MftNUffL OF I^LftNT Histology. ^'FHOMi^£:]^Ki) BlJ-BlE?: .ossjisii QK T4r Cornell XHniversit^ OEPARTMEN^ IRew l^ork State College ot Hgrtculture .£,.£...:b.u.q \^..\>nx...\iv. Date Due MMmJJ'US3S«ak. ,^^^gM'''m^ QK 673.T45 ""'"""""•' '■"'"'* ^limmimiimiu'""^' "' P'^"' histology. 3 1924 001 734 932 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/cu31924001734932 LABORATORY MANUAL PLANT HISTOLOGY MASON B. THOMAS, B. S. Professor of Biology in Wabash College, WILLIAM R. DUDLEY, M. S. Pi-ofessor of Botany in Leiand Stanford, Jr., University. C1?AWF0RDSVILLE, INDIANA; 1894. 594. Copyright, 1894 MASON B. THOMAS. All Eights Eesbkved. The Journal Co., PbintebS, Crawfordsville, Ind. INTRODUCTION. Almost every thoughtful teacher of botany in the colleges and universities of our country is confronted by two problems in con- nection with his laboratory instruction. He is forced to provide a course which shall give the general • student a fair knowledge of what the teacher deems the most important phases of plant Ufe; and on the other hand, if a conscientious instructor, he will encour- age students to advanced work, inaugurating courses which are intended not only to inform the mind, but to train the powers of ' observation, compauison and scientific judgment, and finally pro- duce the investigator capable of pursuing problems of science without aid or admonition, if not without suggfestions from his , professor. Some ten years ago, the present writer, then iu charge of the Histology and Oryptogamic Botany at Cornell University, attempted a revision of his laboratory course in plant anatomy, in order to adapt it to the advanced courses, chiefly in Oryptogamic Botany, ■^hich followed. The result was a small hand-book, privately printed in 1886, entitled, "Anatomy and Histology of Plants," which evidenced the author's desire to impart some special knowl- edge of tissues as a foundation for more serious , work in any subsequent subject involving the use of the microscope. It SQon appeared, however, that better methods in the preparation of soft tissues and delicate organisms must be adopted, if any great advance was to be made toward the solution of problems of structural development. It inay here be mentioned that imbedding and cutting serial sections of delicate plant tissues, had not been, put in practice in m , INTRODUCTION. American botanical laboratories previous to that time, and even in Germany it was but little in vogue. To study better methods of microscopic manipulation in this and other directions, was the object of the writer in spending the year 1887-88 in the German laboratories, where he used for the first time the collodion method of imbedding. It was seen more and more clearly that in the future, students were to be trained to useful work in biological investigation chiefly through a mastery of microscopical technique, and a thorough knowledge of tissues and cell contents with their behavior under the influence of reagents. The changes in methods in the histological course brought about during the four years following 1888, were made to bear upon the work of students taking the courses on the higher and lower Cryptogams, with most excel- lent results. Such changes were included in the plans for a revised manual, carefully drawn up in 1892. Mr. Mason B. Thomas, an undergraduate, then Fellow in Botany in the writer's laboratory, 1888-91, and afterward Profes- of Biology in Wabash College, was invited to assist in this work. During his university course he had been able to render me invalu- able assistance, by refining and abridging the gjrocess of imbedding in collodion, and by devising various laboratory appliances con- nected with it (still remaining in the laboratory at Cornell), some of which are described in his papers published in 1891 to 93, * and detailed at some length in Atkinson's " Biology of Ferns" (1894), particularly in Part II., Chapter I. The exactions of work since 1892, in an entirely new field have obliged me to abandon rewriting the Manual. At my request, Professor Thomas has done this, so far as it seemed necessary. He has also prepared the part on technique (Parb I.), as well as plates, selecting the illustrations from his many beautiful prepara- tions made while at Cornell University and since that time. The fact that some of the best laboratories in this country have adopted , the methods formulated by him makes it particularly appropriate that he should write this part. In it no attempt has been made at an exhaustive treatise but * (1) "The Oollodion Method in Botany;" Rep. Am. Society of Microscopists, 1891; (2) "A Dehydrating Apparatus," Am. Monthly Microscopical Jol., Jan., 1891. (3) " Sectioning Pern Prothallia," The Microscope, Nov. 1893. INTBODUCTIOK vii the matter is presented rather in the form of suggestions to those who may be at the beginning of their work in micro-chemistry or technique. ' ' ' The tests for the different vegetable substances and the gen- eral properties of reagents have been taken from the best authori- ties on those subjects, and carefully tested. I am responsible for the plan of the manual of directions (Part II.), for some of its phrasiology, and for the selections of most of the subjects used for study; and any imperfections in this part must be laid at my door. Nevertheless the plan- has .stood the test of many years thoughtful use in my own laboratory, and more recently in that of Wabash College ; and Professor Thomas shares completely with the writer the belief that such an element- ary course, most thoroughly taught, should be made the founda- tion for advanced' instruction on the, morphology of the higher and lower plants,' and should enter into the education of a student for ■ any independent work in anatomy, physiology, or biology. If other teachers should find the work acceptable, we would remind thfem that a course of carefully prepared lectures should supplement the laboratory work, and we urge them to so present the subject, that the intergradations of tissues may not be overlooked, ' and the larger relations of great tissue masses and their beautiful adaptations to the necessities of the living plant, may be completely understood by the student. No true teacher will allow a student ' to consider these individual studies in an unrelated way. We have cited freely text books and reference works of unquestioned value, such, as are to be found on the book-shelves of every good laboratory, biit we have not made a practice^of referring to original papers^ as it would be for the most part out of ' place in a work of this kind. But this does not release the teacher from the duty of placing the most important papers bearing directly on a subject of study within the reach of the student and requiring him to look them over. . The inconvenience of usrug plates placed at the end of a book will not be great^ and is offset by the fact that they are removed from the unavoidable scrutiny of the student as he is executing his own drawings, but any defect in this or any other direction noticed and, communicated by a teacher may be rectified in another edition. viU ^ INTBODUCTION. Tp the student we would say that they, as .men fitting them- selves for professional or semi-professional scientific careers, have certain duties to them.selves, entirely independent of the formal requirements of the instructor. Their aim should be a complete familiarity with the methods suggested, a comprehensive and scien- tific knowledge of as many facts as possible, and an ability not only to execute but to finally plan their own work, and themselves solve their scientific problems. To this end they should not, even in this elementary course, content themselves with the lines laid down, but should consult all bo^ks suggested in the studies given in the manual, and read carefully the passages to which reference is made. They should miss no opportunity to learn of a new work or an original paper in botany, or any fact concerning the mode of work of any genuine contributor to the literature of the science. We have an especial sympathy with the ambitious student whose superior training or skill enables him to accomplish more than the average students. For him are suggested the additional studies in the hand-book, and he will always find his instructor r^ady to advise him in regard to further reading. Both teacher and pupil should recognize the- fact that in the present day, a sure foundation may ~ be laid in undergraduate years, for a 'subsequent successful profes- sional career, if the pupU thoroughly learns the use of his tools and pursues his chosen science with the zeal that belongs to his time of life. It is with genuine regret that I lay down this work as well as the particular plans which were the motive of it, for broadening and deepening the training of American botanical students; but in doing so, I am sure that in the hands of Professor THbmas it will arrive at a better developiaent than in my oWn, and that his efforts in this field will find nothing but appreciation. The authors wish to express their indebtedness to Mr. E. "W. Olive, Instructor in Biology in Wabash College, for the many ways in which his services have lightened th.e labors in the preparation of this manual. WILLIAM EUSSEL DUDLEY, I August, 1894. Leland Standford, Jr., University. WORKS OF REfEF^ENCE. In the selection of the list of hooks and periodicals below, it has heen the intention to gi^e only the more general ones and those that should be in every botanical laboratory. The list is in no sense intended to be a complete one, and it is expected that the student will have at his disposal, a number at least, from each of the groups. For special or advanced work the original papers and monographs, on each particular subject considered, must be obtained. General Botanical Works. Bastin, College Botany/; Engelhard & Co., Chicago, 1890. Bennett & Murray, Oryptogamic Botany; Longmans & Co., London, 1889. Bessey, Botany for High Schools and Colleges; Holt & Co., N. Y., 1892. Campbell, Structural and Systematic Botany; Ginn & Co., Boston, 1890. DeBary, Comparative Anatomy of. Phanerogams and Ferns; Oxford Press, Lon- don, 1894. JSngler and Prantl, Die Naturlichen Pfiianzen Familien; Englemann, Leipzig; issued in parts and not yet complete. Frank, Lehrbuch der Botanik; Engelmann, Leipzig, /1893. Goebel, Outlines of Classification and Special Morphology; Oxford Pi-ess, Lon- don, 1887. Gray, Structural Botany; Am. Book Co., New York. 1879. Sachs, Gesammelte Abhandlungen ueber PJlanzeuphysiologie; Engelmann, Leipzig, 1893. Sachs, The Physiology of Plants; Oxford Press, London, 1887. Sachs, History of Botany; Oxford Press, London, 1890. Vines, Physiology (if Plants; Cambridge Press, London, 1886. Vines' Text Book of Botany; Swan, Sonnenschein & Co., London, 1894. Laboratory rianuals. Arthur, Barnes, and Coulter, Plant Bissection; Holt & Co.. N. Yj, 1887. Bower, Practical Botany; MacMillan & Co., N. Y., 1891. Davis,, Text Book of Biology; Chas. GrifSn & Co., London, 1893. Dodge, Elementary Biology; Harper & Brothers, N. Y., i894. Dudley, Histology of Plants; Ithaca, N. Y., 1886. Goodale, Structural Botany; Am. Book Co., N. Y., 1885. Huxley & Martin, Practical Biology; MacMilla,n & Co., N. Y., 1889. Parker, Elementary Biology; MacMillan & Co., N. Y., 1891. Sedgwick and Wilson, Biology; Holt & Co., N. Y., 1889. Spalding, Introduction to Botany; Heath & Co., Boston, 1893. Strasburger, Practical Botany; Swan, Sonnenschein & Co., London, 1893. a; WOBKS OP BEFEHENGK Structural and Technique. Atkinson, Biology of Ferns; MacMillan & Co., N. Y., 1894. Methods for treatment of tissues; structure. Bausoh, Manipulation of the Microscope; Rochester, N. Y. Manipulation and care of Instrument. Beale, How to Work With the Microscope; London, 1880. Structure and methods. Beherens, Guide to the Microscope in Botany; Boston, 1885. Carpenter, The Microscope and its Revelations; Philadelphia, 1891. Manipulation and care of instruments; also structure. Clark, Practical Methods In Microscopy; Heath & Co., Boston, 1893. Fry, The Microscope and Microscopical Technology; New York, 1892. Structure, manipulation and methods. Gage, The Microscope and Histology; Ithaca, N. Y., 1894. C?ire and manipulation of instruments ; also methods of mounting. Goodale, Physiological Botany; Am. Book Co. Lee, Mlcrotomists Vade Mecum ; Philadelphia, 1 890. Methods. Strashurger, Practical Botany. ' Van Heurck, The Microscope, Construction and Management; D. Van Nostrand Co., N. Y., 1893. . Botanical flicro-^Chemistry. Poulsen, Mlcro-Chemlstry, Trans, by Trelease; Glnn & Co., Boston, 1886, Zimmermann, Botanical Micro-Technique, Trans, by Humphrey; Holt & Co., N. Y., 1893. Botanical Journals, and Periodical Publications. Annals of Botany; Oxford, Clarendon Press, London. Morphological, Systematic, and Pljyslological. Am. Monthly Microscopical Jol.; Washington, D. O. Micro.scopical methods and histology. Botanical Gazette; Lake Forest, HI. i Morophological, Physiological, and Systematic. Botanisches Centralblatt, Cassel,; contains original work together with Bibli- ography of Current Botanical Literature, 1880 Bulletin of Torrey Botanical Club, N. Y. ; largely systematic. Just's Botanischer Jahresberlcht, Berlin; Bibliography of Botanical Literature, 1873 . Annales des Sciences Naturelles (Botanique); Eed. par A. Brongnlart et J, Decaisne,' Paris, 1884 ;/ chiefly original papers. Botanische Zeiturig, Leipzig; original papers and Bibliography, 1843 CONTENTS. PART FIRST. nicro=Chemistry and Technique. Reagents Alcohor Separation of Inulin Acetle Acid Test for Crystals Alum Ammonia .■ Test for Middle Lamella Anilin Chloride Test for Lignin... Argentic Nitrate Test for Living Protoplasm Chloroform Solvent for Fats, etc Calcic Chloride Clearing Tissue Cuprlc Sulphate Test for Sugars Carbon Bisulphide Solvent for Carotin Carbolic Acid Solvent for Fats, etc Test for Lignin Chromic Acid Solvent for Cell WaU Cuprammonia „ Solvent for Cellulose Cleaning Mixture j Nrtro-Sulphurio Acid Dichromate Mixture Collodion .....1 2 per cent, and 5 percent, solutions... Ether Glycerin Separation of Inulin Hydrochloric Acid Test for Sypochlorin 7 Iodine 7 Test for Starch 7 Test for Cellulose 8 Action on Protoplasm 8 Millon's Reagent 8 Detection of Albuminoids 8 Nitric Acid ; 8 Macerating Agent. 8 Test for Protein Matters 8 Clearing Tissue of Starch 8 Oxalic Acid 8 Bleacliing Tissue 8 Solvent for Pectose 8 Perosmic Acid 9 Fixing and Hardening Agent 9 Pptassic Dichromate 9 Hardening Resin Masses 9 Test for Tannin 9 Potassic Chlorate 9 Macerating Agent 9 Test for Suberin 9 ParafBne :: ,. 9 Melting Points 9 Phosphoric Acid 10 Test for Crystalloids 10 Rosalie Acid , 10 Test for Vegetable Jelly 10 Stain for Sieve Tissue 10 Sugar 10 Test for Protoplasm 10 Pollen and Spore Cultures , 10 Sulphuric Acid : 10 Test for Cellulose 10 Action on Starch 10 Action on Fat Bodies 10 a;it CONTENTS. Ohior-lodide of Zinc 11 Test for Celtalose 11 Test for Tannin ,... 11 Action on Fungus Cellulose 11 Hardening Agents 12 Alcohol 12 Dehydrating apparatus 13 , Picric Acid 15 Chromic Acid 15 Osmic Acid 15 Hardening Fluid 15 Cutting AND Mounting Tissues 16 Free-Hand Sectioning 16 Softening Hard Tissues 16 Use of Pith or Cork 16 Use of ParafBne 17 ParafBne Method , 17 Construction of Paper Boat 18 Microtomes 19 Fixing Sections to Slide 19 Staining, Mounting, etc 20 Collodion Method 21 Hardening, Sectioning, etc 22 !?^ Ether Vapor Bottle 23 Treatment of Delicate Tissues 24 Staining Agents 26 Ammonium Carmine 26 Alum Carmine 26 Bosin 27 Haematoxylin 27 Aniline Colors 27 Methyl Violet 37 . Methyl Green 27 Aniline Blue 28 Magenta 28 Picric Acid 28 Silver Nitrate 28 Clearing Agents 29 Cedar Oil 29 Clove Oil 29 Solvent for Collodion i 29 Use in the Minute Dissections 29 Origanum Oil 30 Sandalwood Oil 30 Carbolic Acid and Turpentine 30 Mounting Media 31 Aluminum Acetate 31 Mounting Algae 31 Balsam 31 Clearing 31 Preparation of Balsam 31 Treatment for Air Bubbles 31 Sealing Mounts 32 Balsam Bottle 32 Carbolic Acid 32 Calcic Chloride 32 Glycerin Jelly 32 Kaiser's Formula 32 Glycerin 32 Use for Fresh Tissues 33 King's Mounting Medium 33 Water..... .' 33 Action on Tissues 33 Cements ...i 34 Gold Size 34 Shellac 34 Ball Cement 34 Asphalt Vajrnish 34 White Zinc Cement 34 Serial SEOTiONiNG 35 Paraffline, and Collodion Sections...... 35 Arrangement on Slide 35 Double Staining , '. 37 Combination of Stains 37 Use of Mordant 37 Fluid Mounts 38 Mounting in Cells 38 Construction of Cells 38 Mounting Fluid 38 Sealing Mounts 39 Dry Mounts 39 Equipping OP Laboratory 40 Case for Reagents 40 Supplies and Their Location...; 40 Waste Vessels 40 Clearer Bottle 41 Collection and Preservation of Mate- rial •!...:. 43 Treatment of Soft Tissues 42 Preservation in Alcohol 42 Preservation in Collodion 43 Preservation on Corks or Blocks 43 The Microscope 44 Description of Instrument 45-46 Methods or Study 47 Care in Observation .„ 47 Directions for Drawing .....47-48 Use of Camera Lucida 48 Drawing Material...4 49 Page of Drawing Book 50 Preservation of Slides 50 Mailing Boxes : 50 Construction of Cabinet 51 Catalogue of Preparations with Sample Card 52 Apparatus Needed..; 53 Microscopes 53 CONTENTS. seiii Microtomes , 53 Keagents 54 Slides, Covers, Brushes, Paper, Dissect- ing Needles, Razor, etc 54-55 Care ov Apparatus 56 Cleaning Instrument, Lenses, etc 56 Testing Tissue with Acids 56 Oiling, Changing Objectives and Ocu- lar....'. 56 Care of Lenses, Focussing 57 Care of Byes 58 Eye Shade R0 Sources of Light 59 Artificial Light 59 Manipdlation of Apparatus 60 Interpreting Appearances 60 Cloudiness on the Lenses 60 Focussing 61 Optical Sections 61 Air Bubbles 62 Oil Globules 62 Magnification 63 Determination by use of a Camera Lu- cida 63 Determination of Ocular Micrometer Ratio 64 Micron 64 Tube Length 65 Practical Exercises 65 PART SECOND. Laboratory Directions. Divisions of the Subject. A. Living Cells, (with Protoplasm and Chlorophyll.) 66 B. Contents of Cells, (the secondary products.) 66 C. Elementary Tissues 66 D. The Primary Meristem 66 E. The Systems of Tissues 66 P. The Thickening of Stems, etc., (secondary growth) 66 A. Study of Living Cells 67 1. Those Livinq Separate prom One Another 67 a. Protoeoceus vlridis 67 b. Mother-cells of Pollen,— Begonia 68 c. Mature Pollen GTains,—.Malvaeeae^. 70 d. Culture of Pollen Grains,— Trodescontto 71 2. Cells in Colonics, Joined Temporarily 72 a. Spirocfj/ra,— (showing Protoplasm, Chlorophyll and Progressive Cell Di- vision) 72 8. Cells Permanently Joined 74 A. Not Forming Tissue : 74 Stamen Hairs of Tradescantia 74 B. Forming Tissue 74 Illustrated by the Majority of the Subsequent Studies...!. 74 xiv CONTENTS. B. Cell Contents 75 1. Starch Grains - 75 a. Potato Tuber,— Solanttm tuberosum 75 t). Garden Pea,— Ptsum satiBum 76 c. Wheat Grain,— Triticttm vuJ^are , 76 Structure of Cereal Grains , 77 ' d. Grain of Indian Corn,— Zea Maj/s 78 e. Grain of Oa.ti,—Avena aativa 78 2. Obystals 78 Crystal Prisms,— Onion Bulb, Allium Cepa 79 Kaphides,— Roots of Many Plants 80 3. Protein Gbandles 80 a. Crystalloids,— Potato Tuber 75 b. Aleurone Grains, Garden Pea, Corn and Wheat Grains 76 Cystoliths 80 Leaf of Ficus elastica , 80 INULIN ; J 80 lioot of Dahlia 80 C. Elementary Tissues „ 82 1. Parenchyma Tissue ; 82 a. Isodiametrlc,— Stem of Qeranium 82 b. Ellipsoidal,— EoOt of Hj/acintTi v 83 c. Irregular and Epidermal,— Leaf of Oeranium , 83 d. Stellate,— Petiole of Pontederia 84 e. Suberous,— Bark of Cork Oak 84 ' Modification of Cell Wall 85 a. Cell Walls with Mucilage,— Seed Coat of Flax 85 b. Cell Walls with Lignin,— 'Fibrous Tissue 85 c. Cell Walls with Cutin,— Epidermis of Cycas leaf 86 d. Cell WaUs with Minerals,— Stem of Bguisefttm 86 Continuity OF Protoplasm , 86 2. COLLENCHYMA TISSUE 87 stem ot Bisgonia ortGercmium 87 Endodebmal Cells 1 87 3. SCLliRENOHYMA TISSUE ; 88 a. Roots ot Dahlia variabilis 88- b. Ivory NTit,—Phytelephasmacrocarpa 88 c. Rhizome of Pteris aguflina ; 89 4 and 5. Pbosenchyma Tissue 89 Prosenchyma Proper 89 Wood and Bast Cells,- Leatherwood,— Dirca palustris 90 Tracheary Tissue 90 Tracheids,— Stems of Horse Chestnut, Moon Seed and Grape Vines 91 Tracheids of Coniferae,— Pinits Strobus 92 Tracheae ; 92 a. Dotted,— Stem of Grape Vine , 93 b. Pitted; c. Spiral; d. Reticulated; o. Sc^lariform i 94 Stem of Castor Oil Bean 94 f. Annular,— Stem of Corn ., 94 g. Trabecular,— Leaf of Juniper 94 6. Sieve Tissue 94 Stem of Cucumber, or Pumpkin : 95 7. Laticipebous Tissue 95 Latex Cells,— Stem or Petiole of Euphorbia 96 Latex Tubes or Vessels, Stem or Petiolp of Celandine 96 CONTENTS. ■ XV Glands and Watbb Pores 96 Glands 96 a. Lemon Skin 96 \>. Leaf of Eucalyptiis 97 c. Eesin,Ducts of Pinus 97 Water Pokes .' 97 Leaf Tooth of michsia ,. 97 D. rieristem Tissue 99 Primary Meristem 99 a. Single Apical Cell., 101 Tip of Eguisetum, or Fern Boot 101 b . Group of Initial Cells 99 Tip of Hyacinth Boot 99 FiBRO-VASOtiLAR Bundles '. :. 103 a. Collateral,— Stem of Moon Seed Vine 103 b. Bicollateral,— Stem of Cucurhita , 104 c. Badial,— Boot of Corn ,, , 106 d. Concentric— Stem of Pteris, Bhizome of iris 107 E. The System ol Tissues 108 1. Epidermal 108 Formation of Stomates,— "Stone Crop," or Sedum tematum 108 Tvichomes.—ShepliercHa Candensis, Nettles, etc 109 Water Pore,— Fucftsia 110 2 and 3. Fibbo-Vascdlab and Fundamental 110 Exogenous Stem 110 Herbaceous,— Becfoiiia,...; 110 Woody,— Moon Seed Vine ^ 110 Endogenous Stem 110 Herbaceous,— Corn 110 Woody,— Smitaa; ;.110 Coniferae,— Ptoits 110 Vascular Cryptogams.. 110 F. Secondary Tliickening ^ 112 Stem of Dicots,— Moon Seed Vine ;. .112 Stem of Monocot,— Smflaa; Htspida 113 Stem of Coniferae,— Ptniis 113 Boots of Monocots,— OrcftMttceoe or Cyperaceae 114 Vascular System of Leaves 114 UxalU 114 Lenticels and Cork Thickening 115 Elder, or Moon Seed Vine '..; 115 LIST OF ILLUSTRATIONS. Figure 1. Dehydrating Apparatus Page 13 3. Paper Boat 19 3. Ether Vapor Bottle 23 4. Microscope 44 5. Transection of Anther of Begonia 50 6. Section of Drawer for Glass Slides 51 7. Eye Shade ', 58 8. Culture Slide 71 xvi CONTENTS. Figure 9. Formation of Pollen Grains, Funfcto OKCtta Plate I 10. Staniea Hairs of Tradesccmtia , ,. I 11. Section of Potato Tuber II IS. Various Forms of Parenchyma Cells II 13. Sclerenchyma cells from Dahlia Root , Ill 14. Hesin Duct from Ptous , III 15. Section of Lemon Peel Ill 16. liOngisection of Boot, Cypripedium pabescens IV 17. Transection of Boot, CvpHi>e, stances, it readily causing them to turn a strong red color. The reagent is, prepared by pouring over some pure mercury an equal quantity by weight of strong HNO3. If the solution is not com- plete, heat the mixture, then pour over it twice its volume of water. After allowing it to stand a few hours, decant the clear portion for use. The reagent will act only when used in fresh solutions. Nitric Acid. Nitric acid is used with potassic chlprate as' a macerating agent. When added alone to tissues, it causes the protein matters to turn a bright yellow. The reaction is made more apparent upon the addition of ammonia. According to Hoehnel, the acid forms a good test for suberin. It is also used for clearing tissue of starch,, causing the grains to swell and soon dissolving them. Oxalic Acid. An alcoholic solution of the acid is very Useful in bleaching sec- tions that have previously been too deeply stained. Dilute aqueous solutions are employed with some stains for various tissues, and a concentrated solution dissolves pectose after treatment with potash. 9 EEAQENT8. Perosmic Acid. This acid is very volatile and has an extremely disagreeable and poisonous odor ; it therefore must be kept in a sealed glass tube. It is usually employed in an aqueous solution of 1 per cent, strength, and this should be kept tightly corked in a dark place. The acid is most useful for killing and fixing at once living pro- toplasm. It is, therefore, very helpful in studying nuclear and cell division, since it prevents immediately all further change. Oils and fats are discolored by the acid, due to its reduction and the deposition of metallic osmium. As a hardening agent it is used with 9 parts of 25 per cent, chromic acid, and it not only hardens, but stains simultaneously meristematic tissue. Potassic Dichromate. Potassic dichromate is often used as a substitute for chromic acid in hardening, and seems to gi^e about the same results. It is used especially for hardening resin masses and sometimes for the detection of tannin. After continued action it colors cells contain- ing the latter substance a reddish brown. Potassic Chlorate. This salt with HNO3 forms Schulze's macerating agent, which is especially, useful in destroying the middle lamella/ and for the isolation of ■ wood cells. The macerating is aided by the applica- tion of heat. Since the fumes arising from the mixture readily corrode metal, it is very important that the operations with the agent be performed in a room that does not contain any\delicate instruments. Schulze's agent .is altio used in the detection of suberin. The suberized cell walls resist for a long time the action of the mixture but finally break down, and a part of the suberin forms eerie acid, which is readily soluble in potassic hydrate, ether, chloroform, etc. Paraffine. Hard and soft paraffine are both useful ; the former with a melting point of about 45 °C., the latter with 33 °C. Different melting points can be secured by mixing in different proportions 10 BEAGENTS. the hard and soft kinds. The meltmg point can be lowered by the addition of chloroform. It must be remembered, however, that before any attempt is made to section imbedded tissue, all of the chloroform must be driven off in the infiltrating oven, otherwise the paraffine will be too soft for support. Turpentine can be used in place of the chloroform and is perhaps quite as good. Phosphoric Acid. Phosphoric acid is sometimes used to remove water from tissues, and when added to any containing crystalloids, it causes the latter 'to swell. Rosalie Acid. This acid is used in connection with sodic carbonate as a test for vegetable jelly, staining it red. It is 'useful in coloring the callosities of sieve-tubes and in bringing out the general structure of cribrose tissue. Sugar. If tissue containing protoplasm is left for some time in a thick syrup of cane sugar and then transferred to Hj SO^, it will turn a red color. The reaction is not always a certain one. A 10 per cent, solution of sugar is very useful for pollen and for spore cul- tures. Sulphuric Acid. Sulphuric acid is used in connectibn with many tests and is also valuable in breaking down cellulose walls, without shrinking the protoplasm. This makes it especially , important in demonstrating the continuity of protoplasm. It is also used in connection with iodine to determine the purity of the cellulose that makes up the cell walls of any tissue. In this test the tissue is first treated with a tincture of iodine, and sulphuric acid is then added. The walls will turn blue if they are composed of pure cellulose. Dilute sulphuric acid causes starch grains to swell, while with the concentrated acid they are dissolved. Pure cellulose walls are likewise dissolved by the acid, while cutinized ones resist its action. Fat bodies are not soluble in it, but they form small refrative drops. 11 BEAOIJNTS. Chlor-iodide of Zinc. This is known as Schulze's reagent and is very useful in detecting the presence of cellulose. This reagent is made by pour- ing over metallic zinc some hydrochloric acid and then evaporating the solution with an excess of zinc present, until it becomes of a thin syrupy consistency. Add as much iodide of potassium as will be taken up, and then iodine until a saturated solution is obtained. Keep the reagent in the dark to prevent the formation in it of hydriodic acid. , Pure cellulose gives with this j*eagent a blue or violet color, ,due to the staininjg by the iodine of the amyloid which is formed by the action of the agent on cellulose. Wood, cork, and cutinized walls are colored yellow, while starch colors blue, but the grains soon become disorganized. Cells containing tannin are colored by the reagent red or violet. Fungus cellulose, unlike ordinary cellulose, remains uncolored by the action of this agent. HARDENING AGENtS. The subject of harderting agents is one of very great import- ance and presents a field in which much yet remains to be learned. The object of hardening is to bring the tissue in a condition io be either sectioned directly without crushing, or to allow it to be infiltrated with some substance that will hold jt firm for cutting. The difficulty to overcome is to find an agent that will harden the tissue without shr-inking it. The method employed "to overcome this is to bring the tissue in contact with the dilute hardening agent, and then gradually increase its strength to prevent a vio- lent action between it and the tissue. The former must always be present in a large excess in ofder that an equilibrium may not be established too quickly. The exact strength of the hardening agent in which the tissue should first be jilaced is a matter of some uncertainty and can only be determined after experimenting with each sort of tissue. After the object is hardened, it should not be left any great length of time in the full strength of the haidening agent as it is liable to become brittle. In arranging the tissue to be hardened it should be carefully trimmed and only the portions that are needed for examination placed in the agent. In the case of large pieces, as, for example, closed pistils, cut the parts open 'to allow the hardening agent to penetrate all parts of the object, otherwise, deterioration will result. Alcohol. This is one of the most frequently employed hardening agents, and for many plant tissues is all that could be desired. J]or most soft material 40 per cent, alcohol is dilute enough to begin the 13 HABDENING AGENT^. hardening. From this strength it is transferred to 50 per cent., 65 per cent., 75 per cent., 85 per cent, and 95 per cent, alcohol respectively, allowing it to remain in each solution for about 24 hours, varying the time according to the nature of the tissue. If it is desirable to preserve the tissue for future examination, the hardening should cease with the 75 per cent, strength, and in this it should be kept until needed, when the hardening may be com- pleted with the 85 per cent, and 95 per cent, strengths. A very convenient apparatus for hardening plant tissues was invented by Schulze, and a modified form of it is recommended for laboratory purposes. It can be made as f pUows: Fig. 1. Dehydrating apparatus. Pig. 1. Apparatus complete, showing dehydrating tube B in place. A. plaster of paris diaphragm. C. Glass rod supporting the ' disk. Pig. 2. Dehydrating tulie. D. Chamois sliin diaphragm. E. Spring holding the diaphragm in place. In a 9x9 Whitall-Tatum museum jar a disk of plaster of paris is supported about 5 c. m. from the top by means of legs made of glass rods. The disk is perforated to allow tubes of various sizes, from 2-4 c. m. in diameter to pass through. These are the so- 14 ~ HABDENING AGENTS. called dehydrating tubes. The plaster of paris diaphragm can be made by first constructing a mould of the desired size, witli a paper bottom and a card-board hoop for the, outside. This must be J placed on a level surface. The plaster of paris is then mixed with water and poured into the mould to about the depth of 1^ c. m. "While it is yet soft the three legs can be inserted near the edge and holes for the dehydrating tubes cut in the disk with a knife or pressed out with glass tubing of convenient size. When the plaster is dry the hoop can be removed and the disk placed in position in the jar, which is then filled with alcohol to within about 2 c. m. of the under side of the plaster. The dehydrating tubes should be about 12 c. m. long and can be made by cutting off the bottom of large test tubes. In one end is placed a diaphragm of chamois skin, which can be fastened in position by means of a spring made of steel wire or ribbon and forded with the chamois skin in the tube. A rubber band around the tubes prevents them from falling through the holes in the disk and enables them to be lowered to any desired depth in the alcohol. The tissue to be dehydrated is packed closely in the dehyrat- ing tube and just enough 50 per cent, alcohol added to cover it. This is then quickly lowered through the hole in the disk, until the two liquids are at a level. After from 12 to 24 hours, by osmosis, the two liquids will be of the same strength. The tissue can then be taken out and placed in the infiltrating bath at once. • This method of hardening has been tried on nearly all kinds of plant tissue, and in almost every case it was found to be successful. For the most del- icate tissues where slow hardening is desired, 5 per cent, alcohol can be placed in the dehydrating tube, and thick chamois skin used for a diaphragm, while for some of the more delicate algae it has been found advisable to use as low as 1 per cent, alcohol in the tube. The strength of the alcohol in the jar can be kept up by add- ing to it from time to time some calcic chloride, which will in no way injure the alcohol. The jar should be tall enough to allow the cover to be kept on while the tubes are in position and thus prevent evaporation. An apparatus of such a form with a dozen dehydrating tubes can be used for months without changing the alcohol. Other har- dening agents such as picric, chromic, acetic, or osmic acids can be 15 HABBENING AGENTS. used with equal success. Not more than 24 hours is necessary for dehydrating and hardening nearly all kinds of plant tissue. The apparatus does away with the transferring of the tissues from bottles containing alcohol of different strengths, and since no such sudden transition occurs, the tissue is less liable to shrink. Many different materials may be used for a diaphragm and almost any speed of dehydrating can be obtained. The apparatus can be made in any size to adapt it for private or general laboratory work. ' Picric Acid. This is to be recommended as a hardening agent. It acts rapidly and energetically and should be used first in dilute solu' tions. About one-fifth per cent, is the proper strength. Tissue hardened in picric acid can be kept in 75 per cent, alcohol until needed. If a little glycerin is added to the hardening agent, the tissue wiU be less brittle. Chromic Acid. This agent can be used for hardening and with it the tissue requires the same treatment as with picric acid. Osmic Acid. This acid is very useful as a fixing agent and is often used for hardening. The tissue must not be kept in it long, as it will become blackened and brittle. Hardening Fluid. See p. 17. Other hardening agents are strongly recommended, but enough has been said to enable one to properly prepare tissue for such studies as may follow in this work. CUTTING fIND MOUNTING TISSUES. The methods of cutting and mounting tissues are indeed numerous and only the more important ones will be revie;wed. Free-Hand Sectioning. Many things can be prepared for study in t!his way. The tis- sue must be firm in order that it may not be crushed under the knife and yet not be too hard or brittle to prevent its cutting readily. The object to be sectioned should, be held between the thumb and fore finger, while the razor should rest on the tip of the finger with the edge inward. Draw the knife, with the edge pressing against the tissue, across the finger keeping the thumb well below the line of cutting. Do not try to cut large sections but make them small and if need be wedge-shaped in order to get a piece thin enough for study. It is usually best to keep the tissue wet with alcohol or water to, make the sectioning easy. Transfer the sections from the razor to the slide with a camel's hair brush. If the object to be sectioned is quite small it can be placed between two pieces of elder pith or cork and sections made through these which shall include in them the sections of tissue. It is often very convenient to fasten the tissue with the cork, or if ^arge enough, directly in a microtome, and section with a microtome knife or razor, keeping the object and knife wet as before directed. This method is a very useful one for the transections of woody stems and firm tissues. These can frequently be softened without shrinking by leaving them for- some time in glycerin, and in the case of the firmest ones, boiling for five minutes or more. Many small objects can be held for sectioning by placing them at once in 17 ' CUTTING AND MOUNTING TI8SU:ES. melted hard paraffins and cooling it quickly by immersing in cold water. The paraffine block can then be placed in a microtome and the object sectioned. This method will apply only to those objects that will not be shriveled by the high temperature of the melted paraffine. For thorough, systematic study some method should be used that will enable sections to be made with accuracy, and if need be, in a series with a known definite thickness. The method should also provide something that can be infiltrated into the tissue and hardened to prevent crushing while being cut. The collodion and paraffine methods are the ones usually used for this purpose. Many modifications of both have been suggested but each method wUl be outlined to apply to the more general kinds of tissues. Experience will assist in modifying them for special cases. J ""'' • Paraffine riethod. Much discussion has arisen in regard to the relative merits of the different paraffine methods, but the general diEferences are more or less of an unimportant nature and beca.use of the dif- erent kind of tissues subjected to treatment. The. most im- portant methods are those of Mohl, (Bot. Gazette, Jan., 1888), and Schoenland (Bot. Gazette, July, 1887), whUe most of the others are modifications of these resulting from the experiments of different workers on the different classes of tissue. The tissue to be treated is first hardened in chromic or picric acid or mixtures of these with other agents. As was suggested by Mohl, the acids act on the tissues in some way and make them much more penetrable to the infiltrating mass. Paraffine does not easily penetrate tissue treate^d with alcohol, yet many recommend it as a good hardening agent even under these conditions. A good mixture for hardening is made from equal parts of 1 per cent, chro- mic ajid, osmic acid, 2 per cent., and acetic acid, 1 per cent. The osmic acid is very useful in many hardening agents for fixing the protoplasm, especially if it is desired to demonstrate karyokinesis, or cell division. Tissue should be kept in this mixture from 24 to 48 hours and then washed with running water 6 to 8 hours, after which it is placed for 12 hours successively in alcohol of 20 per cent., 40 per cent., 60 per cent., 80 per cent., and 95 per cent., 18 CUTTING AND MOUNTING TISSUES. strengths. A Schulze's apparatus is to be recommended for this operation. If alcohol has been used for hardening, the disagreeable process of washing can be omitted. The alcohol is now replaced by some solvent of paraffine, as chloroform, turpentine, clove or cedar oil, xylol, or benzol. Chloroform is readily miscible with paraffine, but is not very penetrating, and therefore requires a much longer time for clearing than some of the other solvents. Further it must be entirely driven off before sectioning, otherwise the paraffine will be too soft to support the tissue. Turpentine is recommended by Mohl, but this is often harmful to delicate struc- tures. Cedar oil is perhaps the best of any of the agents for pene- trating and clearing. Should turpentine be used, as Mohl recom- mends, the tissue is first placed for a few hours in a mixture of equal parts of alcohol and turpentine and an equal length of time in the pure agent, then after a few hours it should be removed, placed in a cold saturated solution of paraffine in turpentine, after which it is placed in a bath of equal parts of turpentine and para- ffine, kept at the temperature of 30° to 40°C. In a few hours the tissue is transferred to pure paraffine, with a melting point of 50°C., and in this it is allowed to remain until thoroughly satur- ated with the imbedding mass. With cedar oil the object is trans- ferred directly from the clearing agent to pure paraffine and left there until infiltrated. The time required for infiltrating any object depends upon the nature of the tissue. For some objects an hour is sufficient, while with others one or two days is required. After the object is infiltrated it is placed in a paper box and pure melted paraffine is poured over it. A convenient form of a paper box can be made as follows : Take a piece of stiff paper of the proportions shown in Fig. 2 and fold -inward along the lines A A and B B, one third of each side. Repeat the operation with the ends, folding them along the lines C C and D D. At each corner fold inward on a line bisecting the angle formed by the lines A A and B B, etc., allowing the crease to follow the lines E E, E'E', etc. Then turn up the sides and ends until they are at right angles with the bottom, and bring back around the ends the portions projecting at the corners. Fold outward the portion projecting above the sides of the box and press it firmly against the end thus holding all the parts in place. 19 CUTTING AND MOUNTING TISSUES. \ D r" -■ T~ S This box will be found most use- _. ful for various purposes in the labor- atory. The object in the box can be D arranged by the use of hot needles, while the paraffine is yet in a liquid state. As soon as a film is formed over the paraffine, it is plunged into cold water to harden quickly and thus prevent the mass from becom- ing filled with air spaces. After the C paraffine has become hard it can be readily removed from the box and r'" . • ^ fastened in a microtome for section- A B ii^g- Pis. 2. Diagram to show the lines of ' folding in making a paper boat. Several different forms of microtomes are highly recommended. Minot's is certainly one of the best. A very inexpensive well micro- tome will answer the purpose. In case the latter is used the block containing the object should be cut down until it will fit in the well where it is fastened by pouring over it pure melted paraffine. It is best that a microtome with movable jaws be used in order that the position of the object can be changed to vary the angle of section- ing. This is especially important in longisections of roobs or stems. If longisections of a root are being made or the whole of an object is to be studied, it is advisable that a series be made and only those need be mounted which contain the structure desired.' la order to stain and clear the sections it is necessary that they be in some way glued to the slide. This ca.a be accomplished in sev- eral ways. Those methods to be especially recommended are as follows: With a camel's hair brush spread over the slide a very thin layer of a mixture of equal parts of clove oil and 2 per cent. coUodion. Place the sections on the slide and press them gently against it with a brush or any soft object. The preparation is then put in an oven with the temperature at 50° C. for 20 minutes, or until the paraffine has melted and the clove oil evaporated. The sections will then have become fastened to the slide and can be stained and washed without danger of loosening. The same result can be more quickly obtained by heating the slide very cautiously 20 CUTTING AND MOUNTING TISSUES. over a flame until the odor of the clove oil has disappeared. Care must, however, be taken not to shrivel the tissue by overheating it. A better method of fastening the sections, is to albumenize the slide by soaking it a short time in a mixture of egg albumen 1 part, water 200 parts, and then allowing the slide to drain until dry. A number should be treated at one time and kept on hand until desired for use. With these slides, by warming the sections until the paraffine melts, the preparation will flatten out and adhere with great tenacity to the albumeniz.ed surface. The albumen will not be effected by staining. After the sections are fastened, the slide is placed in tur- pentine, or better, in xylol to dissolve away the paraffine. This will usually be accomplished in 15 to 20 minutes, when the object is washed with water and is ready for staining. The stain to be used and the method of applying it depends largely on the nature of the tissue and the part one desires to bring out. For general studies haematoxylin works well and with eosin makes a good double stain. If the haematoxylin is used, the slide need be left in it but a few minutes, when it is removed and washed thoroughly with water to take out the surplus stain, dehydrated with 95 per cent, alcohol, cleared for a few piinutes in clearing mixture, and mounted in balsam. If the tissue is to be stained in tqto, before imbedding, Mohl recommends that it be taken from the 60 per centj alcohol, while being hardened, and placed for 24 hours in a solution of alum carmine, after which the hardening can continue. With this treatment, when sectioned, it is only necessary that the paraffine be dissolved away, the tissue cleared with a clearing agent, and mounted in balsam. With haematoxylin or alum carmine the cell wall stains well and the nuclei show very clearly. In double stain- ing, if eosin is applied, the sections need be left in it but 20-30 seconds. If glycerin jelly is to be used as a mounting medium, the sections can be- mounted directly after washing off the surplus stain with water. During the sectioning it may happen that some of the parts of the section will loosen as they are being cut away. In this case they may be held together with collodion applied in a 1 per cent, solution with a camel's hair brush. This is painted over the tissue just before the section is cut, it dries quickly and hold all the parts 21 CUTTING AND MOUNTING TISSUES. in place, while it does not in any way interfere with the staining. It is advisable that all paraffine sections be mounted in balsam. The use of the parafSne method is recommended for the firmer tissues that cannot be held in place by the collodion. The objections to the paraffine method are that heat is em- ployed in infiltrating and this is injurious in some , degree to most tissues.. Further the length of time required to get the tissue, in condition to section is quite extended, and this is often an impor- tant consideration. The operations requiring paraffine are not as clean in the hands of most students as would be desired. The Collodion Method. This method is now coming into general use for nearly all kinds of plant tissue. For the use of collodion for infiltrating we are indebted to Duval, who first published his results in the Journ. de I'Anat., 1879, p. 185. A little later Merkel and Schiefferdecker suggested the use of celloidin, which is merely a patent collodion. Some discussion then arose regarding the rela- tive merits of each, but it rs generally agreed that one has little or' no advantage over the other. The method' as applied to plant tissues is a comparatively new one and many modifications of it are " at present recommended by various workers. The following directions are in the main taken from a report ■ of the author on the method, made before the Am. Soc. of Micro- scopists and published in their proceedings in 1890. The tissue to be treated is first dehydrated and hardened in alcohol. For this purpose a Schulze's apparatus is of the first im- portance, in fact it has been found that some tissue can be har- dened in no other way without shrinking. With its use, from 12-24 hours is sufficient for hardening and dehydrating any plant tissue. The material is taken from the dehydrating apparatus and placed in 95 per cent, alcohol for one hour, to insure complete dehydra- tion. There is then poured over it a 2 per cent, solution of col- lodion. In this it is allowed to remain from 12-24 hours, depend- ing on the nature of the tissue, 24 hours being enough for the very firmest. It is then transferred for the same length of time to a 5 per cent, solution; or the 2 per cent, solution may be allowed to 22 CUTTING AND MOUNTING TISSUES. evaporate until it is of the consistency of the former. After this, it is taken out and arranged in position on a cork or block of wood of convenient size to fit in the jaws of the microtome. By means of a camel's hair brush the material on the cork is covered with successive layers of collodion until it is quite enclosed in the mass. Allow each coat to dry slightly in the air before applying the next. After the tissue is covered it is placed in about 80 per cent, alcohol until hard enough to section. Much difference of opinion exists regarding the proper strength of alcohol to use for hardening the collodion, but 80 per cent, answers very well, and the tissue can be kept in it a long time without deteriorating. After a few hours the collodion will be firm enough for use. Sections of small or deli- cate objects can be cut by allowing them to harden in a block of collodion and then carefully clamping it directly in the jaws of the microtome. For sectioning, any sliding microtome will answer, but one especially adapted for the purpose will enable the object, which can be clearly seen through the collodion, to be inclined in any- desired position and sections taken in any plane. It is also neces- sary that the sections be removed with a long sweeping cut, since a direct cross-cut would tear them. The sections should be covered with alcohol while being removed and then floated from the knife to the slide. (See P. A. Pish^'s modification, Proceedings Am. Mic. Soc. Aug, 1893.) The slower the section is cut, the better it will usually be. Serial sections can be arranged in their proper place on the slide. For fixing to the slide, blow some dry ether vapor on the object (Fig. 3), or add a drop of ether to the side of the prepara- tion. The ether dissolves the collodion and fastens the sections in place. The preparation is then washed with water, stained, the surplus stain washed off with water, the sections dehydrated with 95 per cent, alcohol, cleared, and mounted in balsam. For clearing, the carbolic acid and turpentine mixture is to be recommended. It clears quickly and does not injure the most delicate tissues. For staining, one must use that which seems best adapted for their purpose, but for general study, haematoxylin seems especially adapted for collodion sections. Some difficulty m^y arise in cutting sections that have in them free parts. It sometimes happens that they become detached from the collodion and float away. In this case, the section can be 23 CUTTING ANB MOUNTING TISSUES. Fig. 3. Ether wash-bottle for blowing ether vapor upon collodion or celloidin sections to fasten them to slide. The tube of calcium chloride (CaCIj) is for dehydrating the ether vapor. collodionized as first suggested by Dr. Mark. This is done by coat- ing the tissue before each section is cut, with a thin coat of one per cent, collodion, using a camel's hair brush for thie purpose ; then draw the knife across the tissue very slowly, keeping alcohol dripping on it while the section is being cut. In this way beauti- ful sections can be obtained of material with loose parts, where all will retain their proper position. Care should be taken that none of the sections be cut before coUodionization, for although it nlay not always be necessary to keep the parts in place, yet it is a safe- guard against their displacement. The method given is found to work admirably on very deUcate meristematic tissue. No heat being required, the most dehcate of tissues will not shrink. The shortness of the method commends it for general use. Two dajfs, or even less, is sufficient to go through the whole operation of hardening, infiltrating, and sectioning, near- ly all kinds of plant tissues. The sections after being cut can be easily handled with a camel's hair brush without fear of breaking. i 24 CUTTING A ND MO UNTING TISS UE8. In the case of delicate tissues, like fern prothallia, or the apical cell of Nitella or Chara, some little variation is made from the regular method and a detailed description of the process may be of value. (The Microscope, Nov. 1893.) The material is first placed in 10 per cent, alcohol in a dehy- drating apparatus and allowed to remain for 24 hours, vphen it is taken out and placed for one hour in 95 per cent, alcohol to insure complete dehydration. After this the tissue is placed in a 1^ per cent, solution of collodion and allowed to remain in a tightly corked vial for 12 hours. The cork is then removed, and by slow evapora- tion of the ether and alcohol, the collodion will thicken. When it is of the consistency of ordinary glue, the preparation is poured out of the vial, with the collodion, into a paper boat, of the kind used in paraffine imbedding, or an ordinary watch glass will answer the purpose. The thick collodion is then poured over the tissue and allowed to harden in the air until a firm film- has formed over the surface. After this the mass is placed in a jar of 85 per cent, alcohol and allowed to remain from 5-6 hours until the collodion is quite tough. Then with a thin knife cut out a block of collodion containing the tissue inside. The block can be placed in any desired position on the end of a cork and held while thick collodion is poured over, until it is covered. Each coat as added should be allowed to slightly harden before applying the next, until the operation is completed. After the whole has become firm in the air, it is placed in a jar of 85 per cent, alcohol where it should remain 6 to 8 hours. The operation of sectioning and mounting is the same as outlined for the firmer tissues. In order that the sections may be all arranged the same side up, the block of collodion, which should always be trimmed at the top, can be cut with a notch near one corner, and the notches all arranged with the same relative position on the slide. It will be found that with this method perfect serial sections of any desired thickness can be obtained from objects which are not more than one layer of cells thick, and thus render the prepara- tion of delicate tissues but little more difiicult than that of the firmer kinds found in ordinary stems and roots. ' Many substances for infiltrating have from time to time been suggested, and have met with varying success. The more impor- 25 GUTTING AND MOUNTING TISSUES. tank ones tried are gelatin, gelatin soap, paper, wax, gum arable, and-paraffine mixtures, but it is not necessary to outline other njethods here as the few described in detail wUl enable the student to prepare material for any work suggested in this manual. STAINING AGENTS. Only a few of the more important staining agents will be men- tioned, and directions given for their general use. Ammonium Carmine. This stain is best prepared, as suggested by Hartig. Dissolve a little carmine in water until the mixture has the consistency of paste. Add to this, a little strong ammonia and evaporate the whole to dryness over a water bath. The resulting powder dissolved in water is used for staining. Alum Carmine. , Make a concentrated solution of alum and add to it enough powdered carmine to give it a deep color, (1 gram to 100 c.c. of alum solution). Boil for 10 minutes and, when cold, filter. This agent is much used and is often valuable as a selective st^in. It colors pure cellulose cell walls a bright red, but does not effect those that are liguified or subetized. Picro-Carmine (Gage). Twenty grams of picric acid are dissolved in 200 parts of water, and mixed with 5 grams of carmine in 250 c.c. of strong ammonia. Stir the whole thoroughly and evaporate to dryness. Dissolve the residue in 700 c.c. of water. All of the carmine stains are very useful, easUy handled, and quite selective. Picro-carmine turas protoplasm a yellowish red. 27 STAINING A9ENTS. Eosin. This is a valuable general stain, as it has a great coloring capacity. It stains nucleus and cell wall readily and is much used in double staining. It can be applied either in an aqueous or alco- holic solution ; 1 gram of eosin to 100 c.c. of water is a very con- veilient strength. Haematoxylin. This coloring agent can not be recommended too highly. It has a very wide application and gives uniformly good results. The stain' is made by adding a concentrated solution of haematoxylin crystals in alcohol, cautiously, to a 3 per cent, aqueous solution of alum, until a medium purple color is obtained. The solution becomes darker and better by standing a few days, but deteriorates after a time and will require filtering often. As suggested by Prof. Gage the addition of chloral hydrate and proper sterilizing of the constituents of the stain during its manufacture greatly increases its keeping power. (Proc. Am. Microscopical Society, Jan. 18931 The old stain, if kept in a cool place, is very valuable in stain- ing meristematic tissue. Haematoxylin stains the nucleus a deep blue or purple and is to be recommended for all general work. ' It is sometimes used in staining bacteria and with other stains in double staining. ANILINE COLORS. These colors have of late been very useful in furnishing stains for histological work. To them we owe much for the present valu- able and convenient methods of staining. Only a few of the more important agents can be included in this outline. Hethyl Violet. An aqueous solution of this is much used in staining bacteria. It is also valuable as a selective stain for many plant tissues. riethyl Green. An aqueous solution with 1 per cent, of acetic acid is used for staining the nucleus and chlorophyll grains. As a double stain it is often used on transections of stems in' connection with eosin. Iodine green is, however, preferable for double staining. 28 STAINING AGENTS. Aniline Blue. .This stain is much used in connection with the staining of bacillus tuberculosis, but is also very satisfactory' as a stain for sieve plates and sieve tissue. Cellulose takes a blue color while the sieve plates become azure. Sections treated with this color are liable to fade after a time. The stain is good in .double staining. riagenta. Magenta is used both as a general and selective stain. It should be applied' in an aqueous solution, containing a little acetic acid. The tissue will need to be left in the solution for some time, before the proper depth of staining is secured. ', ■/,■,, Picric Acid. This is a very convenient and useful ground or general stain and is usually applied in weak alcoholic solutions. It must be borne in mind, however, that picric acid will wash out to quite an extent many of the aniline colors, but in use with haematoxylin, borax, or alum carmine, it makes a most excellent general stain. In connec- tion with hydrochloric acid it will readily wash out the carmine colors. ^ •Silver Nitrate. In a dilute alkaline aqueous solution this is often used as a test for living protoplasm, since it colors it black, while dead protoplasm remains unchanged. The reaction is very delicate and positive. Tannic acid also colors less dilute alkaline solutions black, while cells' containing glucose are colored brown. As a general stain the action of silver nitrate is too uncertain for positive directions. CLEARING fJGENTS. The function of a clearing agent is to make the tissue trans- parent by penetrating into all parts of it. It must be a liquid of high refracti'^e index and miscible with balsam, as well as having the pow6r to drive out all of the alcohol. The agents employed are very numerous, but a successful one should replace the alcohol quickly and yet not destroy the tissue by shrinking. The chief clearing agents are the essential oils. Cedar Oil. This oil is a very good clearing agent but is quite slow in its action; however, it does not shrink the tissue nor fade aniline colors. Clove Oil. - This is one of the best clearing agents. The clove oil on the market is usually impure and not suitable for use. The pure oil can be obtained only from reliable dealers: As a dearer, it pene- trates tissue very readily and clears most thoroughly. It has a very high refrative index. Collodion is dissolved by this oil, and it is therefore not safe to use with collodion sections unless proper precautions are taken to prevent the displacement of the disconnected parts. Tissue that remains in clove oil any length of time is liable to become brittle, and this is sometimes very helpful in minute dis- sections. Aniline colors are often faded by the action of this clearer. 30 CLEABINQ AGENTS. Oil of Origanum and Oil of Sandal Wood. These are both to be recommended as clearing agrents, but for many tissues are not wholly satisfactory. Turpentine. Turpentine is much used for clearing paraffine sections, as it dissolves out the paraffin^ and clears the sections at the same time. It is very liable, however, to cause sections cut in alcohol or collodion to shrink. Carbolic Acid and Turpentine. This is to be recommended as the cheapest and at the same time the most satisfactory of all clearing agents. The mixture is made of 3 parts of turpentine and 2 of pure carbolic acid. It clears equally well paraffine, collodion, or alco- holic sections, and needs but ^ few minutes to thoroughly permeate the tissue. It is best to filter the mixture through cotton to remove all particles of dust. The carbolic acid gives the hands an unpleas- ant feeling and they should therefore be kept free from it. MOUNTING MEDIA. Aluminium Acetate. In a saturated aqueous solution this salt forms a good mount- ing medium for many delicate organisms. Most algae can be pre- served in this way without any deterioration, while such objects as the young prothallia of ferns can be kept without shrinking or losing much of the brightness of their chlorophyll or protein granules. Like all liquid mounting media it must be used in a cell, and the slide should lie for 24 hours before sealing. Balsam. Balsam forms a most excellent and substantial mounting medium. Sections mounted in it should be free from- water and this can be easily brought about by the use of alcohol. It is best to begin with alcohol of moderate strength (50 per cent.), and gradually increase it until the tissue is taken from that of 95 per cent, strength. Before mounting, the sections must be cleared of alcohol by the use of turpentine, chloroform, oil of cloves, or bet- ter, a mixture of 3 parts of turpentine and 2 of pure carbolic acid. Balsam is prepared by evaporating over a gentle heat common commercial Salsam of fir until the volatile oils have been driven off and the residue becomes brittle. The portion that remains is then dissolved in cedar oil, xylol, . or chloroform, and filtered through glass wool in a paper funnel.. The medium should, at the ordinary temperature, be of the consistency of a thick syrup. Care must be taken to keep air bubbles from balsam.- If, however, J they get under the cover they can be driven out if the slide be left in a warm place for a few hours. 32 MOUNTING MEDIA. Although not absolutely necessary to seal the cover glass of balsam mounts, it is, nevertheless, a good precaution, as the cover may crack away from the medium and the section be displaced. The balsam should alwayfe be kept in a glass-stoppered bottle, and applied to the slide from a pointed glass rod. Carbolic Acid. Carbolic acid in a 1 per cent, aqueous solution is used as a mounting medium, also carbolic acid crystals in glycerin, f5r some of the firmer tissues. These agents have a tendency to make the sections clear or faded. Calcic Chloride. About one part of this salt to two parts of water is a good mounting medium for many tissues. A little camphor should be added to the solution to preserve it. Qlycerin Jelly. This is extensively used as a mounting medium for small and delicate objects, which might be injured by clearing for balsam. Glycerin jelly mounts will, after a time, become transparent, unless they have previously been stained with' a permanent slain. Care should^ be tp,ken in mounting sections to pi-event air bubbles getting under the cover glass, since they do not disappear as in balsam mounts. Glycerin jelly mounts should be sealed as soon as cold. Many formulae for the , preparation of this mounting medium are in use, several of which seem to be equally good. Kaiser's jelly is easily made and keeps well. It is prepared by soaking one par c by weight of Ijest French gelatine in six parts of distilled water for two hours ; 7 parts of glycerin are then added, and 1 drop of con- centrated carbolic acid for every gram of the mixture. Warm 10-15 minutes, with constant stirring, until the mixture becomes clear, and then filter while warm though wet glass wool in a hot water filter. The jelly will require warming before use. The mixture keeps well for years and is a very convenient mounting medium. Qlycerin. Glycerin is often used as a permanent mounting medium and, with pVoper precautions, gives satisfactory results. To prevent 33 MOUNTING MEDIA. shrinking, sections to be mounted in glycerin should first be placed in a -mixture of equal parts of glycerin and water and allowed to remain a little time, when stronger glycerin is added at intervals, until the section is thoroughly permeated with the pure agent. As this is hygi-oscopic, the tnounts must be sealed at once as they will readUy absorb a quantity of water sufficient to dilute its strength appreciably. The cover can be sealed first with a httle glycerin, jelly, andj after this has hardened, with shellac or asphalt. Glycerin is a very good mlounting medium for studying fresh tissues, as it evaporates very slowly. Sections moui^ted in it can be kept for examination for some time. Glycerin, like glycerin jelly, has the property of making sections clear or transparent. They should therefore be treated with a permanent stain. Only the purest commercial glycerin should be used, and this must be k^pt in a tightly corked bottle, otherwise, it will absorb a sufficient quantity of water to render it almost useless as a mounting medium. Glycerin and Acetic Acid. A mixture of equal parts of glycerin and acetic acid is a very convenient mounting medium for many kinds of tissue. Especially is this true with some of the fungi. In the preparation of this mixture, pure glycerin and concentrated acetic acid should be used. King's riounting Medium. This is good for many fresh-water algae, and like all fluid mounts it must be used in a cell. It can be secured of dealers in microscopical supplies. Water. Water is not infrequently employed as a mounting fluid. To preserve the mounts from deterioration a little camphor Should be added. Water is often used as a medium for the studying of fresh tissue ; but it should be borne in mind, that it may change the nature of the tissue materially, owing to the osmosis between the cell contents and medium. This can be prevented by adding some substance to the water to make its density equal to that of the tis- sue, Or cell contents. Salt is sometimes used, but is by no means efficient. CEMENTS. The number of cements and varnishes is so numerous that one is at a loss to know just what is best to use, but the general char- acters of some of the more common ones wiU be brieflly noted. Gold Size. This cement can be secured of dealers in microscopical sup- plies and is certainly to be well recommended. If used for balsam mounts, it is best to first ring the cover glass with a coat of shellac. Shellac. Shellac is certainly very convenient and seems to be quite dur- able. It is prepared by dissolving solid shellac in alcohol' until the solution is of a medium oUy consistency. A little aniline dye can be added to color the mixture to one's fancy, also a few drops of oil should be used to prevent the cement from cracking. The mix- ture is applied, as are all sealing mixtures, with a small brush and, preferably, by the use of a turn-table. Ball Cement and Asphalt Varnish. These are among the very best of cements and can be obtained from the regular dealers in, supplies. White Zinc Cement. This cement is much used by microscopists for making cells. It is very hard and often shows a tendency to crack. Many very good cements are on sale by reliable dealers, and one has but to convince himself of the relative merits of a few of the more important ones, when he will be able to settle upon some special one well adapted to his purpose. SERIAL SECTIONING. It is often very desirable and indeed quite necessary that the whole of an organ be studied systematically, but to do this requires that the parts not only be placed in a condition to be observed, but also, that the arrangement of parts as sectioned be so^ system- atic as to show the relation of each section to the whole. This can be accomplished by making what are known as serial sections of the object. In this case it is necessary that the sectioning be done in a microtome in order to preserve a uniform thickness. The sections as cut, should be arranged on the slide close to each other and in the same order as they are removed from the object. They should also occupy the same position with reference to the relation of each section to the whole. With the parafSne method when "ribbon sections" are cut, it is only necessary to break off pieces of ribbon and lay them along- side of each other in the order in which they are removed. With the collodion method as the sections are cut, they should be taken up with a camel's hair brush and transferred to the slide. After being arranged in position, they should be sealed by blowing over them a little ether vapor. The slide should be kept constantly wet with alcohol to prevent the sections from drying or shriveling. The arrangement of sections on the slide should be uniform at all times. A very convenient order is to begin in the upper left hand corner and place the sections under each other in a row along the longest axis of the slide. After the row has reached , to within 2J c. m. of the opposite end, a new row is begun at the right of the old one, and at the top of the slide; 5x2^ c. m. cpver glasses will be required. 36 SERIAL SECTIONING. When the sections are to be designated by number,.they should be numbered in the order in which they are placed on the slide. Serial sectioning is very important in any thorough investigation, since it places before the observer all of the object to be studied, in condition for careful searching, enabling one to trace the course of any part in every direction through the object. Serial section- ing certainly saves much time and prevents many misconceptions that might otherwise arise from the examination of a single prepara- tion. DOUBLE STAINING. It is often desirable, in order to bring out more clearly, some part of a specimen, to stain different portions of it with separate colors. This is very easy to bring about if the proper stains are employed. For example, the xylem of a fibro-Tascular bundle can be stained one color and the phloem another. These r«5ults are very important in research as one is enabled to recognize in this way similar tissues in different parts of a specimen where other- wise they might be somewhat difficult to distinguish. Certain stains will color one part of a tissue or special part of a cell, and yet not effect in any way other portions. The combinations of stains that can be used for this purpose are very varied, and the results in the case of some quite uncertain to predict, consequently no general rule can be laid down in regard to the kind of tissue any stain will invariably act upon. With regard to the treatment, in general the selective stain should be applied first, and in dilute solutions. The sections should then be thoroughly washed and the general stain applied, the tissue washed again and mounted directly in glycerin jelly or dehydrated, cleared, and mounted in balsam. Glycerin jelly is per- haps a better miounting medium for double stained tissue, since the alcohol and clearer are often injurious in their bleaching effect. Some combinations of stains whose effects can be depended upon are haematoxylin and eosin or picro-carmine, iodine green and eosin, carmine or haematoxylin with picric acid, and methyl green and ' eosin.- It is often necessary to use a mordant to fix the stain, in which case a saturated solution of alum can be applied to advantage. A little experience with double staiaing will enable one to use it in a way that will be of great assistance to a better interpretation of the character of many tissues. FLUID MOUNTS. riounting in Cells, Etc. It is frequently desirable to mount small objects in such a way as to preserve them without crushing or mutilation. In this case the cover glass must be supported in order that it may not come in contact with the preparation. This is accomplished by mounting the object iii a cell, which can be made in various ways, depending on the nature of the object to be mounted. If the specimen is very small and is to be preserved in balsam or glycerin jelly, it may suf- fice to place around the object a few pieces of broken cover glass to keep the preparation from being crushed by the cover. Seal as in other mounts. If the object to be mounted is quite large, a deep cell must be made. The nature of the material of which the cell should be con- structed must depend on the character of the mounting agent, that is, the material of which the cell is made must not be of a sub- stance" in any way acted upon by the mounting fluid. For many mounting media, cells made of shellac are very convenient, and quite durable. To make the cells, place the slide on a turn table and when the table is revolving, touch the slide with a small brush dipped in shellac. A ring is the result. After the shellac has dried, another ring should be made on the top of the first. Allow this to dry and repeat the operation until the cell is of the desired depth. Several of these cells should be prepared and kept on hand until they may be needed. Before using, paint over the top a very light coat of thin shellac. The object to be mounted should be placed in the center of the cell, and the latter filled completely with the mounting 39 FLVID MOUNTS. medium. To place the cover glass in position, rest one side of it on the edge of the ring and lower it slowly over the object, allowing the surplus mounting fluid to be forced out on one side. Press the cover firmly in position. Before sealing fluid mounts, fasten the coVer in place by applying three or four small drops of sfealing mix- ture at the edge of the cover glass at different points. As soon as dry, these hold the cover in place and allow the mount to be per- manently sealed. Cells may be made of glass, zinc, bone, or cellu- loid rings which can be secured of any regular dealer in micro- scopical supplies ; or rings can be cut from sheet lead, tin, paper, or wax, and fastened in position with shellac or marine glue applied to the base of the cell and then held firm, by the ring, against the slide until dry. Seal around the outside and when ready for use, apply a light coat of sealing mixture to the top, to hold the cover- glass in place. "When the object is mounted, seal the whole prepa- ration as in the case of ordinary mounts. Objects can be easily mounted dry in cells, in which case they should be held in position by fastening them to the slide with a little glycerin jelly, collodion, or gelatine. Fluid mounts should be examined from time to time to repair any that may not have been perfectly constructed. EQUIPPING OF LflBOKflTOI^Y. In the equipping of a laboratory, one must necessarily be con- trolled largely by the material and funds at their disposal, but a few suggestions may serve to lighten somewhat the care of over- seeing so much manipulation. Each student should be provided with a case made by boring two rows of holes about 45 m. m. in diameter in a block of wood 30x14 c. m. and 45 m. m. thick. In this should be placed bottles containing the more general reagents and stains that wiU be needed frequently in the work. It is suggested that the set consist of Iodine, Acetic, Sulphuric, and Hydrochloric acids. Glycerin, Potassic Hydrate, Eosin, Haematoxylin, and Clearer. Another case contain- ing reagents and stains of a more special nature can be placed on a center table easily accessible to aU. The desks^ should all be equipped with wash bottles of alcohol and water, and a general supply of the same agents should be located in a convenient place in the laboratory. These arrangements wUl prevent the student from being compelled to walk about searching for some reagent or supply. Cases should be provided for containing the general store of chemicals and glassware. The books should also be in a con- venient place easily accessible to all. It is much more desirable that the microtomes, hardening and infiltrating apparatus be on a special table which should also be supplied with all the stains and reagents necessary for mounting the sections. A convenient form of a waste vessel over which the sections can be treated is made by fastening, with sealing wax, to a tray or dinner platter, glass rods, parallel and about 4 c. m. apart. The slides with the sections can be laid on these rods and treated 41 EQUIPPING OF LABOBATOBY. with the various reagents, which can be washed off directly into the tray beneath and thus prevent table or hands being soiled by the stains or clearer. It is very convenient to have a small supply of alcohol and water in bottles elevated at a little distance on a shelf, and provided with a siphon having rubber connections below to enable it to be used in various parts of the table. The orifice of the glass tip should be small and the rubber tube provided with a pinch cock to regulate the supply at pleasure. This arrangement will be found very convenient to keep knife and tissue wet while sectioning tissue imbedded in collodion, and also for dehydrating and washing the sections on the slide. For the clearer, a wash bottle should be provided with an enlargement at the inner end of the exit tube filled with cotton, thus preventing any particles of dirt from getting into the clearer used on the shde. The enlargement can be made by cutting off from the end of a glass tube, just small enough to enter the mouth of the flask, a piece about 5 c. m. long. Close one end with a cork and pass through a hole in this exit tube. Then fill the piece of tubing loosely with cotton. A similar bottle will be found very useful for the haematoxylin. (Pig.S.) Many devices will be suggested in the laboratory from time tp time and materially lighten the burdens incident to having the charge of so much laboratory instruction. COLLECTION AND PRESEF^V^TION Of MATERIAL. Much of the material for work in histology must be collected during the summer, or at times when it cannot be used at once, and is therefore to be kept stored away* until needed for study. Some difficulty must necessarily arise in the proper preserva- tion of such material, but if special precautions are taken it can easily be kept in good condition for histological purposes. The methods of treatment vary with the nature of the material. All soft tissue collected, as, for example, leaves, herbaceous stems, etc., should be placed at once in 40-50 per cent, alcohol and hardened in the usual way by the use of a Schulze's apparatus, or by transferring successively, for 24 hours in each, to 50 per cent., 67 per cent., and 75 per cent, alcohol, in which it can be kept a long time without deterioration. The softest tissues such as are found in algae, etc., should be placed first in about 10 per cent, alcohol and hardened by the use of /Schulze's apparatus, or by transferring, successively, for 24 hours in each, to 20 per cent., 30 per cent., 40 per cent., 50 per cent., etc., to 75 per cent, alcohol, where they may be kept as in the case of the firmer tissues. The material thus hardened when ready for use can be dehydrated with 95 per cent, alcohol, infiltrated with 2 per cent, and then 5 per cent, collodion. In the latter solution it may remain indefinitely without shrinking. If the tissue to be sectioned is kept on the cork in alcohol, it will in time become~discolored by the tannin of the cork. The tissue of each sort should be trimmed until only such parts remain as are needed for sectioning. These should be care- 43 COLLECTION AND PBESERVATION OF MATERIAL. fully tied up in pieces of bibulous paper, with the labels written in India ink or pencil, inside. In this way, many different things can be kept in the same jar and thus economize room and solutions. If the material is to be preserved in thick collodion, the stopper of the vessel in which it is placed should make the bottle air tight and should also be held in place in some way, otherwise the evapora- tion of the ether mtly force it out and ruin the ' material by the hardening of the collodion. If it is desired that the tissue be kept on blocks in alcohol any great length of time, in order that it may be ready ~for use at once, hard rubber rods sawed into convenient lengths of about 2 cm. may be substituted for the cork. -As the alcohol does not effect the rods, tissue can be kept in this way any length of time without deterioration. The rubber rods can be obtained of the Educational Supply Co., Boston. Blocks of hard wood have also been suggested for the same purpose. I In collecting material to preserve for class-room work, much ' care should be taken to prevent confusion of labels, etc., and all important data should be included with the notes on each study. THE MICROSCOPE. Fig. 4. Microscope, with the parts lettered to correspond with the description. 45 THE MICBOSCOPE. A. Base of the instrument and part that forms its entire sup- port. This may be in the form of a horse-shoe or a tripod, support- ing at three points. This condition is desirable, as greater solidity is thereby given to the instrument. B. Pillar This is the upright support from the base and usually has a joint at the top, by means of which the instrument may be inclined. C. Arm — This carries all of the remaining parts of the instrument. D. Body.— This is the tube holding the optical parts of the instrument that are above the stage. The raising and lowering of these is controlled either by a gearing or by friction of the outer stationary part with the inner movable one. E. Nose-Piece — A small revolving part fastened to, the lower end of the body and forming an attachment for the objectives. F. Objective — This is the lower of the lens combinations above the stage and is usually screwed into the nose-piece. Most microscope makers use a uniform size of thread and this is known as the society screw. The function of the objective is to form an image of the object. G. Ocular. — The part holding the upper combination of lenses, and fitting into the upper end of the body. The function of the ocular is to magnify the image produced by the objective. H. Draw-Tube — This forms the inner part of the body and moves in an outer sheath. The length of the body may be varied by the adjustment of the tube in its collar. I. Collar — A ring fastened to the upper end of the body and forming a sleeve for the draw-tube. J. Course Adjustment — This is used for raising and lowering the body. It is provided with two large milled heads (K), which revolve a pinion that acts upon a rack, and controls the working of the adjustment. L. Fine Adjustment — Used to raise or lower the body slowly through short distances, in this way obtaining the exact focus. It consists of a milled head with a screw that acts upon the body of the instrument. M. Stage — This is firmly attached to the pillar and is for the support of the object during * examination. Sometimes a 46 THE MICEOSCOFE. movable slide carrier is attached to the stage. . The more expensive instruments are often provided with a mechanical movement that enables the object to be carried in any desired direction by simply, turning two screws loca^ted above or at the side of the stage. F. Clips. — These are to hold the glass slide, on which the object is mounted, firmly against the stage. O. Mirror — This is one of the optical parts below the stage and is for the purpose of illuminating the object, either by throw- ing light through it, or on it from above. One side of the mirror is usually plane and the other concave. P. Mirror Bar — A bar attached to the arm and carrying the mirror. It can usually be swung from side to side to vary the angle of illumination. Q. Sub-Stage Ring — Used to support either the diaphragm or some of the optical parts that are used below the stage. It is often attached to the latter but in the more perfec:^ instruments is borne on a separate bar. S. Diaphragm — This is a disk provided with numerous apertures, of various sizes, and can^ be turned to regulate the amount of light coming from the mirror to the object. Field of the nicroscope — This is the clear area seen by look ing into the microscope, and should be perfectly circular, when the instrument is properly hghted. METHODS OF STUDY. It is very important that the student follow from the begin- ning a system of work that will enable him to utilize the experi- ence of those who may have had years of training and have learned the roads to uniformly good results. A few suggestions may not be out of place. >, Care in Observation. Avoid making hasty conclusions even though the appearances seem to warrant them. Do not consider as proven a condition that can be seen but once and then under difficulties. In important cases, always verify results by trying the study again with other material so that there can be no doubt as to the exact state of things. Never substitute an opinion or inclination for a fact even though it often involves a disappointment, better that than error. ^ Selecting Material. Many of the troubles and difficulties in the way of a proper study can be overcome by taking due precaution in the selection and preparation of material. Too often a section is carelessly made with the hopes that it may show gomething desired, but it is better always to select the material carefully, then section and mount with all the proper precautions'. Directions for Drawing. It is necessary that the student study and thoroughly under- stand the tissue under examination before any attempt is made to reproduce it, otherwise, after the drawing is finished errors in it 48 METHODS OF STUDY. will probably be found, and it will not represent the true relations of parts; Do not draw everything that can be seen, but place on paper enough to represent accurately the general outline and' minute structure of the object that is being studied. Let the relations of the parts drawn be/so clearly indicated that a correct picture of the object can be perfectly formed in the mind from the drawings. The first drawing should usually be an outline sketch of the whole object that is being studied. Then should follow a more detailed drawing of each particular part as examined, and lastly, the minute structure of any special tissue or cell. It is true that such detail will require much time but practice will soon reduce this and make the work seem very easy. It is not best to shade any of the drawings since it may obscure the parts that should be kept prominent. Often in studying the minute structure of some organ, a single section will not give a complete outline of the whole part that is to be represented, in which case, it should be made up from' the several sections that will best show tlje parts desired. Be certain, however, that the relations of parts are thoroughly understood and correctly represented. One is often inclined to make the drawings too small but this should be guarded against, and the sketches made of sufficient size to enable the parts to be clearly seen without close scrutiny. The dr&wings can^ be made free-hand or by the use of a camera lucida. The latter method is necessarily the more accurate but lacks much of the ele- ment of good training given by the former. If the drawings are made with a camera lucida, much of the detail will need to be fiUed in free hand, but the general outline can be made with great ease. The best camera lucidas are of the Abbe pattern which can be used with- out tilting the tube of the microscope.. With it, some difficulty may be experienced in seeing the object and pencil point at the same time, but/this can be overcome by having the paper equally illuminated with the object under the microscope. Other camera lucidas may be used but it is not necessary to outline the working of each in ihese directions. With the Abbe camera lucida, be cer- tain that the drawing paper is at right angles to the axial line as reflected from the mirror, otherwise, the drawing will be distorted. The variation a from perpendicular with the table can be, deter- mined by the use of a semi-circular protractor, which will give the 49 METHODS OF STUDY. angle to which the mirror is tilted. The drawing paper should be raised at an angle with the table twice as great as the mir- ror is depressed below 45°. The drawing board should be made with a hinged part that will support the paper and yet allow it to be raised to ^.ny desired angle. The magnification of the drawing should be determined and indicated underneath it ; (x300) indicates that the drawing is magnified three hundred diameters. Free-hand drawings should be made with special reference to exact proportion, in order that every part may have the same magnification. Pencil drawings should be made on good drawing paper and with sharp-pointed drawing pencils, which had better be of two degrees of hardness, one being quite hard and the other medium soft. The former can be used for detail work and the latter for general outline. The objection to pencil drawings is that they blur by rubbing and are not as clear as those made with ink. It is better to outline ink drawings at first with a pencil and then retrace and fill in the detail with the pen. The different parts of the drawing should be named at the side and the name connected to them with a dotted line. With a little care, this can be done without trouble, and the drawing book will look nfeat and be easily interpreted. Where any part of an object is to be repeated several times in the same drawing, it is only necessary to outline the parts and indicate the repetition. Also where there is to be shown a large mass of cells of uniform nature, the whole should be outlined and a few cells drawn to indicate their general character. Next in importance to the drawing is the description, and this must not be neglected. It should always include a careful outline of the nature and intent of the study, together with a full explanation of every part of the drawing with the relations of each part to the whole object. It is always best to adopt some uniform way of making drawings and keeping notes. The system outlined below will be found very useful and convenient for examination. 50 METHODS OF STUDY. Study of the Transection of the Anther of Begonia. Anther cavity containing pollen mother cells. Mother cells .with young pollen grains. Empty anther cavity. Parenchyma cells of the interior of the anther. Fibro-Tascular bundle extending along the center of the anther. Epidermis of the anther. Xjco. Fig. 5. Sample page of a drawing book. The notes and explanations which accompany the drawings may be kept on separate sheets or pages in the drawing bqok. Preservation of Slides. To the person who makes a collection of microscopic slides, it , becomes a matter of no little importance'us to how they should be arranged in order that they may be best preserved and at the same time available for instant reference. Many kinds of cabinets may be purchased of dealers, but the large ones are quite expensive and beyond the means of many. A very inexpensive way to preserve the preparations is in mailing boxes, which hold 25 slides. These can be filed away but should be kept on end to prevent displace- ment of the mount. After a time, however, the number of boxes accumulates so as to become burdensome ; and furthermore, it is better for slides to be supported from below on each side of the 51 METHODS OF STUDT. cover glass, rather than at the ends. A servicable and convenient cabinet can be made by removing the drawers of an empty spool- case and making a door for the front. The case can then be filled with drawers suitable for holding the slides. ■ To make these cut a board, one-half inch thick and as wide as the case is deep, into lengths one inch shorter than the width of the case. With the aid of a buzz-saw and chisel, the boards can be made so that a section of it will appear as the figure : $■ d-' J^ B ^-i^ — V jr ID y^^ \§/^ ^^§^ - Fig. 6. Diagram showing the construction of a drawer for a slide case. The grooves are to be cut across the grain of the wood. Cut thin strips and glue them in position in the grooves 1 and 2. The distance between these points should be 3J in. The slides rest on the surfaces 3 and 4, and are separated by little partitions, about 1|^ in. apart, glued into a groove made across the board with the saw before the partitions 5, 5 ' were placed in position. The little partitions must necessarily be short, aboon a small bubble. If the rays of light from the mirror pass perpendicularly through the slide, the bubble will have a light center and a uniform dark border. If the mirror is not central, the light spot will be at one side of the center. After taking off the sub- stage attachments, move the mirror bar until it is at an angle with the stage, and observe, when the light passes through the . bubble, the direction from the center which the light spot has taken. It will be away from the side to which the mirror bar was moved. Now beat some cedar oil or oU of cloves, and repeat the experi- ment. Observe the direction taken by the light spot in the oil globule. It will be contrary to that taken by the one in the air bubble, or to the side on which the mirror bar was swung. It is interesting to niix the mucilage with the oil and find an oil gloljule and air bubble side by side. Then study the eifect of oblique light to identify the bubble or globule. It is advisable that the student familiarize himself with the more common objects that he will meet, perhaps, as foreign bodies in his studies. Mounts should therefore be made on a slide dry or in a, drop of water of such objects as spores, dust, cot- ton and- woolen' fibers^ hair, cork, etc., and studies made of them with the high and low powers. MflGNipiCATION. Any student working with a microscope should be able to determine the magnification of his instrument together with the ocular micrometer ratio. Determination of Magnification by the Use of Wollaston's Camera Lucida. Arrange the stage micrometer in position and focus on it until the lines are clearly visible. Place the camera lucida on the ocular and tilt the body of the microscope until the tube becomes horizon- tal. Then raise the base of the instrument upon blocks,, until the distance from the ocular to the table is 25 c. m. Change the mirror until the light is reflected through the tube of the microscope, after which modify the quantity of light falling on the white paper, which should be placed on the table beneath the ocular, until, by looking through the camera lucida toward the table, the lines of the stage micrometer will be seen on the paper below. Mark off with a pencil the distance between the lines of the stage micrometer, as reflected on the paper, and measure with a rule. To determine the magnification, divide the size of the image bj? the size of the object magnified, and the quotient will be the magnification of the instrument, under the one condition. If the distance between the lines on the micrometer is 1-10 m. m., and on the image 50 m. m., then the magnification of the instrument in that condition is 50 H- 1-1 0=500. Determine the magnification with the several combinations of oculars and objectives. The magnification can also be determined by the eye-piece and stage micrometers. 64 MAGNIFICATION. In the measurement of most objects the former micrometer is used. This is a scale ruled on glass and placed in a slit in the ocular, or inside, by unscrewing the upper lens of the combination. To Determine the Ocular flicrometer Ratio. Place the ocular micrometer in position in the slit in the eye- piece, and move the eye lens up or down, until the lines on " the glass are distinct. Now place ito. position- the stage micrometer, and the lines on it will appear below under those of the ^eye-piece micrometer. Move the two scales until the lines of each are parall- el with the other. Measure with the scale of the eye-piece micro- meter, the distance between the lines of the stage micrometer. The ocular micrometer ratio is obtained by. dividing the number of spaces on the eye-piece micrometer, required to cover a space on the stage micrometer, by the value of the divisions of the latter. For example, suppose the markings of the stage micrometer were 1-100 of a m.m., and the numbet of spaces of the eye-piece micro- meter required to cover one space in the former was 5, then, the ocular micrometer ratio would be 5 -=-1 100=500, i. e., the ratio is 500. The value of each division of the ocular micrometer is 1-500 with the above conditions in the microscope. The ratio with the several objectives should be ascertained. The magnification of an object can be determined, by dividing the size of the image, as measured by the eye-piece micrometer, by the ocular micrometer ratio. For example, suppose the dimension of the image of a cell to be 5 divisions of the eye-piece micrometer, and the ocular micrometer ratio is 300, then the size of the cell is 5-r-300=.0166-(-m.m. The size of an object can likewise be deter- mined b^ measuring the size of the image, under the conditions described for determining the magnification of the instrument with the camera lucida, and then dividing the size of the image by the magnification of the instrument. Suppose the size of the image is 5 m.m. on the paper, and the magnification^ of the instrument is 300 diameters, then the real size of the object is 5-;-300 = .0166-(-in.m. The Unit of micrometry as universally used is the 1-1000 of a m.m. This was suggested by Harting in -1859 and called, in 1869, by Listing, the micron. It is designated by the Greek letter fi. The magnification of an object is then always to be given in nji- 65 MAGNIFICATION. crons, and the reduction is simply made by multiplying the actual size of the object by 1-1000. In all measurements with the microscope, the draw tube should be pulled out, until the whole tube of the microscope , is of a certain length. This distance is known as the ''tube length" and varies in microscopes of different makers, as do also the points between which the measurement is made. See Prof. S. H. Gage's Microscopical Methods, p. 10. In order that the student may become familiar with the work- ing of the microscope he should carefully go through the following exercises: 1. Putting the ocular and objective in position, p. 57. 2. Lighting the field of the microscope, with direct and oblique light, p. 62. I 3. Determine the relative position of optical sections, and the manipulation of the fine and course adjustments, p. 61. 4. Study of air bubbles and oil globules with reference to the identifying of each by their appearance, under direct and oblique illumination, p. 62. 5. Study of currents in liquids and their direction upon incli- nation of the stage of the microscope. These currents can be produced by grinding upon a slide, with a knife, a little carmine in water, and covering with a cover glass. 6. Determine the magnification of the instrument with the various combination of lenses, p. 63-64. 7. Dirt or cloudiness on the lenses, p. 60. Smear the objec- tive with' glycerin knd study the appearance. Eemove the glycerin with water. 8. M^ount various objects (hair, cotton and woolen fabrics, bits of. wood, paper, thread, etc.), under a cover-glass in water, and study with the high and low power. In this way one will become familiar with the, adjustments of the instrunient and. the appear- ance of the more common "foreign bodies'' that might be found in ordinary preparations. lExt Saontr. LABORATORY DIRECTIONS. DIVISIONS OF THE SUBJECT: A. Living Cells, (with Protoplasm and Chlorophyll.) B. Contents of Cells, (the secondary products.) C. Elementary Tissues. D. The Primary Meristem. E. The Systems of Tissues. P. The Thickening of Stems, etc., (secondary growth. A. THE STUDY OF LIVING CELLS. For cojivenience of study, cells may be classified as to manner of association as fpUows: 1. Those which live separate from one another. Examples, Unicellular Fungi and Algae, Pollen-grains, Spores, etc. II. Those living in Colonies, i. e., joined temporarily, but which are able to perform all their normal functions if isolated; example, Spirogyra, (known as "Frog-Spawn," "Water-Carpet," "Pond-Scum," etc.) ■ III. Those Y/hich Ivve perrnanently joined t^ o\:hev- ce\\s and which cannot ordinarily perform their normal functions if isolated; (a) they may not form Tisstte; example, JVitella; (b) they may iorm, Tissue/ example, Hoots, Stems, Leaves of JB^lowering Plants. CASE I. Isolated Cells Containing Protoplasm. Illustration: Protococous viridis, Ag. (green slime.) The plant can be found in damp places, growing on the bark of trees, or in the corner of buildings on the brick, or stones of the foundation. In fact so general is the plant distributed that nc) one need have any difficulty in getting material in good condition for study; Pbepaeation First : With a knife remove some of the material from the .substratum and naount in water. Place under the high power of the microscope and observe: — 1. The unicellular plants, often associated in groups'. 2. Their size, shape, and general appearance. 68 TSE STUDY OF LIVING CELLS. 3. The thiti colorless cell wall surrounding a central granular mass of protoplasm. 4. ^ Ghl6rophyll granules distributed through the protoplasm. These are the centers of the vital processes in the cell, and in sun- light by the decomposition of plant food form starch, protoplasm, etc. 5. The nucleus. Stain the preparation with iodine and observe the effect on the wall, and protbplasmic contents. (See p. 7.) Prepare another slide and stain with a fresh solutioni of chlor-iodide of zinc, to observe the effect oi the' thin cellulose cell wall. By pressing on the cover glass, the cell contents can be forced out and the cell wall be made more visible. (P. 11.) Examine several specimens to observe the various stages in the division of the plants into groups of individuals. Many of the small plants, will be seen moving about through the water. This is due to the presence of cilia which, by their rapid movements, propel the individual. Parker's Biology, p. 23 ; Strasburger, p. 214 ; Vines' Text Book, p. 236 ; Campbell's Struc- tural Botany, p. 22 ; Bibliography of the Literature on the Plant Cell, by Dr. A. Zimmermann f Botanisches Centralblatt, Beihefte, 1894. Further Illustration: Pollen-grains and their mother-cells, from Begonia sp . Preparation First : For the mother-cells of pollen. Lay out a perfectly clean glass slide and cover-glass, placing a few drops of distilled water on the former. Select a young staminate flower bud (less than half grown). Moisten the razor edge and make thin sec- tions across the upper part pf the flower buds, and the tips of the contained anthers. From these sections select the thinnegt, espe- cially those of the anthers and remove by means of a camel's hair brush to the slide. Examine these sections with a tripod lens or a dissecting microscope, removing the thicker anther sections and fragments of the perianth. Cover the sections with the cover-glass. The latter should be taken up with the forceps, one edge placed in the water, and the glass lowered, not too suddenly, but so as to allow the water to run along its lower surface without enclosing any air bubbles. Examine first with the low power objective (f in.), 69 THE STUDY OF LIVING CELLS. then with the higher (1-6 in.), using the C eye-piece and draw-tube when necessary. Obseeve: 1. — The outhne of the sections of the anthers/ oblong or quadrate, usually with rounded angles. 2. The cavities near the angles ; — later, these coalesce in pairs, thus forming the two "anther-cells," or pollen-cavities^ 3. The epidermal layer of cells, the layer just beneath, and the irregularly placed cells of the interior of the anther. 4. Isolated rounded cells in the cavities or floating free in the water. These are the mother-cells of the pollens. (a) — The very thin cell wall, its smooth surface, etc., has it perceptible thickness ? (■b).^The protoplasm, of the interior, its characteristics. (c) — Ihe nucleus, its appearance. (d) — The nucleolus. Further Preparation : If (c) and (d) are not clearly demon- straied apply a drop of iodine solution to one side of the cover- glass, and place a piece of filter paper on the opposite side. This absorbs the iodine and water, while the former acts on tne proto- plasra as a staining agent. (The iodine may be removed t)y using water in the same way). Observe the relative amount of color given to the cell wall and the cell contents; especially the effect on the fiucleus after the mothet-cells have remained some time in the stain. Also observe the increased distinctness of parts. From this study determine the constant effects "of iodine on protoplasm and refer to page 7 for the use of this reagent in plant histology. Measure the diameter of the iDother-cells by the use of the camera lucida, or ocular micrometer, selecting those of extrerne and also of average breadth. If practical retain this slide in a moist condition until the next preparation has been examined. It is useless to keep slides of soft tissue containing protoplasm more than a few hours when mounted in water.' A temporary moist chamber can be made by placing a small plate partly fiUed with water on a level surface and covering it with a bell jar. The mount may be laid across a small watch glass inside. Sketch transection of anther from -J objective and a mother-cell from 1-6 objective. (Fig. ^). 10 THE STUDY OF LIVING CELLS. Pbeeabation Second: For young Pollen-geains. (Fig. 9 .) Take a staminate flower somewhat older than the first. (The exact stage will have to be obtained possibly after trying several buds.) Section and prepare as before. Stain with iodine, letting it run under the cover one-third of its diameter. Focus on that region bordering on the stained and unstained portions. Observe: 1. The mother-cells (of pollen grains), floating free in the water, and the exceeding thinness of the walls. 2. The contents of the mother-cells — -four small "pollen grains]'/ or if only three appear focus carefully to ascertain the presence of a possible fourth. ^ 3. Form, of pollens; their nucleus and protoplasmic contents. 4. The readiness with which the protoplasm of the pollen becomes stained, even when the water medium appears scarcely tinged. ' 5. Mother cell wall, almost colorless, although the iodine must have passed through it. Why? 6. Is the place between the mother-cell wall and the contained pollens more tinged than the water medium? Figures of developing pollen grains, Goebel, p. 362; Stras- burger, p. 313 ; Sachs' Physiology, pp. 99, 100. Draw one or two mother-cells with contained pollens. Pkepaeation Third. For mature pollen grains. From the mature anther of an open flower jar out the pollen, letting it fall on a,dry slide. Examine carefully with a microscope, and'then apply water to the side of the cover-glass. Observe: 1. Appearance of the pollen grains when on the drt/ slide. 2. Change of form when the water is applied; the cause? 3. The wall, its relative thickness as compared with that of the mother cells. Measure a large and small grain, the longer and shorter axis. Draw a grain in the dry condition, and one in the moist. Sachs' Botany, p. 15; Bessey, p. 23; Gray's Structural Bot., pp. 256-257; Bot. Centralbl., Beih., 1893, pp. 206-17, 321-54 401-36. Illustration Second: Pollen-Grains from the Order Mal- vaceae, (either Hibiscus or Abutilon.) 71 THE STUDY OF LIVING CELLS. Pbepakation First. This should be similar to that of pre- paration third under Begonia. Observe: I. The short processes, sometimes lobed, covering the face of the grain. II. If the water causes the grain to swell. III. The size, — measuring with the micrometer. Preparation Second: Culture of pollen grain and the study of germinating pollen tubes, Tradescantia or Begonia. SterUize a slide, cover-glass, and ring, for making a cell. This can be done by heating them for some time in an oven at about 128° C, or quicker, by passing them slowly through the flame of an alcohol lamp. Make a 10 per cent, sugar solution and boil it ten •minutes. Then with a platinum wire, cooled after passing through an alcohol flame, place! a small drop of sugar solution in the center of the sterilized cover-glass. Sprinkle on this a very few pollen grains and invert over the ring, which is placed in the center of the slide and held in position by a drop of water at its lower edge. The culture will then have the arrangement shown in Pig. 8. Ee- mote the slide to a moist chamber and allow it to remain in a warm place for a few hours. MiH!}:iiii,nm\\nimiiim},)Vimi;iiiiiiiiah'm!mn,j,uu)})),i::u\i)i,i).im E^g. 8. Showing the method of cultivating pollen grains In a hanging drop. A. Glass slide. B. Glass ring. C. Hang:int; drop containing pollen grains. D. Cover-glass. Examine the pollen grains after a few hours with a 1-6 objec- tive taking care not to break the cover-glass by forcing the lens against it. Observe: 1. The outer and inner coat of the grain; the for- mer is called the extine, the latter the iivtine. 2. Pollen tubes of varying lengths projecting from the several grains, usually but one tube from each grain (there may, however, be several). 3. The tfiin wall of the pollen tube continuous with the intine, and the extine ruptured by the germination. 4. The granular contents of the pollen tube consisting of protoplasm and starch,^ together with the nucleus of the grain, on 72 THE STUDY OF LIVING CELLS. its passage to the end of the tube, from which it goes to perform the ofi&ce oi fertilization when in contact with the oosphere of the embryo sac. If the pollen grains of Malvaceae are used, observe the relation of the pollen tubes to the processes ov protuberences on the grains. The culture slide can be kept, for several days, and the devel- opment of the pollen tubes observed at intervals. For figures of developing pollen tubes see: Bot. Gazette, 1886; Goebel, p. 365; Strasburger, pp. 304, 320; Goodale, p. 429. CASE II. Cells in Colonies, Joined TemporarMy. Showing CELLS in Colonies, also Protoplasm, Chloeophyll in SPIRAL BANDS, and Progressive cell division. Illustration : Spirogyra. This genus of filamentous un- branched aquatic plants belong to the Conjugatae, a- group of Thallophytes (See Sachs' Text Book of Botany, p. 25; Bessey's Botany, p. 232.) Spirogyras, when in the vegetative state, are bright green, and have a silky luster when taken from the water. They vary much in diameter of the filament in difierent species, but are seldom over .15 m. m. They frequently occur in fresh pool's or slow flowing streams, and may be found in winter in pools that do not freeze. Most species pass into a reproductive stage in early summer. But few species are knc(,wn to conjugate in the winter. Preparation First: Place a few of the filaments on a slide in water. Observe : 1. Cells placed end to end. 2. The septa, or transverse partitions, plain in some fila- ments ; possibly in others a box-like area will be observed at the septum. 3. The spiral band or bands, of bright green color, part chlorophyll ;gigment.and part protoplasm. Ascertain if possible the number of bands. To do this, count the number that cross a thread between the two points where it touches the opposite sides of the cell, and this number plus one, will be the number of bands in the cell. 73 THE STUBY OF LIVING CELLS. 4. The irregular clearly cut margin of the hands, and the protoplasmic ridge traversing the roiddle of each. If these are not well defined the cells are not in a healthy condition. 5. Globose masses or corpuscles, in tTie band at intervals. These corpuscles are apparently centers of protoplasm, where starch grains are produced, under proper conditions. The cor- puscles and ' the connecting protoplasmic ridge are regarded by Schmitz as one continuous "chloro-plastid." 6. The transparent roundish area at the middle of each cor- puscle, — the pyrenoid of Schmitz. 7. The cell nucleus if present; its form. It may be more clearly brought out by the application of a little dilute iodine. 8. If protoplasm can be detected in the cell, except in the chlorophyll bands. Peepabation Second: — Stain a preparation with iodine, by placing a drop at the edge of the cover glass. Observe: 1. The effect on the corpuscles, nucleus and pro- toplasm in various parts of the cell. 2. That the cell wall remains unstained or slightly colored although the iodine must have passed thrOughit. 3. The slow action of the iodine on the chlorophyll bands. The cause of this slowness. Peeparation Third: — Stain as before, using 'strong iodine in- stead of the dilute solution. Oeseeve: 1. Contraction of the whole cell contents in some cases. It is a coagulation of the protoplasm, the water being ex- pelled by the iodine. 2. The color given to the different parts. For the study of cell division, keep the plants in a very cool place over night and bring in to the warm laboratory in the morn- ing. The division will begin in a short time. If the plants are kept constantly in a warm place, the division will usually take place at night. In specimens treated as above directed. Observe: 1. The ridge of cellulose being pushed inward from the side walls in some cells. This is a septum in active formation, (by progressive cell division). 2. The chlorophyll, etc., continuing through the aperture of the partly formed septum. Sketch the above appearance. 74 THE STUDY OF LIVING CELLS. Describe the plant and all the phenomena observed, carefully and concisely, including the effects of the staining agents. Sachs' Botany, p. 16; Vines Text Book, p. 118; Parker's Biology, p. 192; Goebel, p. 49; Strasburger, p. 246; McAlpine's Charts, pi. iv; 19th Smithsonian Contribution^ pi. 14 and 15. For method of making permanent mounts of Spirogyra see p. 38. CASE III. This includes by far the largest number of plants existing. They may be considered under two heads. A. Those which do not form Tissue, of which, Chara and JVttella may be taken as a type. The sanae condition exists in stamen hairs, many trichomes, etc. B. Those which form Tissue, as the higher Qryptogamia and the Flowering Plants, which will furnish the illustrations under the subsequent heading of 2\ssues. Case III. (A.) Illustration First: Stamen Hairs of Tradescantia showing PROTOPLASM in MOTION. (Streaming movement.) (Fig. 10.) Preparation. Remove from a perfectly fresh, newly opened flower a stamen with the attached hairs or trichomes. Place them in water taking particular care not to break or injure the trichomes. Observe: 1. The rvwmher waAform of the trichome cells. 2. The faint pink color of the cell sap, due not to colored granules, but to a red pigment held in solution> 3. The nucleus. 4. ^lenAev streams oi moving protoplasm,. Trace their course. 5. Estimate their rate of movement. For figures of the above see Strasburger, p. 29; Sedgwick and Wilson's Biology, p. 30; Bessey's, p. 12. The movement of protoplasm can likewise be studied in the young plant hairs of Gucurbitaoeae, in Nitella, or in Vallisneria. Any of the specimens can be mounted in water and treated the same as Tradescantia. B. CELL CONTENTS. The several illustrations following, represent the secondary products of the cell as distinguished from active protoplasm and its immediate derivaties, the chlorophyll body. The secondary products are found chiefly in the fundamental or cellular tissue, the characteristics of which will be observed. The solid forms of these products will be studied as follows: 1. Starch Qrains. 2. Crystals. 3. Protein granules. 4. Crystalloids. 5. Aleurone grains. Illustration First: Showing Pabenchima Cklls with Stabch. The TuBEE of the Potato. {Solanum tuberosum.) (Fig. 11.) Pbepaeation: Cut a frfesh surface on a tuber, make several thin sections from 2-3 m. m. in breadth and as thin as possible. Mount in water. Observe: 1. Form of \:he parenchymM cells of the tuber. 2. Ovoid or pyriform starch grains, oi y&vioas Bizes, lying in and about the cells. 3. Structure of grains; some plainly showing a hilum, around which appear rings more or less eccentric. The rings are layers of greater or less density. 4. Measuri some of the larger grains. 5. Eun a drop of weak iodine under the cover and note the blue color resulting in the grain, the characteristic reaction for starch. Sketch a tuber cell with contained starch grains. Vines' 76. CELL CONTENTS. Text Book, p. 110; Stiasburger, p. 11; Goodale, p. 49; Bessey, p. 54; Bot. Centralbl, LV. (1893) p. 157. Illustration Second : Showing Crystalloids. Hypodeemal TISSUE of Potato tubee. Peepakation : Sections should be made below the cuticle, but very near the surface, and the parenchyma should contain but a few smalLstarch grains. Observe : 1. The rectangular cells with cell nuclei if present. 2. Crystalloids, cubical in outline, surface plane, or, some- times eroded. 3. Upon the application of iodine, observe yellowish^ tint given to crystalloids indicating their nitrogenous nature. 4. After drawing oflf a considerable portion of the fluid under the cover-glass and subsequently applying a saturated solution of common salt (NaCl), observe the changes in, and final dissolution of, the crystalloids. True crystals are not thus acted upon. Strasbui'ger, p. 25; Goodale, p^ 47; Vines' Text Book, p. 111. Illustration Tliird : Aleurone and Starch. The Garden Pea (seed of Pisum sativum.) Preparation: Separate the cotyledons of the pea, and cut away a portion of one with a knife. Then with the heel of the razor make several small sections from the smooth surface. Mount in glycerin. Observe: 1. The grains of starch; iheiv form and concen- tric lines of structure or stratification. ^ 2. Their lines of fissure. 3. The rninute grains in the cell wit^ the starch, the aleurone 4. The cell structure and intercellular spaces. Draw, showing the above characters; then treat the section with strong iodine, and observe its effect on the aleurone. Goodale, p. 47; Vines' Text Book of Bot., p. 112; Strasburger, p. 18. Illustration Fourth : Stabch, Aleurone, and the Structure or Cereal Grains. Grain of Wheat {Triticum vulgare.) Pbepaeation: By cross-section cut away one-third of the grain. Make several thin sections from this surface, iwhich shall include only a small portion of the coats of the grain, and of the 77 CELL CONTENTS. white interior. The coats are better developed near the groove or Binus of the seed. Mount in glycerin or water. The sections can be more easily prepared by soaking the grains for a few hours in water. Observe: 1. The form of the cells of the interior of the grain. 2. The form of the starch-grains in these cells. The stratifi- cation is not often visible. It can be detected in some upon the application of dilute Chromic Acid. This is a useful agent in, rendering the stratification of starch-grains, cell-walls, etc., more distinct. 3. The form of the Aleurone-cells (or ^'■gluten sacs") in- closed by broad white walls, whose outlines may be traced even on their peripheral side. , The Aleurone-cells lie next to the starch- bearing cells. 4. The minute ydllowish grains of Aleurone filling these sacs. 5. The Seed-coats and the Ovary-coats. (a) The Inner seed coat, (or Secundine) made up of two layers of white homogeneous cellulose, separated by an interrupted gran- ular line qr thin layer. The peripheral of these two layers is regarded as the true " Inner seed-coat." (b) The Outer seed-coat (or Frimine), usually a narrow brown band. In this lies the color pf "red" and "white" wheat. (c) The Endocarp, apparently marked at intervals by oblong transverse pits, pinkish from refracted light. The Endocarp con- sists of a layer of elongated, thick-walled cells, running transverse to the long axis of the grain, therefore parallel to this section. The oblong pits referred to are narrow, nearly-closed passages through the party walls between these cells. (d) and (e) The Exocarp. This is made up of several layiers (usually two) of oblong, thick-walled cells running parallel to the long axis of the grain. The ends of these cells wUl therefore appear in the present section as oblong openings. When first mounted they are not always apparent. These thick-walled cells of the Endocarp, Exocarp, etc., are Sclerenchyma cells, and characteristic of the pro- tective coverings of seeds. 18 CELL CONTENTS. It will be noticed that in the above illustrations the cells inclos- ing starch, etc., belong to the Cellular Tissue. FuBTHEE Preparation : The coats above named and their com- ponent cells vriU be more clearly understood if two or three thin, tarigential sections be made, i. e., sections parallel to a plane tang- ent to the surface of the grain, and extending only to the starch- cells. Trace here the Aleurone cells, the Oute7- seed-coat {Primine), the Endocarp, the Exocarp. If iodine be applied to either the transection or the tangential section, and be followed by strong sulphuric acid, the inner seed-coat, the Endocarp and Exocarp turn a deep blue (the reaction for cellulose), then dissolve, leaving the brown Outer seed-coat entire, thus indicating the corky nature of the latter. The above study should be made with care as all cereals show the same relative arrangement of seed-coats, aleurone-cells, and starch-bearing cells. It will make more clearly understood some of the processes of "high milling," by which the nitrogenous com- pounds (the Aleurone grains), are separated and saved from the bran. The ovary and seed-coats form the bran proper; the aleu- rone-sacs contain most of the nitrogenous compounds or albumin- oids, and the phosphates; the interior cells contain the starch, which makes up the bulk of the "white" flour. For figures illustrating the structure of the coats of the wheat- grain, see Report of the U. S. Com., at Vienna Ex., 1873, vol. 11, p. 4, on Art on Vienna Bread ; Goodale, p. 4:1', 181 ; Strasburger, p. 19; Vine's Text Book of Bot., p. 112; Landois and Stirling, Human Phys., p. 444. Illustration Fifth: Starch. Grain of Indian Corn. (^Zea Mays.) The semi-transparent portion of the dry grain should be used, ^ and the sections made very thin and necessarily minute. The cells of the seed should be observed, but particularly the form of the starch-grains and the peculiar stellate cavities at the center of some when the section is first mounted in water. For figures, see Sachs' Botany, p. 61; Goodale, p. 181; Bessey, p. 55; Strasburger, p. 12; Vines Text Book of Bot., p. 112. Illustration Sixth: Starch in Compound Granules. Grain of Oats. {Avena saliva.) 79, CELL CONTENTS. Remove the palets from the grain or seed, and mount the sec- tions of the later in glycerin to prevent the disorganization of the compound granules. Observe the reticulated appearance of the latter, due to the lines of union of simple grairis of starch; ol^erve the form of the compound granules. (For figures see Goodale'e Botany, p. 49, p, 181; Strasburger, pp. 11 and 12.) With a little care the coats of the above grains may be sec- tioned for examination at the same time as the starch and compared vrith those of the wheat. In Fig. 139 (Goodale) the layer (a) is that of the palet, and should not be represented in connection with the true coats of the grain itself. Sections of seeds may be per- manently mounted by removing the water and adding a drop of glycerin jelly. Seal when hard. , Strasburger, pp. 11 and 12. 1 1. Eaphldes— needle-shaped. Fqrms of Plant Orystals-< 2. Crystal Prisms— prismatic. I 3. Spnaeraphides— compound, spherical. Illustration Seventh: Cevstalb. Illustration for form 2. The outer dried lamina of an Onion bulb, {Allium Vepa.) Peepaeation: Split a thick brown lamina parallel to the sur- face; this may be done by twisting two parts in different direqtions with the hands. The thin margin of the rent including the cells on the convex surface may be mounted in water. Obseeve: 1. The form of the cells in which the crystals lie. 2. The various forms of single crystals, viz: ' Prisms often with modified angles, also hexagons and pyramids if present. 3. The number oi principal faces to the prisms. 4. The double crystals in the form of an oblique cross; triple crystals if present. Do the axes of such crystals pass through one another? Tests i'ob Cetstals:' The greater number of plant-crystals are Calcic Oxalate. A limited number are Calcic Carbonate. Crystals of other salts of lime occur but rarely. Calcic Oxtilafe is not acted on by Acetic Acid. Calcic Oxalate is dissolved by Hydrochloric Acid without effervescence. Calcic Carbonate is dissolved by both acids with effervescence. As the composition of the crystals is uncertain, test veith 80 CELL CONTENT^. Acetic Acid first. If it has no effect, draw it off, and apply Hydro- chloric Acid. Make sure the acids reach the crystals and particu- larly observe the effect on any which may have floated out of the cells. ' I Tests for Calcic Phosphate and Calcic Sulphate are given in Poulsen and Trelease's Botan. Micro-Chemistry, p. 96 ; Zimmer- mann's Microtechnique, pp. 64, 62. The Study of Group I. Maphides will be reserved until later in the outline, in connection with an examination of growing root^, in which these crystals are found in abundance near the tip. Illustration Eighth : Cystoliths. These are the concre- tions composed of cellulose and a salt of lime, and are found chiefly in the epidermal regions of certain Urticaceae. Illustration : Leaf of Picus Elastica (one of the " Caout- chouc Trees.) Peepakation : Select a piece from the upper part of a leaf and mount the thin transection in water. See p. 16. Observe: 1. The m^T^ev JEpidermis, Sk l&yeTc of small color- less cells on the upper surface of the leaf. 2. The 'Hypodermal layer of larger, also colorless cells just- beneath the upper Epidermis. 3. Larger cells at intervals in the hypodermal layer contain- ing the concretions — the Gysioliths, attached to the peripheral side of the cell by a stalk. Apply to this the preceding test for crystals, and demonstrate what salt of hme is present. After the test has been completed, and the acid washed out, apply strong iodine, and observe the skeleton of cellulose re- maining in the Cystolith. DeBary, p. 44, 104-105; Bessey, p. 11; Vines' Text Book of Bot., p. 108; Zimmermann's Microtechnique, p. a;. Inulin. Illustration Ninth : A substance of the same chemical com- position as starch and found dissolved fn the cell sap of many plants : Dahlia root, or Jerusalem Artichoke. Preparation: Make thin transections of apiece of Dahlia root that has beeu lefjt for some days in strong alcohol or glycerin. 81 CELL CONTENTS. Observe : 1. Sphaerocrystals of various sizes, adhering to the cell wallg. The radial and concentric stratification of the crystal ; seen more clearly by the application of nitric acid. Remove the alcohol by water and note that after a little time all of the crjrstals have been dissolved. Sulphuric acid also dis- solves the crystals. Zimmermann's Bot. Microtechnique, p. 78; Vines Text Book of Bot., p. 114; Strasburger, p. 51{ Comptes Rendus cx\i (1893), pp. 514-17. C. ELEMENTflRY TISSUES. TABLE OP TISSUE FOEMS, OR ELEMENTARY TISSUES. I. Parenchyma Tissue. IV. Fibrous Tissue. II. COLLENOHYMA TiSSUE. V. TeACHEARY TiSSUE. III. Sclehenchyma Tissue. ' VI. Sieve Tissue. VII. Laticiperous Tissue. 1. Parenchyma Tissue. Tissue of the Fundamental System, forming the "grou7id work" of plants ; with cell walls of varying thickness, and although of diverse forms yet never fitrous, as dis- tinguished from Prosenchyma. The more ordinary forms to be considered are : 1. Isodiametric. , 4. Irregular . 2. Stellate. 5. Epidermal. 3. Ellipsoidal. 6. Suberous. Illustration: Isodiametric cells from Geranium St.ems {^Pelargonium inquinans), Pith of most Stems. Preparation First: A thin transection of the stem can be made free hand as directed on p. 16 and mounted in water or glycerin. Orserve: 1. In the center of the section, thin walled isodi- ametric oells of varying size. 2. Nuclei if present. 3. Contents of pu,renchyma cells in various parts of the section. 4. The irregular spaces between the cells, — Inter-cellular spaces. (Fig. 12.) 5. By careful focusing near the outer edge of the section 83 ELEMENTABY TISSUES. .where tlie cell walls are somewhat thickened, the primary partitiQn between contiguous cells: The middle lamella or intej- -cellular substance, as it is sometimes called. This lamella is of much the same chemical composition as the remaining cell walls of the tissue, and undergoes modification with it. ' PiiEPAKATioN Second: Ellipsoidal cells, from the root of any herbaceop-S plant; — those of the Hyacinth grown in water are easily obtainable. For this study a longisection of a root should be prepared in the manner directed for the previous study, or better, after the directions on p. 21. Observe: 1. Thin walled ellipsoid cells, beneath the epider- mal layer, making up the main body of the root — the cortex, — and extending to the central cylinder. 2. The cells toward the apex of the root are more isodia- metric. 3. The cell contends, together with the general characters of the cortical, tissue. , Bastin's Bot., p. 152. Preparation Third: Epidermal and Irregular Parenchyma cells from the leaf of Scarlet Geranium. Remove a portion of the epidermis of the lower surface of the leaf. This may be done by raising it at a cut margin and stripping it back. It will come away as a transparent membrane. Mount in water with the external surface of the epidermis uppermost. Observe: 1. T^he irregular ouiXme ai^he chXarXesB epidermal cells. 2. Stomates, with elliptical openings, at intervals. 3. Trichomes in various stages growing from the epidermis. The elongated cylindrical cell or ceUs composing them; the globose glands at the apex of some. (Fig. 25.) This illustrates the fact that cell walls, when not acted on from without, will develop with curved surfaces. Bastin's Bot., p. 156.^ Prbparaton Fourth: Make thin cross-sections of small por- tions of leaves, hardened and partly bleached, in 50 per cent, alco- hol. In these the chlorophyll bodies retain their form and some of their color. The green tint imparted to the alcohol shows the read, ihess with which the chlorophyll pigment is extracted l)y its solv- eixts. While sectioning, the razor and the leaf must be kept sup- 84 ELEMENTABY 'TISSUES. plied with alcohol to prevent access of air. Mount in alcohol or glycerin. Observe 1. Epidermis of the upper and lower sides of the leaf, appearing almost colorless as seen in section. 2. The thickened (cutinized) outside walls of the cells, which are in very close contact with each other. 3. The palisade cells (ellipsoidal) , and the underlying layers of irregular parenchyma cells making up the mesophyll of the leaf. These cells »form the main body of nearly all leaves. Note the large irregular intercellular spaces in connection with them, also their contents in the sections of fresh tissue.' 4. jStomates in section over small intercellular spaces. Preparation Fifth: Stellate pArenchvma from stems and Petioles of many aquatic plants. Ziiliacece and J'ontederiacece furnish excellent material for this study. Make several thin transections from the stein or petiole of Pontederia and mount in water. Observe 1. The firm regular epidermal parenchyma with slightly cutinized walls. 2. The loose isodiametric cells making up the interior of the section. 3. Large air-passages occupying a considerable portion of the section. 4. Certain spaces filled with stellate cells, having their project- ing ends in contact and forming a sieve-like plate across the cavity. (Fig 12.) The function of these plates is probably to form a support for the cross running fibro-va;scular bundles, and at the same time allow the free passage of air through the stem (Bot. Jahresbericht; 1, 196.) 5. The large nuclei and granular protoplasmic contents. Preparation SixtB: Suberqus cells, from the "Cork Oak," Quercus Suber, (commercial cork.) Mount several very thin sections in water; mount others in alcohol on the same slide. Suberin, the peculiar substance of cork cells, repels water, and the comparison between the two prepara- tions should be made as- soon as possible. 85 ELSMENTABY TISSUES. Observe: 1. If there is a larger amount of air imprisoned in one preparation, than in the other. 2. The form and color of the cells, their thin walls resem- bling the parenchyma previously studied. Apply concentrated chromic acid to the section and note the effect. Suberin will enable the cork to resist for a long time, (often many days and perhaps continuously) , the action of the acid, while all other modifications of cellulose are readily dissolved by it. fhe probable absence of cellulose from suberized walls, as pre- sented by Eugene Gilson, is interesting to note in this connection, (La suberine et les cellules du liege. La Cellule, etc., p. p. Carony, T. 1890 p. 63.) See Zimmermann's Microtechnique, p. 148. MODIFICATON OF CELL WALLS. Those that produce changes in the chemical composition ot the cell wall may be classified as follows: 1. Mucilaginous Modi^eation. 2. Ligtiification. 3. Cutinization. 4. Mineralization. Illustration First: Cell walls containing Mucilage. Seed Coats of Flax or Linseed. Make a thin tangential section of the seed/ and place for a few minutes in water. Observe: 1. The ready absorption of water, and consequent thickening of the cell wall. 2. Its gelatinous consistence in seeds that have been left for some time in water. In many cases the modification goes so far as to convert the cell wall into a gum, soluble in water. Examples of this can be found in the Plum, Cherry, or Peach trees. Vines' Text Book of Botany, p. 107; Goodale,/p. 34; Strasburger, pp. 99, 340. Illustration Second : Cell walls containing Lignin. Fibrous tissue of the stem of any woody Phanerogams, or Vascular Cryptogams. Make a thin section of a woody stem and mount in a few drops of cuprammonia. Note the walls are not dissolved, which would be the case, if they were pure cellulose. (P. 5.) To a fresh section add iodine and sulphuric acid, or chlor- 86 , ELEMENTABY TISSUES. iodide of zinc, and observe that the cell walls are colored' yellow or brown — the characteristic test for Lignin. What would be the reaction with pure cellulose? Zimmermann's Microtechique, p. 143 ; Goodale, p. 36 ; Vines' Text Book of Bot., p. 107 ; Strasbur- ger, pp. 59, 119. Illustration Third : Cell walls containing Cutin. Epidermis of most plants, and in many tissues where cell walls are to be strengthened, or protection secured. Make thin transections of the leaf of Pinus Sylvestris or Cycas, and mount in water. Observe: 1. The small epidermal cells with outer walls much thickened, usually in layers by the formation of cutin. The cutin can be removed by leaving the tissue in strong chromic acid for some time. After this, wash the section with water and add chlor-iodide of zinc. Note the blue color, the reaction for cellulose, forming the greater per cent, of the inner part of the wall, while the outer part consists of nearly pure, cutin. Vines' Text Book of Bot., p. 107 ; Goodale, p. 38 : Strasburger, p. 58 ; DeBary, f. 78. Illustration Fourth: Cell walls containing minerals, in CRYSTALLINE or AMORPHOUS forms. (Stems of Cereals, and many Sedges.) The substance most frequently present is Silica. To test this, ignite the specimen in a platinum dish, treat with nitric acid, and again ignite. The silica remains behind, and often retains the microscopic form of the tissues. This is true with the stems of Equisetum (horsetails.) Crystals of Galciutn salts cd,n be found in the ceU walls of the bast tissues of the willow and many Gymnosperms. For further study of the subject, examine Vines' Text Book of Bot., p. 108; Goodale, p. 39 : DeBary, p. 102. Continuity of Protoplasm. Illustration: Stem of Aesculus (common horse-chestnut.) Preparation: By the use of a knife remove the outer dark colored and inner green bark (periderm), from a young growing stem, about 1 cm. in diameter. With a sharp razor make thin tangential sections of the exposed whitish tissue (cortex), and place in a solution of iodine in potassic iodide until brown. Wash 87- ELEMENTABY TISSUES. thoroughly with water and add strong sulphuric acid. After a few minutes repeat the operation of washing. The acid repders the cellulosfe cell-wall transparent, and the protoplasmic strands can be seen connecting the masses of, contigu- ous cells. If this does not show clearly stain the preparation with aniline blue. The sections can be permanently mounted in a drop of glycerin jelly. Str/asburger, p. 371 ; Quart. Journ. Micros. Science, 1882, p, 365, 1883, p. 151; Bot. Gazette, 1889, p. 8^. II. CoIIenchyma Tissue. This tissue is composed of parenchyma like cells with "walls thickened at the corners, or points of contact, and usually tapering at the ends. They form cylinders of tissue beneath the epidermis in many herbaceous stems and petioles. The cells often contain chlorophyll and are sometimes capable of division. Illustration: Collenohyma cells from a mature stem of the Begonia, or Geranium. Pkeparation First: Transections of a stem 3 to 4 m.m. in diameter should be cut free hand, sfiained with haematoxylin, and mounted in glycerin jelly ; or better, hardened in alcohol, infiltrated with collodion, sectioned, and mounted as directed on p. 21. Observe: 1. Thin walled cells forming the epidermal tissue of the stem. 2. Inside of this a ring of glistening thick walled cells form- ' ing a zone about the stem. 3. The latter, the collenchyma tissue is composed of cells with walls comparatively thin along the lateral surfaces, but thickened at the angles. This tissue is very strong and contributes greatly to the strength of the stem. ' Make a longitudinal section of the stem and examine the cells of this zone. De Bary, p. 119; Strasburger, p. 106; Goodale, p. 65. Endodermal Cells. , A modified form of thick walled parenchyma is found in the Endodermis, in most roots a single layer of cells, surrounding the fibro-vascular bundles of the central cylinder. (Pigs. 17, 22.) 88 ELBMENTABY TISSUES. The walls of the cells are thickened and often folded at the points of contact between contiguous cells of the sheath. They are strongly suberized, and have a clear glistening appearance. Illustration: Endodermis of the central cylinder in Hyacinth or CORN root, grown in water. Make a thin transection of an old root, and prepare as for the previous study. Observe: 1. Near the centqr of the root surrounding the Jibro-vascular bundles a single layer of cells thickened only at the points of contact, but the walls uniformly suberized, j;hus giving , them a glistening appearance, — the endodermis. De Bary, p. 121 ; Vine's Text Book of Bot., p. 165; Strasburger, p. 136. Ill Sclerenchyma Tissue. The cells under this tissue form vary from the short isodiametric " Stone cells, " to elongated fibres, with thick walls, passing by gradations into fibrous tissue on the one hand, and into cellular tissue on the other, (refer to parenchyma). They will be here con- sidered as distinct elements. Illustration: (of ''Stone cells), Pleshj roots of Dahlia variab- ilis. Preparation First: Longitudinal sections should be' made just beneath the brown epideriiiis. The zone containing the cells will be apparent by cutting or scraping off the epidermis with a knife, when the sclerenchyma tissue will be found as a hard gritty layer. Mount in water. Observe: 1. The thin walled parenchyma, making up the main body of the fleshy root. 2. Groups of Sclerenchyma cells with ver-y thick walls; the slender canals, often branched, running through the wall- from the central cavity. 3. The meeting of the canals of adjacent cells. 4. The form of the canal openitigs as seen on the surface of the cells. (Pig. 13.) _ Preparation Second: Sclerenchyma cells from the " Ivory NUT," (Phytelephas macrocarpa) often found made into buttons, umbrella handles, etc. Some of the more common nuts may be used. 89 ELEMENTAl^Y TI88-UE8. With an old razor or sharp knife, small sections of sufficient thinness can be obtained from the surface of the nut. These may be mounted in water for study, or in balsam for permanent preser- vation. . Observe: 1. The tissue of the shell composed entirely of sclerenchyma cells. 2. The narrov] cavities of the rectangular cells. 3. Very thick but clear cell walls; cell contents. 4. The unbranched canals passing through the walls at var- ious places. Enlargement of the canals at their place of union between con- tiguous cells. .In many cases the septum between the two is not absorbed. 6. The peculiar branches of the canals at the end walls of the cells. PEEPAEATfoN Thied: Sclebenohtma CELLS from the underground stem of Pteris aquilina, (common brake). Place some of the un- derground stem between two pieces of cork, and fasten in the jaws of a microtome. With a strong razor kept wet with alcohol cut thin transections and mount in glycerin jelly for permanent preser- vation. Obseeve : 1. The bands of dark reddish brown tissue, ex- tending across the section. These bands are composed of scleren- chyma cells and assist greatly in strengthening the stem. (Fig. 23.) 2. With the high power, the laminated tfiick walls of the _ cells. 3. The branching canals, through these walls. 4. If the ends of canals of neighboring cells are in contact. 5. By careful focusing, the ends of these canals at the bottom of some of the cells. Vines' Text Book of Bot., p. 133 ; Strasburger, p. 146; De- Bary, 132 ; Goodale, p. 63 ; Sedgwick and Wilson, p. 76. IV and V Prosenchyma (in its widest sense.) IV. Peosenchyma (proper) or Fibrous Tissue. (a) JBast-cells, — in the bark, (derived from J'hlcBm,.) (b) Typical Wood-cells — in the wood, (derived from Xykm). 90 ELEMENTABY TISSUES. V. Traoheary Tissue. , (c) Tracheids — like Wood-cells in form, and like ves- sels or Trachece in structural marhings. (d) TrachscB—'' Vessels " or " Ducts." Type 1 includes Dotted and Banded Ducts, passing into Pitted and Scalarif arm JixictB by gradations. Type 2 includes Spiral, Annular, and Meticulated Vessels and their gradation. See Goodale,, p. 59 ; DeBary IV. * Illustration : For Prosenchyma (in its widest sense.) Bark and Wood of Leatherwood (Dirca palustris.) Preparation for (a). Eemove the brown cuticle from a branch of ZieatKerwood, also the loose cells beneath. Make two or more very tbin longitudinal sections of the bark, pulling one end of each section from the branch instead of cutting it. Many of the silky bast-fibers Will thus be free at the end. Mount in water. Another mode, is to place bast tissue in nitric a9id and potassium chlorate, and heat for a few minutes. The bast fibres can then be separated under a dissecting microscopy. Use a 3-4 and then a 1-5 in. objective. Orserve : 1. The very long bast-fibres; trace out a free one, and measure its' length. 2. What kind of cells, if any, occur with the bast. 3. The clear unmarked walls of bast ; the tapering ends when not broken. Preparation for (b) and (c). Make (1) very thin, tangential sections of wood ; (2) very thin transections of the branch includ- ing wood and bark. Mount in water and stain with haematoxylin. In the longitudinal section: Observe: 1. The short unmarked, fusiform wood fibres or typical wood-cells whose ends over-lap. Measure the longest. 2. The fusiform cells marked by spiral lines, dots, bordered pits, etc. — Tracheids; see Goodale, Fig. 78, 79; Bessey, p. 81; Bastin, p. 162 ; Vines' Text Book pf Bot., p. 182. * It will be observed that the classification of Prosenchyma tissue is not the one usually presented. It is believed that by adhering closely to this one in these studies, a correct idea can be obtained of the development of the more perfect forms of ducts from the simple cells. It Is desirable that the classifications of others be compared and the points of difference noted. 91 ELEMENTABY TISSUES. 3. Occasional Ducts (long tubes marked with bordered pits, bands, etc.) ' 4. Rectangular cells of medullary rays, if present. To what tissue-form do they belong? In the transection: Obsekve : 1. Ends of wood-cells in very regular radial rows ; thickness of their walls, etc. 2. The larger openings (ducts), marking each years growth. 3. Thin plates of medullary tissue. 4. The bark with ends of 'bast appearing. 5. Pith at center. Bastin, p. 159 ; Goodale, pp. 88, 89. Notice the brittleness of wood, and strength of bast, corres- ponding' to the difference in the length of cells in each. ' Usually wood-cells are less regularly distributed than in the above. PeeparatioS for (a), (b), (c), and (d). Make a radial longitudi- nal section (i. e., alongitudinal section passing the center of the pith). Around the pith is a sheath of Spiral or Heticulatect vessels (see Type 1). In the woody tissue of the se(3tion are Jr'itted vessels (see Type 2). Observe : 1. The Spiral Ducts. 2. That the spiral appearance is due to the thickened ridge deposited on the inner surface of a thin-walled tube. 3. If there are any ' Meticulated or Annular vessels in the medullary sheath. 4. That the Pitted vessels are furnished with "bordered pits", — the outline of the pit or cavity being nearly circular, and the "lumen" or central aperture oblong. Vines' Text' Book of Bot., p. 134; Goodkle, p. 85; Bessey, p. 74; Strasburger, p. 129. V. Tracheary Tissue (continued). Illustration : Sections of the stems of Cukeant, Hoese Chestnut, Moon Seed and Geape Vines, are to be mounted to serve as material for the study of both Tracheids and Tracheae. Note carefully the distinction between the two, the former being " like •mood cells in outline and like Vessels or Tracheae in structural markings.''^ Bead carefully DeBary, pp. 164-166. 92 ELEMENTARY TISSUES. Preparation: Suitable sections can be obtained by fastening small portions of these stems in the jaws of a micAtome and sectioning with a stout razor. Several radial-longitudinal sections of each should be obtained in order to be certain of including the proper portion (xylem) of the fibro-vascular bundle. Other material than that suggested above can be used, but such common plants have been selected as are found to show the desired structure. These specimens should be examined with reference to the study of the general forms indicated in the illustra- tions of Tracheae which follow.> Vines' Text Book of Bot., p. 135 ; Goodale, p. 82 ; DeBary, p. 165. The Tracheids of Coniferae. Illustration : Wood of White Pine {Piiius Strobus). The seasoned sap-wood of a young Pine answers the best for the study. Preparation First: Make several thin longitudinal sections at right angles to the ""grain" (the annual layers.) Mount in water. , Observe : 1/ The Tracheids, oblong and fusiform like wood fibres, but shovviag " ftorc^erec? ^ite " at intervals. This form of Tracheid is found throughout the Gymnosperms, fossil and living. 2. The outer and inner ring of the bordered pit. By focus- ing, the inner ring or outline of the lumen on the opposite side of the pit may be seen. 3. The rows, of rectangular cells occasionally crossing the Tracheids at right angles. These are portions of the Medullary Mays. Preparation Second: (1.) Make several thin longitudinal sections parallel to the "grain." (2.) Make several thin cross- sections. Mount (1) and (2) in water under the same cover. Observe in (1.) 1. The small cavities, occurring along the common- wall of two Tracheids. These are single and lens-shaped — the bordered pits seen in section. , 2. Look for the middle lamella or " limiting membrane "- which originally separated the two halves of the pit and was con- tinuous with the common-wall of the two Tracheids. 3. The row of roundish or angular cells— three to six in number occasionally seen between Tracheids. These are largei" B3 ELEMENTARY TISSUES. than the sectioned pits and are the cells of the Medullary Bays in cross-section. ' Observe in (2): 1. The form of the Tracheids in cross-section. 2. The "middle-lamella" in the cell-wall of each. 3. The Bordered-pits and Medullary Rays in the section. 4. The Besin-passages in section. These are large openings surrounded by irregular thin-walled cells containing resin. - These passages often occur running transversely, following the larger medullary rays. (Fig. 14.) 5. The annual layers of tissue (seen best with a low power) marked by alternating layers of larger and smaller cells, and '. crossed at right angles by the Medullary rays. (Strasburger pp. 115, 128a.) 6. The entire absence of true 2'rachea^, these being found in Gymnosperms only next the pith. In structure Tracheids seem to be intermediate between Fibrous tissue and Tracheae. Bessey, pp. 25, 26 ; Goodale, p. 83 ; DeBary, pp. 159, 160 ; Vines' Text Book of Bot., p. 200 ; Strasburger, p. 56. V. (d) Tracheae. Illustration: Sections of stems for the study of Tracheae, — Vessels or Ducts. The following general forms will be examined: (a) Dotted. (c) Spiral. (e)- Scalariform. (b) Pitted. (d) Reticulated. (f) Annular. For these studies the woody tissue is to be treated as directed for the previous illustration, but in the case of herbaceous stems the tissue should be hardened in alcohol and carried through the method for mounting, outlined on p. 21. Preparation First : (a) , Lolngisection of Grape vine stem. Observe: 1. In the region of the section containing the fibro- vascular tissue, the large well developed ducts with pitted surfaces. 2. These dots are true openings allovsfing free communication between contiguous cells. Goodale, p. 29 ; Bastin, p. 162. Preparation Second : (b) Radial longitudinal section of the stem of Castor Oil Bean, 1 cm. in diameter (thin section mounted in water). Observe: 1. Rectangular cells oipith and cortex. 2. In the vascular region;' large thick walled ducts, with, the surface coyered with bordered pits. 91 ELEMENTABY TISSUES. 3. The orifice of the pit, consisting of two "acutely diverging la/mellae.'" De Bary, p. 167 ;, Sachs' Physiology, p. 137; Besspy, p. 27. Peepakation Third : (c), (d) and (e) Radial longitudinal sec- tion of the stem of Begonia, Bean, Banana, or better the material used in Prepaeation Second. In this preparation, Observe : 1. Large thin walled ducts with thickened spiral markings. 2. At various places the spiral bands broken loose from the wall of the duct. 3. In portions of the preparation some ducts may appear in longisection, in which case, the ends of the thickened bands may be clearly seen. 4. The steepness and direction of coils, also the number of bands. 5. Their branching at various places, often forming in some vessels a reticulation. This is the origin of the reticulated ducts. Excellent material for their illustration can also be found in the Cucvirbitaceae and Impatien's. 6. Certain ducts with the broad openings, between the reti- culations, arranged one above the other in the form of a ladder — Scalariform Vessels. Care must be taken to prevent confusing these with the pitted vessels which they closely resemble. De- Bary, p. 158. , Peepaeation Poueth: (f) Longisection of the stem of coen. Zea Mays. Observe: 1. Lying next the large spiral vessels of the bun- dles, certain ducts strengthened by transverse thickenings in the walls in the form of rings, — the Annular Vessels. It is to be noted that these are but a modification of the spiral ducts. De- Bary, p. 156; Strasburger, p. 90; Bessey, p. 82; Sachs' Text Book of Bot., p. 114 ;— Physiology, pp. 91, 136. A, modified form of Annular Tracheae exists in the Trabecular Duct, which has its walls strengthened by transverse bars^ across the cavity. Good illustrations of this can be found in the fibro- vascular bundles of the Juniper leaf. Bastin, p. 163 ; De Bary, p. 156. 95 ELEMENTABY TISSUES. VI. Sieve Tissue. Illustration : Stem of Cucurbitaceae (Cucumber or Pumpkin). Pbepakation: This material should be hardened in alcohol and treated as directed on p. 21. Both longitudinal and transverse sections are to be cut, stained with haematoxylin, aid several of each permanently mounted. In the longisection, portions must be secured that contain some of the fibro-vascular tissue. In the lat- ter section on the outer and inner edge of the bundle, Observe : 1. The thin walled sieve tubes, recognized by their sieve like plates, forming septa at tlie ends, aud not infre- quently on the lateral walls. , 2. The protoplasmic contents of the tubes, frequently denser near the ends. 3. The structure of the sieve plates. 4. The sieve pores, of varying sizes, Usually largest' at the ■ center. 5. A clear glistening bluish mass surrounding the sieve plate, ^ contents. The latex consists of a watery fluid with various albuminoids, organic acids, or alkaloids, in solution. The sus- pended matter may consist of proteid compounds, accompanied sometimes with starch grains. Latex tubes or vessels. Illustration : Stem or Petiole of Chdidonium majus (Celandine) Stylophorum diphyllum, (Poppy.) In sections obtained as for the previous study : Observe : 1. Thin walled profusely branched tubes, often anastomosing, and extending through the various tissues of the stem. These latex tubes are formed from rows of cells which be- come united by the absorption of the partition between, or by its perforation to allo\y free communication. The walls of these cells are usually thin, but frequently become thickened in the form of striations. 2. The effect of iodine on the latex tissiiet Goodale, p. 94 ; Vines' Text Book of Bot., p. 141 ; DeBary, p. 189 : Bessey, p. 75 ; Strasburger, p. 104 ; Sachs' Text Book, p. 86-7. QIands and Water Pores. Illustration First : Subepidermal Glands of the " Lemon SKIN." Preparation First: Harden, section, and mount a piece of " lemon peel, ^^ after the method on p. 21. Observe : 1. Small cells of the epidermis, often containing crystals. , 2. Near the outer portion of the section large cavities in the tissue — The Glatids or reserviors of lysigenous origin, i. e.,' formed by the breaking down of cells. 97 ELEMENTABY I'lSSUES. 3. Thin-walled cells, with large nuclei, lining the cavities. In some cases the cells are much disorganized. 4. Crystals in the adjoining tissue. 5. Fragments of fibro-vascular bundles sc&ttered through the preparation. (Fig. 15.) Preparation Second: Make a tangential section and compare with the previous study. Illustration Second: Glands in the leaf of JEuealyptus. Preparation First: Transections of a mature leaf can be pre- pared as directed for the previous study. Observe: 1. Large glands located on both upper and lower sides of the section. 2. The cavity of the gland. 3. The large thin-walled cells, partly disorganized, lining the gland, and the smaller thicker-walled ones just outside. 4. The t^esophyll and. palisade cells surrounding the gland. 5. The flattened epidermal cells above. Preparation Second: Make transections of the young leaf of Eucalyptus and trace the formation of the gland, which results f rpm the breaking down and absorption of the mother cells. Stras- burger, p. 164, DeBary, p. 201, "Vine's Text Book of Botanj', p. 40, Goodale, p. 98. The Resin Ducts of Pinus were examined in the study of the Tracheids of Coniferae. Water Pores. Illustration: Leaf Tooth from Fuchsia. Preparation: Several sections should be made serially, and must include those through the apex of the leaf tooth, (p. 35.) In the section passing thi:ough the center of the tooth : Observe: 1. The club-like enlargement at the outer edge. 2. The epidermis, consisting of small cells, interrupted at the apex and forming a circular opening — the pore. 8. Two or three layers of chlorophyll bearing cells beneath the epidermis. 4. The large region of the center of the section occupied by 'ihB. spiral marked tracheids. 5. The water cavity between the pore and the long parenchyma 98 ELEMEWTABY TISSUES. cells beneath. The section should be preserved for reference in connection with the study of the leaf structure, of which this is a type of a very large group. j Bessey, p. 105, DeBary, p. 52, Vine's Plant Phys., p. 91. D. MERISTEM TISSUE. Meeistem, or Generating Tissue, is usually classified under two heads, viz: — I. Primary Meristem, giving rise to the Peimary Stbucture in plants, such as young cellular tissup, and found at the apices of young thallomes, stems and roots, and at the apex and base of young leaves. II. Secondary Meristem, or Cambium, giving rise to the Secondary Steuctuee, as the thickening growth of stems, roots of DiOOTYLEpONS, or ExOGENS. The Secondary Meristem, as Procambium, forms a part of every fibro-vascular bundle in its earliest stage. > The Primary Heristem. Type A. Of a jingle Apical or Initial Cell. Found in most of the higher Cryptogamia. Type B. Of a Group of Initial Cells. This is characteristic of Phanerogams. , Illustration, Type B: Root from a cultivated Hyacinth grown in a jar of water. Peepaeation Fiest: Trans - and longitudinal sections prepared after the method on p. 21, also for serial sections, p. 35. (Fig. 16.) In the longitudinal section, observe the general arrangement of the different layers of cells, viz: 1. That the several series of cells converge into a rounded cone at a point just back of the apex. , This point is the center of the so-called ^^ Initial 6rTOt«p" which constitutes the Primary Meris- tem. It is impossible to say just how far raeristematic cells extend from this point. 100 MERISTEM TISSUE. 2. The Galyptrogen, a row of cells on the apical side of the Initial Group, producing rows of cuboidal cells radiating towards the apex of the root, the outer ones becoming rounded and loosely coherent. 3. The Dermatogen, arising from the Initial Group curving to the right and left and ultimately producing a double layer of cuboidal or flattish cells bounding the section on each side. In large roots it consists of more than two rows of cells. 4. The Plerome, axial rows of oblong or cuboidal cells form- ing a central band immediately back of the Initial Group. 5. The Periblem — thin-walled cells of various forms in several rows between the Plerome and Dermatogen. 6. That the differentation into the above named layers occurs at a very early period in the development of the root-tissues. They retain their original distinctness after passing over into permanent tissue when they are named as foUows : The Calyptrogen gives rise to the Root-Gap. ' The Dermatogen gives rise to the Epidermis. • The Periblem gives rise to the Cortex or Oortioal Parenchyma. The Plerome gives rise to the Axial or Central Cylinder. 7. The first appearance of Spiral Vessels on the right and left margin of the Plerome. Trace them backwards. They repre- sent the beginnings of the Fibro-Vascular Bundles which always arise within the Plerome in both stem and root. 8. The two rows of cells on the peripheral side of the Spiral Vessels: The inner (the Pericambium) belongs to the Plerome, the outer (the Endodermis) belongs to the Periblem or Cortex. The Endodermis is sometimes called the "Plerome-sheath" or "Bundle-sheath." 9. The peripheral layers of cells of the Plerome itself, extend- ing back from the Initial Group to the beginning of the Pibro- Vascular Bundles. These are termed collectively the Procambium, — as it is from such cells that the tissues of the Bundles themselves arise by fission and differentiation. 10. Occasional dark lines along the lateral walls of the Cortex cells. These are due to cylinders of air in narrow intercellular spaces. (See 2 under observations below on the cross-section.) 101 MEBISTEM TI88VE. 11. The JRaphides or Needle-shaped Crystals, in black bun- dles in many cells of the Cortex, and in this tissue only. To a fresh section apply the tests for Crystals given on p. 3. In the Cross-Section: (Fig. 17.) Observe: 1. The Epidermis, and outer zone of two or, three layers of cells ; form oi^ latter. 2. The zone of the Cortex cells, vrith frequent small intercell- ular spaces. (See obs. 10 above). 3. The Endodermis layer of the Cortex with a dark spot on the common wall between adjoining cells, due to a minute fold in the wall. ^ 4. The Axial Cylinder. a! The Fericambial layer. b. The Bundles five to ten in number ; the Xylem mass (that including the large duct openings) alternating with the thin-walled Phloem, after the usual plan in roots. See Pig. of Acorus in Sachs' Botany, p. 115 ; De Bary, pp. 10,348 ; Strasburger, p. 184 ; Good- ale, pp. 105, 112; Vines' Text Book of Bot.', p. 147. Make a sketch of ,a cross-section and the longitudinal section, showing the zones and layers above mentioned. Also an enlarged drawing of two ^fylem rays, the intermediate Phloem, Pericambium, and Endodermal layers. A' comparison should be made with the roots of a Dicotyledon (Radish, Pea, etc.), treated after the same manner as directed above. With reference to the development of the various parts of the root froln distinct initial ffroups, several types have been described by Janczewski, Flahault, and others. See Goodale, p. 107; De- Bary, p. 7. Type A. A SINGLE APICAL OK INITIAL CELL. Illustration : Boot of Fern (Pteris) or Equisetum. Pkepahation : The same as for the previous studj', except that serial logitudinal sections must be made in order to insure the presence of the desired parts. See p. 35. In the median section, Observe : 1. The cone-shaped root cap covering the tip of the root, not unlike the one in type B. 2. Just beneath the cap and at the center of the root, the 102- , , MEBISTEM TISSUE. large trilateral pyramidal cell with its convex base turned toward the root-cap, — the apical cell. ^ 3. A transverse partition cutting off the outermost portion, which becomes the initial cell of the root-cap, — Dermatogen. 4. Segments cut from the innermost faces of the cell and, by longitudinal partition, developing into the tissues of the Plerome aixd Periblehi. Strasburger, p. 188 ; Vines' Text Book of Bot., p. 150 ; De- Bary, p. 18. In the transection. Observe : 1. The irregularly ttickened walls of the epidermis. 2. The cortex consisting, on the peripherial portion, of dark brown parenchyma and merging into sclerenchyma toward the plerome. 3. The bundle sheath between the cortex and fibro-vacular. bundle. 4. The pericambium, a narrow sheath ( just inside the bun- dle sheath) of parenchyma cells filled with protoplasm. 5: The regular ^adiM bundle of the root. If time will admit a, comparative study should be made of the apex of the stevis of Monocots, Dicots, and higher Cryptogams. The preparation of the tissues for study would be the same as that recommended for the roots. DeBary, p. 19 ; Strasburger, p. 177 ; Vines' Text Book of Bot., p. 146. Fibre- Vacular Bundles. The following general classes are noted : A. Collateral. B. JBicollateral. C. Radial. D. Concentric. Illustration A : Stem of Begonia, Geranium, Moon Seed Vine, or Smilax. Priepaeation First: The herbaceous stems should be pre- pared in the usual way, the woody ones may be sectioned by plac- ing them between pieces of cork in the jaws of a microtome. Transection and longisection should be mounted together. 103 MEBI8TEM TISSUE. In the preparation of the Moon Seed Vine, {^Menispermum Oanadense) a stem of one year's growth should be selected. (Fig. 27.) , Examine with a hand lens, and observe the general manage- ment of the tissue systems in a cross-section. 1. Fith, (Fundamental Tissue.) 2. Fibro-vascular bundles, (outline of each is nearly circular). 3. Epidermis. Obseeve : 1. The Epidermis; The external walls of its cells being thickened very greatly, and a light olive in color, the inner and lateral walls being of ordinary thickness and the cell cavity small. * 2. The- Cortical Parenchyma; of two kinds of cells, both containing chlorophyll. (a) The outer are regular, closely packed and thick-walled. (b) The inner are thinner-walled, larger and looser, passing into the oblong cells of the Medullary Rays. 3. The Fihro - Vascular Bundles : The Phlmm is sharply distinguished from the Xylem, form- ing a strictly Collateral Bundle. In the Phloem : ' (a) The Crescent-Shape<^ band of thick-walled cells on the peripheral side of the Phloem ; the lamellate structure of the walls. (b) The squarish outline of Phloem cells adjoining the thick- walled Xylem. Some of them somewhat thickened (lignified). (c) The larger cells lying between (a) and (b). They are irregular in outline, thin-walled and stain but slightly. In the Xylem: (d) The larger duct openings between which the smaller cells (tracheids, etc.,) appear. (e) The thick-walled cells of the axial region of the xylem forming a continuous band, or "bundle sheath," bounding the axial side of the fibro-vascular bundles. (f) Between the Xylem, and Phloem several of narrow, thin- walled cells — the Cam,bium. This is the region of growth, in sec- ondary thickenings. In the radial-long itudhial sections trace out the groups of 104 MERISTEM TISSUE. cells corresponding to those observed under the previous prepara- tion, and demonstrate the Tissue Forms in each. Drawings should be made sufficiently in detail to show the character of the above-mentioned groups of cells in both trans- and longitudinal section. DeBary, pp. 319-339, Strasburger, p. 107, Bessey, p. 117, Vine's Text Book of Bot., pp. 174-182. Compare with closed collateral bundle of a monoeot, (Fig. 19, 28.) Illustration B: Prom the stem of Cucurbit Pepo. Preparation First: ' Eadial longitudinal and transverse sec- tions are to be prepared. Orserve: 1. The Fibro- Vascular Bundles: the smaller ones opposite the external ridges of the stem; the larger alternating with the smaller, and occupying the ridges projecting into the central cavity of the stem. To the naked eye the bundles appear as ganglia of smaller cells. 2. The larger openings in the middle area of the larger bun- dles, 3 to 8 in number. 3. A group of smaller openings on the axial side\df the larger ones. This middle area is the xylem,. ,4. Two nearly semi-cireular or sometimes cresoent-shaped areas of tissue, one on the axial, and one on the peripheral, side of the xylem. Both of these are masses of Phloem. Focusing upon the larger openings of these areas, sieve-like plates may be observed in some, forming septa. (Sachs' Text Book, p. 113.) 5. Form of cells in other parts of the bundle. 6. The thin- walled parenchyma, forming the greater part of the stem and surrounding all the bundles. This is the tissue of the Fundamental systetn. 7. The Epidermis of the Stem. 8. The '■^ Intra- Cortical Ring": — -a band of several layers of thick-walled cells, but faintly colored, and separated from the epi- dermis by slightly stained cells. 9. In the ' above-named slightly stained band note the sepa- rated areas of Collenchyma cells. Bessey, p. 30. Collenchyma cells, as previously noted, are recognized by the thickening of the walls at the angles. 105 MEBI8TEM TISSUE. In the middle longitudinal section of one of the bundles : Observe: 1. The Fundamental IHssue (Parenchyma), on the axial side of the bundle beyond its limits. In the Axial Mass of Phloem: ,2. The Sieve- Ttibes; known by the Sieve-like plates forming the septa or occasionally seen on the lateral walls. See Sachs', p. 113 ; Bessey, p. 78. 3. The Protoplasm enclosed in these tuh&s. 4. The Fihrous-cells, fusiform in outline, scattered among the Sieve-tubes or occurring in a band next the SpiraljDucts. In the Xylem, of the Bundle: Observe: 5. The Spiral Ducts — one narrow with a loose spiral ; another broader with a close spiral, occasionally becoming reticulated. 6. A Sealariform duct; see Bessey, jj. 27, (Fig. 18 ;) some- times this approaches (in this plant) a Pitted Vessel in structure. 7. The Pitted Vessels— -very broad. a. The shallow pits on the walls in transverse rows showing a "border" and " lumen." b. The ridges on the walls between rows of pits. c. The frequent occurrence of rings marking the partially absorbed septa. 8. The thin-walled cambiform cells, with septa oblique or at right angles to the lateral walls. This layer separates the Xylem of the Bundle from the Phloem, on the peripheral side of the bundle. In these bundles it will be noted there exists a p>ermanent band of cambium,, which insured the continued growth of the vascular tissue. The bundle is therefore said to be an open one. In the closed bundles the procambium tissue becomes differ- entiated into permanent wood and bast, and the bundle undergoes no further differentiation. The latter condition is the case in the stems and roots of most Monotiots, and Pteridophytes. Vines' Text Book of Bot., p. 177. Strasburger, p. 83 ; Bessey, p. 121. In the Peripheral Mass of Phloem: 9. Another group of Sieve- Tubes. 10. A few J3ast Fibres. Trace in the cross-section, the tissues, cells, etc., correspond- 106 MERI8TEM TISSUE. ing to those observed in longitudinal section, particularly noticing what belongs to the PUoem, and what to the Xylem. This Bundle is not only a Collateral Bundle (Bessey, p. 120), but a Bicollateral Bundle, (Fig. 20) i. e., one having a layer of Phloem tissue (Sieve-tubes, etc.), on the axial, as well as on the peri- pheral part of the Bundle. De Bary, p. 319. Sketch so much of the cross-section as shall include one Bundle and the tissue extending from it to the Epidermis ; also the middle longitudinal section studied. The drawings should be on a scale to show the characteristics of each tissue-form. Illustration C: Badial Bundle in the root of Hyacinth, or Corn. 'ii'-"!V'-';'°"' ;!^l. r;,''--,:|t-,-" V-ie r T.G^A. T,B,-B. PiQ. 10. stamen hair of Trcidescanlia. Pig. B. Stamen hair showing general form and relations of cells. A, cell of proximal end; B, nucleus. (j{20.) Pig. A. Cell much enlarged. A, outer wall of cell; B, nucleus and contained nucleolus; 0, stream of living protoplasm. (x380.) Plate II. Fig. 11- Section ol Potato tuber. Solanumtubernsum. A, brown epidermis; B, rectangular cells ,iust beneath epidermis; 0, hypodermal cells containing crystal- i!oi(Js(P); D, starch bearing parenchyma; E, starch grains. (x80). (Modified alter Landois and Stirling.) Tie^.E. Ti(\,C, Fc(,D. Fig. 12. Various forms of parenchyma cells. Fig. A. Stellate from the stem of Pontederia; Pig. B. Ellipsoidal and rectangular from the root of HyacintTi ; Kig. C. Cylindrical from the Hyacinth root, (a trichome, or root hair); Fig. D. and E. Isodi- ametric and globose from the cortex and pith of a Oeraniium stems. A. Cell cavity; B, Intercellular space. (About x 100.) Plate III. --4— A tA,'3J- T^ia.A. T'(k.S. FIG. 13. "Grit cells,- (sclerenchyma), from the Dahlut ^°°^-A:\^Jl^^°ilf?^ B, thin walled cells surroundins the sclerenchyma; and E opening of canals in the cell wall; D, canals in section Fig A (x300 ) Fig. B. (xluO.) f;<5.a. M.B.T. Fig. 14. Transection of the leaf of rinus sylvestris A, Cavity of the gland; B, thick walled epidermis; C, parenchyma of the raesophyll; D, bundle sheath; E, large thin-walled cells lining the gland. (x300.) Fig. 15. Transection of "Lemon peel." Fig. B, Slightly enlarged to show gen- eral relations of parts. (x5); F. Fragments of fibro-vascular bundles; G. interior of the "peel." (x30.) Fig. A, More highly magnified to show the character of a gland and the surrounding parts; A, Cavity of the gland; B, epidermis; V, crystals, abundant in the parenchyma (D;, .surrounding the gland; B, thin-walled cells, usually mpre or less disorganized, lining the gland, Plate IV. Fig. 16. Longisection of root, Cypripedium pubescens. A, B, Root-cap, com- pletely covering the tip of the root; 0, central or plei'ome cylinder, showing the thln-walled procambiura cells which laler develop into the elements of the flbro- vascular bundle; U, Initial group of meristcmatic cells from which originates the various tissues of the root; E, cortex developing from the periblem of the meris- tematic region- at the tip of the root; F, plerome or bundle sheath composed of a single layer of modified parenchyma cells, having the walls uniformly suberized but folded or thickened at their points of contact; G, epidermis having its origin in the dermaiogen at the apex just outside of the periblem. (xiO.) Plate V. M.B.T. Fig. 17. Transectioa of the root of Cifpripedium pubescens. A. epidermis; tex; C, bundle slieath ; D, xylem ray; E, phloem. S.-e Fig. 16 for parts la Ion tion. (x30). cortex ; section. Plate VI. Fig. 18. Transection of an open, collateral fibro-vascular bundle from the stem of Begonia nUida. A, thln-wallea cortical parenchyma surrounding the bundle; B, large thick-walled ves.^els of the xylem; 0, thin-walled cells of the cambium; 1), sieve duct of the phloem; E, wood pareuchyma of the xylem. (x350.) Plate VII. Fig. 19. Tranverse sectio-i of a closed, collateral vascular bundle from th" stem of Indian Corn, Zca Maj/s. A, thin-walled p-irencliyma surrounding the bundle; B, large pitted vessels in section; C, sclerenchymatous cells forming a shpath about the bundle; D, phloem of the bundle, with sieve-tubes and bast-fibres; E, spiral or annular vessel in section; F, intercellular space of lysigenous origin. (x200). Plate nil. Fig. 20. Transection of a blcollateral fibro-vascular bundle from the stem of Cucurhita Pepo. A, Parencli.vm;i cells of the fundamental system surrounding the bundle; B, B, large thick-walled vessels of the xylem; C, C, sieve-tubes of the phloem; D, D, thin-walled cells of the cambium. (xl.iO.) Plate IX. Fig. 21. Transection of the root of Spii^anthes cernua. Showing the general structure of the root with the relations of the various parts. A, root hair; B, xjlem of the radial bundle;