in 95% alco- hol indefinitely). Material is then placed in absolute alcohol to remove all trace of water (dehydration). Dehydration takes from thirty minutes to an hour or more, according to the size of the object, and it is well to change the alcohol once or twice if there is much material. From absolute alcohol the material is carried into a clearing agent, clove oil or xylol being the simplest. Clove oil removes the absolute alcohol rapidly and is the better clearing agent following anilin dyes, but should never be used after hematoxry- lin, for its acid quality fades that stain. Xylol acts more slowly and does not affect hematoxylin stains. Microtome sections on the slide are handled much more rapidly through the alcohols and clearing agents, as is described in Sec. 199. With delicate material xylol is always the safest clearing agent, because it mixes more slowly and less violently with absolute alcohol. The danger of shrinkage is lessened greatly by preparing three mixtures of absolute alcohol MOUNTING IN BALSAM AND GLYCERIN 201 and xylol: (1) one fourth xylol and three fourths absolute alcohol ; (2) half and half xylol and absolute alcohol ; (8) three fourths xylol and one fourth absolute alcohol. Even the most delicate material of alge and fungi can generally be carried without shrinkage into pure xylol if passed through these grades, being left an hour or more in each. Once in xylol, material is safe from shrinkage, and it may be left in this reagent indefinitely. It is absolutely essential for good results that the dehydration be perfect. Small objects should be examined throughout the process to determine the exact time of any cell shrinkage, which may be corrected with greater care. After being in clove oil a short time (from five to fifteen minutes), or in xylol a much longer time (several hours), the material is transferred to Canada balsam on the slide. The balsam should be so diluted with xylol that it drops readily from a glass rod. A cover glass is then gently lowered over the object with the point of a needle. The balsam will gradually harden as the xylol dries out. Air bubbles need give no concern ; they will work out to the edge of the cover glass as the balsam hardens. When balsam thickens in its bottle xylol should be added; the cloudiness which may develop will soon pass away. 188. Mounting in glycerin. Material is transferred from water to a con- siderable quantity of a 10% aqueous solution of glycerin in a watch glass. This should not cause shrinkage. The watch glass is then protected from dust and the water allowed to evaporate until the solution is about as thick as pure glycerin. The material is now ready to be mounted and will be so soft that it can be easily teased apart. A small drop of the solution is placed on the slide, the material arranged in it, and a clean cover glass with one edge resting on the slide is carefully lowered with a needle until the glycerin runs out to the edge on all sides. This must be done so carefully that no bubbles of air are inclosed. Practice will determine the amount of glycerin necessary to fill the space under the cover glass, which should not be more than five eighths the width of the slide. The less glycerin the better. The glycerin should not run out beyond the edge of the cover glass, although a small amount may be wiped away with a cloth moistened in alcohol. On no account must the glycerin be allowed to run over the edge of the cover glass; such a prepara- tion is worthless.” The slide is now ready to be sealed, or it may be laid away to allow the glycerin to become somewhat more dense. The best cement is gold size. This should not be so thick that it cannot be easily spread with a brush. If too thick, thin with oil of turpentine. The gold size is generally applied at the edge of the cover glass while the slide is whirling on a turntable. A thin ring should be laid three eighths of an inch wide, half on the slide and half over the edge of the cover glass, thus seal- ing the glycerin within a chamber. The sealing will not be perfect unless 202 BOTANICAL MICROTECHNIQUE the cover glass and slide are absolutely dry, that is, free from any glycerin. The first ring should be thin and allowed to dry thoroughly before the second ring is applied. More may be added if necessary. Properly sealed prepara- tions will last indefinitely, but the sealing is a delicate operation and re- quires some experience. Glycerin jelly is for some subjects as good a mounting medium as glycerin, and the preparations are more durable. Transfer material from a rather thick solution of glycerin to a drop of melted jelly on a warm slide, arrange with needles and carefully lower a warm cover glass over the mount, taking care not to inclose air bubbles. It is necessary to work quickly. The cover glass may be sealed with a ring of gold size and thus strengthened. 189, Venetian turpentine. The difficulty of properly sealing glycerin preparations, together with their fragile nature, is the chief objection to the glycerin mount. A method of mounting in Venetian turpentine has recently been perfected by Chamberlain.1_ By this process material may be brought without danger of shrinkage into a medium (Venetian turpentine) which hardens like balsam and requires no sealing. The technique is somewhat long and the staining methods special, but the results are striking. The staining, so far as we have seen preparations, does not bring out the finest details of protoplasmic structure as well as such stains as iron-alum hematox- ylin, safranin, and gentian violet. For details of this method the reader is referred to Chamberlain. IMBEDDING IN PARAFFIN 190. The paraffin method of sectioning. There are several methods of sec- tioning plant tissue, all of which have their limitations, because plant struc- tures range from those of, great delicacy, as among the thallophytes and bryophytes, to the firm and hard tissues of the sporophyte generation of the pteridophytes and spermatophytes. Very firm or hard tissue cannot be cut in paraffin, and sections may be made free-hand (Sec. 194) from fresh or pre- served material, but are better cut in celloidin (Sec. 195). Softer structures, such as anthers, ovule cases, and many developing organs of the seed plants, together with the gametophyte generations of the pteridophytes and almost all structures in the bryophytes and thallophytes, are sectioned most effect- ively by the paraffin method. Its advantages are that sections can be cut very thin, that they can easily be arranged serially on the slide, and that they can be stained with greater precision. Sectioning in paraffin is pre- ceded by the process of imbedding, which involves the preliminary processes of dehydration and clearing, and infiltration. 1 Methods of Plant Histology, p. 79, 1905. IMBEDDING IN PARAFFIN 208 191. Dehydration and clearing. The material to be cut is passed carefully through the grades of alcohol to 95% (Sec. 178). It should remain in 95% alco- hol for at least several hours or more, and is then placed in absolute alcohol, generally in a vial, and this should be poured off and renewed after an hour or two. The material should be left in absolute alcohol from four to eight hours or over night, unless the object be very small. It ought then to be free from water (dehydrated) and ready for the clearing agent, which will remove the absolute alcohol and also dissolve the paraffin, so that the latter may re- place the former throughout the tissue. The clearing agents most frequently used are chloroform and xylol. Chloroform acts more rapidly, but there is less danger of shrinkage with xylol. However, the chief danger of shrinkage lies in imperfect dehydration. Three mixtures of the clearing agent (chloroform or xylol) vith absolute alcohol are necessary to insure the gradual replacement of the latter by the former. These are (1) one fourth clearing agent, three fourths absolute alcohol ; (2) half and half clearing agent and absolute alcohol ; (3) three fourths clearing agent and one fourth absolute alcohol. The material is passed through these mixtures in the above order and then into the pure clearing fluid, either chloroform or xylol. When chloroform is used the material need not be left more than from four to eight hours in each mixture, and less if the object be small. If xylol is used, the material should be ‘left at least twelve hours in each mixture, and a longer time will do no harm. It should not remain in pure chloroform more than twelve hours before paraffin is added, and a shorter time is generally better; but it may be left in pure xylol a longer time, and even a day or more with advantage. Besides removing the absolute alcohol the clearing agent renders the tissues more transparent, that is, ‘‘ clears’? them. 192. Infiltration. Small pieces of paraffin are now added to the chloroform or xylol to the point of saturation and beyond. At this time the vials may be placed on the top of the oven, where they will be warmed, thus allowing more paraffin to dissolve. The best form of paraffin bath is a square or rectangular hot-water oven, with a door at the side and one or more shelves within. This should be heated by gas or by an electric coil with a thermostat arrangement to keep the oven at a constant temperature of about 52°C. The temperature may run as high as 56°, or probably higher if the dehydration has been perfect, but in genera] the temperature should be kept low. Material in chloroform and paraffin is placed in the bath and the vial un- corked. The chloroform will be driven off after a number of hours, leaving the material in pure melted paraffin. This process should not be hastened ; a day or two in the paraffin bath will generally give the most satisfactory results. Tasting the paraffin is the best test of the removal of the chloroform ; should 204 BOTANICAL MICROTECHNIQUE it be at all sweet there is chloroform still present. The chloroform must be entirely driven off before imbedding, otherwise the paraffin will not cut well. Material in xylol and paraffin must be treated differently from that in chloroform. Xylol cannot be removed easily by heat. Consequently the ma- terial must be transferred through solutions with less xylol in them until it is carried into pure paraffin. The simplest way is to pour off solutions and add melted paraffin, keeping the vials in the bath. The mixtures of paraffin and xylol may be saved and used again or simply left in the bath to gradually purify as the xylol is driven off. Finally the material is placed in two or three changes of pure paraffin to remove the last trace of xylol. It will do the material no harm to remain several days in the mixtures of paraffin and xylol, and structures with thick walls or coats (such as the megaspores of Selaginella)must be left sometimes for weeks before infiltration is completed. 198, Imbedding. The material is now in pure melted paraffin and ready to be cast in a cake. Most subjects can be cut in paraffin, which melts at a relatively low temperature, 50°-52° C. Others require a hard paraffin with a melting point of 56° or higher. In general it is better to imbed in a medium paraffin and plan to cut in a room at a cool temperature. Petri dishes are good receptacles for the casting, or paper trays may be used. Two L-shaped pieces of metal on a glass plate are convenient, since the size of the mold may be readily adjusted to the object. The interior of the receptacle should be smeared with glycerin to prevent the paraffin from sticking. The melted paraffin is poured into the receptacle with the material and the latter is then arranged with a heated needle. Finally the receptacle is gently lowered into a vessel of cold water, so that the paraffin is cooled quickly, which prevents its crystallizing, but it cannot be entirely immersed until the paraffin has solidified over the top. When cold, the cake may be cut up into blocks of convenient size which are ready for cutting (Sec. 196). Material that is perfectly imbedded will be preserved indefinitely in a form that gives no further trouble, and for this reason it is often desirable to run material into paraffin instead of keeping it in alcohol. SECTIONING 194. Free-hand sectioning. Free-hand sections are, as a rule, sufficiently satisfactory for general studies of the tissues of spermatophytes and pterido- phytes. The technique is as follows. The object is held between the thumb and finger of the left hand, or, if small or soft, it must be placed between two flat pieces of pith. The razor is held in the right hand and is drawn across the object with the edge towards the operator and the blade sliding on the forefinger of the left hand. There should be water on the upper edge of the SECTIONING 205 razor, and as the sections are cut they should slip into the water and float in it. When a number of sections have been cut, they may be removed with a brush to a watch glass of water. It is, of course, impossible to cut good. sections with a dull razor. A small hand or table microtome is frequently of great assistance, taking the place of free-hand sectioning. The object is held between pith in an adjustable clamp, and the razor slides over a glass plate. A large number of sections, sufficient to supply a class, may thus be easily cut from such an object as a piece of stem or leaf. Some methods of staining free-hand sections have been outlined in Secs. 183-186. Those of preserved material in alcohol require no further treatment before being placed in the stain, but sections of living material must be fixed before they can be satisfactorily stained. If the tissue is firm, the simplest method is to place the sections directly into absolute alcohol, when after an hour or so they may be stained. If, however, the tissue is delicate, or the cells contain much protoplasm, it is best to fix in medium chrom-acetic acid (Sec. 172) for two to twelve hours, washing for an hour or more in several changes of water. Such sections may be stained at once or run up into alcohol. 195. Sectioning in celloidin. As previously stated (Sec. 190), very firm and hard tissues such as characterize the sporophyte generation of pterido- phytes and spermatophytes cannot be cut in paraffin. Exact work is fre- quently only possible through sections cut in celloidin. Furthermore, large sections of stems, roots, etc., can only be cut by this method. The tech- nique is, however, somewhat long, and for the purposes of general studies free-hand sections, or those cut on a hand microtome, are likely to prove sufficiently satisfactory. A detailed account of the celloidin method as em- ployed in botany is given by Plowman, Botanical Gazette, Vol. XXXVII, p. 456, 1904; and in Chamberlain’s Methods of Histology. 196. Sectioning in paraffin. Sectioning in paraffin is only possible for structures of reasonably soft tissue and not very large. The advantages of the method are that the sections may be cut much thinner than in celloidin or free-hand, that they may easily be arranged serially, and that they may be stained with greater precision. The method is very generally applicable throughout the thallophytes and bryophytes and for the gametophyte gen- eration of the pteridophytes and spermatophytes, together with the softer tissues of many organs and developing structures of the sporophyte genera- tion of the latter groups. Paraffin material is cut on a microtome. The most convenient instru- ment is the rotary microtome of the Minot type, of which there are several forms on the market. The sliding microtome of the Jung-Thoma type is also excellent, and while not so rapid as the rotary is sometimes more accu- rate for the most exact work. 206 BOTANICAL MICROTECHNIQUE The material, imbedded in the paraffin cake, is cut out in a small block, which is fastened by heat to a metal holder for the rotary microtome or to small wooden holders for the sliding ones. The block should be arranged so that it will be cut as nearly as possible in the correct plane. The paraffin is then trimmed around the object so that the cutting edge is square or rec- tangular, with parallel edges. The block is then adjusted by a mechanism so that the face which is to strike the knife is exactly parallel to its edge and so that the object will be cut in the desired plane. Cutting in paraffin is not successful unless the sections run off the knife edge in an unbroken ribbon. There are a number of conditions necessary to obtain this result. The knife must be very sharp and the edge without nicks (at least where the cutting is done), which will split the ribbon lengthwise. It is useless to attempt to cut with a poor or dull knife. Ifa ribbon after running smoothly begins to split or show conspicuous lines, draw the finger upwards along the edge of the knife. The difficulty may have been caused by some hard particle lodged against the edge, which is thus removed. The edge of the knife must be clean; grease or paraffin may be removed with xylol applied by a brush or with a soft rag. The ribbon should run straight. If it begins to curve, trim the block unevenly so that it will come off the knife straight; a curved ribbon is generally due to differences in the texture of the two sides of the object. Sometimes sections roll up or fail to stick together in a ribbon. This gen- erally means that the paraffin is too hard for the temperature of the room. The cutting must be done in a warmer room or the material reimbedded in a softer paraffin. Cutting the sections in the sunshine of a window instead of in the shade will often remedy the difficulty. A more fre- quent difficulty is a crushing of the sections together. This means either that the knife is not sharp, that its edge is not perfectly clean, or that the paraffin is too soft for the temperature of the room. If the paraffin is too soft (as is commonly true in summer temperatures), the block and knife may be cooled in ice water or the material reimbedded in harder paraffin. It is time wasted to attempt to cut when the ribbon is not running smoothly; find out where the trouble lies and remedy it. The finest quality of hone is necessary for sharpening microtome knives, and it should never be used for any other purpose. The Belgian stones are ‘the best. There are also some good carborundum hones. The razor is gently passed back and forth on the hone with the edge forward, and strop- ping is not necessary or desirable if the hone is of the best quality. Soapy water is one of the best lubricants of the hone. Sections are best cut from 7-10 micromillimeters thick for general histo- logical work, but must be cut 5 or less for the finest details of protoplasmic structure. A micromillimeter (also called a micron) is a thousandth of a STAINING ON THE SLIDE 207 millimeter. As the ribbon comes off the microtome knife it is removed in convenient lengths and laid in a series from left to right on a clean piece of paper. The ribbons may be kept indefinitely under a bell jar, but they are best mounted as soon as convenient, since they will collect some dust no matter what precautions are taken. The ribbons are made to adhere to the slide with a fixative of the follow- ing formula (Mayer’s albumen fixative) : white of egg 50 ce. glycerin 50 ce. sodium salicylate 1 gram Mix well and filter. The sodium salicylate is an antiseptic and the fixative will keep for several months. Place a very small drop on the slide and with the tip of the little finger spread the thintiest film that can be laid on evenly over it. Then cover the film of fixative with water and place the ribbons cut to the proper lengths upon the water, arranged as desired. Warm the water gently over a flame; the paraffin will soften and the ribbons will expand and become perfectly smooth. The paraffin should not be allowed to melt. Drain the water off carefully and arrange the ribbons, which will now lie in a film of water, over the fixative. Put the slide aside to dry. It is frequently convenient to warm the ribbons in the water by placing the slide on the top of the paraffin bath and then to dry the slide in the same way, protected from too much heat by several thicknesses of blotting paper. The preparations are not ready for staining on the slide until perfectly dry. They may be kept thus indefinitely, but it is best to stain soon, since the surface of the ribbons will inevitably collect dust. A great saving of time can be secured by preparing a number of slides at a time and carry- ing them simultaneously through the above processes and those of staining on the slide. STAINING ON THE SLIDE 197. Preparation for staining on the slide. The dry slides with the ribbons adhering to the fixative may be placed in the bath to melt the paraffin, or they may be gently heated over a flame (with the ribbon side up), a process which must be managed carefully so as not to scorch the sections. The slide is then placed upright in a well of xylol (Stender dishes are convenient), which should not be near the flame. The xylol will dissolve the melted paraffin in a minute or so. The slide is then taken out of the well (the under side wiped off) and either placed in a well of 95% alcohol or a stream of alcohol is run over it from a pipette or wash bottle. The slide is now ready to be placed in the staining wells, of which there are various forms, but Stender dishes are satisfactory. 208 BOTANICAL MICROTECHNIQUE 198. Bleaching after osmic acid. Material fixed in chrom-osmo-acetic acids (Flemming’s fluids) is always blackened by the osmic acid. This blackening must be chiefly or wholly removed before staining. Microtome sections, after the solution of the paraffin with xylol and rinsing in 95% alcohol (Sec. 197), are placed in wells of 5-10% hydrogen peroxide in 70% alcohol. The bleaching is generally effected in an hour or less, but may require longer. Stronger solutions of hydrogen peroxide can be used if necessary, but it is safer to employ them weak. As soon as the gray or black tint is removed the slide is rinsed in 95% alcohol and is then ready for the stain. Small objects which are not to be sectioned (such as filamentous alge) are treated in the same manner in watch glasses. - 199. Staining on the slide. The best stains for the details of protoplasm are iron-alum hematoxylin, or safranin followed by gentian violet. Perhaps the most successful combination is safranin, gentian violet, and orange G, — a combination known as Flemming’s triple stain, the use of which is, how- ever, one of the most difficult of the staining methods. Delafield’s hema- toxylin, and safranin followed by Delafield’s hematoxylin, are among the best general stains for tissues, and methyl green followed by fuchsin is also good. A. Iron-alum hematoxylin. This method follows the same outline as is given in Sec. 182 for staining in bulk. The slide is taken from 95% alcohol, dipped in 35%, and placed in iron alum (best used in 1% solu- tion) for from two to four hours; it is then rinsed in distilled water and left in hematoxylin from four to eight hours or over night. From the hematoxylin it is returned to the iron alum to extract the stain, and this process must be watched with care, the preparation being examined from time to time under the microscope. Atthe proper point of extrac- tion the slide is placed in a large dish of tap water, where it must remain for fifteen minutes or more. It is then dipped in 35% alcohol (if desired) and left two or three minutes in a well of 95% alcohol (it may remain indefinitely in the 95%). Finally the slide is taken from the 95% alco- hol, drained, and some absolute alcohol is poured over the sections from a small bottle and rapidly drained off, and the slide placed as soon as possible in a well of xylol (clove oil should never be used). The preparation must remain in the xylol half an hour or more, since the xylol and absolute alcohol do not mix rapidly, after which it is removed, the superfluous xylol drained off, and the sections mounted in balsam. Minute bubbles on the slide after having been in xylol indicate that the dehydration has not been perfect, and they must be removed by absolute alcohol and the slide again placed in xylol. B. Iron-alum hematoxylin and safranin. The hematoxylin stain may be extracted by iron alum until it remains practically in the nucleus alone. STAINING ON THE SLIDE 209 Then after passing through tap water it may be placed in a solution of safranin (Sec. 184) until the protoplasm and cell walls are slightly stained, after which it is carried through absolute alcohol into xylol. C. Delatield’s hematoxylin. This stain is excellent for tissues, since it colors the cell walls sharply, as is not done by iron-alum hematoxylin. Follow the outline given in Sec. 183. D. Safranin, gentian violet, and orange G. It is not necessary to use the orange G in this combination, known as Flemming’s triple stain, but the best results have been ‘obtained with it. The technique of this method of staining is difficult, but it gives perhaps the most effect- ive staining for the study of protoplasmic structure, especially during nuclear division. It is impossible to give more than a general programme of the method, since the time limits necessary to obtain satisfactory results vary with different material and must be tested experimentally. The slide is transferred from 95% alcohol to a well of safranin. The alcoholic solution mixed with an equal part of water is good, as is also anilin safranin (Sec. 184). After remaining in safranin from four to twenty-four hours (over night is generally convenient), the slide is placed in 50% alcohol and the stain extracted until it remains in the nucleolus and chromatin alone. Acid alcohol (Sec. 184) may be used to extract the stain more rapidly, but it is generally not necessary. The preparation is then placed in a well of gentian violet. A satu- rated aqueous solution is good, or a 1% solution is generally strong enough. The slide is left in gentian violet as short a time as possible to obtain good results, and this can only be determined by trial. Some- times merely dipping it in the stain is sufficient ; other material may require a number of seconds, or even minutes. On removal from gen- tian violet the slide is drained and rinsed in 50% alcohol, and then absolute alcohol is poured over the sections, followed by a few drops of oil of cloves placed in the center of the preparation. The oil of cloves may be replaced with cedar oil or xylol to avoid possible fading of the stain. The secret of success with gentian violet is not to stain the nucleolus and chromatin so deeply that the’ stain will not wash out. They should be left red and the other protoplasmic structures blue. If the nucleolus and chromatin come out blue, the slide has been left too long in gentian violet. In our practice the best results have come with very short treat- ment in strong gentian violet (frequently only a dip, or a few seconds timed by the watch), followed directly by absolute alcohol. The oil of cloves may be depended upon to remove much of the gentian violet. But, as previously stated, material differs very greatly in its reaction to gentian violet, and each subject requires experimentation and a critical 210 BOTANICAL MICROTECHNIQUE examination at various stages in the process of staining, to correct errors. The commonest mistake is to overstain with gentian violet. Should orange G be brought into the combination, the slide after re- moval from gentian violet is rinsed in water and placed in a 1% aqueous solution or a dilution of this strength. It is left from ten to thirty sec- onds in this stain and then treated with absolute alcohol and cleared in oil of cloves as described above for the gentian violet. The orange G, if successfully used, will give a grayish tinge to the cytoplasm, while the spindle fibers (kinoplasm) will be»blue and the nucleolus and chro- matin red. The combination, when successful, is the most striking stain known for nuclear figures. Slides should not be destroyed if the results are not up to expecta~- tions. If the protoplasm is uniformly blue, structures may still show fairly well, and if the stain is too weak, the balsam may be removed _with xylol and the slide stained again. It is not necessary to use orange G, and in our own practice this stain is generally omitted. . Safranin and Delafield’s hematoxylin. This combination, applied as outlined in Sec. 185, is excellent for the staining of tissues, without much regard for the details of protoplasmic structure, such as nuclear figures, etc. Other stains. Other anilin dyes besides safranin and gentian violet are frequently used in staining on the slide. Fuchsin (Sec. 186, A), methyl green followed by fuchsin (Sec. 186, B), and erythrosin after hematoxy- lin, or blue or green anilin stains (Sec. 186, C) give good results for the study of tissues. They are especially satisfactory for the differentiation of structure in fibro-vascular bundles. ; CULTURE METHODS THE CULTURE OF ALG 200. Culture in aquaria. The most convenient forms of aquaria are shallow glass dishes eight to ten inches wide, battery jars six to eight inches wide, or other large glass receptacles. These should be loosely covered with pieces of heavy glass to keep out the dust, and should not be filled more than two thirds full of water. It is not generally necessary to aérate the water, and cultures should rather be left to themselves to grow the forms with which they are stocked, or to develop whatever types may appear. It is always interesting, and frequently surprising, to see what growths will develop of their own accord in aquaria. There should be no metal in contact with the water of aquaria, and copper is especially poisonous. Some alge, such as the water net, Hydrodictyon, species of Gidogonium, Coleochate, Chara, Oscillatoria, and numerous one-celled forms grow readily in aquaria. Other types are more difficult to cultivate, as Spirogyra and other pond scums, and it is almost impossible to keep the red or the brown marine alge alive for any length of time. Terrestrial species of Vaucheria frequently grow luxuriantly over the earth of the flowerpots in greenhouses. Alge are more likely to survive in aquaria when kept in the water of the ponds and ditches from which they came. Such water may be filtered, and the aquaria should be stocked with only a small amount of the alge. It is not desirable to have animals such as snails or crustacea in the aquaria, for there is almost sure to be present a sufficient quantity of microscopic forms to preserve a balance of animal and plant life. The aquaria are best placed outside the room on window ledges, except in freezing weather, and they should have very little, if any, direct sunlight. A. Knop’s solution. There are a number of culture solutions. One of the best known is that of Knop, made as follows : potassium nitrate 1 gram potassium phosphate 1 gram magnesium sulphate 1 gram These three salts are dissolved in one half liter of rain water or fresh tap water, that is tap water which has not been standing in metal pipes. To this is added a solution of 4 grams calcium nitrate in one half liter of similar water. There will be formed an insoluble precipitate of 211 212 CULTURE METHODS calcium phosphate which is left in the fluid. This makes a .7% solution of the salts, which is too strong for general purposes. It is better diluted with an equal quantity of water (making a .85% solution), or, for delicate algze, with two liters of water (making what is approximately a .2% solu- tion of the salts). Alge are placed directly in this culture solution, and many of them do well in it. B. Moore's solution. Moore reports that the following solution (which is a modification of one of Beyerinck’s) is much more satisfactory than that of Knop: ammonium nitrate .o gram potassium phosphate .2 gram magnesium sulphate .2 gram calcium chloride -1 gram iron sulphate trace These salts are dissolved in a liter of water. For blue-green alge the amount of ammonium nitrate should be doubled, and 1-2% of glucose may be added with benefit. C. Cane sugar. A 2-4% solution of cane sugar is an important fluid, since some alge —as Vaucheria, Hydrodictyon, and Spirogyra — will gener- ally fruit after a few days when transferred to it from pond water or cultures solutions and exposed to bright light or moderate sunshine. 201. Cultures on agar-agar. Pure cultures of unicellular algze may be grown and isolated on agar-agar mixed with a nutrient solution. Moore recom- mends his modified Beyerinck’s solution (Sec. 200, B), with double the amount ofammonium nitrate and 2% of glucose. To a liter of this solution (heated to boiling) 5 grams of agar is added, and after its liquefaction the fluid is poured into small Erlenmeyer flasks of 100 cc. capacity, or other small dishes which may be tightly covered; on cooling, the liquid will stiffen to a moist jelly. Pure cultures may be easily established in such vessels, and if protected from drying will flourish for years. THE CULTURE OF FUNGI 202. Cultures in moist chambers. Fungi will grow in abundance upon a great variety of substances when kept damp in moist chambers. The most convenient form of a large moist chamber is a rather low bell jar five to six inches high, set in a dish of water. The substance is placed on some sup- port, such as a zine rack, so that it is raised above the surface of the water, the evaporation of which keeps the air in the interior of the bell jar moist. It is well also to line the interior of the bell jar with moist filter paper in con- tact with the water below. Cultures upon large pieces of bread and cheese are CULTURE OF FUNGI 2138 best arranged for in this manner. Smaller moist chambers may be made by placing filter paper above wet Sphagnum on the bottom of shallow glass dishes, such as crystallizing dishes, three or more inches high, covered by a piece of glass. The substance to be used as the substratum is placed on the filter paper, which is kept moist by the Sphagnum. Such chambers are well suited to cultures on small pieces of fruit or other vegetable matter, and on the dung of various animals. Cultures on horse dung are best made in larger dishes or under bell jars. 203. Pure cultures on potato agar. Most saprophytic fungi may be cul- tivated on potato agar, which is one of the simplest and most satisfactory of the culture media. To make potato agar, pare two or three medium-sized potatoes, cut into thin slices, place in a stewpan, and cover with tap water. Allow the water to simmer gently for one half hour, or until the potatoes are soft but not dis- organized. Do not let it boil. Strain the liquid, which should be as clear as possible. Add enough tap water to make one half liter, and place in a flask with 10 grams of agar-agar cut up in small pieces. Heat the flask in a steam sterilizer until the agar has melted and mixed with the culture fluid. Clean about thirty test tubes (six inches long and three fourths of an inch across), rinse, drain, and dry. Fit cotton plugs of a good quality into the dry test tubes. They should enter the tubes at least an inch and project somewhat beyond. Place the tubes fitted with cotton plugs in a dry-air sterilizer and expose to a temperature of 140° C. for an hour; this will kill all spores of fungi, including bacteria, in the tubes or on the cotton. The tubes are best handled in a receptacle made of heavy wire netting, and of a size which will slip into the steam sterilizer. Pour the melted potato agar into the test tubes by means of a funnel, removing the cotton plug carefully and holding between the fingers while fill- ingeach. The tubes should be filled up about one and one-half inches from the bottom. It is very important that no agar become smeared around the top of the test tube where the plug isinserted. The filled tubes, carefully plugged, are now sterilized twice a day (morning and night) on three successive days in the steam sterilizer for an hour each time. Thisis necessary to render the tubes free from bacteria, for the spores of bacteria are not generally killed by the temperature of 100° C., but they germinate quickly in the potato agar, and the vegetative bacteria produced by them are then killed by that temperature. After the sterilization of the third day the tubes are taken out and laid on a table inclined against a board so that the surface of the hot fluid agar runs three or four inches up the sides of the tubes. As they cool, the agar stiffens in this position, forming a long, slanting surface in each tube, which is now ready for inoculation. 214 CULTURE METHODS Transfers of spores are made to the tubes by means of a piece of stiff platinum wire about two inches long, set in the end of a glass rod when melted in a flame. The lower end of the rod and the wire are sterilized in a flame and the point of the wire is touched to a single spore-bearing hypha in a culture. The cotton plug is then carefully removed from a tube and held between the fingers while the point of the wire is drawn over the sur- face of the agar. It is best that the tube be held inverted while being inoc- ulated so that dust may not enter. The plug is replaced quickly and the inoculated tube is laid aside to await developments. If but a single spore- bearing filament has been touched, the culture will probably be pure. Should an impure culture develop, transfers may generally be made from it to another tube. Tubes with cultures may be prevented from drying out too rapidly by cutting off the tops of the cotton plugs and coating the ends of the tubes with paraffin. Potato agar may be poured into Petri dishes and sterilized in the same manner as the test tubes. Such dishes are excellent for studies upon the bacteria as outlined in Sec. 100. : 204. Hanging-drop cultures. These are used for the study of germinating spores, pollen grains, and other subjects. The spores are placed in a drop of the culture fluid on a cover glass, which is then arranged so that the drop hangs down from the lower side in a small moist chamber on a slide. The chamber may be ‘made of a ring of glass cemented on the slide with wax or gold size (Van Tieghem cell), over which the cover glass fits and is sealed with vaseline. A little distilled water in the bottom of the chamber will save some evaporation from the drop of the culture fluid. A more temporary but also effective chamber may be made by cutting a square hole about one half inch in diameter in the center of a piece of card- board one inch wide, one and one-half inches long, and one eighth of an inch or more thick. The cardboard is boiled and pressed closely on the slide. The culture drop is then placed in the center of a cover glass an inch square or slightly smaller. This is inverted over the hole in the cardboard so that the culture drop hangs down in the center and the cover glass is then pressed closely against its wet surface. Water is added from time to time to the edge of the cardboard to keep it moist, and the slide when not being studied may be placed in a moist chamber, which will hinder the cardboard from drying rapidly. - The culture fluids vary with the subject. Boiled decoctions of horse dung are good for the germination of many fungal spores. Decoctions of decayed wood are used for the spores of slime molds. Solutions of sugar (83-30% in tap water) are employed in the germination of pollen grains (see Experiment XLII) ; 1.5% of gelatin may sometimes be added to advantage to the sugar solutions. Spores of mosses and ferns germinate readily in sweetened water. CULTURE OF LIVERWORTS AND MOSSES 215 THE CULTURE OF LIVERWORTS AND MOSSES 205. The culture of liverworts. Aquatic liverworts, such as Ricciocarpus natans and some species of Riccia, will sometimes grow fairly well in large glass aquaria, but they must have pure air and considerable water. They will do much better in tanks or cement basins in greenhouses. The terrestrial liverworts, such as Marchantia, Lunuldria, Conocephalus, and related types, grow readily on damp soil in greenhouses or in large vessels covered with glass. These forms and various mosses are frequently present in ill-kept greenhouses. They will not do well in very bright light, preferring shaded situations, and must have abundant moisture in the earth. If convenient, it is well to cultivate them on soil from their habitats. 206. The culture of protonemata. Moss spores germinate readily, and it is not difficult to obtain luxuriant cultures of protonemata. These frequently appear over the surface of earth in flowerpots in greenhouses, and then somewhat resemble the more common growths of Vaucheria. Cultures of the spores are conveniently made in bulb pots or other wide pots set in sau- cers of water. After filling the pot with earth to an inch from the top, it is well to heat it for two or three hours in a steam sterilizer to kil] fungi which might be troublesome, but this is not absolutely necessary. The spores of many of the common mosses of the fields will grow readily, but species of Funaria are especially satisfactory. The spore cases may be crushed over a sheet of paper to remove the spores, which are then gently blown over the surface of the earth. The top of the pot is covered with a piece of glass and the culture is watered from the saucer below. The earth should be merely moist, not wet, for too much moisture may result in the death of the culture by ‘‘ damping off’? from the growth of fungi. The earth will shortly become covered with a growth of green filaments. After two or three weeks, buds will be developed, followed by the appearance of the leafy moss plants. 207. Moss cultures. A thick growth of leafy moss plants generally arises from the protonemata as described above. These plants will in time develop sexual organs, the antheridial plants being easily distinguished by the rosette of expanded leaves around the yellow or orange-colored clusters of anther- idia. The archegonia will not be fertilized if the culture is watered entirely from the saucer below. When sporophytes are desired the culture must be flooded with water, first closing with a cork the opening in the pot below. It should remain flooded for an hour or more, after which the water may be allowed to run off. All ripe archegonia and antheridia will have opened, and the eggs, having been fertilized, will develop sporophytes. By succes- sively flooding the culture at intervals, sporophytes in various stages of development may be obtained. 216 CULTURE METHODS THE CULTURE OF FERNS 208. The culture of fern prothallia. Fern prothallia may be cultivated even more easily than moss protonemata. The method is essentially the same. The spores of common greenhouse ferns will germinate readily, but the pro- thallia of some present abnormalities due to apogamy (Principles, Sec. 311), so it is better to sow the spores of some of the wild ferns, such as Pteris aquilina, species of Onoclea, Aspidium, Polypodium, etc. Such spores gen- erally retain their vitality for a year or more. The spores of Osmunda, on the contrary, and also those of Hquisetum live only a few days and must be sown at once at maturity, but then give very luxuriant cultures. The spores, crushed out of their sporangia on a piece of paper, are blown over the surface of earth in bulb pots or shallow dishes. These are covered with glass and the pot is set in a saucer and watered from below. The earth in the pot may be sterilized with advantage (Sec. 206). The culture should not be kept too moist, for there is danger of the prothallia damping off. Prothallia will begin to develop antheridia in three or four weeks and will be full-grown in six weeks. Care should be taken not to sow the spores too thickly, at least in portions of the dish, for crowded growths of prothallia remain dwarf and only develop antheridia. Growths of young fern sporophytes are obtained by flooding a culture of mature fern prothallia for an hour or more, closing the bottom of the pot tem- porarily as described in Sec. 207. In a few weeks the first leaves of the young fern plants will appear, growing up in the notch of the large fern prothallia. 209. Water ferns. The floating water ferns Salvinia and Azolla, like the floating liverworts (Sec. 205), will grow in glass vessels if they have pure air and plenty of water, but they do much better in large tanks or cement basins in greenhouses. Marsilia is hardy and grows well in tanks or basins. Sal- vinia and Azolla may be kept thus over winter, and in the spring, when placed in ponds out of doors, will generally do well. Marsilia is easily intro- duced into ponds, where it forms thick growths in shallow water. Salvinia is not uncommon under cultivation in water-lily ponds of city parks. THE CULTURE OF SEED PLANTS 210. Directions for the culture of seed plants. It is hardly worth while to give an account of the manner in which such seed plants as are needed for studies and demonstrations are best cultivated. The advice of a competent florist will be found more helpful than any set of printed directions. Das Pflanzenmaterial fiir den botanischen Unterricht, by Dr. P. Esser, I. Teil, Cologne, 1903, gives much useful information. Its price is Marks 8.20. MATERIAL, APPARATUS, AND SUPPLIES LISTS OF PREPARATIONS FOR THE MICROSCOPE 211. Slides of value in studies on the plant cell. Spirogyra. To show the nucleus: filaments. fixed in weak chrom- aeetie acid (Sec. 172, A), stained in iron-alum hematoxylin (Sec. 182), mounted in glycerin (Sec. 188) ; or stained in Magdala red and anilin blue and mounted in Venetian turpentine (Chamberlain, Methods of Histology, p. 81, 1905). Root tip of onion or hyacinth. For the study of cell and nuclear division : fixed in medium chrom-acetic (Sec. 172, B), or weak Flemming (Sec. 178, A), sectioned in paraffin from five to seven micromillimeters thick, stained with safranin and gentian violet (Sec. 199, D). Pollen mother cells of the lily or related types. Also excellent. subjects for the study of cell and nuclear division (see Lilium in next section). 212. Slides of value in type studies. he following list of preparations is merely suggestive; many other subjects may be added. It is a mistake to suppose that permanent preparations are necessary for type studies. They will, however, at times be of great assistance. The accumulation of class preparations requires time and patience and their proper use demands judgment. There is much danger in giving slides to students before they are prepared to understand from studies on living or preserved material the topography and significance of the structures shown in the preparations. Volvox. Whole colonies, safranin or iron-alum hematoxylin, carried with care into balsam (Sec. 187), the cover glass supported by two strips of paper to prevent crushing. Ulothri«, or other alga, forming zodspores. Tron-alum hematoxylin and safranin (Sec. 182), carried with care into balsam. Gidogonium. Iron-alum hematoxylin and safranin, glycerin, or carried with care into balsam. Coleochete. Fruiting thallus, Delafield’s hematoxylin (Sec. 183), balsam. Fucus. Paraffin sections of odgonial conceptacles to show histological de- tails, safranin and gentian violet, or D.’s hematoxylin. _ Sporodinia or Rhizopus. Zygospores with mycelium slightly stained with D.’s hematoxylin, glycerin, or balsam. Albugo. Paraffin sections of (1) blisters, D.’s hematoxylin ; (2) tissue with odgonia, safranin and gentian violet. 217 218 MATERIAL, APPARATUS, AND SUPPLIES Peziza or other cup fungus. Paraffin sections of fruiting surface, safranin and gentian violet. Puccinia. Paraffin sections (1) across sori of teleutospores and uredo- spores ; (2) cluster cup on barberry, D.’s hematoxylin. Coprinus or other gill fungus. Paraffin sections of gills, cut rather thick, showing basidia with spores, D.’s hematoxylin. Marchantia. Paraffin sections (1) of thallus, safranin and D.’s hematox- ylin (Sec, 185) ; (2) antheridial receptacles, D.’s hematoxylin; (8) arche- gonial receptacles for archegonia and sporophytes, safranin and D.’s hematoxylin. Porella. Paraffin lengthwise sections (1) of antheridial branches, D.’s hematoxylin ; (2) archegonial branches for sporophytes, safranin and D.’s hematoxylin. Anthoceros. Paraffin lengthwise sections of medium-sized sporophytes attached to small pieces of the gametophytes, safranin and D.’s hematoxylin. Funaria, Mnium, or Atrichum. Paraffin sections (1) of the tips of anther- idial and archegonial plants; (2) medium-sized spore cases for spore-bearing tissue, D.’s hematoxylin. Webbera. Protonema, common in greenhouses, frequently forms buds in great numbers, safranin, carried with care into balsam. Aspidium. Paraffin sections of sorus, D.’s hematoxylin. Pteris aquilina. (1) Cross and lengthwise sections of rhizome (Sec. 194) mounted on the same slide, safranin and D.’s hematoxylin. Pteris or other fern. Paraffin lengthwise sections of (1) root tip; (2) large prothallia to show archegonia and occasional developing sporophytes, safranin and D.’s hematoxylin. Pinus. (1) Cross sections of needle, safranin and D.’s hematoxylin; (2) Sections of wood, cross, radial, and tangential, mounted on the same slide, safranin and D.’s hematoxylin, or methyl green and fuchsin (Sec. 186, B); (8) paraffin lengthwise sections of ovules from a year-old cone, safranin and gentian violet or safranin and D.’s hematoxylin. Lilium or related types. (1) Paraffin cross and lengthwise sections of anthers, showing pollen mother cells in stages of nuclear and cell division, also sections of mature anthers ; (2) paraffin cross sections of ovule cases of various ages to show the development and structure of the embryo sac and ovule ; (3) double fertilization in embryo sac; (4) development of embryo and endosperm ; all stained with safranin and gentian violet, or safranin, gentian violet, and orange G (Sec. 199, D). Sambucus. Paraffin cross sections of mature anthers to show gameto- phyte nuclei in the pollen grain, safranin and gentian violet. Capsella. (1) Paraffin lengthwise sections of tip of growing raceme to show stages in the development of the flowers, D.’s hematoxylin ; (2) paraffin LISTS OF PREPARATIONS 219 lengthwise sections of ovules after fertilization, showing embryo in embryo sac, safranin and D.’s hematoxylin. 213. Slides of value in histological studies on the seed plants. ROOTS Corn root tip. Lengthwise section, showing plerome, periblem, and der- matogen, safranin and Delafield’s hematoxylin. Tradescantia root tip. Lengthwise section, showing general root struc- ture and nuclear division, safranin and gentian violet. Smilax root. Cross section, showing cortex, endodermis, and radial bundles, safranin and D.’s hematoxylin. STEMS Hippuris shoot. Lengthwise section of stem apex, showing meristem, origin of lateral shoots and leaves, safranin and D.’s hematoxylin. Indian corn stem. Lengthwise section, showing sieve tubes, companion cells, annular tracheids and sclerenchyma, safranin and D.’s hematoxylin. Pumpkin stem. Lengthwise section, showing sieve tubes and companion cells and vessels, safranin and D.’s hematoxylin. Menispermum stem. Cross section, showing separate bundles of a climbing stem with large vessels for conducting water, safranin and D.’s hematoxylin. Brasenia stem. Cross section, showing typical structure of an aquatic stem with large air cavities and scanty vascular system, D.’s hematoxylin. Dodder on golden-rod stem. Cross section, showing penetration of stem tissues by haustoria, safranin and D.’s hematoxylin. LEAVES Cycas revoluta. Cross section, extremely xerophytic leaf structure with thick cuticle, highly developed palisades, and depressed stomata, safranin and D.’s hematoxylin. Peperomia. Cross section, typical of water storage in the leaf outside of the photosynthetic tissue, D.’s hematoxylin. Silphium laciniatum. Cross section, vertical leaf with a palisade layer near each surface, D.’s hematoxylin. Potamogetm. Cross section, submerged leaf with thin epidermis, no sto- mata nor palisades, large air cavities and scanty vascular system, D.’s hematoxylin. Water buttercup. Cross sections (1) of aérial leaf; (2) submerged leaf, D.’s hematoxylin. 220 MATERIAL, APPARATUS, AND SUPPLIES SUGGESTIONS ON MATERIAL FOR THE STUDY OF PLANT HISTOLOGY 214. Histological material of the seed plants. Air passages. Rootstock of sweet flag (Acorus); stem of Juncus, Myrio- phyllum, Scirpus; leafstalks and flower stalks of pond lily and of Nuphar. Aleurone grains. Seeds of almond, Brazil nut, castor bean, and nutmeg. Bast fibers. Stem of flax, of hemp, of linden (young twig) ; leaf of Carlu- dovica, esparto grass, palm, pineapple. Bundles, closed. Stem of asparagus, corn, green brier (Smilax) ; flower stalks or leaves of Yucca jfilamentosa; petioles of fan palm leaves (cut if necessary from the handle of a palm-leaf fan). Bundles, open. Young stems of Aristolochia, Begonia, Clematis, evening primrose, Menispermum ; stems of sunflowers or other large composites. Cambium. See Bundles, open. Cambium, cork (phellogen). Young twigs of Abutilon (greenhouse species) and of elder. Central cylinder of root. Roots from bulb of hyacinth or onion ; roots of Acorus, Actea, Smilax, Veratrum. Chlorophyll bodies. Best seen in leaves of moss, as Funaria or Mnium, or in fern prothallia ; thin sections of any green leaves or upper epidermis torn off, with the tops of the palisade cells attached ; large and distinct in leaves or bracts of pineapple. Chromatophores or chromoplasts. Surface sections from sepals of Tro- peolum, pulp of fruit of Crategus, asparagus, or rose, root of carrot; many alge, such as Spirogyra, Ulothrix, desmids, diatoms, Ectocarpus, Nema- lion, ete. Collenchyma. Young stems of Aristolochia Sipho; stems of begonias, Salvia, and most Umbellifere. Cork. Young twigs of Ailanthus, sweet gum (Liguidambar), cherry, currant, Cytisus Laburnum, Euonymus alatus; ordinary bottle corks (from Quercus Suber) ; cortex of potato tuber. Cuticle. Leaves of Agave, Aloe, Cycas revoluta, Ficus elastica, Yucca, fila- mentosa. Embryo sac. Lily, buttercup, and.their allies. Epidermis. See under Cuticle. Also thin and easily peeled epidermis of iris, lily, hyacinth. Fertilization and development of embryo. Young fruit of Capsella, Mono- tropa, Pyrola, Veronica, lily, buttercup. Growing point. Tip of stem of Myriophyllum or Hippuris ; buds of lilac, Crategus, Viburnum. MATERIAL FOR STUDY OF PLANT HISTOLOGY 221 Hairs and scales. Glandular hairs, ‘“‘ geraniums,’’ most Labiate, sundew, tomato; ordinary unbranched hairs, leaves and stems of most Borraginacee, Gnaphalium, seeds of cotton ; scale-like hairs, leaves of Hlwagnus, olive, Shepherdia; star-shaped hairs, leaves of Matthiola; stinging hairs, stem of nettle ; T-shaped hairs, leaves and stems of Artemisias; branched hairs of mullein. Laticiferous tissue. Root of chicory, of dandelion ; stem of Euphorbia splendens, of lettuce ; bract of Ficus elastica. Leaf structure. Hydrophytic: EHlodea, Potamogeton, submerged leaves of Sagittaria ; mesophytic: most deciduous trees and shrubs; xerophytic: see under Cuticle, —also bearberry, crowberry, Elewagnus, holly, oleander, mistletoe, olive. Lenticels. Young twigs of birch, cherry, elder, and sumac. ‘ Leucoplasts. Pseudo-bulbs of Phajus grandifolius, rootstocks of Iris ger- manica. Nuclear division (mitosis). Pollen mother cells of lily and its allies ; cells of root tip of onion or hyacinth. Nuclei. Epidermal cells of many leaves; growing points of roots or stems; hairs of roots, stamen hairs of Tradescantia, hairs of stem of cucum- ber ; internodes of Tradescantia; bulb scale of onion; pollen mother cells of lily and its allies. Oil and resin glands. Aments of the hop; hairs and emergences on leaves of any aromatic Labiate ; leaves of Eucalyptus globulus, of Ruta; rind of lemon or orange. Oil as reserve material. Oily seeds, as almonds, Brazil nuts, cacao seeds, castor beans, peanuts, squash seeds, sunflower seeds. Palisade cells. Leaves of beech, Cycas, English ivy, Ficus elastica, holly, Japan quince, mistletoe, oleander, poplar, privet, willow, yucca. Pollen tubes. Pollen of snowdrop (Leucojum), sweet pea, Tropwolum, tulip. Root, dicotyledonous. Beans and other Leguminose, Composite, grapevine, primrose, Ranunculus; very young roots of most hardwood shrubs and trees. Root, monocotyledonous. See Central cylinder of root. Also asparagus, Aspidistra, corn and other large grasses, Iris, sedges, Smilax. Rootcap. Monocotyledonous: corn and other grasses, Iris; dicotyledo- nous: bean, pea, sunflower, Tradescantia grown in water. Root hairs. Most roots of very young seedlings from seeds sprouted on wet paper in a slightly damp atmosphere, Tradescantia grown in water. Sclerenchyma fibers. See Bast fibers and Wood fibers. Secondary thickening. Twigs two years old and more of coniferous and of hardwood trees. 222 MATERIAL, APPARATUS, AND SUPPLIES Seeds. With endosperm: buckwheat, castor bean, four-o’clock, all grasses, morning-glory, honey locust ; without endosperm?: all Cucurbitacew, most Leguminose, all Rosacee. Sieve tubes. Stems of Cucurbitacece, young stems of grapevine. Starch. Rootstocks of arrowroot (Maranta), of Canna; seeds of beans, buckwheat, corn, oats, rice, wheat ; stems of Huphorbias ; tubers of potato. Stem, dicotyledonous. See Bundles, open. Very young twigs of most hard- wood shrubs and trees. . Stem, monocotyledonous. See Bundles, closed. Rootstocks of grasses and sedges ; stem of bamboo and of rattan. Stomata. Leaves of Aloe, of Crassulacee, Cycas revoluta, Ficus elastica, Iris, Liliacee, oleander. Stone cells. Allspice fruit, clove flower stalk, oak bark, pear-fruit stalk. Tracheids. Wood of any coniferous shrub or tree. Vessels. Leaf of banana; peduncle of banana, of yucca; root of Acorus, of Iris; stem of corn, evening primrose, grapevine, rattan, Ricinus, sunflower. Water-storage system. Leaves of Agave, Aloe, Ficus elastica, Mesembryan- thacee, Peperomia; stems of Cactacew. Wood fibers. Fibrous hard wood, as alder, birch, hickory, linden, locust, magnolia, poplar (Populus). Wood parenchyma. Wood of apple, bladder nut, hawthorn, linden, pear, red cedar, rose. ; i Rererences. Strasburger-Hillhouse, 6; Strasburger, Noll, Schenck, Karsten, 1; Tschirch, 74. APPARATUS FOR THE LABORATORY 215, Apparatus. The stocking of a laboratory with apparatus is a matter of time and experience, depending upon the character of the work given. The following list is therefore merely suggestive : Aquarium jars. Large battery jars or museum jars are also good. Balances, large, capacity 2, gram to 2 kilograms, with weights. Balances, small, capacity 1 milligram to 100 grams, with weights. Battery jars, glass, quarts and gallons. Bell jars from five to six inches high, and one or more tall ones. Blotting paper. Bottles, dropping. Bottles, glass stoppered, assorted. Bottles, ordinary wide mouthed, assorted. Boxes, small, wooden, for germination experiments. 1].e. with the reserve-material practically all contained in the embryo, even if traces of endosperm remain. APPARATUS FOR THE LABORATORY 228 Camera lucida. Chemical thermometers, registering 100° C. and above. Clinostat. Clock glasses. Corks. Crystallizing dishes. Culture jars, large and small flat glass dishes. Dishes, ordinary plates and saucers, Evaporating dishes. Eyepiece micrometer. Flasks. Flowerpots, ordinary, and bulb pots, with saucers. Funnels, glass, assorted sizes. Glass growing case (see Wardian case). Graduated cylinders, 10, 100, 500 cc. Hones, one rough for scalpels, one Belgian or carborundum for microtome knives. Imbedding oven. Lead, thin sheet. Mason butter jars for preserved material. Microtomes. Museum jars, very useful but expensive. Petri dishes. Pipettes (medicine droppers). Printing frames, photographic. Printing paper, ordinary photographic and for blue prints. Razor strops. Retort stands. Sand. Sawdust. Stage micrometer. Stender dishes. Sterilizer, steam. Test tubes. Thermostat. Thistle tubes. Tools, such as hammer, saw, file, screw-driver, pliers, monkey wrench, cork borers, etc. Tubing, glass assorted and also rubber. Tumblers. Wardian case (glass growing case), Ganong, 7, p. 82. Wash bottles. Watch glasses, solid. 224 MATERIAL, APPARATUS, AND SUPPLIES CHEMICALS FOR THE LABORATORY 216. The supply of necessary chemicals in a laboratory will depend upon the character of the work. This list is therefore merely suggestive. Acids, commercial acetic, glacial acetic, chromic, hydrochloric, nitric, osmic, sulphuric. Alcohol, commercial and denatured. Alcohol, absolute. Ammonia. Ammonia sulphate of iron (iron alum). Benzine. Calcium nitrate. Calcium oxide (quicklime). Calcium sulphate (plaster of Paris). Canada balsam. Chloroform. Chlorzine iodine (see Sec. 169). Ether. Fehling’s solution (see Sec. 170). Ferric chloride. Formalin. Glycerin. Grafting wax. Hydrogen peroxide. Iodine. Magnesium sulphate. Mercury. Millon’s reagent (see Sec. 170). Oil of cloves, cedar oil, olive oil. Paraffin, hard and soft. Potassium chloride. Potassium hydroxide (caustic potash), 5% and 15% solutions (see Sec. 169). Potassium permanganate, Potassium phosphate, acid. Sodium chloride (common table salt and also c.p.). Stains, acid fuchsin, eosin, erythrosin, gentian violet, hematoxylin, iodine, methyl green, orange G, phloroglucin, safranin (directions for making up these stains are given in Secs. 169, 170, 181-186). Sugar, cane. Vaseline. Water, distilled. Xylol. DEALERS IN SUPPLIES 225 DEALERS IN MATERIAL, APPARATUS, AND SUPPLIES 217. Material and slides. Plant material, both preserved and dried, and prepared slides for the microscope are offered by the following dealers, from whom price lists may be obtained. A. Cambridge Botanical Supply Company, Cambridge, Mass. Preserved and living material, slides. See also Sec. 218, B, and Sec. 219, A. B. Miss E. M. Drury, 45 Munroe Street, Roxbury (Boston), Mass. Slides and preserved material. C. Marine Biological Laboratory (Botanical Supply Department), George M. Gray, Woods Hole, Mass. Preserved material of thallophytes and bryophytes, mounted sheets of marine alge. D. H. M. Phillips, 19 Warriner Avenue, Springfield, Mass. Plant mate- rial, especially fungi. E. St. Louis Biological Laboratory, St. Louis, Mo. Slides and preserved material. See also Sec. 219, B. F. Williams, Brown & Earle, 918 Chestnut Street, Philadelphia, Pa. Slides. See also Sec. 218, K. G. Queen & Company, 1010 Chestnut Street, Philadelphia, Pa. Slides. See also Sec. 218, L. 218. Apparatus and supplies. Microscopes, physiological and other appa- ratus, glassware, general botanical supplies, etc., are sold by the dealers listed below from price lists which may be obtained from them. A. Bausch & Lomb, Rochester, N.Y. Microscopes, apparatus especially for plant physiology, glassware, chemicals, and general supplies. B. Cambridge Botanical Supply Company, Cambridge, Mass. Physio- logical apparatus, instruments, special botanical equipments, notebooks, general botanical supplies. C. James T. Dougherty, 409 West 59th Street, New York City. Reichert microscopes, apparatus, glassware, and chemicals. D. Eimer & Amend, 205 Third Avenue, New York City. Glassware, chemicals, and many instruments, general importers. E. L. E. Knott Apparatus Company, Ashburton Place, Boston, Mass. Apparatus and supplies for general and special purposes. F. Kny-Scheerer Company, 225 Fourth Avenue, New York City. Appa- ratus, chemicals, and supplies, importers. G. Ernst Leitz, 30 East 18th Street, New York City. Microscopes, appa- ratus, glassware, chemicals, and general supplies. H. Spencer Lens Company, Buffalo, N.Y. Microscopes, apparatus, glassware, chemicals, and supplies. I. The Scientific Shop, 822 Dearborn Street, Chicago. Zeiss microscopes, special pieces of apparatus, photographic and projecting instruments. 226 MATERIAL, APPARATUS, AND SUPPLIES J. Whitall, Tatum & Co., New York City. Glassware. K. Williams, Brown & Earle, 918 Chestnut Street, Philadelphia, Pa. Beck microscopes, apparatus, glassware, and general supplies. L. Queen & Company, 1010 Chestnut Street, Philadelphia, Pa. Micro- scopes, apparatus, glassware, and general supplies. 219. Lantern slides and charts. Lantern slides are sold singly or in sets by : A. Cambridge Botanical Supply Company, Cambridge, Mass. B. St. Louis Biological Laboratory, St. Louis, Mo. There are a number of sets of charts on the market. The most com- plete are: C. Kny. Botanische Wandtafeln, a series of 105 so far published, price 355 Marks. Paul Parey, Hedemannstrasse 10, Berlin, S. W. D. Frank-Tschirch. Wandtafeln fiir den Unterricht in der Pflanzen- physiologie, a series of 60, price 180 Marks. Published also by Paul Parey (see above). A new set of charts has recently been announced, which promises well. E. Baur-Jahn. Tabule Botanice, to appear in series of five, 25 Marks a series. Published by Gebriider Borntraeger, Dessauerstrasse 29, Ber- lin, S.W. ¥*1, 2. 3. BIBLIOGRAPHY [Six books which in the opinion of the authors should be in every botanical library have been double starred. ] GENERAL TEXTS Strasburger, Noll, Schenck, Karsten. A Text-Book of Botany. Second revised translation by W. H. Lang. The Macmillan Company, New York, 1903. $5.00. The original Lehrbuch der Botanik ap- pears in frequent revised editions. G. Fischer, Jena. Marks 8.50. Kerner-Oliver. Natural History of Plants. Henry Holt & Company, New York, 1905, two volumes. $11.00. Campbell. A University Text-Book of Botany. The Macmillan Com- pany, New York, 1902. $4.00. LABORATORY MANUALS, METHODS OF TEACHING, ETC. #46 7. 8. 9. 10. 11. 12. 13. . Strasburger-Hillhouse. Handbook of Practical Botany. The Mac- millan Company, New York, 1900. $2.60. Ganong. The Teaching Botanist. The Macmillan Company, New York, 1899. $1.10. Lloyd and Bigelow. The Teaching of Biology in the Secondary School. Longmans, Green & Company, New York, 1904. $1.50. Detmer-Moor. Practical Plant Physiology. The Macmillan Com- pany, 1898. $3.00. Ganong. Laboratory Course in Plant Physiology. Henry Holt & Company, New York, 1901. $1.00. Darwin and Acton. Practical Physiology of Plants. University Press, Cambridge, 1897. $1.25. Caldwell. Handbook of Plant Morphology. Henry Holt & Com- pany, New York, 1904. $1.00. Osterhout. Experiments with Plants. The Macmillan Company, New York, 1905. $1.25. 227 228 BIBLIOGRAPHY MORPHOLOGY General See 1, 2, and 3. 16. Goebel. Outlines of Classification and Special Morphology of Plants. Clarendon Press, Oxford, 1887. $5.25. Alge 17. Oltmanns. Die Morphologie und Biologie der Algen. G. Fischer, Jena, 1904-1905, two volumes. Marks 36.50. 18. West. Treatise on the British Fresh-Water Alga. University Press, Cambridge, 1904. $3.50. 19. Murray. An Introduction to the Study of Sea-Weeds. The Macmil- lan Company, New York, 1895, $1.75. Fungi 20. De Bary. Comparative Morphology and Biology of the Fungi, My- cetozoa, and Bacteria, Clarendon Press, Oxford, 1887. $5.50. 21. Massee. A Text-Book of Fungi. The Macmillan Company, New York, 1906. $2.00. 22. Fischer-Jones. The Structure and Functions of Bacteria. Clarendon Press, Oxford, 1900. $2.10. Bryophytes and Pteridophytes 23. Campbell. The Structure and Development of Mosses and Ferns. The Macmillan Company, New York, 1905. $4.50. G'ymnosperms 24. Coulter and Chamberlain. Morphology of Spermatophytes: Part I, Gymnosperms. D. Appleton & Company, New York, 1901. $1.75. Angiosperms 25. Coulter and Chamberlain. Morphology of Angiosperms. D. Apple- ton & Company, New York, 1903. $2.50. | Tschirch, see 74. PHYSIOLOGY **31. Pfeffer-Ewart. The Physiology of Plants: Vol. I, General Intro- duction, Properties of Cells, Nutrition; Vol. II, Growth, Causes of the Shape of the Plant Body, Variation and Heredity, Power of Resistance to Extremes; Vol. III, Movements, Production of Heat, Light, and Electricity, Transformations of Energy. Clar- endon Press, Oxford. Vol. I, 1900, $7.00; Vol. II, 1903, $4.00; Vol. III, 1906, $7.00. Detmer-Moor, see 9. BIBLIOGRAPHY 229 32. Peirce. Teaxt-Book of Plant Physiology. Henry Holt & Company, New York, 1903. $2.00. 33. Haberlandt. Physiologische Pflanzenanatomie. Engelmann, Leip- zig, 1904. Marks 21. 34, Sorauer-Weiss. A Popular Treatise on the Physiology of Plants. Longmans, Green & Company, New York, 1895. $3.00. Ganong, see 10. Osterhout, see 13. Darwin and Acton, see 11. 35. Darwin, Charles. The Power of Movement in Plants. D. Appleton & Company, New York, 1900. $2.00. TAXONOMY 3 ~General 36. Engler. Syllabus der Pflanzenfamilien. Gebriider Borntraeger, Berlin, 1905. Marks 4.50. **37. Warming-Mobius. Handbuch der systematischen Botanik. Gebrider Borntraeger, Berlin, 1902. Marks 8. Slime Molds 38. Macbride. Zhe North American Slime Moulds. The Macmillan Company, New York, 1889. $2.25. “Alge 39. Engler and Prantl. Die natiirlichen Pflanzenfamilien . I. Teil, Ab- teilung 1a, Schizomycetes, Schizophycex, Flagellata; I. Teil, Abteilung 1 6, Peridinales, Bacillariacee ; I. Teil, Abteilung 2, Conjugate, Chlorophycex, Pheophycer, Rhodophycee. Engel- mann, Leipzig. Oltmanns, see 17. West, see 18. Murray, see 19. Fungi 40. Engler and Prantl. Die natiirlichen Pflanzenfamilien: I. Teil, Ab- teilung 1, Myxomycetes, Phycomycetes, Ascomycetes; I. Teil, Abteilung 1**, Basidiomycetes, Fungi imperfecti. Engelmann, Leipzig. : Massee, see 21. 41. Atkinson. Mushrooms. Henry Holt & Company, New York, 1903. $3.00. Lichens 42. Schneider. A Guide to the Study of Lichens. Bradlee Whidden, Boston, 1898. $1.90. 230 BIBLIOGRAPHY Liverworts and Mosses Gray, see 46. 43. Grout. Mosses with Hand-Lens and Microscope. Published by the author, 860 Lenox Road, New York City, 1903-1906. $3.75. Campbell, see 23. Ferns and Fern Allies Gray, see 46. 44. Underwood. Our Native Ferns and Their Allies. Henry Holt & Company, New York, 1899. $1.00. 45. Clute. The Fern Allies of North America North of Mexico. Frederick A. Stokes & Company, New York, 1905. $2.00. Campbell, see 23. Seed Plants : 46. Gray. Manual of Botany of the Northern United States. American Book Company, New York. 47. Gray, Watson, Robinson. Synoptical Flora of North America. A monographic treatment of I; Gamopetale (Smithsonian Institu- tion, 1886, $2.50); II, Polypetale, Vol. I, Fas. 1 and 2, Ranun- culacee to Polygalacee (American Book Company, $5.20). 48. Britton. Flora of the Northern States and Canada. Henry Holt & Company, New York. $2.25. 49. Gray. Field, Forest, and Garden Botany. American Book Com- pany, New York, 1895. $1.44. 50. Sargent. Manual of the Trees of North America (exclusive of Mexico). Houghton, Mifflin & Company, Boston, 1905. $6.00. 51. Chapman. Flora of the Southern United States. American Book Company, 1897. $3.60. 52. Coulter. Manual of Rocky Mountain Botany. American Book Company, New York, 1885. $1.62. ECOLOGY AND PLANT GEOGRAPHY 56. Schimper-Fisher. Plant Geography. Clarendon Press, Oxford, 1903. $14.00. 57. Warming-Graebner. Oekologische Pflanzengeographie. Gebriider Borntraeger, Berlin, 1902. Marks 8. 58. Pound and Clements. Phytogeography of Nebraska. University of Nebraska, Lincoln, Nebraska, 1900. $2.00. 59. Clements. Research Methods in Ecology. University Publishing Company, Lincoln, Nebraska, 1905. $3.00. 60. 61. 62. 63. 64.. **66. 70. 71. 72. 74 BIBLIOGRAPHY 231 Wiesner. Biologie der Pflanzen. A. Héldner, Wien, 1901. Marks 10.20. Ludwig. Lehrbuch der Pflanzenbiologie. Enke, Stuttgart, 1895. Marks 16. Knuth-Davis. Handbook of Flower Pollination. Clarendon Press, Oxford, Vol. I, 1906. $6.75. Beal. Seed Dispersal. Ginn & Company, Boston, 1898. 35 cents. Darwin, Charles. IJnsectivorous Plants. D. Appleton & Company, New York, 1900. $2.00. EVOLUTION Darwin, Charles. The Origin of Species. D. Appleton & Company, New York, 1906. $2.00. . De Vries. Species and Varieties: their Origin by Mutation. Open Court Publishing Company, Chicago, 1905. $5.00. . Bailey. The Survival of the Unlike. The Macmillan Company, New York, 1899. $2.00. . Darwin, Charles. Variation of Animals and Planis under Domesti- cation, D. Appleton & Company, New York, 1900, two volumes. $5.00. Campbell. Lectures on the Evolution of Plants. The Macmillan Company, New York, 1899. $1.25. ECONOMIC BOTANY United States Department of Agriculture.. Procure lists of publica- tions from Superintendent of Documents, Government Printing Office, Washington, D.C. Important papers appear frequently, which may be obtained at small cost. Agricultural Experiment Stations. These issue many important circulars, bulletins, and reports. For addresses of the staff pro- cure the Organization Lists of Agricultural Experiment Stations from the Superintendent of Documents (address above). Price of list, 10 cents. . Bailey. . Plant Breeding. The Macmillan Company, New York, 1906. $1.25. . Tschirch. Angewandte Pflanzenanatomie, Erster Band. Urban & Schwarzenberg, Wien, 1889. $4.50. 232 75. 76. 77. 78. 79. 80. 81. 82. 83. 86. 87. 88. BIBLIOGRAPHY Roth. First Book of Forestry. Ginn & Company, Boston, 1902. 75 cents. : Pinchot. A Primer of Forestry: Part I, The Forest; Part IT, Practical Forestry. United States Department of Agriculture, Bureau of Forestry, Bulletin 24: Part I, 1899, 35 cents; Part II, 1905, 30 cents. Tubeuf-Smith. Diseases of Plants. Longmans, Green & Company, New York, 1897. $5.50. Freeman. Minnesota Plant Diseases. Published by the University of Minnesota, Minneapolis, 1905. Conn. Agricultural Bacteriology. P. Blakiston’s Son & Company, Philadelphia, 1901. $2.50. Conn. Bacteria, Yeasts, and Molds in the Home. Ginn & Company, Boston, 1903. $1.00. Lafar-Salter. Technical Mycology. J. B. Lippincott, Philadel- phia: Vol. I, 1906, $4.00; Vol. II, 1903, $2.50. Whipple. The Microscopy of Drinking Water. John Wiley & Sons, New York, 1905. $3.50. Willis. Practical Flora. American Book Company, New York, 1894. $1.50. MISCELLANEOUS Carnegie Institution. Procure lists of publications from the Car- negie Institution of Washington, Washington, D.C. Important papers appear frequently, which may be obtained at moder- ate cost. United States Department of Agriculture, see 71. Agricultural Experiment Stations, see 72. Winslow. Elements of Applied Microscopy. John Wiley & Sons, New York, 1905. $1.50. Jackson. Glossary of Botanical Terms. J.B. Lippincott Company, Philadelphia, 1906. $2.00. APPENDIX 1. Laboratory notes. Much of the value of laboratory work must always depend upon the way in which the notebooks are inspected. Those with detached leaves, which may be assembled in some kind of binder, are to be preferred; and the instructor may certify his accept- ance of the notes, page by page, either during the laboratory period or immediately afterward. In general, unsatisfactory notes should be rewritten from new studies of the material in question or (better) of equivalent forms. Valuable data of any sort secured during the year’s work should be preserved for subsequent use. In this way, for example, the optimum conditions for many of the phenomena of plant life may be ascertained by assembling the results of the best student observers. Duplicates of photographs and drawings may be kept in the botanical museum. 2. Students’ preparations. In all cases in which the student finds it advisable to make for himself a set of preparations, e.g. of slides to illustrate any histological topic, such preparations should be treated as a part of his laboratory record. A duplicate series for the laboratory can usually be made with little extra effort. In the same way preserved material illustrating the life history of the plant, its variations as a whole, or the modifications of its parts under varying environmental conditions, may be added to the museum or other general collections. 3. Sources of material. Aside from the obvious sources of material for morphological and still more for histological study (such as the wild seed plants of the region, gardens, and greenhouses) there are others not less valuable. A very large number of admirable subjects for histo- logical work can be had from the wholesale druggists, particularly those who make a specialty of « botanical” remedies. Almost any part of the plant body may be used in medicine, and many officinal substances, such as calisaya bark, the young wood of Solanum dulcamara, the root- stock of Acorus, the fruits of many Umbellifere, and a great number of other substances, to be had of dealers in drugs, form excellent material 233 234 APPENDIX for study. Seeds, bulbs, and rootstocks in great variety can be had of the seedsmen, and much useful material may be found among the fruits and vegetables sold by marketmen or provision dealers all the year round. Dealers in fine lumber and cabinet woods can supply many kinds which are of histological interest. 4. Collection of material at the proper season. In many cases the useful- ness of material depends largely on its being collected at just the right stage of maturity or at some particular season. Experience will show the instructor at what time he can in his special locality best lay in the stock for his year’s work. A few examples only need here be mentioned. Green corn (best of the large “dent” variety), just passing out of the milky condition, should be collected and boiled in water for about twenty minutes. It is then to be sliced from the cob, cutting deeply enough to preserve the embryos uninjured. The grains thus prepared may be preserved in alcohol indefinitely for study of the entire embryo. Bean fruits of all ages, from the newly fertilized pistil to the fully developed but not dry pod, should be collected in season and put in preservative fluid, such as formalin, to illustrate the development of a fruit. If convenient, series of stages in the development of other fruits, e.g. apple, strawberry, orange, should be secured. Indian corn plants should be dug up at various stages of growth and the lower part of the stem, with the attached roots, dried and preserved, after being thoroughly washed. These will illustrate secondary roots and successive steps in the formation of aérial roots. Pieces of corn stem of various ages show the development of bundles and of the very hard sclerenchyma of the rind. Bits of Aristolochia or other stem used to illustrate open bundles should be collected in early summer, as soon as these are found to be well developed. A series of such stems cut at short intervals through- out the growing season is valuable. Leaves of deciduous trees or shrubs should be collected as soon as fully grown and again when about to fall, and preserved in alcohol for the study of translocation of starch before falling. Series of herbarium sheets and of specimens in preservative fluid should be secured to show the seasonal cycle of such plants as Erythro- nium, Sanguinaria, and many similar forms. 5. Field work. Many botanical topics can best be taught and some can only be taught in the field. It may be found desirable to interweave APPENDIX 235 instruction in ecological topics during field trips primarily undertaken for other purposes. So much depends on the nature of the region in which the work is done, the maturity of the class, the amount of time available for out-of-door study, and other circumstances, that only a few general principles for field work are here suggested. (a) Every expedition should have one definite purpose. If many incidental subjects come up, they must not interfere with the primary object of the trip. (6) All observations must be exact. If it is desired to ascertain the composition of a plant formation, the number of species and individuals occurring in measured units of area should be ascertained by actual count. Temperatures of air, soil, or water should be noted with a good thermometer. Tlumination (in shaded areas) should be measured at several periods, e.g. two hours after sunrise, noon, and two hours before sunset. In some cases it may be practicable to determine the relative moisture of the soil of several stations for at least the driest period of the year. Such results should be kept on record and may be taken into account by all classes which thereafter discuss the vegetation of those areas. (c) The distribution of vegetation forms must be studied with refer- ence to extreme conditions. Xerophytic structure does not always serve as a protection against a low annual rainfall but rather against severe periods of drought. (ad) Reasoning on ecological topics should be conducted with great caution. It is not safe to assume that attractiveness in any part of the plant body, as in the sweet cambium layer of the white pine or the inner bark of the slippery elm, is always of use to the plant. It may be highly disadvantageous. On the other hand, such “defenses” as the thorns on the trunk of the honey locust tree are of little or no conceiv- able use (on the branches they may be advantageous). 6. The study of Spirogyra. It is important to proceed slowly when students begin to use the compound microscope, and to make sure that correct habits of work are formed. The power to visualize or interpret objects studied with the compound microscope should be carefully trained. Simple models may be constructed. Thus the structure of the cell of Spirogyra may be demonstrated very effectively with a glass jar to represent the cell wall, a paper bag for the living plasma membrane, 236 APPENDIX strips of paper properly wound inside to represent the chromatophore, and some object suspended in the center to illustrate the position of the nucleus. A simple model of this character made with the codperation of the class will greatly assist the students to a clear understanding of the study outlined in Sec. 56. Whenever possible, a study of the Ameba or Euglena in comparison with the cell of Spirogyra will be found very helpful in making clear the characteristics of a protoplast. Stained preparations of Spirogyra filaments (Sec. 211) may help to an understanding of the cell structure, but in general it is better that the student handle living material and with the aid of a few simple re- agents (such as iodine and the salt solution) discover the facts for himself. 7. Cultures of Ameba. Cultures of Ameba may be made in an aqua- rium jar overstocked with filamentous alge. Introduce sediment from a pool of Oscillatoria with a mass of the alge; growths of Scenedesmus are also likely to have Amebe. Place the jar near a window but just out of the direct rays of the sun. The filamentous algz will decay and after a few weeks a coating will be formed on the sides of the jar. From this coating or from the sediment on the bottom of the jar Amebe can usually be obtained in quantity. They are generally most abundant on the sides nearest the light. In preparing material for the class gather a quantity of the slime and sediment with a pipette and place in a small bottle. After a few hours the water at the top will frequently contain so many Amebe that the students may readily find them in mounted drops. 8, Nuclear and cell division. Nuclear and cell division may also be studied from preparations of the pollen mother cells of the lily (Sec. 141) and in sections of various growing tissues such as stem Hips; developing ovules, embryo sacs of lily, etc. 9. Cultures of Euglena. A culture of Euglena in a shallow dish with leaves and sediment at the bottom may be allowed to slowly dry up by evaporation. The Euglene will pass into the encysted condition. This dish of dried material may then be placed aside and will keep indefi- nitely. If water be added, the encysted Euglene will come forth again in the motile condition. 10. Spherella and Volvox. If stones are placed in a dish swarming with Spherella, the motile cells will settle upon them and pass into a APPENDIX 237 resting stage in which the cells develop heavy walls and the contents become reddish. Such stones may be dried, and when placed again in rain water will develop anew generation of green motile cells. Cultures may thus be carried along from season to season. The odspores of Volvox will fall to the bottom of an aquarium among the sediment and after a time produce a new generation of Volvoz colonies. 11. Hydrodictyon. Hydrodictyon may be readily grown in aquaria, where it will form successive generations of nets. Material cultivated in diluted Knop’s solution, one half to one per cent (Sec. 200, A), is likely to form zoéspores after a few hours if transferred to pond or tap water. Nets placed in a five per cent solution of cane sugar (Sec. 200, C) are likely to produce gametes in a few days. 12. Coleochete. Coleochete frequently appears in aquaria, forming green disks on the sides of the glass. These may readily be detached with a scalpel and gathered with a pipette, and show the vegetative structure of the plant excellently. We have never known the alga to produce sexual fruit in aquaria. 13. Diatoms. The shells of diatoms in polishing powders and earths, or masses of fresh material, may be cleaned as follows. Heat in a small porcelain evaporator with c.p. sulphuric acid and slowly drop in a few crystals of sodium nitrate. After cooling rinse the sediment thoroughly in water, decanting frequently. It may then be dried on a slide and mounted permanently in balsam. 14. Bacteria. It is much more important that the bacteria be studied for the appearance of the growths en masse than that detailed micro- scopical examinations be made of these minute organisms. The micro- scopical examination of the “fur” from the teeth gives one of the most striking assemblages of forms. The cultures are best made by groups of students working with a set of Petri dishes in common. Excellent cul- tures of bacteria may: be obtained in Petri dishes on potato agar as a substratum (Sec. 203), and these are preferable to cultures on slices of potato, but the preparation of agar requires more time and considerable laboratory equipment. 15. The bread mold. Individual cultures of bread mold may be readily made by the students and are more convenient for study than a ‘large culture in common. Place small pieces of bread in watch glasses and set the latter over wet blotting paper in saucers, covering each with a tumbler. To make sure of good growthis inoculate the pieces of bread 238 APPENDIX with spores from an old culture. These small cultures can be readily handled and studied with the hand lens, especially portions of the mycelium which may grow out over the surface of the watch glass. The bread mold is almost invariably followed by extensive growths of the green mildew, Penicillium. 16. Lichens. When time is limited it is more important that the lichen be studied than such Ascomycetes as Microsphera or Peziza, since most lichens show excellently the fruiting characters of the sac fungi. The remarkable associations of fungi and alge to form these composite organisms gives the lichens peculiar interest. A variety of lichens should be collected, dried, and fastened to cards for comparative studies of growth habits and form. 17. Gill fungi. The study of gill fungi must frequently be made at times inconvenient or impossible for field trips. The laboratory work may then be upon material preserved in strong alcohol or on edible forms sold in the markets (the commonest is Agaricus campestris). Horse manure placed under a bell jar will give excellent material of some small and delicate species of Coprinus, the development of which will prove interesting. 18. Diagrams and formule illustrating alternation of generations. The difficult subject of alternation of generations may generally be made ‘clearer if the chief phases in the life histories of the types studied are represented by a series of diagrams. It is helpful if the diagrams are drawn with colored pencils; thus the gametophyte phases may be rep- resented in yellow and the sporophyte phases in green. Life-history formule may be constructed after the manner suggested in the Princi- ples, pp. 222, 278, 319, 336, and 375. 19. Mosses. Field studies of the mosses may not be possible at the time the type is studied in the laboratory. However, dried material is generally very satisfactory for habit studies on a variety of forms, and the greenhouses and cultures (Sec. 207) may be depended upon to fur- nish living material. Moss spores will generally germinate in sweetened water. : 20. Ferns. A variety of ferns may be easily cultivated in the labora- tory, which with herbarium material will give a very good idea of dif- ferent forms of stems and leaves, A simple study of stem structure may _ be made by cutting across stems and whittling them lengthwise. Such an examination will make clear at least the position and importance APPENDIX 239 of the rigid tissue and fibro-vascular bundles. Fern spores may be germinated in sweetened water. 21. The pine. The pine is one of the best types of seed plants, easily available, to make clear the relationships between pteridophytes and spermatophytes. The general homologies between the staminate cone and the cones of the club mosses and horsetails are easily under- stood, as well as the homologies between the pollen grain with its inclosed male gametophyte and the microspores of pteridophytes with their male prothallia. The female gametophyte of the pine, also, is readily compared with the reduced female prothallia of heterosporous pteridophytes. For these reasons the life history of the pine is more easily understood with reference to the life histories of pteridophytes than is the life history of an angiosperm (such as the lily) where the gametophytes are much more reduced in structure. ; The pine is also excellent for the study of the tissues of a tree with annual growth from acambium ring. The structure of pine wood is one of the best exercises in the interpretation of cell structure, and the study outlined in Sec. 138, C, 5, is often given as a laboratory problem. In a course outlining the evolution of life histories in plants the pine is as important a type as Selaginella, the fern, or the moss, besides having in itself a remarkably interesting morphology in relation to peculiar life habits. 22. The lily. There are perhaps no types more convenient than those of the lily family for the study of the gametophytes of the angio- sperms. It is, however, not easy to present the life history of angio- sperms in full from the study of a single type in a general course. It is probably better to use various forms which may be especially favorable for particular phases ; as, for example, the lily for the development of the pollen, but the elder for the male gametophyte; the lily for the development of the embryo sac together with fertilization, double fertilization, and the origin of the endosperm, but the shepherd’s purse for the development of the ovule and embryo. 23. The shepherd’s purse. This plant would be almost perfect for a complete study of an angiosperm life history were it not for the small size of the flowers, anthers, and pistil, and in consequence the minute- ness of the gametophytes. However, the shepherd’s purse is one of the best types for the study of floral development (notwithstanding certain irregularities) and the development of the ovule and embryo. GLOSSARY? Actinomorphic (ray shaped). Having star-like or radiating symmetry. Zcidium. A fructification peculiar to certain rusts producing ecidio- spores, — a cluster cup. Alternation of generations. The alternation in a life history of a sexual generation or gametophyte with an asexual generation or sporophyte. Anemophilous (wind loving). A term applied to plants which are pol- linated by the wind. Angiosperms (vessel seed). Plants which have the seeds inclosed in an ovary or seed case. Anther (flowering). The part of a stamen which bears pollen. Antheridiophore (antheridia bearer). In the liverworts a specialized receptacle bearing antheridia. Antheridium. The male sexual organ producing sperms or antherozoids in the groups below the seed plants; also called an antherid. Antherozoid. See Sperm. Antipodal (against the foot). The term applied to three cells at the base of the embryo sac. Apical. Pertaining to the apex or tip. Apical cell. A terminal cell which constitutes a growing point. Apogamy. The development of an egg without fertilization, or the devel- opment of a sporophyte generation as a bud-like outgrowth from the gametophyte. Apospory. The suppression of spore formation and the development of a gametophyte generation directly from the sporophyte. Apothecium. In sac fungi, Ascomycetes (including lichens), the open cup- or saucer-shaped fructification in which the sacs or asci lie ex- posed in a membrane or hymenium. Archegoniophore (archegonia bearer). In the liverworts a specialized receptacle bearing archegonia. Archegonium (beginning of offspring). The many-celled female sexual organ prodycing an egg, characteristic of the bryophytes, pterido- phytes, and some gymnosperms. -Archesporium (beginning of a spore). The cell or cells constituting the tissue from which the spores of bryophytes and pteridophytes are ultimately derived and also their homologues, the pollen grains. 1 Most of the nouns in this glossary form their plurals by the addition of s or es, like ordinary English nouns. Those ending in ws, unless otherwise stated, form the plural in i, as nucleus, plu. nuclei. Those ending in ium form the plural in ia, as untheridium, plu. antheridia. 241 242 GLOSSARY Ascocarp (sac fruit). The fructification in which asci are formed. ; Ascogonium (sac offspring). The female sexual organ of the sac fungi, or Ascomycetes. Ascospores (sac spores). The spores developed in the ascus. Ascus (a sac). One of the spore-producing cells of an ascocarp ; each ascus generally develops eight spores. Asexual spore. One having no immediate relation to sexual cell unions. Association. One of the ecological unit groups (smaller than a plant formation) of which the formation is sometimes made up. Axial. Concerning or belonging to the axis. Axil (the armpit). The upper angle formed by the junction of the leaf and stem. Axis (an axle), An imaginary central line about which organs are developed or ranged. Basidium (a little pedestal). A spore-bearing cell in the basidia fungi. Bast, hard. The fibrous portion of the phloém. Bast, soft. The portion of the phloém composed of sieve tubes. Bilateral symmetry. The arrangement of parts in corresponding right and left halves, — zygomorphism. Bisexual. Term used of flowers having both stamens and pistils. Bract. A modified leaf of an inflorescence. Bryophytes (moss plants). The great group composed of the liverworts and mosses. Bulb. A short subterranean stem or bud with fleshy scales. Calyptra (a veil). The cap covering the developing spore case of a moss (and also a liverwort), formed by the enlargement of the archego- nium after fertilization. Calyx (a cup). A collective term for the sepals or outer members of the perianth. Cambium (to change). The meristematic layer which in dicotyledons lies between the xylem and phloém parts of each . fibro-vascular bundle and forms a very thin cylinder joining wood and bark. Canal cells. A row of cells in the neck of an archegonium above the egg. Capsule (a box). A dry, dehiscent seed vessel. Carpel (fruit). The simplest form of seed-bearing organ; a simple pistil or one of the parts of a compound pistil ; morphologically a megasporophyll. . oes (fruit offspring). The female sexual organ of the red alge. oreo (fruit spore). A spore developed in the cystocarp of a red alga, Cell (a small chamber). A unit of protoplasm ; a protoplast (with or without a cell wall). Cell wall. The carbohydrate membrane, generally cellulose, by which plant protoplasts are usually inclosed, GLOSSARY 243 Cellulose. The carbohydrate material of which cell walls are formed. Central cylinder. The stele or portion of a root or stem which is inclosed by the primary cortex. Chlamydospore (cloaked spore). A thick-walled resting cell or spore. Chlorophyll (leaf green). The ordinary green coloring matter of plants held in the chloroplasts, or chromatophores. Chlorophyll bodies. Masses of protoplasm (in seed plants usually minute disk-shaped bodies) colored green by chlorophyll, — chloroplasts. Chloroplasts (green molded). Chlorophyll bodies; plastids containing chlorophyll. Choripetalous (separate petal). Having the petals separate. Chorisepalous (separate sepal). Having the sepals separate. Chromatin (color). The deeply staining substance contained in the nucleus which forms the chromosomes. Chromatophore (color bearer). Any large green, brown, or red proto- plasmic body, especially characteristic of the cells of alge. - Chromoplast (color molded). Plastids of other colors than green (as red, brown, yellow, etc.), a term used in contrast to chloroplast. Chromosomes (color bodies). Readily stained bodies within the nucleus, composed of chromatin and appearing most conspicuously during nuclear division. Cilium (an eyelash). A vibrating fibril attached to a zoéspore or sperm. Cladophyll (branch leaf). A branch with the form and functions of a leaf, called also a cladode and phylloclade. Class. A taxonomic group composed of orders. Cleistogamous (closed marriage). A term applied to fertilization occur- ring in unopened flowers. Closed bundle. A fibro-vascular bundle which contains no cambium and is consequently incapable of further growth. Cenocyte (a vessel in common). A multinucleate cell, generally of large size. Coenogamete (a gamete in common). A multinucleate gamete, gen- erally of large size. Collenchyma. Parenchyma cells with walls thickened, usually at the angles. Columella, plu. columelle (a small column). The persistent axis of cer- tain spore cases and spore fruits. Companion cell. An elongated cell associated with a sieve tube. Conceptacle. A pit-like cavity in the rockweeds containing the sexual organs. Cone. *The sealy fruit of such conifers as the pines, spruces, etc., also of the lycopods and horsetails, — a strobilus. Conidiophore (conidia bearer). A generally upright stalk upon which conidia are borne. Conidium (dust). An asexual spore of a fungus, generally formed in the air. Coniferous. Cone bearing. 244 GLOSSARY Conjugation. The sexual union of similar gametes to form a zygospore or zygote. Cork. Protective tissue in the outer portions of the bark. Corm (a trunk). The bulb-like fleshy base of some stems. Corolla (a small crown). A collective term for the petals or inner mem- bers of the perianth. Cortex. The bark or rind. Cotyledon. An embryo leaf borne by the hypocotyl. Cuticle (the outer skin). The exterior layer of the epidermis. Cystocarp (bladder fruit). The fruit of the red alge resulting from the fertilization of the carpogonium. 7 Cytoplasm (cell plasm). The general protoplasm of the cell exclusive of the nucleus and plastids. Deciduous (to fall). Falling when their function is performed, as the leaves of most hard-wood trees in temperate climates. Dehiscent (to yawn). Opening spontaneously when mature, as anthers, to discharge pollen, or as capsules, to discharge spores. Dermatogen (skin producer). The*layer of cells around growing points from which the epidermis is derived. Determinate. A term applied to stems where the growth in length is determined by the presence of a winter bud, and to an inflorescence where there is a terminal flower bud. Diageotropic. Growing horizontally under the influence of gravity, as some branches do. Dicotyledonous. Having two seed leaves or cotyledons. Dimorphous flowers (two forms). Flowers which have two forms, as long and short styled. Diecious (two households). Unisexual, the male and female sexual organs borne by separate individuals. Dorsiventral (back, belly). Having upper and lower faces, as in most leaves. Drupe (an olive). A stone fruit, as a peach or plum. Ecology (household discourse). The study of plants in relation to their surroundings. The term is often made to include much of the subject-matter of ecological plant geography, or the distribution of plants on the earth with reference to environment. In this sense it is very frequently used in connection with the study of plant formations. Egg. A nonmotile female gamete, generally large in comparison with the sperm. Egg apparatus. A group of three cells at the micropylar end of the embryo sac, consisting of the egg and two synergids. Elater (a driver). A spirally thickened elongated cell or other filamen- tous structure developed to assist in expelling spores from a spore case. GLOSSARY 245 Embryo (a rudimentary animal). The rudimentary plantlet, as in the seed. Embryo sac. The cavity which contains the female gametophyte of a seed plant and later the embryo and endosperm (if present) in the seed. Emergence. An outgrowth from the surface of a plant not (like a hair) arising solely from the epidermis nor (like a thorn) from a bud. Endosperm (within the seed). A parenchymatous tissue formed within the embryo sac and often developed into the principal mass of reserve material in the seed. Entomophilous (insect loving). A term applied to plants that are pol- linated by insects. Enzyme (in yeast). An unorganized or soluble ferment (such as dias- tase) which is not associated with any organism. Epidermis (upon the skin). The cellular skin or covering of the plant body inside the cuticle. Epigynous, A term applied to flowers in which the stamens and perianth appear to grow from the top of the ovary. Epiphyte (upon a plant). A plant which grows upon other plants but not parasitically, — an air plant. Eusporangiate. A term applied to pteridophytes the sporangia of which arise from a group of cells. Family. A taxonomic group standing between genus and order. Fertilization. The fusion of two sexual cells, especially the fusion of the sperm with the egg. Fiber. A slender, thick-walled cell, many times longer than its width. Fibro-vascular. Composed of fibers and vessels, as a fibro-vascular bundle. Filament (a thread). The stalk of a stamen bearing the anther. Fission. The process of cell division by a gradual pinching in two of the cell. Flower. An assemblage of organs in the seed plants necessary for ferti- lization, often with protecting envelopes. The flower of the angio- sperms when bisexual usually consists of a perianth, stamens, and pistil or pistils. Foot. A portion of the sporophyte set apart to absorb water or nourish- ment from the gametophyte. Formation. An ecological term denoting a well-defined assemblage of plants characteristic of a given kind of station, as a peat bog. Frond (a leaf). The leaf of a fern, generally both vegetative and spore producing in its functions. ' Fruit. The ripened seed case and its contents, or, in a broader sense, a spore-producing structure of the lower plants. Fundamental tissue. The general ground tissue (mostly undifferentiated) in which fibro-vascular bundles and other specialized tissues arise. Funiculus (a little rope). The stalk by which the ovule or seed is at- tached to the placenta. 246 GLOSSARY Gametangium (gamete vessel). The organ which produces gametes. Gamete. A sexual reproductive cell which ordinarily must fuse with another gamete in order to live. Gametophyte (gamete plant). The sexual plant in an alternation of generations, producing sexual cells or gametes (see Sporophyte). Gemma, plu. gemme (a bud). An asexual reproductive body, generally many celled, rather characteristic of the bryophytes. Generative cell. The cell within the male gametophyte of seed plants from which the two sperm nuclei are developed. Genus, plu. genera (a race). The taxonomic group composed of related species. Geotropism (earth turning). The action of gravity in directing growth. Gill. A flat spore-bearing plate on the under side of a mushroom or toad- stool. i Glume (a husk). A chaffy bract on the inflorescence of grasses. Grain. Such a seed-like fruit as that of the grasses, —a minute, roundish body, as a starch grain. Ground tissue. See fundamental tissue. Growing point. The meristematic tip of the root or stem from which the tissues are produced. Guard cell. One of the cells (usually two in number) which serve to open and close a stoma. Gymnosperms (naked seeds). Plants (as the Coniferw) which have no closed ovaries, so that the seeds are borne naked, usually on scales. Halophyte (salt plant). A plant which habitually grows in saline soils, as on sea beaches or in salt marshes. Haustorium (a drawer). A sucker-like absorbing organ of a parasitic plant. Heliotropism (sun turning). The action of light in directing growth. Hermaphrodite. Having both forms of sexual organs together in the same structure; bisexual. Heterocyst (unlike cell). In the blue-green alge a large cell, empty or almost empty of protoplasm. : Heterogamy (unlike gametes). The condition where the pairing gametes are different in form and structure, as the egg and sperm. Heterospory (unlike spores). The condition in which a sporophyte pro- duces spores of two sizes, microspores and megaspores. Hilum. The scar on a seed showing its point of attachment to the funiculus or the placenta. Holdfast. An organ of attachment developed by certain alge. Homologous (similar discourse). Of one type, though differing in form and function. Homospory (similar spores). The condition in which a sporophyte pro- duces spores of the same size. Hormogonium (chain offspring). A portion of the filament of a blue-green alga reproductive in function. GLOSSARY 247 Host. A plant or animal which nourishes a parasite. Hybrid (a mongrel). The offspring obtained by the action of the pollen of one species on the pistil of another. The term is also used for the offspring of any cross. Hydrophyte (water plant). A water plant. Hygroscopic (moisture seeing). Expanding or shrinking readily under the influence of moisture. Hymenium (a membrane). An expanded fruiting surface of a fungus. Hypha, plu. hyphe (a web). The filament of a fungus. Hypocotyl. The portion of an embryo or very young seedling between the cotyledons and the root. Hypogynous. A term applied to flowers in which the stamens and peri- anth grow from beneath the ovary. Indeterminate. A term applied to stems where the growth in length is indefinite because no terminal bud is formed, and to an inflorescence where there is no terminal flower. Indusium. In ferns a protective outgrowth from the leaf covering a cluster of sporangia or sorus. Inferior ovary. See Epigynous. Inflorescence (flowering). The manner in which the flowers are arranged in the flower cluster. Intercellular. Between the cells or among them. Internode. The portion of the stem between two nodes. Involucre (a wrapper). A ring of bracts surrounding several flowers or their flower stalks. Irritability (easily excited). Sensitiveness to stimuli, such as light, heat, gravity, etc. Isogamy (equal gametes). The condition where the pairing gametes are similar in form and structure. Lamina, plu. lamine (a layer). The blade of a leaf. Leaf trace. The group of fibro-vascular, bundles which connects the veins of the leaf with the fibro-vascular system of the stem. Lenticel. A roundish or lens-shaped spot on young bark, marking the former position of a stoma. Leptosporangiate. A term applied to pteridophytes in which the sporan- gium arises from a single epidermal cell. Leucoplast (white molded). A protoplasmic body found in cells in the interior of the plant body, often serving as a starch builder, —a colorless plastid. Lignin (wood). The thickening material deposited in cell walls to pro- duce woody tissue. Locule (a little compartment). A cavity or chamber, as of an ovary. Medullary (belonging to the marrow). Related to the pith, as the med- ullary rays. 248 GLOSSARY Megasporangium (large spore vessel). The sporangium which develops megaspores. Megaspore (large spore). The larger one of the two sorts of spores pro- duced by heterosporous pteridophytes; it gives rise to a female gametophyte. Megasporophyll (large spore leaf). A leaf bearing megaspores. Meristem (divisible). Formative, rapidly dividing tissue such as cam- bium or the cells of growing points. Mesophyll (middle leaf). The entire parenchyma of the leaf, inside the epidermis. Mesophyte. A plant adapted to live with a moderate amount of soil water and humidity. Micropyle (small gate). The small opening between the integuments leading to the nucellus of an ovule. Microsporangium (small spore vessel). A sporangium which develops microspores. Microspore (small spore). The smaller of the two sorts of spores pro- duced by heterosporous pteridophytes; it gives rise to a male gametophyte. Microsporophyll (small spore leaf). A leaf bearing microspores. Mitosis (a thread or web). The process of indirect nuclear division characterized by the presence of a spindle. Monocotyledonous. Having only one seed leaf or cotyledon. Monecious (one household). Having the male and female sexual organs borne separately by the same individual. Morphology (form discourse). The science of the form and structure of an organism. Mutation. A decided and abrupt departure in the offspring from the characters of the parent, often sufficient to constitute a new species. Mycelium (fungus growth). A mass of vegetative fungal filaments, or hyphe. Nastic. A term applied to movements produced by all-round (not one- sided) stimuli. The opening and closing of such flowers as the cro- cus, tulip, etc., are thermonastic movements. Nectary. The organ in which nectar is secreted. Node (a knot). The part of a stem which normally bears a leaf or group of leaves. Nucellus (a little kernel). The portion of the ovule within the integu- ments and containing the embryo sac. Nucleolus (diminutive of nucleus). A small readily stained body, gen- erally present with the chromatin, in the nucleus; also called a nucleole. Nucleus (a kernel). The organ of the cell containing the chromatin and nucleolus. Odgonium (egg offspring). The cell in the thallophytes which develops the egg; also called an odgone. GLOSSARY 249 Odsphere (egg sphere). An egg cell. Odspore (egg spore). A fertilized egg which develops a heavy wall and passes through a period of rest before germinating. Open bundle. A fibro-vascular bundle which contains cambium and is consequently capable of further growth. Operculum, plu. opercula (a cover). In mosses the cover of the spore case. Order. A taxonomic group composed of families. Osmosis (a thrusting). The diffusion or interchange of liquids through membranes. : Ovary. The ovule-bearing part of the pistil. Ovule. The undeveloped structure which after fertilization becomes the seed, Palisade cells. Elongated parenchyma cells of a leaf, which lie beneath the epidermis with their long axes at right angles to the leaf surface. Palmate (like the palm of the hand). With veins or sinuses radiating like fingers. Parasite. An animal or plant that obtains its food from some other liv- ing organism, called its host. Parenchyma. Tissue composed of nearly globular cells or polyhedral cells the diameters of which are approximately equal, as pith. Parietal (a house wall). Pertaining to a wall, as a placenta on an ovary wall. Parthenogenesis (virgin generation). The development of an egg or other gamete without the process of fertilization. Pathogenic (disease offspring). Producing disease. Pedicel (a little foot). The stalk on which an organ is borne, especially the flower stalk of each separate flower in a cluster. Peduncle (a little foot). The flower stalk. Perianth (around the flower). A collective term for calyx and corolla taken together. Periblem (clothing). The part of the meristem at the growing apex of a root or shoot, immediately beneath the epidermis. It develops into the cortex. Pericambium. See Pericycle. Pericycle. The outermost layer of the central cylinder of a root. Perigynous. A term applied to those flowers in wl.ich the stamens and perianth appear to grow from around the wall of the ovary. Peristome (around the mouth). In mosses the circle of tecth or segments surrounding the opening of the spore caxe. ; Perithecium (around a case). In sac fungi, Ascomycetes (including lichens), a cavity containing the szcs or asci. Petal (a flower leaf). A leaf of the corolla. Petiole (a little foot). A leaf stalk. Phloém (bark). The soft portion of a fibro-vascular bundle, — the bast. In dicotyledons the part outside of the cambium, — the inner bark. Photosynthesis (light putting together). The process of manufacture of carbohydrates, such as starch and sugar, from water and carbon 250 GLOSSARY dioxide. It is carried on by the chromatophores and chloroplasts acted on by the energy of sunlight. Physiology. The science of the action and functions of organisms. Pinnate (a feather). Having leaflets arranged along two sides of a main leaf axis. Pistil (a pestle). The simple or compound structure (composed of one or more carpels) which in angiosperms contains the ovules. Placenta. The ovule-bearing portion of the interior of the ovary. Plageotropic (oblique turning). Assuming an oblique direction under the influence of gravity, as most secondary roots. Plasma membrane. The limiting membrane of a protoplast. Plasmolysis (that which is formed, loosing). A separation by osmotic action of the protoplast from the cell wall. Plastid (that formed). A protoplasmic body usually with a special func- tion. The term is used collectively for chloroplasts, chromoplasts, and leucoplasts. Plerome (that which fills). That part of the meristem near a growing point which is surrounded by the periblem and develops into the central cylinder. Plumule (a little feather). The primary leaf bud of an embryo seed plant. Pod. A dry, many-seeded, dehiscent fruit. Pollen (fine flour). Minute grains developed in the pollen mother cells of the anther and essential for the fertilization of the ovule. The locules of the anther are morphologically microsporangia and the pollen grains are microspores. Pollen tube. The structure which is developed from the inner coat of the pollen grain and serves to carry the sperm nuclei into the embryo sac of the ovule. Pollination. The transference of the pollen to the stigma or to the naked ovule of the gymnosperms. Prosenchyma. Tissue composed of elongated cells. Proteid. Any one of a group of nitrogenous compounds of which albu- men is an example. Prothallium (before a young shoot). The gametophyte developed from the spore of a pteridophyte. Protonema, plu. protonemata (first thread). A filamentous growth devel- oped from the spore of a moss, from which the leafy moss plants arise. Protoplasm (first formed). The living part of the material of the plant or animal body contained in the cells. Protoplast. A unit of protoplasm, or cell, with or without a -cell wall, Pteridophytes (fern plants). The great group composed of the ferns, horsetails, and club mosses. Pyrenoid (resembling a kernel). Minute bodies imbedded in the chro- matophores, which act as centers of starch formation. GLOSSARY 251 Receptacle. The extremity of the flower stalk, on which the fioral parts are borne; in Composite the common receptacle bears the head of flowers, — any structure carrying sexual organs. Rhizoid (resembling a root). A root-like filament in the lower plants. Rootstock. A somewhat root-like stem, usually nearly horizontal and dorsiventral, extending either above or under ground. Saprophyte (rotten plant). A plant that lives on dead organic matter. Scape (a stem). A leafless peduncle arising from the ground. Sclereid (hard). See Stone cell. Sclerenchyma. Rigid or strengthening tissue, composed of thick-walled cells, often having the form of fibers. Secondary growth. The growth which takes place in gymnosperms and woody dicotyledons from the development of the cambium cylinder. Seed. The fertilized and matured ovule. Seed plant. A member of the highest division of the plant kingdom, characterized by producing seeds. Sepal (a covering). A leaf of the calyx. . Sieve tubes, or Sieve cells. Soft bast or phloém cells with perforated sieve plates in their walls. Species. A kind of plant or animal, one of the taxonomic subdivisions of a genus. Sperm. A male gamete, generally very small and motile in comparison with the egg. Spermatia. Non-motile sperms, as in the red alge. Spermatophytes (seed plants). The great group composed of seed plants. Spermogonium. In the rusts a cup-shaped receptacle producing minute cells (spermatia) believed to be sperms no longer functional. Spindle. A mechanism consisting of delicate fibrils concerned with the distribution of the chromosomes during nuclear division (mitosis). Sporangium (spore vessel). A spore-producing case. . Spore (seed). A term applied to a variety of one- or few-celled repro- ductive bodies characteristic of groups below the seed plants. Sporidium (diminutive of spore). A spore produced by a promycelium. Sporogonium (spore offspring). The sporophyte generation of the liver- worts and mosses, sometimes called the fruit. Sporophyll (spore leaf). A leaf which bears spores. Sporophyte (spore plant). The asexual plant in an alternation of gen- erations producing asexual spores (see gametophyte). Stamen, The pollen-bearing organ of seed plants; morphologically a microsporophyll. Stele (a pillar). The central cylinder of a stem or root. Sometimes a stem has more than one plerome strand at the growing point and so develops several cylinders and is called polystelic. Stigma (a spot or mark). The portion of the pistil (destitute of epider- mis) on which the pollen lodges and germinates. 252 GLOSSARY Stoma, plu. stomata (a mouth). An opening through the leaf epidermis which serves for transpiration. The stomatic apparatus consists of the stoma and its guard cells. Stone cell. A hard cell with its walls much thickened by secondary deposits, as the grit cells of the pear. Strobilus (a fir cone). A cone-like cluster of sporophylls. Style (a pillar). An elongation of the pistil above the ovary, bearing the stigma. Suspensor. In seed plants and club mosses a structure arising from the fertilized egg, which pushes the developing embryo deep into the tissue of the gametophyte or endosperm. Symbiont. An organism living in a condition of symbiosis. Symbiosis (living together). The condition in which two or more organ- isms are living in intimate physiological relationship. Sympetalous. With the petals appearing as if grown together by their edges. Synergids (co-workers). Two cells accompanying the egg at the micro- pylar end of the embryo sac, the group of three constituting the egg apparatus. . Synsepalous. With the sepals appearing as if grown together by their edges. Taxonomy (order, law). The study of classification. Plant taxonomy is often called systematic botany. Teleutospores (end spores). The resting spores (chlamydospores) of the rusts, producing a promycelium. i Testa (a shell). The outer coat of the seed. Tetraspore (four spore). An asexual spore characteristic of the red alge, usually produced in groups of four. Thallophytes (thallus plants). The great group composed of the alge and fungi. Thallus (a young shoot). A simple vegetative body without differentia- 5 tion into roots, stem, or leaves. Tissue. A definite region of similar cells with the same functions, Trachea, plu. trachee (the windpipe). See Vessel. Tracheid (trachea-like). An elongated cell with closed ends and the walls with secondary thickening, as the pitted cells of coniferous wood. Trichogyne. A delicate filamentous extension from the carpogonium, specialized to receive the sperms. Tropophyte. A plant which is mesophytic during part of the year and xerophytic during the remaining part, as most deciduous trees. Turgor. The inflated or distended condition of a cell which is full of liquid. Unisexual. Having only male or female reproductive organs, Uredospore (blight spore). A spore of the rusts for rapid multiplication (summer spore). GLOSSARY 253 Variety. A subdivision of a species. Vein. A fibro-vascular bundle of a leaf, petal, or other thin and flat organ. Venation. The manner in which veins are distributed. Venter. The swollen basal portion of an archegonium containing the egg. Vernation. The manner of unfolding in buds. Vessel. A tube or duct made of separate sections but continuous from the absorption of the cross partitions. The walls have various thickening deposits, often spiral or ring-formed. Volva (a wrapper). An envelope inclosing a young toadstool and rup- tured by the growth of the latter, portions sometimes remaining as scales on the top of the cap and sometimes as a cup at the base of the stalk. Xerophyte (dry plant). A plant which can live with a scanty supply of water. Xylem (wood). The wood or inner part of a fibro-vascular bundle, the portion within the bundle cambium. Zone. In ecology a band of any given plant formation, usually bounded by other bands representing other formations, as about a pond, a salt spring, etc. Zodspore (animal spore). A ciliated and therefore motile asexual spore. Zygomorphism ee form). The arrangement of parts in corresponding right and left halves; bilateral symmetry. Zygospore (yoke spore). A sexually formed spore resulting from the fusion of similar gametes (isogamy) ; also called a zygote. INDEX Acorn, 71 Agaricus, type study, 116, 117 Albugo, type study, 109 Alcohol as a preservative, 195, 196 Aleurone grains, 27 Alge, culture of, 211, 212 Ameeba, type study, 80 Anabeena, type study, 86 Anthoceros, type study, 125 Apparatus, dealers in, 225, 226 Apparatus for the laboratory, 222, 223 Aspergillus, type study, 111, 112 Bacteria, culture of, 102-104 Bacteria, type study, 104, 105 Balsam, mounting in, 200, 201 Basidia fungi, field work on, 110 Bean pod, 69, 70 Bean seed, 18, 19 Bleaching after osmic acid, 208 Blue-green algze, field work on, 84 Bread mold, type study, 107, 108 Buds, 48-50 Buttercup flower, 67, 68 Capsella, development of embryo, 5 Capsella, development of flower, 164 Capsella, development of ovule, 164, Caraway fruit, 70 Carnivorous plants, 167, 168 Carnoy’s fluid, 195 Cell division, study of, 81, 82, 160, 161 Cellulose, 23 Chara, type study, 96 Charts, 7, 226 Chemical compounds recognition of, 23, 24 Chemicals for the laboratory, 224 Cherry fruit, 73 in plants, Chrom-acetic acid, 191-193 Chrom-osmo-acetic acid, 198, 194 Cladophora, type study, 91 Classes, ecological, 175 Club mosses, field work on, 182, 133 Coleochete, type study, 92 Competition among plants, 174 Composite, type study, 186, 187 Convallaria, type study, 179, 180 Cork, 42, 47 Corn grain, 19, 22, 23 Corn stem, 39, 40 Cycad, type study, 151 Dehydration, 203 Delafield’s hematoxylin, with, 198, 199, 209 Desmids, type study, 93 Diatoms, type study, 94 Dock fruit, 70, 71 staining Ectocarpus, type study, 97 Elder, study of male gametophyte, 163, 164 Elm leaf, 51 Eosin, staining with, 197 Equisetum, type study, 142-145 Erigeron, type study, 187 Erythronium, type study, 180, 181 Erythrosin, staining with, 200, 210 Euglena, type study, 83 Fern, type study, 183-189 Ferns, culture of, 216 Ferns, field work on, 132, 133 Ficus elastica leaf, 56, 61, 62 Fixing, 191-195 Flemming’s fluids, 193, 194 Flower of angiosperms, 64-68 Formalin as a preservative, 196 Fruit of angiosperms, 69-74 Fruit of angiosperms, development of; 73, 74 255 256 Fuchsin, acid, staining with, 199, 210 Fucus, type study, 98, 99 Funaria, type study, 127-182 Fungi, culture of, 212-214 Gentian violet, staining with, 209 Germination of seeds, 17-21 Gill fungus, type study, 116, 117 Gleocapsa, type study, 84 Glycerin, mounting in, 201, 202 Glycerin jelly, mounting in, 202 Green alge, field work on, 87 Growing point of stem, 50, 61 Halophytes, 176, 177 Hanging-drop cultures, 214 Heliotropic movements, 54, 55 Horse-chestnut seed, 19 Horsetails, field work on, 182, 183 Hydrodictyon, type study, 89° Hydrophytes, ‘175, 176 Imbedding in paraffin, 202-204 Invasion among plants, 174, 175 Iron-alum hematoxylin, staining with, 197, 198, 208 Iron-alum hematoxylin and saf- ranin, staining with, 208, 209 Isoetes, type study, 150, 151 Killing, 191-195 Knop’s solution, 211, 212 Lantern slides, 7, 226 Lathyrus, type study, 184 Leaves, 51-63 Leguminose, type study, 183, 184 Lemon fruit, 71-78 Lichen, type study, 112, 118 Lignin, 28, 24 Liliacez, type study, 179-181 Lily, development of embryo sac, 162 Lily, development of endosperm, 163 Lily, development of pollen, 160, 161 Lily, fertilization and double fertili- zation, 163 Lily leaf, 56, 56 Liverworts, culture of, 215 Liverworts, field work on, 117, 118 Lycopodium, type study, 145, 146 INDEX Maple leaf, 52 Marchantia, type study, 119-123 Marine alge, field work on, 97 Marsilia, type study, 139-142 Material, dealers in, 225, 226 Material for plant histology, 220-222 Material, preservation of, 195, 196 Mesophytes, 175, 176 Methyl green, staining with, 200, 210 Microscope, compound, construction of, 10, 11 Microscope, compound, use of, 12-14 Microsphera, type study, 110, 111 Mineral constituents of plants, 33, 34 Mitosis, study of, 81, 82, 160, 161 Moore’s solution, 212 Moss, type study, 127-182 Moss leaf, cell structure of, 80 Mosses, culture of, 215 Mosses, field work on, 117, 118 Nastic movements of leaves, 53, 54 Nemalion, type study, 100, 101 Nitella, type study, 96 Nocturnal position, 53 Nostoc, type study, 86 Nuclear division, study of, 81, 82, 160, 161 Cidogonium, type study, 91, 92 Oil, 24-26 Onion, 47, 48 Orange G, staining with, 209 Oscillatoria, type study, 85 Osmosis, 36, 37, 77 Oxygen making, 57 Parasites, 167 Pea seed, 19 Penicillium, type study, 111, 112 Peziza, type study, 112 Physcia, type study, 112, 113 Pine, type study, 151-159 Plasmodium, type study, 84 Plasmolysis in Spirogyra, 77 Pleurococcus, type study, 87, 88 Pollen tubes, 68, 69 Pollination, 169-172 Polysiphonia, type study, 101, 102 Pond scums, type study, 93 Porella, type study, 123-125 INDEX Potato agar, cultures on, 218, 214 Potato tuber, 46, 47 Propagation by roots, 36 Propagation, vegetative, 172, 173 Protection of plants from animals, 168 Proteids, 24, 26, 27 Prothallia, culture of, 216 Protonema, culture of, 215 Protoplasm, circulation of, 81 Prunus, type study, 182, 183 Puccinia, type study, 114, 115 Ranunculacez, type study, 181 Reagents, general, 188, 189 Reagents, special, 190, 191 Rhizopus, type study, 107, 108 Ricciocarpus, type study, 118 Robinia, type study, 183, 184 Root, 28-83 Roots, physiology of, 34-37 Rosa, type study, 181, 182 Rosacee, type study, 181-183 Sac fungi, field work on, 110 Safranin, staining with, 199, 209 Safranin and Delafield’s hematoxy- lin, staining with, 199, 210 Safranin, gentian violet, and orange G, staining with, 209 Saprolegnia, type study, 108 Secondary growth of stem, 43, 44 Sectioning in paraffin, 205-207 Sectioning free-hand, 204, 205 Seed plants, culture of, 216 Seeds, dissemination of, 178, 174 Selaginella, type study, 146-150 Shade leaves, 55 Slides for histology of seed plants, 219 Slides for study of plant cell, 217 Slides for type studies, 217-219 Slime mold, type study, 83, 84 Smut, type study, 114 Spherella, type study, 88 Sphagnum, type study, 126 Spirogyra, cell structure of, 75-77 Spirogyra, zygospore formation, 78, 79 Sporodinia, type study, 108 Squash seed, 17, 18 257 Staining in bulk, 197-200 Staining on-the slide, 207-210 Starch, 23-25, 383, 47, 57-59, 63 Starch in leaves, 57-59 Stem, dicotyledonous, structure of, 41— Stem, monocotyledonous, structure of, 39, 40 Stem, work of, 45-48 Storage of food in seeds, 21-27 Strawberry fruit, 73 ~ Successions among plants, 175 Sun leaves, 55 Supplies, dealers in, 225, 226 Taraxacum, type study, 186, 187 Temperature, effect on absorption of water, 34, 35 Tolypothrix, type study, 86 Tomato fruit, 71 Transpiration, 60-63 Trillium flower, 64-66 Tropzolum leaves, starch in, 57, 58 Tropeolum, study of, 15-17 Tulip flower, 66, 67 Twigs, 37-39 Ulothrix, type study, 89, 90 Ulva, type study, 91 Ustilago, type study, 114 Vaucheria, type study, 94-96 Vernation, 50 Violacee, type study, 185 Volvox, type study, 88 Water cultures, 33, 34 Water, movement of, in leaves, 63 Water, percentage of, in plant body, 33 Water, rise of, 34, 35, 45, 46, 68 Windsor beans, 35, 36 Xerophytes, 175, 176 Yeast, culture of, 105, 106 Yeast, type study, 106 Zonation, 177-179 Zoospores, formation and habits of, 91 ANNOUNCEMENTS PRINCIPLES OF BOTANY By JOSEPH Y. BERGEN, recently Instructor in Biology in the English High School, Boston, and BRADLEY M. DAVIS, Head of the Department of Botany in the Marine Biological Laboratory, and recently Assistant Professor of Plant Morphology in the University of Chicago 12mo. Cloth. vii+555 pages. Illustrated. List price, $1.50; mailing price, $1.65 A LABORATORY AND FIELD MANUAL OF BOTANY (In preparation) By JOSEPH Y. BERGEN and BRADLEY M. DAVIS RINCIPLES OF BOTANY is a work especially suited for college and normal-school classes and for those high schools that are equipped to give more than an average course in this subject. It claims superior merit in the mate- rial which it offers for a consecutive series of studies of representative spore plants, so treated as to outline the evolutionary history of the plant world. Some of the characteristics which make this volume superior to others of its kind are: I. That it presents more adequately than any other on the market the subject-matter demanded by the College Entrance Examination Board and the state universities of the middle and far West. II. That no other botany of its scope leads up to the more difficult portions of the subject in so easy and untechnical a manner. III. That it is the first book of its class to present ecology as a con- nected subject and not as a series of snap-shot studies. IV. That it is the only botany which gives a clear idea of the dis- tribution of vegetation in the United States, with the reasons for it. V. That it is better and more fully illustrated than any other book for beginners in botany. With decidedly mature students a half year’s course may be framed from the book. GINN & COMPANY Pus tisHErs BERGEN’S FOUNDATIONS OF BOTANY By JOSEPH Y. BERGEN Recently Instructor in Biology in the English High School, Boston and Author of ‘* Elements of Botany °” 412 + 257 pages. Illustrated. List price, $1.50 mailing price, $1.70 A HANDBOOK FOR THE USE OF TEACHERS To accompany Bergen’s Foundations of Botany Flexible cloth. 64 pages. List price, 30 cents; mailing price, 35 cents NE of the notable text-books on our list is “ Foundations of Botany” by Mr. Bergen, whose “ Elements of Botany” has come to be the most widely used recent text-book on the subject in the higher schools and academies of the country. The “Foundations of Botany” is sufficient to prepare for any college or university which accepts botany as an entrance requirement. It offers an extended and comprehensive course for schools that wish to devote an entire year to the subject, and provides the teacher who has only a minimum amount of time with the distinct advantage of a considerable option as regards the kind and amount of work which he shall present to his classes. It represents the simplest and most practical methods of botany teaching, combining a standard text liberally illustrated with a complete course in laboratory work and a key for the study of. systematic botany. The treatment of structural and physiological botany is unusually full and yet has special reference to the necessary limitations of work in secondary schools. The flora includes seven hundred species, and is the only recent short and thoroughly usable and intelligible flora of the central and northeastern states. The descriptions are written in the very simplest language consistent with scientific accuracy. GINN & COMPANY Pus isHers BERGEN’S ELEMENTS OF BOTANY REVISED EDITION By JOSEPH Y. BERGEN, Recently Instructor in Biology in the English High School, Boston Including Key and Flora for Northern and Central States. 12mo. Cloth. 283 +257 pages. Illustrated. List price, $1.30; mailing price, $1.45. Without Key and Flora. List price, $1.00; mailing price, $1.10. Issued also in three special editions with a Key and Flora for each: Pacific Coast Edition, Southern States Edition, and Rocky Mountain Edition. List price, $1.30 each; mailing price, $1.45. ERGEN’S “Elements of Botany,” Revised Edition, is designed to furnish a half-year course in the subject for students in secondary schools. It covers all the ground which ordinary classes can traverse in the time indicated, and presents only those topics which are essential to an elementary course in the science. It differs from the earlier editions of the “Elements” mainly in the greater stress laid on the topics of ecology and cryptogamic botany, in the somewhat abbreviated directions for histological work on seed plants, and in the greatly improved quality of the illustrations. Minor changes will be found on almost every page. THE BOOK IS CHARACTERIZED By the natural method of presentation, introducing the pupil first, as Professor Huxley recommended, to the comparatively familiar forms and processes of plant life. By the sparing use of technical terms, employing these only when they are indispensable for the sake of clearness or of brevity. By the treatment of the structures and the functions of plants consecutively, not in widely separated portions of the book. By the intimate combination of laboratory work with discussion, taking pains, however, not to tell the pupil, either in words or by means of illustrations, what he is to see before he sees it for himself. By the accuracy of the illustrations in detail, the half tones being used only to give gen- eral effects, never for minute organs or structures. By the fact that four special keys and floras have been prepared to accompany the text. This allows the student in any part of the country to obtain practice in the determi- nation of species of phanerogams, and to get a practical idea of their relationships and classification by means of a simply written and inexpensive flora of his own region. BOTANY NOTEBOOK To accompany Bergen’s Text-Books on Botany, and for general use in Botanical Laboratories or for Secondary Schools. Square 4to. Cloth. 144 pages. List price, 45 cents; mailing price, 60 cents. ERGEN’S Notebook was prepared with the particular view of minimizing the amount of routine dictation for both teacher and pupil without doing any of the latter’s thinking for him. Not only will it save time and trouble but it will also lead the pupil to per- form neat and accurate work. GINN & COMPANY PuvuBLIsHERs TEXT-BOOKS ON SCIENCE FOR HIGHER SCHOOLS AND COLLEGES Bergen and Davis’s Principles of Botany . . Bergen’s Elements of Botany. Sco Edition) Bergen’s Foundations of Botany 5 Blaisdell’s Life and Health Blaisdell’s Practical Physiology . . Brown’s Physiology for the ‘Laboratory . Byrd’s Laboratory Manual in Astronomy . Davis’s Elementary Meteorology . Davis’s Elementary Physical Geography Davis’s Physical Geography . . Gage’s Elements of Physics. (Revised Edition) . Hastings and Beach’s General Physics Higgins’s Lessons in Physics . Hough and Sedgwick’s Human Mechanism Linville and Kelly’s Zodlogy : CE OE ie 8 Meier’s Herbarium and Plant Description. With direc- tions for collecting, pressing, and mounting specimens McPherson and Henderson’s epapin’ | pk ie of Chem- istry . Millikan and Gale’ s First Course i in Physics E Moore’s Laboratory Directions for Beer in Bac- teriology . Nichols, Smith, and Turton’s Manual of Experimental Physics ap far MOR be, Oo Cae Bae gs Norton’s Elements of Geology . Pratt’s Invertebrate Zodlogy. . Sabine’s Laboratory Course in | Physical Measurements. (Revised Edition) . : Seller’s Elementary Treatise on Qualitative Chemical Analysis . TT ee ae eee 8 Stone’s Experimental Physics Ward’s Practical Exercises in “Elementary Meteorology Wentworth and Hill’s Text-Book of Physics . ‘ Wentworth and Hill’s Laboratory Exercises in Elemen- tary Physics. (Revised Edition) Williams’s Elements of Chemistry Pate Aer os Young’s Elements of Astronomy . . . .... . Young’s General Astronomy ..... .. .- Young’s Lessons in Astronomy. (Revised Edition) Young’s Manual of Astronomy ee a eC a List Mailing price price oe 50 $1.65 1.30 1.45 1.50 1.70 90 1.00 1.10 1.20 75 85 1.25 1.35 2.50 2.70 12 1.40 1.25 1.40 1.12 1.20 2.75 2.95 QO 1.00 2.50 2.65 1.50 1.70 60 -70 1.25 1.40 1.25 1.40 1.00 1.05 -90 1.00 1.40 1.95 1.25 1.35 1.25 1.30 75 80 1.00 1.10 I.I2 1.25 I.1§ 1.25 25 +30 1.10 1.20 1.60 1.75 2.75 3.00 1.25 1.40 2.25 2.45 GINN & COMPANY Pus tisuers in i sit iit iH iH litt erp i! Hit nih HIE SHH a i Ht f a +) i i Hi i i ; | i if i iH hit bil Ht Ht ‘th i i re state ! ih HN H th Hf Hit Hi a ee i ! HHH tN Ht Sa ee HHIHR IRE AA iit i Doe RE Ta en na HAA Ha i i i ii i