inate Pe 2 i Lire Ee! i eked ibs oe A aa sail Ses See Sa e eee Hats He ce ‘ see mgt br ae Ee Aig peat Lae SS Pea Sep ee ineeret ae Ley eee epee et Tae Peer Eby ATt i ae f He Built ie bees iH eu q t ee ae peal oat cia yaaa OU metata aey ce oe it i ae : eee Be ee Sar cffrecu] Ss 0 oo ACTER q HERE ny Mew Dork SHtate College of Agriculture At Cornell University Uthaca, 2. DB. Librarp pial research; vegetab Cornell University 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/cu31924003009747 Methods IN Microscopical Research METHODS IN Microscopical Research Vegetable Histology BY ABRAHAM FLATTERS, F.R.MLS., Lecturer and Demonstrator in Microscopy at the Municipal School of Technology, Manchester. SHERRATT AND HUGHES 65 LONG ACRE LONDON WwW. 27 ST. ANN STREET MANCHESTER 1905 + Preface. THIS work is the outcome of the Microscopical Research Class conducted by me during the first session held in the new Municipal School of Technology, Sackville Street, Manchester, 1902-3. The course of instruction consisted chiefly in working out the internal structure of the higher vegetable types. The notes used were those I had written down from time to time in my private laboratory work. The photomicrographs are all from specimens collected, fixed, cut, stained, mounted, and photographed by myself, the reproductions from which are hand-painted, direct from the microscopical productions, by my artist assistants, under my supervision. At the end of the session I was asked by several of my students to have the notes, formule, and methods of manipulation printed and, if possible, illustrated. To do this meant an enormous amount of additional work, especially in the production of suitable photomicrographs for publication, most of the sections for which had to be specially cut and stained to meet the requirements for reproduction in colours. In the publication of this work I believe a long-felt want will be supplied, as I have endeavoured to lay down a system of work, which, if followed out by the student, will enable him successfully to carry the work through, from the collecting’ of the specimen to the finished microscopical preparation. Had such a work been at my service twenty years ago, I feel sure that I should have been spared years of persistent hard work and many disappointments. The chief idea in the production of this work is to simplify the methods of microscopical research, in so far as relates to the specimens dealt with. There is not a single difficult problem in the work, if the student will make himself vi PREFACE acquainted with the art of cutting and staining sections. I trust the work will prove a help to the artisan and labourer students in microscopy, of which there are not a few, and to such I would say, be not discouraged, the author of this work issued from your ranks. To Prof. F. E. Weiss, D.Sc., F.L.S., my thanks are due for his assistance and suggestions relating to the structural parts of the photomicrographs, ABRAHAM FLATTERS. 20, CHURCH ROAD, LONGSIGHT, September, 1905. Contents. REMARKS ON THE WORK. Table for diluting alcohol - Conversion scale ti Table of specific gravities - e Diagram of graduating and narcotising vessel Remarks on clearing oils Remarks on celloidine Acidulated alcohol, use of - Bleaching solution, use of : CHAPTER I. Collection and preservation of specimens The “Flatters” collecting vasculum Reagents used for killing and fixing tissues Flemming’s fluid Chromo-acetic acid solution - Action of fixative on spirogyra Corrosive-picro-formaldehyde solution PAGE ABR wWwWwWNN HY! CO ONN AWN vill CONTENTS Corrosive sublimate Picric-acid solution Formaldehyde Action of fixatives on the root of Acorus calamus CHAPTER II. TOOLS AND METHODS OF WORK. Tools required in the dissection of animals and plants The old “Flatters” microtome The “Flatters” microtome, how to use Paraffin bath, and imbedding or moulding tubes Form of section-knife The art of cutting sections The operator at work Preparing specimens for sectionising The celloidinising method The paraffin method The Rocking microtome The moulding frame Mounting paraffin sections The art of staining Mounting and finishing the slide Mounting outfit Types of 3x1 slips Types of rings for cell-making Method of placing on cover slips Method of building up cell for a balsam mount Cleaning, ringing, and finishing slides Operator at work Types of finished slides 14 15 17 20 21 21 22 23 24 25 26 27 29 29 31 32 33 34 35 36 36 37 38 CONTENTS CHAPTER III. STAINS, REAGENTS, MOUNTING MEDIA, CEMENTS, ETC.: FORMULZ AND MANIPULATION. No. Borax carmine formula (Grenacher) No. a 2 Fe (Woodward) No. Hematoxylin ,, (Kleinenberg) - No. . ; (Delafield) - No. . ammoniated Brazilin formula Picro-carminate of ammonia Aniline-picrate and Gossypimine formula Zz ° SOE 20 NT OY Ga a eos Hematoxylin 5 s . Aniline blue formula . Malachite green formula . Safranin formula ZAAZzZzAz4 go 999 one Oo . Eosin 5 MOUNTING MEDIA. Canada balsam - Gum dammar Glycerine jelly Farrarit’s medium Glycerated water : Carbolised water Bleaching solution formula CEMENTS. Brown cement formula Oxide of zine 53 Asphaltum - - ix THEIR 31 51 x CONTENTS CHAPTER IV. INSTRUCTIONS FOR THE PREPARATION OF TYPES. Root structure Stem _,, Leaf Floral _,, Cell 5 and cell contents General Remarks on the Work. NINETY-TWO per cent. alcohol is the “ordinary” methylated spirits of commerce, free from the mineral additions as supplied by chemists. The strength of alcohol, as sent out from the distillery, varies from 88 per cent. to 92 per cent. In making up percentages, therefore, it is safest to calculate on a 90 per cent. strength, unless the student can obtain its specific gravity by means of the hydrometer. Percentages for all ordinary purposes may be obtained by volume as shown in the following table. Table for Diluting Alcohol. 100 volumes of alcohol of per cent. 333 gee | 99 85 8 75 7o 65 60 35 50 Ass =o Require addition of volumes of water. 85 68 — — — — — — — 80 13'8 68 — — — — — 75 21'9 14°5 72 ae ai = oa = = 70 311 23°1 15°4 76 — 65 405 330 247 164 SA 60 | 537 445 354 265 176 orgs = = 55 679 579 481 383 286 I9g0 5. _ 50 847 739 630 $24 417 313 25 Io4 — 45 | 1053 933 814 695 578 461 345 229 I14 40 1308 1173 I104'0 90°8 77°06 64°5 514 38°5 25°60 35 163°3. 1480 13299 1178 102°8 87°9 7371 58°3 43°6 30 | 2062 1886 711% 1536 1364 1189 I01'7 84°5 67°5 25 2661 245°2 2243 2035 1828 1622 I41°7 I21'2 1007 20 35538 3298 3040 2783 252:°0 2270 2014 1760 1506 15 | 5053 4710 4369 4028 2688 3349 3011 26773 233°5 to | 80475 7537 7029 6522 6016 5511 5006 4502 399°9 To obtain any required quantity of any given percentage of alcohol from the above table, take for example, required 250 cc. of 60 per cent. of alcohol, how much go per cent. alcohol will be required. 1500: 250::100 = 1662 cc. MICROSCOPICAL RESEARCH Commercial methylated spirits may be used for scientific purposes when it is not necessary to use it for making up lower percentages by the addition to it of water, on the addition of which it becomes turbid, and specimens or sections when placed in it cannot be observed. For permission to use “ordinary” methylated spirits, free from the mineral additions, a permit must be obtained from the Excise authorities, for which purpose information may be obtained from any of the Excise offices. be found useful by the Student in working out formula, etc. :— To convert Grammes into Grains Fe 3 ue % ozs. Avoir. ss A Kilogrammes 3 Pounds es x Grains 3 Grammes x i Avoir., ozs. rf > me 5 Troy, ozs. - a Fa PA Cubic centimetres (cc.) » Imperial fluid ozs. i 3 Litres 3 Fe » . Fluid ozs. » Cubic centimetres (cc.) Pints re Litres ” ” x x x x The following table of factors for converting from one scale to the other will 15°432 0'03527 2°2046 0'0048 28°35 31104 0'0352 35°2 28°42 0568 28°42 cc.=I1 fluid ounce. 56834 cc.=one pint. 1000 cc.=35'196 fluid ozs. Absolute alcohol of Commerce is generally 99'4 to 99°957%. The sp. gr. of Ether used in the Celloidinizing process is ” of Chloroform used in the Paraffin and Celloidinizing process is of the Oil of Cloves used in this work is of the Oil of Cajeput used in this work is of the Oil of Bergamot used in this work is "720 I I 490—1°495 ‘052 ‘921 884 GENERAL REMARKS 3 The term “Pure Benzol” used in this work signifies Rectified Benzol free from Thiophen. Cap to oil cup. Oil cup and narcotic carrier. Regulating tap. Graduating tube. Oil drop. Alcohol containing specimens. A Graduating and Narcotising vessel, for graduating specimens from a light to a heavy fluid, and for narcotizing zoophytes, &c. Clearing Ottls. Oil of Cajeput is recommended as a clearing agent for stained sections, because of its neutral action on the stains used. 4 MICROSCOPICAL RESEARCH Oil of Bergamot is used as a clearing agent for all celloidinized sections on account of it having no appreciable action on the celloidine. Celloidine is dissolved when placed in contact with oil of cloves or cajeput. Oil of Cloves, being slower in its action than either of the above, is generally used for clearing tissue in bulk, and for graduating delicate objects out of alcohol, which would be contracted or destroyed by transferring them direct from alcohol to the oil (Fig. A). Cellordine. Schering’s celloidine chips are recommended for use; it is put up in one ounce sealed boxes. Celloidine is soluble in a mixture of equal parts of absolute alcohol and ether, and is used for “celloidinising” animal and vegetable tissues, when it is required to bind or hold together any loose parts which we may desire to examine in their natural positions. Celloidine does not “infiltrate” the tissues, but only surrounds and binds, or holds them in position during the manipulative processes. Acidulated Alcohol. Hydrochloric acid, two drops (minims). 92 per cent. alcohol, one ounce. This solution is found useful in reducing the colour of overstained sections, Sections stained with carmine or haematoxylin may require a stronger solution for their reduction, or a prolonged soaking. Aniline-stained sections seldom require the use of acid alcohol, alcohol free from acid will generally be found sufficient for the purpose. Sections on which acid alcohol has been used must in all cases be rinsed in alcohcl free from acid before passing them on to the next process, ‘ Bleaching Solution. Sections must never be bleached when it is desired to retain the cell-contents, but only when it is necessary to cleanse the sections of resinous matter so as to admit of better staining. After bleaching, the sections must in all cases be freed from the chlorine by prolonged washing in water, or by reagents, before staining. COLLECTING, FIXING, &c. 5 Cuaprter I. Collecting, Fixing, and Preservation of Specimens. Tue ultimate aim in microscopical research should be the acquisition of knowledge. Among the chief factors leading to this attainment are the methods adopted for collecting, fixing, and preservation of specimens to be operated on and studied. In the commencement of work let it be understood that every process a specimen is put through must be for the purpose of preparing it for the next process, and that every process must be carried out in its proper sequence from collecting the specimen to. the finished microscopical slide. In all cases the specimens must be collected at the time and under the conditions best suited to the end for which they are intended. The phenomena of life, the cycle of life from the single nascent cell from which it springs through all the manifold changes that take place in building up the individual plant or animal to its maturity, its reproduction, its decadence and disintegration, the ultimate separation of its elements and return to the earth and air from which it took its birth, should form the basis and goal of our investigations; and the importance of collecting the necessary materials at the proper time and under suitable conditions that these phenomena may be thoroughly examined, cannot be too strongly emphasised. It is necessary, therefore, that the student in practical microscopy should 6 MICROSCOPICAL RESEARCH have some knowledge of field and garden botany, and of natural history generally. Day by day we see plants spring from the earth, become bigger and bigger, and send out ramifications in every direction, but how few stop to study the phenomena of their growth; how does the plant or animal grow is the question that the microscopist has to answer; and herein lies the necessity for the use of chemical killing, fixing, and preservative agents in microscopical investigations, so that the dead tissues of plants and animals shall as nearly as possible present to the observer their natural conditions as when in a living state; thus enabling the investigator to trace out all the varied changes that take place during the life cycle of the plant or animal under observation. Many methods have been devised for this purpose, among which the following have proved efficient, and will answer for the killing, fixing, and preservation of the specimens used to illustrate this work. Fic. 1.—Collecting Case, containing six 34 in. x 1} in. tubes for fixatives. The most simple preservative is methylated spirits of 92 per cent. in strength, into which may be placed immediately as collected, such specimens as roots, stems, leaves, leaf or flower-buds, ovaries, seeds, COLLECTING, FIXING, &c: 7 &c., when it is intended only to show the position of the various parts of which such specimens are composed. By the action of the spirits on the specimens, resinous substances will be dissolved out, and the spirits become discoloured. The latter should now be poured off and the specimens covered with fresh spirits, in which they may remain indefinitely. For the fixation of cells, cell-contents, nuclear division, &c., of both plants and animals some specific fixative must be used, so as to fix and preserve them in their natural condition. As a general rule the reagents best suited for this purpose are :— Osmic acid. Chromic acid. Acetic acid. Picric acid. Corrosive sublimate. Formaldehyde. AAR YS = These are combined to suit the purposes for which they are intended, the formulz found most useful are made up as follows :— 1. Plemming’s Flurd. 25 cc. of 1 per cent. chromic acid, 10 cc. of 1 per cent. glacial acetic acid, 55 cc. of water, and immediately before use add 10 cc. of 1 per cent osmic acid. For use this solution should be taken into the field and immediately the specimens are collected they should be cut into as small pieces as possible compatible with the purpose for which they are required and placed directly into the solution, and allowed to remain there sufficiently long for it to completely penetrate the tissue, This will vary from one B 38 MICROSCOPICAL RESEARCH to twenty-four or more hours, according to the size and density of the pieces dealt with. The specimens should now be soaked in water for four to six hours, if small, and for twenty-four or more hours, if large, to free them as much as possible from the acids, the water being changed at intervals during the process; or a much better method is to ze up the specimens in a muslin cloth, place them in a dish, and allow a stream of water to pass through them. The specimens should now be graduated through 25 per cent., 4o per cent., 60 per cent., 75 per cent., 85 per cent., and 92 per cent. alcohol, in which they may remain for future use. The specimens must stand in each strength of alcohol for one or two hours during the first few changes, and for eight to twelve hours during the latter two or three changes to prevent contraction of the tissues. The method of “graduating” applies in every case when it is necessary to transfer dedzcate specimens (or sections) from a dense to a light medium, or vce versa. 2. Chromo-acetic Acid Solution. 1 gram chromic acid, 1 cc. glacial acetic acid, ;Stock solution. 98 cc. water. This solution is much cheaper than Flemming’s fluid, and may be kept ready for use in quantities suitable for one’s requirements. It is the best all-round fixative for general purposes. The solution is a strong one, and should be weakened by the addition of water when it is desired to fix very delicate objects, or weaker solutions may be made up to suit individual requirements. In my own work it is used in its full strength for large pieces of tissue, developing vegetative buds, or male and female cones of pinus, vegetative buds and fertile spikes of COLLECTING, FIXING, &e 9 equisetum, &c. For flower or leaf buds take ‘wo parts of the solution and add one part of water, and for more delicate objects still (spirogyra) I take egual parts of the solution and water (Fig. 2). Fic. 2,—Spirogyra, fixed with equal parts of the chromo-acetic solution and water, magnified 80 dia. A, frond in normal vegetative condition; B, in conjugation. This solution, with careful modifications, some knowledge relating to the nature of the objects under manipulation, and common sense, will serve as a fixative for, practically, the whole range of vegetable histology. After fixation the specimens must be treated as described above. 3. Corrosive-prcro-formaldehyde Solution. Boiling water, 100 cc., = : : : Dissolve and add Corrosive sublimate, 2°5 grams, | Picric acid, 1 gram. Allow to cool, and immediately before use add Formaldehyde, 40 per cent. 10 cc. This solution fixes vegetable and animal cells and their contents, perhaps, more completely than any known fixative. I have used it for 10 MISCROSCOPICAL RESEARCH the fixation of developing pine buds, but have discarded it in favour of the chromo-acetic solution. It fixes everything so completely that the resinous substances in the cells, even after cutting the sections extremely thin, is so dark that the albuminous contents can scarcely be determined. After fixing, the specimens must be thoroughly washed, and afterwards graduated to 92 per cent. alcohol, in which they may remain for future use. 4. Corroswe Sublimate. (A) A saturated solution in water. (B) A saturated solution in 75 per cent. alcohol. The water solution is used chiefly for the fixation of delicate specimens of the lower cryptogams. For use take 100 cc. of the solution and to this add 1 cc. glacial acetic acid. Specimens will fix in this solution in ten to fifteen minutes. After fixing, the specimens should be washed in several changes of water, and then graduated to 92 per cent. alcohol, when the nature of the specimen will allow this to be done without contraction of the tissue, in which case the specimens should be placed direct into a solution made up of eight parts distilled water and one part each of 92 per cent. alcohol and pure glycerine. This solution will serve also for permanently mounting the specimen. When the alcoholic solution is used, which is applicable for general purposes, the specimens must be washed in several changes of 75 per cent. alcohol, to which a few drops of iodine solution, commercial, should be added, so as to liberate any corrosive that may have crystallised in the tissue, which if not removed may completely spoil the finished preparation by the appearance of metallic masses. When the iodine solution is added the alcohol takes on a brownish colour, which, however, soon clears, and if the addition of the iodine be COLLECTING, FIXING, &c. II continued, the washing may be considered complete when the alcohol no longer clears, but retains the brown colour. The specimens may now be transferred to 92 per cent. alcohol, in which they may remain indefinitely. 5. Pucric Acid Solution. A saturated solution of picric acid in 92 per cent. alcohol. This solution is used chiefly for fixing large pieces of tissue, its action being very rapid. The specimens must remain in the solution for several hours, or until the solution has completely penetrated through them; they must then be transferred to alcohol, which must be changed at intervals for a day or two to wash out the acid as far as possible. Vegetable tissue takes up picric acid very quickly and retains it so persistently that even after prolonged and_ repeated washings, it is scarcely possible to eradicate the acid until the tissue is sectionised, and the sections passed through two or three changes of alcohol. 6. Formaldehyde. Formaldehyde, 40 per cent., 3 cc. Water bay an OF ce, This is also made up in one to five per cent. solutions, according to requirements. A three per cent. solution being, perhaps, the most useful for all-round work. It is suited to most delicate objects, but is used more frequently in animal than in vegetable work. A two per cent. solution of formaldehyde may also be used as a mounting medium, but a stronger solution has a tendency to turn the objects opaque after they have been mounted for some weeks, and on this account I prefer carbolised water as a mountant. 12 MICROSCOPICAL RESEARCH The reason for the use of “fixatives” has already been stated. General rules only can be laid down for the guidance of students, the ‘‘absolute” is gained only by actual experience. Collecting specimens at the proper time and under suitable conditions, with a due regard to their fixation and after treatment, is of the greatest importance in histological work, as on this more than anything else depends the success of the after operations,—no after manipulative skill can rectify bad fixation and preservation. It is only after we have gained a knowledge of the nature of things that we can decide readily as to the use or non-use of fixatives, whether in any case their use is imperative, or altogether unnecessary. In all cases this will be indicated when dealing with the “type preparations” used to illustrate this work. The action of a true fixative is clearly illustrated by the photomicrograph Fig. 3 A, and an unsuitable one is shown at B. Both sections are prepared from the same specimen, one being fixed with the chromo- acetic solution, the other placed direct into 92 per cent. alcohol. The former is perfectly fixed, the cell-contents are preserved, and no contraction or shrinkage of the cell-walls has taken place. In the other there has been a complete breakdown and disorganisation of the entire system of cells and their contents, brought about by the rapid “dehydrating” action of the alcohol on the tissues. The specimen is an aquatic one, the cells of which are filled with water and the tissues are too delicate to resist the action of the alcohol, hence their collapse and destruction. On the other hand, where the vascular system of the specimen is more highly developed, and when grown in dry soil, it may be plunged direct into 92 per cent. alcohol without any apparent injury to the tissues, as is clearly illustrated by the roots of Ranunculus and Zea mais (Plate 1, Figs. 1 and 4). COLLECTING, FIXING, &c. ERS ERA ET, BL es oy oe Fic. 3.—Transverse sections through root of “ Acorus calamus” magnified 60 diam. A. Fixed with chromo-acetic solution, and graduated to g2 per cent. alcohol. B. Placed direct into 92 per cent. alcohol immediately when collected. 13 14 MICROSCOPICAL RESEARCH Cuapter II, Tools and Methods of Work. THE apparatus and instruments requisite for use in microscopical research vary in accordance with the requirements of each individual worker. The acquisition of a complete outfit for all-round research work is an expensive business, but much good work may be done with comparatively simple and inexpensive tools. In all cases the worker’s Fic. 4.—Tools required in the dissection of animals and plants. outfit should be as simple as possible compatible with the work it is intended to undertake. The following list will be fairly complete for ordinary purposes, viz., a microscope with rin., 3in., and in. objective, TOOLS AND METHODS OF WORK 15 microtome and section knife, paraffin bath and _ spirit lamp, turntable, forceps, needles, scissors, dishes and watch glasses, a graduated 100 cc. measure, pipettes, sable brushes of various sizes, wire clips, tin rings for building up cells, 3in. by rin. plain slips, cavity slips, circular and oblong cover-slips, mounting block, stains, balsam, glycerine jelly, clearing-oils, bottles for specimens, incubator or drying apparatus. Good microscopes are supplied by the leading opticians, but some difficulty may be experienced in obtaining a suitable microtome from this source, as each type appears to have been designed for one branch Fic. 5.—The old “Flatters” microtome, from which the present microtome was evolved. of work only, and that chiefly animal histology. I, therefore, describe somewhat fully the instrument I have used in my botanical and textile work since 1892. It is the outcome of several previous instruments, all of them more or less deficient in some respects. See Figs. 6 to 9. 16 MICROSCOPICAL RESEARCH Fic. 7.—Showing knife-plate turned aside to Fic. 6.—Showing Microtome ready for use. allow the easy removal of the paraffin, after use. = Fic. 8.—Showing underside of oblong top : : and side view of carrier of same. Fic. 9.—Vertical section TOOLS AND METHODS OF WORK 17 THE “FLATTERS” MICROTOME. The microtome is made of brass, the tube or well is 3in. deep by rin. in diameter internally, the spindle is of the same length, the screw having twenty-eight threads to the inch. The spindle is fitted with a thumb-screw at the lower end to admit of the toothed discs being easily changed. A spring stop, the tension of which can be adjusted, works in the teeth of the disc, thus ensuring a series of sections of uniform thickness. Three discs, divided as follow, are found to be sufficient for ordinary purposes, viz. :— No, 1. 72 teeth, giving sections zo'scin. in thickness No. 2. 54 ae io rosin. Approximately. No. 3. 43 i is reovin. ms The latter being the one mostly employed for general purposes, and which when moved two notches gives sections sovin. in thickness. Numbers 1 and 2 are used chiefly for the production of sections of animal tissue and of textile fibres. To ascertain the thickness of the sections obtained, multiply the notches in the disc used by the number of threads per inch on the spindle. The knife plate, 2in by 4#in., is made of hardened brass polished ‘dead flat,” and has an aperture the same diameter as the tube, tapering slightly to the top to prevent the specimen from turning or rising while the sections are being cut; it is attached at one end to the headstock by a stout screw, and is securely held in position by a reliable catch, which is clamped under the headstock. The specimen to be cut is placed in the well of the microtome and melted paraffin wax, melting point 130 deg. F., poured in; this is allowed to set, and the superfluous wax is then removed. The “candle” so formed is then moved upwards by turning the toothed disc. The sections are cut by passing the knife 18 MICROSCOPICAL RESEARCH obliquely over the knife-plate, which is always kept moist with alcohol. The instrument is fitted with an oblong top, very useful for the production of sections from specimens which are too large for the ordinary well; it fits on top of the microtome (Fig. 10), and is held in position by a series of clamps. The aperture is din. wide by rin. long by thin. deep, The carrier fits into the tube of the microtome and is actuated by the spindle in the usual way. Fic. 10.—The Microtome, with the “oblong top” attachment set up for use. The specimens to be cut are embedded in the well of the microtome in various ways according to their form and nature, the pieces to be cut must have one surface or edge levelled so that it will stand vertical on the carrier, and should be placed a little out of centre towards the cutting side, or operator, so that the knife has not to travel so far over the aperture before coming in contact with the specimen. As the specimen to be cut is surrounded with hard paraffin it is advisable to remove it from the front of the specimen, that the TOOLS AND METHODS OF WORK 19 knife may not be impeded by having to pass through it (Fig. 11). Very small objects that require some support to keep them in position during the time the paraffin is setting may be arranged on a piece of cork (which has been previously boiled in paraffin to expel the air) with a series of pins, these are withdrawn after the paraffin is set hard. Fic. 11.—Well of Microtome, showing specimen embedded and paraffin removed from the cutting surface. When there are a number of specimens to be sectionised, imbedding tubes, or moulds, Figs. 12 B may be employed; these should be about three-quarters of an inch high and of the same diameter as the well of the microtome, their inner surface must be smeared with glycerine to facilitate the removal of the blocks when the imbedding is complete,— these can be pushed out of the tubes and placed in the well of the microtome as required, a narrow groove must be cut longitudinally along the surface of each block to allow the air to escape from the well of the microtome, as it is being pushed in. A large series of imbeddings 20 MICROSCOPICAL RESEARCH may be made in this way, and the blocks placed in the stock-jars, where they can remain indefinitely. The specimens to be cut must in all cases be imbedded in the well of the microtome in such a manner that the section-knife shall pass through the exact zone of tissue required for examination. A Ns Wee vee ek eer ener go fl ae ns Vu e ae : L Fic. 12.—Paraffin bath with tripod and bunsen. A. Vertical section. B. Imbedding or moulding tubes. TOOLS AND METHODS OF WORK 21 The section-knife best suited for general use in botanical work is one having a straight handle with a fixed stout blade three to three and a half inches long, and slightly hollow-ground on both sides; the knife may be set on any ordinary stone, with the use of water and fine emery or carborundrum powder. A very small amount of this powder is placed on the stone with a few drops of water, and first rubbed into a very fine paste with an old disused section-knife. The knife for use is now set on this by passing it lightly over the stone edge forward, the traverse being from apex to base, and base to apex. The back and edge of the knife must be parallel to each other, and in true line from back to edge. The Art of Cutting Sections. To make a complete study of a solid mass of tissue, sections must be cut at different planes to each other, viz., ‘‘transverse,” “radial- longitudinally,” and “tangential-longitudinally.” The transverse section Fic, 13.—Diagram of dicotyledonous stem. 1. Tangenital area passing through the bast fibres. 2, Tangenital area, passing through the xylem. 3. Radial line, passing through the pith. 22 MICROSCOPICAL RESEARCH is taken at s7ght angles to the axzs of growth. The radial longitudinal section is éaken through the axis of growth. The tangential longitudinal section may be varzed in its direction between the central axzs and the circumference in accordance with posztzon of the speczal tissue it is desired to examine. Each section when placed under the microscope will present to the observer a surface cut at right angles to each other, and without which a complete study of the tissue could not be made. It is, therefore, of the greatest importance that the student should A B Fic. 14.—Photographs of the operator in the act of cutting sections. A, showing the point of the knife in contact with the specimen. B, showing the position of knife at-completion of cut, with the section resting on the blade and the paraffin curled over in front of it. ‘The movement of the cut is a sliding one, its traverse being from right to left, the full length of the blade having been used in the operation. thoroughly understand the principle of section-cutting, as on this, more than anything else, depends his success or failure in the production of satisfactory preparations. The melting point of the paraffin used for imbedding purposes is 130 degs. Fahr. When new it has a tendency to “crumble” when the TOOLS AND METHODS OF WORK 23 sections are being cut, but its consistency may be modified by the addition of a little vaseline, and is much improved by constant and repeated use. There is no other medium required for imbedding ordinary roots and stems, and many of the leaves, seeds, &c., even the cellular aquatic roots and stems are imbedded for cutting in this way. The specimen is taken out of the stock jar, placed in position in the well of the microtome, and held in position with a needle while the melted paraffin is being poured in. After the “candle” so formed is set hard, the superfluous paraffin is removed from the knife-plate with an old section knife, which completes the operations preparatory to section-cutting. Preparing Specimens for Sectionising. Most of the delicate tissues and all specimens having loose parts require some special method of treatment before they can be sectionised with success. The methods best adapted for this purpose are the Fic. 15.—Diagram showing the various positions of floral parts in a lily, the dotted line being the line of cut for celloidinising purposes. 24 MICROSCOPICAL RESEARCH “paraffin” and the ‘“‘celloidinising” processes. Under treatment with paraffin, the tissues of plants and animals become infiltrated and form a solid block. With the celloidinising process the farts of the specimens only are surrounded, held in position and supported during the sectionising and succeeding manipulations, the celloidine adhering to the section when cut is treated as being a part of the section itself, and is mounted as such on the finished slide. On the other hand paraffinised sections are fixed on the slide with albumen and the paraffin dissolved out. Specimens it is intended to celloidinise must have their parts so exposed as to admit of the access of the celloidine, and to facilitate this operation the afex of anthers, ovaries, leaf and flower buds are cut away, the cut being sufficiently low to expose all the parts it is intended to keep zz sztw when the sections are taken (Fig. 15). The Cellotdinising Methoa. 1. Place the specimens in absolute alcohol for twelve to twenty-four hours, according to size and density of specimens; if they are large the alcohol should be changed once or twice. It is important that the specimens should be thoroughly dehydrated before passing them on to the next process. 2. Place the specimens in ether, allow to stand twelve hours; change as above, if necessary. 3. Place the specimens in equal parts of Adsolute alcohol and ether, to which add half an inch of Schering’s celloidine chips, this will dissolve in four to six hours, and penetrate the interstices of the specimen. A little of the celloidine chips must be added day by day until the sodutzon becomes sufficiently concentrated to form a TOOLS AND METHODS OF WORK 25 selly-like mass; each addition of celloidine must be ‘horoughly dissolved before the next is added, stir or shake occasionally to facilitate the process. 4. Transfer the specimens from the mass with /ixe-pornted forceps and drop them one by one into chloroform. There must be sufficrent celloidine adhering to the specimens to form a protective coat. The action of the chloroform is to coagulate or solidify the cellocdine and form a sold block. At first the specimens will fHoat on the surface of the chloroform, but as they become solidified they will szz& to the bottom of the vessel; the process will be complete in six to eight hours, when any surplus celloidine adhering to the specimens may be removed, and the specimens placed in 92 per cent. alcohol, where they may remain indefinitely. In actual practice I find it preferable to use two parts of ether and one part of absolute alcohol, rather than equal parts of each,—-the specimen works out more solid and is better to cut, celloidine also has a less tendency to take up the stains. The specimens are imbedded in the well of the microtome with melted paraffin, in exactly the same way as the uncelloidinised specimens; the sections are placed direct into 92 per cent. alcohol, from which they may be stained, then cleared with oil of derzgamot, and transferred to a thin solution of balsam and benzol, out of which they may be mounted when convenient. The reason for the use of oil of dezgamot, in clearing celloidinised sections, is that it has no appreciable action on the celloidine, whereas oil of cazeput and oil of cloves dissolve the celloidrne, and by their use the sections would be destroyed. To Infittrate Specimens with Paraffin. Specimens of either vegetable or animal tissue which cannot be cut sufficiently thin by the celloidinising method and by means of the 26 MICROSCOPICAL RESEARCH ordinary microtome must be infiltrated with paraffin, and cut by means of some mechanical arrangement. The ‘Cambridge Rocking Micro- tome” is well suited for this purpose, its traverse movement being actuated to radon of an inch, and by its use sections have been obtained in my laboratory of animal tissue (suprarenal gland of Cat) cut to rs'o0, sv'v0, and rotov of an inch in thickness for “test” purposes for Mr. J. E. Storey, of the Manchester Microscopical Society. For ordinary purposes sections seldom require cutting thinner than sy0 of an inch in thickness. Fic. 16.—The Cambridge “Rocking” Microtome. The primary method of infiltrating vegetable tissue with paraffin is the “Chloroform” or the ‘“Xylol” process, either of which has but little injurious effect on the tissue if properly conducted. I give here full details for the Chloroform method, but Xylol may be substituted for chloroform by those who prefer it :— 1. Transfer the specimens from 92 per cent. into Adsolute alcohol, allow to stand twelve to twenty-four hours according to size and density of specimen, change the alcohol once or twice during the time. 2. Place the specimens in equal parts of Absolute alcohol and chloroform, and allow to stand twelve hours. TOOLS AND METHODS OF WORK a9 3. Place the specimens in chloroform; allow to stand twelve hours. 4. Place the specimens in a saturated solution of paraffin in chloro- form; allow to stand twelve to twenty-four hours or longer according to size of specimen. Melting point of the paraffin should be about 130 degs. Fahr. 5. Transfer the specimens to paraffin (free from chloroform) melted in the water-oven—the heat must not be higher than one degree above the melting point of the paraffin; allow the specimens to remain in the water-oven for two or three hours, change the paraffin at intervals. By this means the chloroform is evaporated, or thrown off, and the tissues become infiltrated with pure paraffin. It would perhaps be preferable to use several tubes containing melted paraffin and transfer the Fic. 17.—Moulding frame, formed of two L-shaped pieces of brass, with arms one and a half inches long and half-inch angles. specimens from one to the other, allowing them to stand a short time in each, care being taken that the heat is only just sufficient to keep the paraffin in a fluid condition. 6. Have ready a quantity of freshly-melted paraffin, pour this into the mould (Fig. 17), place in the specimen, and arrange it in 28 MICROSCOPICAL RESEARCH position for cutting, with hot needles, and as soon as the “block,” or mould, is just set on the upper surface plunge the entire mould into cold water; by this means the block will be cooled uniformly. If this operation be carried out successfully the block, when removed from the mould, will appear uniformly transparent. Should this not be the case one of three things will have taken place :— (1) The specimen has not been properly dehydrated; (2) The chloroform has not been completely evaporated ; (3) The block may have been cooled too slowly. Either defect will be indicated by a milky opacity in the centre of the block surrounding the specimen; the only remedy is to re-melt the paraffin and re-mould. 7. The block containing the specimen should now be trimmed into a square; warm the paraffin in the orientator, or carrier of the microtome, and also the base of the block containing the specimen, press them gently together; then seal the joint with a hot needle, and the preparation is ready for sectionizing. After the sections are cut they may be fastened to the slide with Mayer’s albumen solution. Formula. White of egg, 50 cc. Pure glycerine, 50 cc. | Shake well to dissolve. Filter for use Salycilate of soda, 1 grain TOOLS AND METHODS OF WORK 29 Place a drop of the solution on the slide and smear it evenly over the surface, and then wipe as much off as possible either with the hand or with a clean piece of non-linty cloth. Now place a few drops of water on the slide, or float the section on to it, warm gently over the spirit lamp, when the paraffin and the section will become smoothed out and free from wrinkles. Now tilt the slide and allow the water to drain off, guiding the sections into position with a needle while doing so. The slide should now be placed in the warm incubator for an hour or two to dry. The paraffin may now be removed by gently heating the slide over the spirit lamp to the melting point of the paraffin, and placing direct into xylol, which completes the operation by dissolving out all traces of paraffin, and by the heating operation the albumen will be coagulated, and the section cemented on the slide. The slide with the sections should now be rinsed in one or two changes of alcohol and it is ready for staining. After staining, place the slide in xylol for ten minutes to clarify the section, drain off the surplus xylol, and mount the section in ‘xylol balsam.” Staining. The art of staining consists in differentiating the various tissues of which plants and animals are composed, the operations being governed by the chemical affinities of the tissues to be stained, and these vary considerably in accordance with the age of the tissues themselves. No tissue will take up a stain unless there is an affinity between the tissue and the stain, and no simple tissue will take up two stains unless the cells composing it are nucleated. A preparation composed of two or more tissues may be stained in two or more colours, but two or more colours cannot be put into a preparation unless there is a tissue for each of them, for example, developing vegetable tissue may be differentiated 30 MICROSCOPICAL RESEARCH to the extent of half a cell-wall, the juncture of the cells first showing a slight lignification, and having an affinity for a stain giving a lignin reaction, while the central portion of the cell-wall is still unlignified and takes up a stain having an affinity for cellulose; each stain gradually merging into the other and in each case showing the deepest in colour at that particular part having the greatest affinity for it. The production of a correctly stained section does not consist so much in putting the stain into the tissues, as in washing it out; the finest preparations being those that have been overstained rather than otherwise and the colour stripped out again, or reduced to the required tint by the use of acid-alcohol or by a prolonged soaking in alcohol, When a nuclear preparation only is required, the reducing operation must be continued until the colour is removed from all the tissues except the nucleus. When it is desired to double stain a preparation composed of two or more tissues, it must be placed first into the stain having an affinity for the primary ground tissue, and treated exactly as if staining only for one colour, and then placed in the second or counter-stain. After staining with the second colour, the sections should be rinsed quickly in two or three changes of alcohol, and immediately the first colour stands out clearly the process must be stopped by transferring the sections to the clearing oil. As a simple example of staining, let us take Grenacher’s formula of borax carmine, which is practically a permanent stain for developing tissue. To remove this stain from tissue for which it has an affinity requires the use of acidulated alcohol, or to be left for weeks in alcohol free from acid, or placed direct from the alcohol into water when diffusion currents would be set up and the carmine liberated. On the other hand Malachite green, which is a counter stain to carmine, having an affinity for formed or lignified tissue, may be TOOLS AND METHODS OF WORK a5 removed from the tissue by a comparatively short washing in alcohol without the aid of acids; it follows, therefore, that in all cases the sections must be stained first with the primary or most permanent colour, and secondly with the counter-stain, out of which they must be rinsed quickly and transferred to the clearing oil as described above. Protoplasmic and nucleated tissue takes up stains more readily than formed or dead tissue. A diffuse stain affects all the elements of a preparation, while a specific stain affects only certain elements, or stains some elements more deeply than others. A nuclear stain is one which has a strong affinity for the nucleus, and which is retained by it when all the surrounding tissue is freed from colour. Grenacher’s formula of borax carmine and Kleinenberg’s formula of Hematoxylin are diffuse stains ; whereas on the other hand Brazilin and Delafield’s formula of Hzmatoxylin are decidedly nuclear and specific stains, and for permanent work are perhaps the most useful stains known to us, with Safranin, Gossypimine, and Malachite green as counter stains. Staining will vary in accordance with the nature of the tissue dealt with and its prior treatment, if, for example, we prepare sections from a lateral branch, one eighth of an inch in diameter, of the Lime tree, collected at the end of March when the tree with all its ramifications is infused with new life, they will take up the stains much more readily and retain them much more persistently than if collected and prepared in December when the tree is in a more or less dormant state. Mounting and Finishing the Shade. After the sections have been stained and cleared, they should be allowed to stand for a short time in a thin mixture of Benzol balsam; 23 MICROSCOPICAL RESEARCH this not only hardens the sections, but also frees them from the clearing oil, which, if not removed before the sections are permanently mounted, diffuses out afterwards and discolours the balsam; a common cause of the dirty brown appearance of so many commercial slides. The sections may remain in the benzol balsam indefinitely, if needs be, without deterioration in colour, as in this condition they are practically mounted. In my own work I have sometimes a dozen of these ‘“‘balsam-pots,” each containing half a gross of sections waiting for days Fic. 18.—Complete staining and slide-mounting outfit. A. Storing cabinet. B. Microscope, mounting blocks, section dish, drying tray, &c. and in some cases weeks before they are permanently mounted, the rims are smeared with balsam and a glass cover placed on them, which practically seals them protecting the sections from dust, and the benzol from evaporation. The sections are mounted permanently by placing a drop of ordinary Benzol balsam on the centre of a plain slip, the section is lifted out of the thin benzol-balsam and placed in this; and then a cover slip is placed on the section, the whole being clamped in o TOOLS AND METHODS OF WORK 33 position by means of the wire “‘spring-clip” (Fig. 18 B), until the slide is baked, or has set hard by the evaporation of the benzol. In mounting solid specimens on cavity slips, the cavity must first be filled with balsam, the specimen placed in this, and then a cover slip, whether round or oval, one size larger than the cavity is placed in position. In this case the spring-clip must be applied with only just sufficient pressure to keep the cover in position during the drying operation, or e ; rs) “4 a sede 00 Fic. 19.—Types of 3in. by 1in. slips, used for mounting microscopical objects. A. Plain slip. 1-5. Round cavity slips. 6-11. Oval cavity slips. there is a danger of the cover being depressed, by which means some of the balsam would be squeezed out, and when the clip was removed the cover would again spring up, causing suction thereby and consequent air-bubbles to find their way into the cavity. When it is required to mount large entire specimens in fluid and the ordinary cavity-slip is not suitable to this requirement, a cell should be built up round the cavity with tin rings. 34 MICROSCOPICAL RESEARCH The slip must be placed on the turntable, and a ring of brown cement run round the cavity (Fig. 20, B, 5-6) ; this must be allowed to set hard, then another layer of the cement should be applied on the top, into which the tin ring must be imbedded, and another cement ring to seal it completely to the first. This must be allowed to dry thoroughly, and, if necessary. the process repeated until the cell is built up to the required height, so that the specimen can be enclosed without having to use pressure upon it. A light “¢acky” ring of brown cement should now Fic. 20.—A. Rings for building up cells. A”. Cardboard rings made out of old post cards, used for dry mounting. B”. Tin rings for balsam or fluid mounts. 1. Upper side of caps for same. 2. Underside of caps showing the flange. B, 1-6. Cells at various stages of construction. 4. With black background for opaque mount. 5 and 6. Cells surrounding a cavity to give depth. ‘be applied to the ¢op of the cell, the cell filled with the mounting fluid, and the specimen arranged. in position, (care being taken that the fluid stands slightly above the margin of the cell), the cover slip must now be applied to the left margin of the cell and gently lowered into position, at the same time flushing out the superfluous fluid. Now place the mount on the turntable, which is set in motion, and a little pressure applied to TOOLS AND METHODS OF WORK 35 edge of the cover slip with the forceps to imbed it firmly into the ‘‘¢achy” ring (Fig. 21, A and B). _ If this be done carefully no difficulty will be experienced with air bubbles. A little of the fluid may have been pressed out of the cell by this operation, this must be absorbed with filter paper, and a fresh ring of cement applied to seal up the junction of cover and cell. The preparation must now be allowed to dry A. B. Fic. 21.—A. Method of placing cover slip on a fluid mount. B. Turntable in action and cover slip being pressed into position. C. Mounting plate on turntable carrier. thoroughly for some days, when it should be rung with old gold size, and while this is still moist a protective cap (Fig. 20, A, 1-2) must be placed over the top of the cell, and a fresh stout ring of gold size applied over the entire structure, and the preparation again put away to dry. A ring of “zinc oxide” made with the proper proportion of “old gold size” may now be applied; this must over-/ap all the previous rings, and if the cements be properly made and properly applied the preparation will stand for a lifetime. 36 MICROSCOPICAL RESEARCH The method of building up a cell for a balsam mount is much more simple than the above. Balsam is placed in the cavity; then the specimen is arranged in position, a tin ring is cut across and each half arranged round the cavity with a small space left between them (Fig. 22). A cover slip is placed on the ring and the whole held in position by a spring clip. If the cell is not already full of balsam more may be run under the cover slip through one of the openings in the ring. The preparation must be allowed to dry sé/owéy; and after the superfluous balsam is removed, the cell should be protected with several layers of brown cement, and ultimately finished off with zinc oxide. Fic. 22.—Tin ring divided for building up balsam mounts. Cleaning, Ringing, and Finishing the Slides. Balsam preparations are dried by being placed in a water-oven for sixteen hours at a temperature of 140 degs. Fahr., or in the absence of the water-oven, should be put aside in a warm place for a few days to evaporate the benzol and set the balsam. If the water-oven be used for this work, the slides, after drying, must remain in it until it has gradually cooled down; they should then be taken out, the spring clips removed, and the superfluous balsam cleaned off with the “hot-knife.” The hot-knife may be made out of an old table-knife by reducing the point end to a chisel edge, which must be square. This is heated in the bunsen flame and then lightly passed round the edge of the cover slip carrying the balsam with it (Fig. 23 A). TOOLS AND METHODS OF WORK ay A. Fic. 23.—-Hot-knifing and ringing slides. A. Hot-knifing. B. Putting on the first cement ring. Fic. 24.—Turntable for ringing ovals, run by clockwork. 1. Guiding pointer, under which the brush is held when ringing. 2. Governor plates of clock. 3. Decentralising motion. 4. Stop-catch. 38 MICROSCOPICAL RESEARCH The smears left by this operation may be removed with a soft cloth moistened with 92 per cent. alcohol. The superfluous jelly, on glycerine jelly mounts, may be removed by washing the slide in cold water with a stiff tooth brush. The slide should now be placed on the turntable and the preparation sealed up with a brown cement ring, as already described. When the brown cement is dry, a ring of zinc oxide may be applied, and on the top of this a finishing ring of asphaltum, or a single black finishing ring may be applied over the brown cement one; the slide should now be labelled and named for use. Fic. 25.—Slides in various stages of cleaning and ringing. 1. Before hot-knifing. 2. First ring of brown cement. 3. Finished with zinc oxide and asphaltum. 4. Oval mount, finished same as number 3. STAINS, REAGENTS, &c. 39 Cuapter III. Stains, Reagents, Mounting-meara, Cements, etc., ther Formule and Manipulation. Tue following formula and manipulative methods are those daily practised by me in the production of animal and vegetable preparations. The list is a very limited one, but will be found sufficiently extensive to cover the requirements of the general worker. The specialist may extend the formule and methods to meet his own wants. No. I. BORAX CARMINE.—Grenacher. Best carmine... ... ... ... ... Sgrams. Borax ...0 60.0 eee eee ee ee eee)=©)- Oc YAMs., Water vc wen us aie ate ad eg, 480kcO: Alcohol 92% .... ... we wee eee 480 ec. Dissolve the carmine and the borax in the water by heat. When cold add the alcohol; allow to stand a few days to ripen; filter for use. ManiIpuLarion. 1. Pour off the alcohol and cover the sections with the stain, and allow to stand fifteen to thirty minutes. 2. Pour off the stain and rinse the sections with acidulated alcohol (two drops of Hydrochloric acid to one ounce or 30 cc. of 92% alcohol); then rinse with alcohol free from acid. Now allow the sections to stand in alcohol for thirty minutes, changing the alcohol during the interval if necessary. 3. Pour off the alcohol and cover the sections with oil of cajeput, and allow to stand fifteen minutes to clarify. 4. Transfer the sections to thin balsam and benzol, and when convenient mount the sections in|balsam. D 40 MICROSCOPICAL RESEARCH The use of hydrochloric acid in process 2 is to remove the surplus stain out of the sections, and if the sections are thick and deeply stained the acid wash must be prolonged until the colour is reduced to the required tint. The acid should never be used if the colour of the section can be reduced to the proper tint by the use of alcohol alone. Two drops of acid to one ounce of alcohol is a useful standard to work with, but in many cases six to eight drops to the ounce will be required; on the other hand the merest trace of acid will remove some stains completely from the sections. Carmine and Hematoxylin are the two chief stains for which the acid should be used; nearly all other stains may be reduced to the required tint by the use of alcohol alone. No. 2. BORAX CARMINE.—Woodward. Best carmine ... ... ... «. « Sgrams. Borax ... Gee ots ae 12 grams. Water Be he. ae ee ae oe SD ee Aleohol 92% sc. ae see nee oie BBO Be. Mix the ingredients and filter for use. The first filtrate. The crystals left on the filter paper must now be washed into 960 ce. of water, dissolve by heating to boiling point; when cold filter for use. The second filtrate. The first filtrate is a weak stain and slow in its action, but it is specially useful for staining entire specimens such as pinne of ferns, spikes of selaginella, etc., and is specially applicable for staining marine zoological specimens. The second filtrate is more rapid in its action, and is useful for staining sections or specimens that are to be mounted in glycerine jelly; the colour being practically permanent in that medium. MANIPULATION FoR THE First FILTRATE. 1. Pour off the alcohol and cover the specimens with the stain, and allow to stand from one to six or eight hours, according to the size and density of the STAINS, REAGENTS, &c. 41 specimen, which must be examined at intervals, to ascertain the progress of staining, and when deep enough in colour, the specimens should be rinsed in two or three changes of alcohol. 2. Place the specimen in Absolute alcohol and allow to stand for twelve hours to thoroughly dehydrate them, and if they are large change the alcohol once or twice during the interval. 3. Pour off the alcohol and cover the specimens with oil of cloves, allow to stand until clarified. 4. Transfer the specimens into thin balsam and benzol, and allow to stand for several hours to free them from the oil of cloves. 5. Mount the specimens in balsam in cavity slips, without pressure. MANIPULATION FOR THE SECOND FILTRATE. Specimens or sections to be stained from water. 1. Pour off the water and cover the specimens or sections with the stain, allow to stand—in the case of sections, for fifteen to thirty minutes,—in the case of specimens, one to three hours according to their size and density. Examine the speciméns from time to time, and when deep enough in colour, rinse in two or three changes of 25% alcohol. If the specimens are to be mounted in balsam they must be graduated to 92% alcohol, and then placed for several hours in absolute alcohol to thoroughly dehydrate, and treated for mounting as described above for the first filtrate. If the specimens are too delicate to admit of mounting in balsam, prepare them as follows for mounting in glycerine jelly. After staining and dehydrating, place the specimens, first, into one part pure glycerine and two parts 92% alcohol; secondly, into equal parts of glycerine and alcohol; thirdly, into pure glycerine; allow the specimens to stand an hour or two in each. 2. Mount the specimens in glycerine jelly. No. 3. HEMATOXYLIN.—Kleinenberg (modified). Hematoxylin ... ... ... «. IJgram. Calcium chloride... ........... 8 grams, in 40 cc. of water. Alum vee eee vee vee 683 Grams, in 60 cc. of water. Alcohol 92% ia) eo ate Gye “RIO CE. 42 MICROSCOPICAL RESEARCH Dissolve the alum and the calcium chloride in their respective quantities of water by heat. Mix the two solutions, and immediately add 700 cc. of the alcohol slowly; allow to stand for an hour; filter. Add the hematoxylin dissolved in the remaining 70 cc. of alcohol, and place the bottle in the sunlight for a few days to ripen; filter for use. MANIPULATION. 1. Pour off the alcohol and cover the sections with the stain, and allow to stand fifteen to thirty minutes. 2. Pour off the stain and rinse with accidulated alcohol, then with alcohol free from acid. 3. Pour off the alcohol and cover the sections with oil of cajeput, and allow to stand fifteen minutes to clarify. 4. Transfer the sections to thin balsam and benzol. 5. Mount the sections in balsam. No. 4. HA MATOXYLIN.—Delafield. Nouctesar StTaIn FoR VEGETABLE AND ANIMAL TISSUES. 200 ce. of a saturated solution of ammonia alum. 2 grams of hematoxylin in 12 ce. absolute alcohol. Mix the two solutions, and expose to light and air for a week. Filter, and add to the filtrate. : 50 ce. pure glycerine. 50 ce. of methylic alcohol (wood spirits). Allow the solution to stand in the sunlight until the colour develops. Refilter for use. MANIPULATION. Sections to be stained from water. 1. Pour off the water and cover the sections with the stain, allow to stand ten to thirty minutes according to requirements, or nature of the tissue under treatment. STAINS, REAGENTS, &c. 43 2. Pour off the stain, and rinse the sections in two or three changes of 25% alcohol, then graduate to 92% alcohol. If the sections are overstained, or if the staining be too diffuse, treat them with the acidulated alcohol until the stain is removed from all parts except the nucleus. The staining may be made more precise in its action if the stain be diluted with several times its own volume of water, and the time in staining be prolonged accordingly. 3. Clear the sections, if uncelloidinized, in oil of cajeput. 4. Transfer the sections to thin balsam and benzol, and when convenient, 5. Mount the sections in balsam. No. 5. AMMONIATED HA:MATOXYLIN.—Enbrlich. NvuciteaR STAIN FOR VEGETABLE AND ANIMAL TISSUES. Hematoxylin ... ... ... 0... ... 15 grams. Ammonium carbonate ... ... ... 1d grams. Absolute alcohol 86) wane ave ive OOD CE: Place in a large bottle and shake occasionally for three days, leaving the stopper out during the intervals. Place the solution in an open dish, and allow it to evaporate to dryness at the temperature of the air. Dissolve the crystalline product obtained in Alcohol 92% ... 0... 0. uu. ue) 750 ec. Pure glycerine 6. as yp an ae TOO GE. Pure water... ... we ue we) 750 Ce. Glacial acetic acid... ... 2... 0. 0...) 5 ee. Ammonia alum... ... ... ... «. 15 grams. Filter for use. MANIPULATION. Sections to be stained from 25% alcohol. 1. Pour off the alcohol and cover the sections with the stain, allow to stand fifteen to thirty minutes. 44 MICROSCOPICAL RESEARCH 2. Pour off the stain and rinse the sections in two or three changes of 25% alcohol, and graduate to 92%. Reduce the colour if necessary with acidulated alcohol. 3. Pour off the alcohol and cover the sections, if uncelloidinized, with oil of cajeput; allow to stand ten to fifteen minutes to clarify. 4, Transfer the sections to thin balsam and benzol, and when convenient 5. Mount the sections in balsam. No. 6. BRAZILIN. NucLear STAIN FoR ANIMAL AND VEGETABLE TISSUES. A. The mordant. Tron Alum... ... 0 0. ue ae ae) gram. Aleoliol 75% vei cus ass’ dee 2s 100 ce, Dissolve and filter for use. B. The stain. Brazalian crystals ... ... 2... ... I gram. Alcohol 75% ... w.. ee ee we 100 8. Dissolve and filter for use. MANIPULATION. 1. Pour off the alcohol and cover the sections with the mordant, A; allow to stand for an hour. 2. Pour off the mordant and rinse once with 75% alcohol. 3. Pour off the alcohol and cover the sections with the stain, B; and allow to stand three, six or more hours, according to nature of tissue dealt with. The thinner the section the longer it should remain in the stain. 4. Pour off the stain and rinse the sections in several changes of 92% alcohol. Reduce the colour if necessary with acidulated alcohol. 5. Clear the sections, if uncelloidinized, with oil of cajeput. 6. Transfer the sections to thin balsam and benzol, and when convenient 7. Mount the sections in balsam. STAINS, REAGENTS, &c. 45 No. 7. PICRO-CARMINATE OF AMMONIA. A Picric acid crystals 9 grams in 400 cc. of 92% alcohol. Best carmine 2 grams in 40 ce. of liquid ammonia. Dissolve the two mixtures, and then add to the carmine solution, 400 cc. of water, and then add to this the picric acid solution. Filter for use. B A saturated solution of ammonium picrate in 92% alcohol. MANIPULATION. 1. Pour off the alcohol and cover the sections with A, and allow to stand one hour. 2. Pour off A, and rinse the sections quickly with alcohol. 3. Pour off the alcohol and cover the sections with B, and allow to stand fifteen minutes. 4. Pour off B, and rinse the sections two or three times with alcohol. 5. Pour off the alcohol and cover the sections with oil of cajeput to clarify. 6. Transfer the sections to thin balsam and benzol. 7. Mount the sections in balsam. No. 8. ANILINE PICRATE AND GOSSYPIMINE. For Dovusir STAINING, VEGETABLE, AND ANIMAL TISSUES. Sections to be stained from water. A Aniline blue crystals... ... ... 3 grams. Pierte acid crystals ... 4.0 ss «. 2 prame. WARGP vce: nde’ ee. a8 eee “eke os SBD: Dissolve the aniline blue crystals in the water, then add the picric acid; filter for use. 46 MICROSCOPICAL RESEARCH B Gossypimine crystals ... ... ... 2grams. Water wk cee uae eee we 900 cc. Alcohol 92% ... ... .. «se «. 600. Dissolve and filter for use. MANIPULATION. 1. Pour off the water and cover the sections with A, and allow to stand thirty minutes. 2. Pour off A, and rinse the sections in two or three changes of 25% alcohol, using a trace of acid to reduce the colour if necessary. 3. Pour off the alcohol and cover the sections with B, and allow to stand thirty minutes. 4. Pour off B, and rinse the sections quickly with 75%, finishing off with 92% aleohol. 5. Clear the sections, if uncelloidinized, in oil of cajeput. 6. Transfer the sections to thin balsam and benzol. 7. Mount the sections in balsam. In working with aniline blue and gossypimine, it may be found advisable to reverse the order of manipulation by staining first with B; this will be found to be the case when the tissues dealt with are fully formed or highly lignified, z.e., that stain must be used first for which the tissues have the greatest affinity, and retains it the most persistently. In the knowledge, therefore, of the nature of the tissues dealt with lies the secret of all good staining. No. 9. HAMATOXYLIN AND GOSSYPIMINE. To double stain sections with hematoxylin and gossypimine, substitute hematoxylin for aniline picrate and manipulate as for No. 8, using acidulated alcohol to reduce the hematoxylin colour if necessary. STAINS, REAGENTS, &c. 47 No. 10. ANILINE BLUE. A SinGLe STAIN FOR VEGETABLE TISSUE. Aniline blue crystals... ...0 2... ... gram. Alcohol 92% i ae aes ave eve 800 COs Dissolve and filter for use. MANIPULATION. 1. Pour off the alcohol and cover the sections with the stain; allow to stand thirty minutes. 2. Pour off the stain and rinse the sections in two or three changes of alcohol ; using the acidulated alcohol to reduce the colour if necessary. 3. Clear the sections, if uncelloidinized, in oil of cajeput. 4. Transfer the sections to thin balsam and benzol, and when convenient 5. Mount the sections in balsam. No. II. MALACHITE GREEN.—A Counter Stain. Malachite green... ... ... ... ... I gram. Aleohol 92% nit ae naw saw vee 800 CC: Dissolve and filter for use. MANIPULATION. j. After staining with carmine, formula No. 1, 2. Pour off the alcohol and cover the sections with the malachite green stain, and allow to stand fifteen to thirty minutes. 3. Pour off the stain and rinse the sections quickly in two or three changes of alcohol; and as soon as the carmine stain stands out clearly pour off the alcohol and 4. Cover the sections with oil of cajeput to clarify. 5. Transfer the sections to thin balsam and benzol, and when convenient 6. Mount the sections in balsam. 48 MICROSCOPICAL RESEARCH No. 12. SAFRANIN.—A Counter Stain. SatraniG: see Gy. ack kek ee ys «SB preImis, Water sitet “hha seat yg! Use” Giron BOO es Alcohol 92% ... ... 01. oe. w. 540 ce. Dissolve by heat, when cold filter for use. Manrrvxation. 1. After staining with hematoxylin formula No. 3, 2. Pour off the alcohol and cover the sections with safranin, and allow to stand thirty minutes. 3. Pour off the stain and rinse the sections quickly in two or three changes of alcohol, and as soon as the hematoxylin colour stands out clearly, pour off the alcohol and 4. Cover the sections with oil of cajeput to clarify. 5. Transfer the sections to thin balsam and benzol, and, when convenient, 6. Mount the sections in balsam. No. 13. EOSIN.—A Counter Stain. HOSin 3% wes Ge ai Oi ee ae Dram: Alcohol 92% ie lay wae aaa egy BOO Ce) Dissolve and filter for use. Eosin may be employed as a counterstain with either aniline blue or hematoxylin, the manipulation being exactly the same as for Nos. 11 and 12. MOUNTING MEDIA, etc. CanaDA Batsam. Canada balsam is supplied commercially in its crude condition containing essential oils and similar substances, which must be eliminated by evaporation before the balsam can be used for microscopical purposes. STAINS, REAGENTS, &c. 49 Place the balsam in an evaporating pan and gently heat over a compound bunsen flame, stirring frequently, until when a drop of the hot balsam is allowed to fall into a vessel of cold water, it at once sets brittle. The balsam should now be allowed to cool, but not to set hard, when it may be thinned down to the required consistency with pure rectified benzol, or with pure aylol as required. Filter for use. Gum Damar. Make a saturated solution of gum dammar in rectified benzol, and filter for use. GLYCERINE JELLY. Finest French gelatine ... ... 0... 0... Loz. Water eis! lang) Gite aie ska ene wer, “6 lOg8. Pure glycerine, by weight ... ... ... 7 ozs. Soak the gelatine in the water for two hours; then add the glycerine, warm until dissolved, and then add 1% of pure carbolic acid as a preservative, and filter for use with the aid of a hot-water jacket. Farrant’s Merprum. Arsenious acid ... ... ... ... .. Igram. Waher eas Sd. Gh, GR Gam . ais SO Gum acacia... ... ... «.. «.. 180 grams. Pure glycerine was aan ce ace 100 Ge Dissolve the acid and the gum in the water by heat; then add the glycerine, and filter for use. GLYCERATED WATER. Distilled water... ... 1... 0. ... 225 ce. Pure @lyeerme ea. ver wee see ee TOG, Corrosive sublimate ... ... ... ... lgram. Filter for use. 50 MICROSCOPICAL RESEARCH CARBOLIZED WATER. Distilled water... ... ... 0... ... 800 ce. Pure carbolic acid ... ... ... ... ... lee. Filter for use. BieacHine SoLvurion. A Chlorinated lime ... ... 1... ... ... )=©=64 028. Water We nate, Aas voles Aioee vite tan, LOvezs: Stir at intervals for an hour. B Sodium carbonate ... ...0 1... ... «ee = 4028. Water 666 See kee ie kas kas as Ooze, Dissolve. Pour off the clear liquid from A, and to this add B, and allow to stand for an hour to settle; filter for use. Sections must always be placed in water before they are bleached. MANIPULATION. 1. Pour off the water and cover the sections with the bleaching solution, allow to stand five to thirty minutes, or until the sections are bleached white. 2. Rinse the sections in several changes of water, or allow them to soak until quite free from the lime solution. 3. Transfer, or graduate if necessary, the sections to alcohol, ready for staining. The washing process may be facilitated by the addition of a few drops of hydrochloric acid to the water. STAINS, REAGENTS, &c. 51 Brown CEMENT. A. Best orange shellac pulverised ... 1... 6.0.0... we wee) 4 008. Alcohol: 92% ss. aie ee aes aes aaa ee aw oe Gas: BOOKER, Bb. A saturated solution of pure rubber in petroleum ether... 60 ce. C. Best gold size... ... .. .-» 120 ce. Add B and C to A. Filter for use. OxIDE oF Zinc. Make a saturated solution of gum dammar in pure benzol; filter this into a mortar, and add as much oxide of zinc as the solution will take up by pounding. Now thin down to the required consistency with pure benzol, and filter through fine muslin. Now add to the solution as much gold size as will impart to it a creamy tint, when shaken up. ASPHALTUM. A saturated solution in pure benzol. Filter for use. 52 MICROSCOPICAL RESEARCH CuHaptTer IV. Type preparations. ROOT STRUCTURE. Roor oF Burrercvr, ‘Ranunculus acris,’ !/,, of an inch in diameter. ; Fixed with 92% alcohol. Cut transverse sections !/,.. of an inch in thickness. Place the sections in alcohol. Manipulate Nos. 3 and 9. Compare the preparation with Fig. 1. Plate I. Roor or Ping, ‘Pinus sylvestris,’ 1/,, of an inch in diameter. Fixed with 92% alcohol. Cut transverse sections !/,,,, of an inch in thickness. Place the sections in alcohol. Bleach the sections as per instructions. Manipulate Nos. 3 and 12. Compare the preparation with Fig. 2. Plate I. Root or Yettow Iris, ‘Iris Pseudoacorus,’ } of an inch in diameter. Fixed in 92% alcohol. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 3 and 12. Compare the preparation with Fig. 3. Plate I. TYPE PREPARATIONS 53 Root or Inpran Cory, ‘Zea maize,’ 3/,, of an inch in diameter. Fixed in 92% alcohol. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 4. Plate I. A. B, Fie. 26.—Germinating box. A. 3, wood box, with strips of wood laid across the open top, over which a strip of flannel is let into folds. 1, loose ends of flannel leading to vessels containing water, which feeds the seed by osmosis. 2, grains of barley in the folds of the flannel. B. The seeds after being germinating for 6 days. Devetorinc Rapicies or Bar.ey. Place some grains of barley in the germinating box, Fig. 26, allow them to germinate for twelve hours (the flannel must be damp when the grains are placed in). Now take out the grains, and with a sharp section knife cut them in half transversely and place the germinal half direct into the chromo-acetic solution, and allow them to ‘fix’ for twenty-four hours; now wash and graduate to alcohol as directed (see collecting and fixing of tissues). Celloidinize the specimens. Cut transverse sections !/,,., of an inch in thickness through the radicle end of the germ; place the sections in alcohol. Manipulate No. 6. Compare the preparation with Fig. 5. Plate IT. 54 MICROSCOPICAL RESEARCH Entire Puant or Duckweep, ‘Lemna minor.’ After fixation and washing :— Ist. Place the specimen in equal parts of alcohol, glycerine, and water; allow to stand six hours. 2nd. Place the specimens in two parts of pure glycerine and one part each of alcohol and water; allow to stand three hours. 3rd. Place the specimens in pure glycerine; allow to stand one hour. 4th. Mount the preparation in glycerine jelly. Compare the preparation with Fig. 6. Plate IT. STEM STRUCTURE. Youne Srem or Bean, ‘ Vicia faba.’ Fixed with chromo-acetic solution. Cut transverse sections 1/,,, of an inch in thickness. Place the sections in alcohol. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 7. Plate III. Srem or Burrercop, ‘Ranunculus acris.’ Fixed with 92% alcohol. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 8. Plate ITI. Stem or Wovunpwort, ‘Stachys sylvatica.” Fixed with 92% alcohol. Cut transverse sections !/,,. of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate No. 7. Compare the preparation with Fig. 9. Plate III. TYPE PREPARATIONS 55 Stem or Tamus, ‘Tamus communis. Fixed with 92% alcohol. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 10. Plate III. Stem or Marestalt, ‘Hippuris vulgaris.’ Fixed with the chromo-acetic solution. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 11. Plate IV. Srem or Limnantu, ‘Limnanthemum nympheoides.’ Fixed with the chromo-acetic solution. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Manipulate Nos. 3 and 12. Compare the preparation with Fig. 12. Plate IV. Stem or SunFLower, ‘Helianthus annuus.’ Fixed with 92% alcohol. 1. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. 2. Cut tangential longitudinal sections 1/,,, of an inch in thickness. Place the sections in alcohol; the line of cut must pass through the inner margin of a primary bundle. Bleach the sections. Manipulate Nos. 1 and 11. Mount both the sections on one slide. Compare the preparations with Figs. 13 and 14. Plate IV. 56 MICROSCOPICAL RESEARCH Stem oF Lrue-tTreeE, ‘Tilia europea.’ Fixed with 92% alcohol. a. Cut transverse sections !/,;, of an inch in thickness. Place the sections in alcohol. b. Cut tangential longitudinal sections 1/,,., of an inch in thickness, passing through the phloem. Place in alcohol. ce. Cut tangential longitudinal sections 1/,,., of an inch in thickness, passing through the xylem. Place in alcohol. Bleach the three series of sections. Manipulation for a Nos. 1 and 11; for 6 and c, Nos. 3 and 12. Mount all the sections on one slide, as in the order named. Compare the preparation with Figs. 15,16 and 17. Plate V. Stem or Draczna, ‘Cordyline rubra.’ Fixed with 92% alcohol. Cut transverse sections !/,., of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 18. Plate V. Stem or Pine-TReE, ‘Pinus sylvestris.’ Fixed with chromo-acetic solution. For development and formation of parts. . Cut longitudinal-median sections through apex of young stem, 1/499) of an inch in thickness. b. Cut transverse sections !/,,, of an inch in thickness, half-inch below growing point. . Cut transverse sections 1/,,, of an inch in thickness, two inches below growing point. d. Cut radial longitudinal sections/,,, of an inch in thickness, two inches below growing point. Place each group of sections in alcohol, as cut. Manipulation for a, No. 6. Manipulation for b, c, d, No. 9. Compare the preparations with Figs. 19, 20, 21, 22 Plate VI. Also Fig. 23. Plate VII. Q gy TYPE PREPARATIONS 57 StrucTURE oF TrmBer, OLD Woop or Pryz, ‘ Pinus sylvestris.’ Prepare half-inch cubes of old wood of pine, Fig. 27. Steep the cubes in tepid water for twenty-four hours to soften. A. B. Fic. 27.—A. Diagram of a cross section of a timber tree, showing the position from which to cut out a cube for sectionizing, a, central axis of growth, 6, cube. B. Cube after removal from A, 6. a, transverse surface, 0, radial-longitudinal surface, c, tangential longitudinal surface. a. Cut sections !/,5,. of an inch in thickness from the transverse surface of cube. b. Cut sections 1/1... of an inch in thickness from the radial longitudinal surface of cube. c. Cut sections 1/,,5. of an inch in thickness from the tangential longitudinal surface of cube. Place the three series of sections in water as cut, separately. Bleach all the sections, and transfer to 92% alcohol. Manipulation for a and c, Nos. 3 and 12. Manipulation for b;——. 1. Place the sections in equal parts of alcohol, glycerine, and water; allow to stand six hours. 2. Placa the sections in two parts of pure glycerine and one part each of alcohol and water; allow to stand three hours. 3. Place the sections in pure glycerine. 4. Mount the sections in glycerine jelly (without staining). Compare the preparations with Figs. 24, 25, 26. Plate VII. 58 MICROSCOPICAL RESEARCH Stem oF Inpran Corn, ‘Zea mais.’ Fixed with 92% alcohol. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Cut radial longitudinal sections '/,,. of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Figs. 27 and 28. Plate VIII. Racuts AND RuizoME oF Fern, ‘ Pteris aquilina.’ Fixed with chromo-acetic solution. Cut transverse sections of rachis !/,,, of an inch in thickness. Place the sections in alcohol. Cut longitudinal sections of rhizome 1/;,, of an inch in thickness. Place the sections in alcohol. (The line of cut for the longitudinal section must pass through both of the crescent-shaped sclerenchymatious bands, Fig. 38. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparations with Figs. 29 and 30. Plate VIII. Fic. 28.—Diagram of a T\S. of rhizome of Pteris, showing the position of vessels. 1-5 position of the five scleriform vessels. a, the two crescent-shaped sclerenchyma- tious bands. The dotted line indicates the direction of cut in taking the sections. TYPE PREPARATIONS 59 Stem or Horseratt, ‘ Equisetum telmateia.’ Fixed with chromo-acetic solution. Cut transverse sections of stem !/,,, of an inch in thickness, immediately below the vegetative bud. Place the sections in alcohol. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 31. Plate IX. VEGETATIVE Bup or Horseratt, ‘Equisetum telmatia.’ Fixed as above. After celloidinizing the bud, a. Cut transverse section 1/,,, of an inch in thickness. Place the sections in alcohol. b. Cut radial longitudinal section !/,,, of an inch in thickness. Place the sections in alcohol. c. Cut radial longitudinal section '/,;,, of an inch in thicknéss, through an apical or lateral growing point. Manipulation for a, No. 1. Manipulation for b, Nos. 1 and 11. Manipulation for c, No. 6. Compare the preparations with Figs. 32—34. Plate IX. LEAF STRUCTURE. Lear or Pry, ‘Pinus maritimus.’ Fixed with 92% alcohol. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 35. Plate X. Lear or Marram Grass, ‘Ammophila arundiacea.’ Fixed with 92% alcohol. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 36. Plate X. 60 MICROSCOPICAL RESEARCH Lear or Orcuis, ‘Cypripedium Sp.’ Fixed with 92% alcohol. Cut transverse sections 1/4), of an inch in thickness. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 37. Plate XI. Lear or Cycas, ‘Cycas revoluta.’ Fixed with the chromo-acetic solution. Cut transverse sections !/,;, of an inch in thickness. Place the sections in alcohol. Manipulate No. 8. Compare the preparation with Fig. 38. Plate XI. LEar OF Borre-BRUSH, ‘Metrosideros verni.’ Fixed with the chromo-acetic solution. Cut transverse sections !/,,,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 8. Compare the preparation with Figs. 89—40. Plate XI. Lear or TrapEscanttA, ‘ Tradescantia discolor.’ Fixed with the chromo-acetic solution. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 8. Compare the preparation with Fig. 41. Plate XII. CurticLte rrom Lear or Monkry-puzzze, ‘Araucaria imbricata.’ Preserved in 92% alcohol. a. Cut the leaves into half-inch squares with a sharp section-knife. b. Place the pieces in a test-tube with a mixture of equal parts of nitric acid and water. TYPE PREPARATIONS 61 c. Boil over the bunsen flame for two or three minutes. d. Pour the contents of the test-tube into cold water, shake well for two or three minutes. e. Pick out the separated cuticles and place them in clean water to wash for an hour or two. If the intermediate tissue has not been completely removed, the boiling process must be repeated, at the same time great care must be taken not to carry the boiling beyond this point, or the guard cells of the stomata will also be removed. After washing the cuticles f. Transfer to 92% alcohol. Manipulate No. 6. Compare the preparation with Fig. 42. Plate XII. StomaTa IN GREEN STEM, SHEE Oak, ‘Casuarina equisetifolia.’ Developing twig 1/,,1n. diameter. . Fixed with chromo-acetic solution. Cut transverse sections !/,.,, of an inch in thickness. Place the sections in alcohol. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 43. Plate XIT. Sting or Nerrue, ‘ Urtica dioica.’ Fixed with the chromo-acetic solution. With a sharp scalpal remove longitudinal strips of cuticle, with its stings, from the midrib of the leaf, divide the strips as required and place in alcohol. Manipulate Nos. 1 and 11. Compare the preparation with Fig. 44. Plate XII. Puyiiopium or Acacta, ‘Acacia decurrens.’ Fixed with chromo-acetic solution. Cut transverse sections '/..9, of an inch in thickness. Place the sections in alcohol. Manipulate No. 8. Compare the preparation with Fig. 45. Plate XIII. 62 MICROSCOPICAL RESEARCH CLADODE oF Butcuer’s Broom, ‘ Ruscus aculeatus.’ d Fixed with chromo-acetic solution. Cut transverse sections !/,., of an inch in thickness. alcohol. Manipulate No. 8. Place the sections in Compare the preparation with Fig. 46. Plate XIII. Lear-bup or Brescu, ‘ Fagus sylvatica.’ Fixed with the chromo-acetic solution. Celloidinize the specimen. Cut transverse sections !/,;,;, of an inch in thickness. in alcohol. Manipulate No. 1. Place the sections Compare the preparation with Fig. 47. Plate XIII. Lear-bup or Asu Tres, ‘ Fraxinus excelsior.’ Fixed with chromo-acetic solution. Celloidinize the specimen. Cut transverse sections !/,,, of an inch in thickness. alcohol. Manipulate No. 1. Place the sections in Compare the preparation with Fig. 48. Plate XIII. Lear-sup oF Sycamore, ‘Acer Pseudo-platanus.’ Fixed with chromo-acetic solution. Celloidinize the specimen. Cut transverse sections !/,., of an inch in thickness. alcohol. Manipulate No. 1. Place the sections in Compare the preparation with Fig. 49. Plate XIV. TYPE PREPARATIONS 63 Derrorr1ation 1n Sycamore, ‘Acer Pseudo-platanus.’ Fixed with 92% alcohol. Specimens collected about the middle of September. Cut longitudinal median sections !/,,, of an inch in thickness, of a lateral branch, passing through the opposite leaf-stalks of the current year, and the developing leaf-buds of next year. Place the sections in alcohol. Bleach the sections. Manipulate Nos. 1 and 11. Compare the preparation with Figs. 50 and 51. Plate XIV. FLORAL STRUCTURE. FLoweEr-bup or Poppy, ‘Papaver rheas.’ Fixed with 92% alcohol. Celloidinize the specimen. Cut transverse sections '/,., of an inch in thickness. Place the sections in alcohol. Manipulate No. 1. Compare the preparation with Fig. 52. Plate XV. FLOWER-BUD OF WALLFLOWER, ‘Cheiranthus Cheiri.’ Fixed with 92% alcohol. Celloidinize the specimen. Cut transverse sections !/,., of an inch in thickness. Place the sections in alcohol. Manipulate No. 1. Compare the preparation with Fig. 53. Plate XV. FLOWER-BUD OF DANDELION, ‘Taraxacum officinalis.’ Fixed with chromo-acetic solution. Celloidinize the specimen. Cut transverse sections 1/,,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 1. Compare the preparation with Fig. 54. Plate XV. 64 MICROSCOPICAL RESEARCH FLOWER-BUD oF PurRpPLE Irs, ‘Iris Germanica.’ Fixed with 92% alcohol. Celloidinize the specimen. Cut transverse sections !/,,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 1. Compare the preparation with Fig. 55. Plate XV. Marvre Fiower-sup or Laity, ‘ Lilium croceum.’ Fixed with chromo-acetic solution. Celloidinize the specimen. Cut transverse sections 1/,,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 1. Compare the preparation with Fig. 56. Plate XVI. ImMATURE FLOWER-BUD oF Laity, ‘ Lilium croceum.’ Fixed with chromo-acetic solution. Dissect out the anthers and the ovary. Celloidinize the specimens. a. Cut transverse sections 1/,)) of an inch in thickness of the anther. Place the sections in alcohol. b. Cut transverse sections !/,,,) of an inch in thickness of the ovary. Place the sections in alcohol. Manipulate No. 6. Compare the preparations with Figs. 57—58. Plate XVI. CaTKIN oF SaLiow, ‘Salix caprea.’ Fixed with chromo-acetic solution. Celloidinize the specimen. Cut longitudinal median sections 1/,,, of an inch in thickness. TYPE PREPARATIONS 65 Place the sections in alcohol. Manipulate No. 8. Compare the preparation with Fig. 59. Plate XVI. Entire Frower or tHe Waueat, ‘Triticum vulgaris.’ Fixed with the chromo-acetic solution. Specimen collected before emerging from the sheath, and dissected out. Manipulate No. 1. Compare the preparation with Fig. 60. Plate XVII. Stigma or Wueat, ‘Triticum vulgaris.’ Fixed with chromo-acetic solution. Specimen dissected out of its sheath about ten days prior to its natural time of opening. Manipulate No. 1. a. Place the stigma in equal parts of alcohol, glycerine, and water. b. Place the stigma in two parts pure glycerine, and one part each of alcohol and water. c. Place the stigma in pure glycerine. Allow to stand in each solution of a and 6} for an hour. d. Mount the specimen in glycerine jelly. Compare the preparation with Figs. 61—62. Plate XVII. CELL-STRUCTURE AND CELL-CONTENTS. Place some grains of wheat, ‘Triticum vulgaris,’ in the germinating box, and allow them to germinate for sixteen hours. Fix with the chromo-acetic solution, and then graduate to 92% alcohol. (See Fig. 26). Cut longitudinal-median sections of entire seed, 1/,., of an inch in thickness. Place the sections in alcohol. Manipulate No. 1. Compare the preparation with Fig. 68. Plate XVIII. 66 MICROSCOPICAL RESEARCH Emsryo or Wueat after germinating twenty-four hours. Fix with the chromo-acetic solution, then graduate to 92% alcohol. Cut longitudinal median section of embryo !/,5., of an inch in thickness. Place the sections in alcohol. Manipulate No. 6. Compare the preparation with Fig. 64. Plate XVIII. Fic. 29.—Ventral and dorsal view of a grain of wheat, the dotted line indicates the median area from which sections must be taken. Seep or WHEAT, germinated for twelve hours, fixed and graduated as above. Cut transverse sections through endosperm !/,5,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 6. Compare the preparation with Fig. 65. Plate XVIII. Empryo or Wueat, after twenty-four hours germination. Fixed with corrosive-picro-formaldehyde solution, then graduated to 92% alcohol. Cut longitudinal median sections of embryo !/,5). of an inch in thickness. Place the sections in alcohol. Manipulate No. 6. Compare the preparation with Fig. 66—67. Plate XIX. TYPE. PREPARATIONS 67 Emsryo or Wueat, collected before fertalization. Fixed with chromo-acetic solution. Cut longitudinal sections !/,,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 6. Compare the preparation with Fig. 68. Plate XIX. Emspryo or Wueat, collected after fertilization. Fixed with chromo-acetic solution. Cut longitudinal sections 1/,;, of an inch in thickness. Place the sections in alcohol. Manipulate No. 6. Compare the preparation with Fig. 69. Plate XIX. WHeat-Rust, ‘Puccinia graminis.’ Uredospores, on leaf of thistle. Fixed with 92% alcohol. Celloidinize the specimen. Cut transverse sections of leaf !/,,., of an inch in thickness. Place the sections in alcohol. Manipulate No. 10. Compare the preparation with Fig. 70. Plate XX. Wueat-rvst, ‘Puccinia graminis.’ Teleutospores on stem of wheat. Fixed with 92% alcohol. Celloidinize the specimen. Cut transverse sections of the stem !/,,., of an inch in thickness. Place the sections in alcohol. Mount without staining. Compare the preparation with Fig. 71. Plate XX. 68 MICROSCOPICAL RESEARCH Wueat-rvst, ‘Puccinia graminis.’ Aicidiaspores on leaf of barbery. Fixed with 92% alcohol. Celloidinize the specimen. Cut transverse sections of leaf !/,;,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 10. Compare the preparation with Fig. 72. Plate XX. Entire plant of ‘Peziza convexula.’ Fixed with 92% alcohol. Celloidinize the specimen. Cut vertical sections of plant !/,;, of an inch in thickness. Place the sections in alcohol. Manipulate No. 8. Compare the preparation with Fig. 73. Plate XX. SEA-WEED. Conceptacles of ‘Fucus vesiculosus.’ Fixed with chromo-acetic solution in sea water. Celloidinize the specimens. Cut transverse sections of male conceptacle, !/,.9, of an inch in thickness. Place the sections in alcohol. Cut transverse sections of female conceptacle, !/,5.. of an inch in thickness. Place the sections in alcohol. Manipulate No. 8. Compare the preparations with Fig. 74—75. Plate XXI. Ferry, entire thallus of ‘Gymnogramma sp.’ Fixed with chromo-acetic solution. Manipulate No. 1. Compare the preparation with Fig. 76. Plate X XI. TYPE PREPARATIONS 69 Fern, thallus of ‘Gymnogramma sp.’ Fixed with chromo-acetic solution. Infiltrate the specimen with paraffin. Cut vertical sections !/5,5, of an inch in thickness, with the mechanical microtome. Mount the preparation as described for the paraffin method. Manipulate No. 6. Compare the preparation with Fig. 77. Plate XXI. SrarcH in stem of tradescantia, ‘Tradescantia discolor.’ Fixed with chromo-acetic solution. Cut transverse sections of stem 1/,., of an inch in thickness. Place the sections in alcohol. ‘Manipulate No. 3 and Gossypimine (8B). Compare the preparation with Fig. 78. Plate XXII. Srarcu in stem of pellionia, ‘Pellionia Daveauna.’ Fixed with chromo-acetic solution. Cut transverse sections of stem 1/,,) of an inch in thickness. Place the sections in alcohol. Manipulate No. 3 and Gossypimine (8B). Compare the preparation with Fig. 79. Plate XXII. APICAL-CELL in root of fern, ‘Aspidium Filix-mas.’ ? Fixed with chromo-acetic solution. Apex of developing roots collected about the middle of May. Infiltrate the specimens with paraffin. Cut longitudinal median sections !/ 99) of an inch in thickness. Mount the sections as described for the paraffin method. . Manipulate No. 6. Compare the preparation with Fig. 80. Plate XXII. 70 MICROSCOPICAL. RESEARCH RapHIDEs in aerial root of monstera, ‘ Monstera deliciosa.’ Fixed with chromo-acetic solution. Infiltrate the spemen with paraffin. Cut longitudinal median sections of apex of root /559) of an inch in thickness. Mount the sections as directed for the paraffin method. Manipulate No. 6. Compare the preparation with Fig. 81. Plate XXII. Enbosrerm in seed of castor-oil plant, ‘Ricinus communis.’ Fixed with 92% alcohol. Remove the kernel from the husk, and divide in half transversely with a sharp section-knife. Celloidinize the specimens. Cut transverse sections !/,.., of an inch in thickness. Place the sections in alcohol. Manipulate No. 6. Compare the preparation with Fig 82, (unstained). Plate XXIII. LariciFERovs VESSELS in root of ‘Scorzonera hispanica.’ Fixed with chromo-osmo-acetic solution. Cut longitudinal sections '/,,. of an inch in thickness. Place the sections in alcohol. Manipulate No. 3. Compare the preparation with Fig. 83. Plate XXIII. LaTIcIFERovs VESSEL in stem of “ Euphorbia splendens.’ Fixed with absolute alcohol. Cut longitudinal sections of stem!/,,,, of an inch in thickness. Place the sections in alcohol. Manipulate No. 1 or 6. Compare the preparation with Fig. 84. Plate XXIII. TYPE PREPARATIONS 71 KaryoxInesis in root of onion, ‘Allium cepa.’ Fixed with chromo-acetic solution. Infiltrate the specimen with paraffin. Cut longitudinal sections / 5.5) of an inch in thickness. Mount the sections as directed for the paraffin method. Manipulate No. 6. Compare the preparation with Fig. 85. Plate XXIII. 72 Fie. 1. Fria. 2. Fic. 3. Fia. 4. MICROSCOPICAL RESEARCH PLATE I. ROOT STRUCTURE. Transverse section. Root of Buttercup. ‘Ranunculus acris’ x75dia. Fixed in 92% Alcohol. 1. Ground tissue containing starch. 2. Endodermis.. 3. The four phloem groups. 4. The four xylem groups. Stained Hematoxylin and Gossypimine. Transverse section. Root of Pine. ‘Pinus sylvestris’ x7hdia. Fixed in 92% Alcohol. Stained Hematoxylin and Safranin. 1. Cortical tissue. 2. Phloem. 3. Cambium. 4. Secondary wood. 65. Primary wood. 6. Resin duct. Transverse section. Root of Iris. ‘Iris pseudoacorus’ x50dia. Fixed in 92% Alcohol. Stained Hematoxylin and Safranin. 1. Primary ground tissue. 2. Endodermis. 3. Large pitted vessels. 4. Phloem elements, stained purple. 5. Xylem elements, stained red. 6. Developing lateral rootlets. 7. Central axis of root. Transverse section. Root of ‘Zea mais” x35dia. Fixed in 92% Alcohol. Stained Borax Carmine and Malachite Green. 1. Cortical tissue. 2. Endodermis. 3. Xylem elements, stained green; phloem elements, stained pink. 4. Pith. 5. Developing lateral rootlets in various stages. S, . ae a -. et Ar KY OA s8@=~ a ee Bi : We? ? 74 Fra. 5. Fre. 6. MICROSCOPICAL RESEARCH PLATE II. ROOT STRUCTURE. Transverse section through radical end of a grain of barley, after twelve hours’ germination. Fixed with Chromo- Acetic solution. Cut 1/,;,,.1m. Stained with Brazilin. 1. Aleurone layer. 2. Endosperm, containing starch. 3. The pericarp. 4. Primary nucleated tissue of scutellum. 5. Cells to form epidermis (dermatogen). 6. Cells to form cortical tissue (periblem). 7. Cells to form vascular system (plerome). 8. Central vessel of root. 9. Absorptive layer of embryo. A. The four lateral roots. B. The primary or tap-root. Entire plant of ‘Lemna minor,’ common Duckweed x 12dia. Fixed with Chromo-Acetic solution. 1. The fronds contain- ing bundles of raphides (shown as dark spots). 2. Root. 3. Root-cap. 4. Developing root. ’ Kos ces yee AT ee Se altefi ea ta = as rrr Sa 76 Fic. 7. Fic. 8. Fic. Fig. 9. 10. MICROSCOPICAL RESEARCH PLATE III. STEM STRUCTURE. Transverse section through young Stem of Bean. ‘Vicia faba’ x 35 dia. Stained with Borax Carmine and Malachite Green. Fixed in the Chromo-Acetic solution. 1. Primary ground tissue. 2. Cambiformtissue. 3. Phloem. 4. Xylem elements. 65. Pith. 6. Traces to first leaf. The line of cut being immediately below the first leaves. Transverse section. Stem of Buttercup. ‘Ranunculus acris’x18dia. Fixed in 92% Alcohol. Stained with Borax Carmine and Malachite Green. 1. Cortical tissue. 2. phloem elements. 3. Xylem elements. 4. Central hollow cavity of stem. 5. Primary ground tissue. 6. Primary vascular bundles. 7. Leaf trace bundle. 8. Cambium. Transverse section. Stem of Wound-wort. ‘Stachys sylvatica’ x 20dia. Fixed in 92% Alcohol. Stained with Picro-carminate of Ammonia. 1. Cortical tissue. 2. Cam- bium. 3. Xylem elements. 4. Primary xylem bundles. 5. Primary ground tissue. Transverse section. Stem of Tamus. ‘Tamus communis’ x25 dia. Fixed in 92% alcohol. Stained with Borax Carmine and Malachite Green. 1. Cortical tissue. 2. Scleren- chymatous ring. 8. Xylem elements. 4. Primary ground tissue. 5. Central cavity of stem. 78 Fic. Fig. Fic. Fic. 11. 12. 13. 14. MICROSCOPICAL RESEARCH PLATE IV. STEM STRUCTURE. Transverse section. Stem of Marestail. ‘Hippuris vulgaris’ x15dia. Fixed in Chromo-Acetic solution. Stained Borax Carmine and Malachite Green. 1. Cortical tissue. 2. Central axis of stem. 38. Xylem elements. 4. Cambiform tissue. 5. Phloem elements. Transverse section. Stem of Limnanth ‘Limnanthemum nympheoides’ x 20 dia. Fixed in Chromo-Acetic solution. Stained Hematoxylin and Safranin. 1. Cortical tissue. 2. Idioblasts. 3. Intercellular space. 4. Xylem elements. 5. Central axis of stem. 6. Primary ground tissue formed of radialy elongated cells. Transverse section of Sunflower. ‘Helianthus annuus’ x20dia. Fixed in 92% Alcohol. Stained Borax Carmine and Malachite Green. 1. Cortical tissue. 2. Primary ground tissue. 3. Cambium ring. 4. Xylem elements. 5. Phloem elements. Tangential longitudinal section. Stem of Sunflower. ‘Helianthus annuus’x65dia. Cut !/,.,,in. The line of cut passing through the inner margin of a primary bundle. 1. Primary ground tissue. 2. Fibro-vascular bundle. 3. Spiral vessel. 80 Fia. Fic. Fig. Fic. 15. 16. 17. 18. MICROSCOPICAL RESEARCH PLATE V. STEM STRUCTURE. Transverse section. Stem of Lime Tree, ‘Tilia europea’ x40dia. Fixed in 92% Alcohol. Stained with Borax Carmine and Malachite Green. 1. Cortical tissue. 2. Primary medullary ray. 3. bast (phloem). 4. Cambium-ring. 5. Xylem elements, of about three years’ growth. 6. Central axis of pith. 7. Medullary sheath containing protoxylem elements. Tangential longitudinal section. Stem of Lime Tree. ‘Tilia europea’ x 25dia. The line of cut passing through the ‘ bast area.’ Cut 1/,...in. Stained Hematoxylin and Safranin. 1. Ground tissue (medullary ray). 2. Bast fibres. Tangential longitudinal section. Stem of Lime Tree. ‘Tilia europea’ x50 dia. The line of cut passing through xylem elements. Stained with Hematoxylin and Safranin. 1. Connective tissue. 2. Pitted vessels. 3. Medullary ray. Transverse section. Stem of Dracena. ‘Cordyline rubra’ x50 dia. Fixed in 92% Alcohol. Stained Borax Carmine and Malachite Green. 1. Cortical tissue. 2. Cambiform zone in which new bundles are being developed. 3. Secondary vascular bundles in various stages of development. 4. Primary vascular bundles. ioe) a Fy poe A = Vs 1 a . ‘ £33 \ bs 3 Sa car a Se eas — iota ; 82 Fia. 19. Fre. 20. Fia. 21. Fie. 22. MICROSCOPICAL RESEARCH PLATE VI. STEM STRUCTURE. Longitudinal median section of apex of Stem of Pine. ‘Pinus sylvestris.’ Fixed with the Chromo-Osmic-Acetic solution x 75 dia. Stained with Brazilin. 1. Zone of apical tissue in active state of division. 2 and 3, axillary buds in various stages of development. 4, primary nucleated tissue. Transverse section, half-inch below growing point. Stem of Pine x75dia. Stained with Aniline-picrate and Gossy- pimine. Tissue fixed as above. 1. Cortical tissue formed of loose cells. 2. Bast. 3. Cambiform tissue. 4, Xylem elements before the beginning of secondary thickening. 5. Primary medullary ray.. 6. Primary ground tissue. Transverse section, two inches below growing point. Stem of Pinex35 dia. Tissue fixed and stained as in Fig. 20. 1. Cortical tissue. 2, Phloem elements. 3. Cambiform tissue. 4. Xylem elements, after the commencement of secondary thickening. 5. Resin passage. 6. Central ground tissue. 7. Developing branch. Radial longitudinal section. Stem of Pinex35dia. Area as in Fig. 21. 1. Cortical tissue. 2. Phloem elements. 3. Xylem elements. 4. Primary central tissue. 5. Cambium. 6. Developing branch. ae} ter it et fg oa fe DAN vo St z (iin py Peon : Tia Re, oa q (on 4 ai! £8) «AM oh Lag Bees He ye RNs he as Ajo S| Y eet isa SCC eR TaB ES Le ee Nee iriezienaces D> eatin a hte? eth we t Y ae, Lyd ha\y dso: [I are ‘ ‘e' e AY er 2 oH 84 Fia. 23. Fia. 24. Fia. 25. Fie. 26. MICROSCOPICAL RESEARCH PLATE VII. STEM STRUCTURE. Transverse section through resin passage. Stem of Pine x76 dia. Tissue fixed as above. 1. Primary ground tissue. 2. Thickening band of prosenchyma. 3. Secreting cells of canal. Transverse section. Old Wood of Pine x 35 dia. Cut?/j995 in. Unstained. 1. Spring growth of the wood. 2. Autumn and winter growth. 3. Resin canal. 4. Medullary ray. Radial longitudinal section. Old Wood of Pine x 75 dia. Cut !/,oo0in. Unstained. 1. Bordered pits in surface view. 2. Medullary ray in radial view. 3. Cell-wall in radial view. Tangential longitudinal section. Old Wood of Pine x 75 dia. Cut 1/is.im. 1. Bordered pits in transverse section. 2. Medullary ray in section. 3. Cell-wall in tangential view. ie) ESET eo EE BAT mag i iene SA TR eR Ge EAS ETI : Ses Sp hte Nie cern RS se Fl e ie peace 4 ne pate er AGRE lt BEE BT arco AR | cong cee eT =| mei CG ieee Sl OE ce RS rey perenne cnet an sce BER eet RUE AR Sree - ae ee eee eer Eng erent ng SEES OS regis ance ens “s ys ay oO, ae oe See eee ee AN ae OS 86 Fia. 27. Fia. 28. Fic. 29. Fic. 30. MICROSCOPICAL RESEARCH PLATE VIII. STEM STRUCTURE. Transverse section. Stem of Indian Corn. ‘Zea mais’ x25dia. Fixed in 92% Alcohol. Stained with Borax Carmine and Malachite Green. 1. Epidermal tissue. 2. Vascular bundle. 3. Bundle-sheath. 4. Phloem. 5. Large pitted vessel. 6. Annular vessel. 7. Primary ground tissue. Longitudinal section passing through a primary vascular bundle. Stem of Indian Corn. ‘Zea mais’ x 50 dia. Stained as in Fig. 27. 1. Primary ground tissue. 2. Large pitted vessels. 3. Annular vessel. 4. Spiral vessel. Transverse section through Rachis of Fern. ‘Pteris aquilina’x40 dia. Fixed in Chromo-Acetic solution. Staining as in above figures. 1. Primary ground tissue. 2. Endodermis. 38. Bast sheath. 4. Bast. 5. Scalariform vessels. 6. Spiral vessels. Longitudinal section of Rhizome of Fern. ‘Pteris aquilina’ x60 dia. Fixed and stained as above. 1. Primary ground tissue. 2. Sclerenchymasheath. 3. Bast. 4. Spiral vessels. 5. Scalariform vessels. edly WRARUV RS OEL SE IHATUTETT {i 88 Fia. Fig. Fic. Fig. dl. 32. 33. 34, MICROSCOPICAL RESEARCH PLATE IX. STEM STRUCTURE. Transverse section immediately below vegetative bud of “Equisetum telmateia’x 75 dia. Tissue fixed in Chromo- Acetic solution. Stained with Borax Carmine and Malachite Green. 1. Nucleated cortical tissue. 2. Bast. 3. Isolated annular vessels in intercellularspace. 4. Primary nucleated tissue. Transverse section through vegetative bud of ‘Equisetum telmateia’x10dia. Fixed as above. Stained Carmine. 1. Central axis of stem. 2. Zone of vascular bundles. 3 and 4. Leaf-sheaths in various stages of development, the outer layer dividing into separate leaves. Longitudinal median section of an older vegetative bud than above x30 dia. Fixed as above. Stained with Borax Carmine and Malachite Green. 1. Central ground tissue. 2. Nodal diaphragm. 3. Secretion cells. 4. Vascular system. 5. Lateral bud. 6. Cavity of vascular bundle, 7. Growing point of lateral bud. 8. Caniculi in formation. Longitudinal median section through growing point of lateral bud in Fig. 33 (7). 1. Meristem tissue. 2. Bud sheath. 8. Apical cell. 4. Zone of dividing nucleated tissue. Cut !/,9..in. Stained Brazilin x 120 dia. , te Te AS € ¢ soit re) 5 CO ee ae | : 2 a® | 1 eas fete) Fie. 35. Fia. 36. MICROSCOPICAL RESEARCH PLATE X. LEAF STRUCTURE. Transverse section through Leaf of Pine. ‘Pinus maritimus’ x46 dia. Fixed in 92% Alcohol. Stained with Borax Carmine and Malachite Green. 1. Epidermis. 2. Hypo- derm. 3. Assimilating tissue. 4. Endodermis. 5. Trans- fusion tissue. 6. Phloem. 6a. Stomata. 7. Xylom. 8. Resin duct. Transverse section through Leaf of Marram Grass. ‘Ammophila arundiacea’x35dia. Fixed and stained as above. 1. Epidermis. 2. Assimilating tissue. 38. Wood vessels. 4. Stiffleafhairs. 5. Phloemofbundle. 6. margin of leaf. out ery, i oH uf sire , g2 Fic. Fia. Fic. Fie. ov. 38. 39. 40. MICROSCOPICAL RESEARCH PLATE XI. LEAF STRUCTURE. Transverse section. Leaf of ‘Cypripedium sp’ x20 dia. Fixed in 92% Alcohol. Stained with Borax Carmine and Malachite Green. 1. Lower epidermis of leaf. 2. Primary ground tissue. 3. Vascular bundle. 4. Water storage cells of upper epidermis, having their upper and under walls cut away. Transverse section. Leaf of Oycas. ‘Cycas revoluta x35 dia. Fixed with the Chromo-Acetic solution. Stained with Aniline-picrate and Gossypimine. 1. Lower epidermis of leaf. 2. Ground tissue. 3. Sclerenchymatous fibres. 4, Bast elements of bundle. 5. Phloemelements. 6. Stomata. 7. Palisade layer containing chloroplasts, stained blue. Transverse section. Leaf of Bottle-brush. ‘ Metrosideros verni’x 60 dia. Fixed with Chromo-Acetic solution. Cut!/oss,in. Stained with Aniline-picrate and Gossypimine. 1. Palisade layer, continuous on upper and under side. 2. Stomata. 3. Guard cells. 4. Primary ground tissue. 5. Xylem elements of bundle. 6. Phloem elements. 7. Small vascular bundles. 8. Oil gland. Part of Fig. 39x 160 dia. 1. Cuticle. 2 and 3. Stoma and guard-cell. 4. Primary vascular bundle. 5. Vascular bundle in longitudinal section. 6. Palisade cells containing chloroplasts, stained blue. . 94 Fie. 41. Fig. 42. Fia. 48. Fia. 44. MICROSCOPICAL RESEARCH PLATE XII. LEAF STRUCTURE. Transverse section. Leaf of Tradescantia. ‘ Tradescantia discolor’ x 85 dia. Fixed with Chromo-Acetic solution. Stamed with Aniline-picrate and Gossypimine. 1. Lower epidermis. 2. Primary ground tissue. 3. Dense layer of assimilating tissue. 4. Vascular bundle. 5. Air cavity. 6. Stoma. 7. Upper epidermis, showing vertical walls of thecellsonly. 8. Mucilaginous lining ofthe cell. 9. Nucleus of cell. 10. Upper cuticle of leaf. Cuticle. Leaf of Monkey-puzzle. ‘Araucaria imbricata’ x60 dia. Stained with Brazilin. 1. Cuticularised tissue. 2. Stomata. Transverse section. Green Stem of ‘ Casuarina equisetifolia’ x50 dia. Fixed in Chromo-Acetic solution. Stained with Borax Carmine and Malachite Green. 1. Cortical tissue. 2. Leaf-trace bundles. 3. Cambium layer. 4. Xylem elements. 5. Primary medullary ray. 6. Central axis of stem. 7. Stomata bordering the stem cavities. Epidermis and stinging-hairs from midrib of young Leaf of Nettle. ‘Urtica dioica’x 35 dia. Fixed with Chromo- Acetic solution. Stained with Borax Carmine and Malachite Green. 1. Cuticularised epidermis of midrib. 2. Enlarged base of sting. 3. Cavity of sting, filled with irritant fluid. 4. Capitate apex of sting. 5. Stiff hairs of midrib. see oe aglaey & @ saad ge 22 eee Boas | 8 @e &©@e & «&: ‘eo ee & 2 qee Ss & | se -e5e soe: eee 5 ae Sia: “28 X=) Saga! eee 6 xe Ss eeseess _@4se¢o0 ‘esse 1 SS (es. Se wei "ies 1 tare me os =e oe. = Se £ eae ‘O: & os i a> ae) e mA es 2). & se ~¢ > a — S86 Ee 8 4 = a ga i) — | —_— 2 ese 8 30 ee ee oe -e ~~ oso | s ge os a — ) 2s seecess i] 4 96 Fie. 45. Fia. 46. Fia. 47. Fia. 48. MICROSCOPICAL RESEARCH PLATE XIII. LEAF STRUCTURE. Transverse section. ‘Phyllode.’ ‘Acacia decurrens’ x 60 dia. Fixed with Chromo- Acetic solution. Stained with Aniline-picrate and Gossypimine. 1. Epidermis. 2. Stoma. 3. Palisade layer, containing chloroplasts, stained blue. 4. Vascular bundle. 5. Smaller vascular bundles—a, xylem elements of bundle; 6, cambium; c, phloem, all having become lignified. Transverse section. ‘Cladode’ of ‘Ruscus aculeatus’ x35 dia. Fixed with Chromo-Acetic solution. Stained with Aniline-picrate and Gossypimine. 1. Epidermis. 2. Ground tissue (spongy parenchyma). 3. Primary vascular bundle. 4. Stoma. 5. Smaller vascular bundle. Transverse section. Leaf Bud of Beech Tree. ‘Fagus sylvatica’x25 dia. Fixed with Chromo-Acetic solution (Celloidinised preparation). Stained with Borax Carmine. 1. Protective bracts or scales. 2. Internal bud _ scales. 3—7. Complete young leaf in section—3, leaf-blades; 4, midrib; 5, 6, 7, lateral veins. Transverse section. Leaf Bud of Ash Tree. ‘Fraxinus excelsior’ x25 dia. Fixed with Chromo-Acetic solution. Stained with Borax Carmine. 1.—2. Imbricate bud scales. 3. Petiole of oldest leaf, leaf-blade being cut away in previous sections. 4. Vascular system of same. 4a. Petiole of secondary leaf, the complete leaf inclosed in dotted outline. 6—7. Developing leaves. ar i ue 98 Fie. 49. Fie. 50. Fie. 51. MICROSCOPICAL RESEARCH PLATE XIV. LEAF STRUCTURE. Transverse section. Leaf Bud of Sycamore. ‘Acer pseudo- platanus’x 25 dia. (Celloidinised preparation). Stained Borax Carmine. 1. Imbricate bud scales. 2. Developing leaf ‘outlined.’ 3. The midrib. 4. Lateral veins of leaf. Longitudinal median section of lateral branch of Sycamore, passing through Leaf Stalk of current year and Developing Bud of next yearx16dia. Fixed in 92% Alcohol. Left half only of the section isshown. 1. Pith. 2. Xylemelements of branch. 3. Cambium. 4. Phloem. 5. Cortical tissue. 6. Central axis of developing branch. 7. Zone of meristem tissue. 8. Budscales. 9. Leavesin development. 10. Xylem of developing branch. 11. Petiole of leaf. 12. Ground tissue ofsame. 13. Thevascularsystem. 14. The separating or absciss layer,’ which forms during the period of vegetation, and through which ‘defoliation’ takes place in Autumn (leaf-fall). Longitudinal median section of a complete Branch of Sycamore, showing the position of the opposite petioles of the current year and the axillary buds for next year’s vegetative period. 1. Central axis (pith). 2. Petiole of current year. 3. Central axis of next year’s branch. 4. Separating layer. 5. Vascular bundle leading to leaf. 6. Xylem elements of branch. 7. Cambium. 8. Phloem. 8a. Cortical tissue. 9. Bud Scales. 10. Developing leaves. 11. Growing point of branch. The action of separation has taken place from the point marked X. TOO Fria. 62. Fia. 53. Fia. 54. Fia. 55. MICROSCOPICAL RESEARCH PLATE XV. ‘ FLORAL STRUCTURE. Transverse section. Flower Bud of Poppy. ‘ Papaver theas’xl5dia. Fixed in 92% Alcohol (Celloidinized preparation). Stained with Borax Carmine. 1. Calyx. 2. Corolla. 3. Anthers containing pollen. 4. Base of Stigmatic Cap. 5. Wall of Ovary. 6. Placenta. Transverse section. Flower Bud of Wallflower. ‘Cheiran- thus Cheiri’ x 25 dia. Fixed in 92% Alcohol. Stained with Borax Carmine (Celloidinized preparation). 1. Calyx. 2. Corolla. 3. Anthers. 4. Capsule (Silique). Transverse section. Flower Bud of Dandelion. ‘Taraxacum officianalis’ x 35 dia. Fixed with Chromo-Acetic solution (Celloidinized preparation). Stained with Borax Carmine. 1. Bracts of the involucrum. 2. Pappus (modified calyx). 3 Tubular corolla. 4. Five ‘syngenesious’ anthers. 5. Style with three vascular bundles. Transverse section. Flower Bud of Iris. ‘Iris Germanica’ x25 dia. Fixed with 92% Alcohol (Celloidinized prepara- tion). Stained with Borax Carmine. 1. Calyx. 2. Corolla. 3. Petaloid stigmas. 4. Anthers, 5. Fringe of hairs running longitudinally along the median plane of each sepal. cr ae 102 Fira. Fic. 57. Fic. Fig. 56. 58. 59. MICROSCOPICAL RESEARCH PLATE XVI. FLORAL STRUCTURE. Transverse section. Flower Bud of Lily. ‘Lilium croceum’ x10 dia. Fixed with Chromo-Acetic solution. Stained with Borax Carmine. (Celloidinised preparation.) 1. Calyx. 2. Corolla. 3. Anthers. 4. Ovary. Transverse section of an Anther Lobe of Lily. ‘Lilium croceum’x6Q0dia. Fixed with Chromo-Acetic solution. Stained with Brazilin. 1. Fibrous layer. 2. Zone of smaller nucleated cells. 3. Tapetal layer. 4. Pollen grains. Transverse section. Ovary of Lily. ‘Lilium croceum’ x75 dia. Fixed with Chromo-Acetic solution. Stained with Brazilin. 1. Outer wall of ovary. 2. Inner wall. 3. Cavity of ovary. 4. Embryo-sac. 5. Central axis. Longitudinal median section. Catkin of Sallow. ‘Salix caprea’xl5dia. Fixed with Chromo-Acetic solution. Stained with Aniline-picrate and Gossypimine. 1. Central axis (pith). 2. Vascular system. 38. Anthers. 4. Floral Scales (bracts). 5. Nectary. S ‘ s 4% (o0n® ep%a* 104 Fie. 60. Fie. 61. Fia. 62. MICROSCOPICAL RESEARCH PLATE XVII. FLORAL STRUCTURE. Flower, entire of Wheat. ‘Triticum vulgaris’ x 15 dia. Fixed with Chromo-Acetic solution. Stained with Borax Carmine. 1. Ovary. 2. Ovule withembryo-sac. 3. Enlarged base of flower. 4. Main shafts of stigma. 5. Lateral branches of stigma. 6. Anthers, having already opened and shed some of their pollen. 7. Filament of stamen. (Specimen dissected out of its sheath about ten days prior to its natural time of opening.) One shaft of a Stigma, from a flower collected and prepared as above. 1. Shaft of the stigma. 2. An isolated pollen grain, with its tube. 3. Pollen grain, its tube penetrating a stigma, its course being indicated by dotted lines. Portion of above Stigma (Fig 3 in dotted outline) x 180 dia. 3. The pollen grain. 4. Its tube; the dotted outline indicates its course. 5. Nucleus of stigmatic cell. 106 Fie. 63. Fia. 64. Fic. 65. MICROSCOPICAL RESEARCH PLATE XVIII. CELL STRUCTURE AND CELL CONTENTS. Longitudinal median section through a grain of Wheat. ‘Triticum vulgaris’ x10dia. (Germinated in ‘milk-warm’ water for sixteen hours.) Fixed with Chromo-Acetic solution. (Unstained.) 1. The pericarp. 2. Aleurone-layer. 3. Endosperm (starch containing cells). 4. Remains of stigma. 65. Scutellum. 6. The growing apex of stem. 7. Developing root. 8. Plumule sheath. 9. Root sheath. Embryo of Wheat in longitudinal median section, after germinating twenty-four hoursx40 dia. Fixed as above. Stained with Brazilin. 1. Scutellum. 2. Developing root (examine transverse section of radicals, Plate II., Fig. 5). 3. Root-cap. 4. Root sheath. 5. A developing lateral, or secondary roolet. 6. Growing apex of stem. 7. Leaves in various stages of development. 8. Plumule sheath. 9. Ligule. Transverse section through a grain of Wheat x76 dia. Fixed as above. Stained with Brazilin. 1. Pericarp of seed. 2. Crystal. 3. Aleurone layer. 4. The endosperm cells containing starch. 108 Fic. 66. Fia. 67. Fia. 68. Fic. 69. MICROSCOPICAL RESEARCH PLATE XIX. CELL STRUCTURE AND CELL CONTENTS. Longitudinal: median section through Apex of Embryo of Wheat after twenty-four hours’ germination in ‘milk-warm’ water. Cut !/,..,in.x80dia. Fixed with Corrosive-Picro- Formaldehyde solution. Stained with Brazilin. 1. Axis of embryo.2. Growing point of stem. 38—4. Leaves in various stages of development. Longitudinal median section through radical end of Embryo of Wheat, after twenty-four hours’ germination in ‘milk-warm’ water. Cut !/,,.,in.x80dia. Fixed and stained as above. 1. Central axis of developing root. 2. Apex of root. 3. Protective root-cap. 4. Root-sheath. 5. Cells to form Epidermis (dermatogen). 6. Cells to form cortical tissue (periblem). 7. Cells to form the vascular system or root (plerom). . Longitudinal section through Embryo of Wheat (before fertilisation) x 35 dia. Fixed with Chromo-Acetic solution. Stained with Brazilin. 1. Spongy tissue of ovary. 2. Ovule. 3. Young embryo. Longitudinal section through Embryo of Wheat (after fertilisation). Prepared as above. 1. Spongy tissue of ovary. 2. Embryo. 3. Disposition of cells to form seed coat. WNas ids. Leet PS, Rtaniugane 2 La POs 2, monies Fae z orn oS . SS She <5 SOTA eget st Va ae OS, Site PSs 21 Pea ae cy rH £ ; ae . 14. IIo Fic. Fig. Fic. Fic. 70. 7 7 ~ 1. 2. 3. MICROSCOPICAL RESEARCH PLATE XX. CELL STRUCTURE AND CELL CONTENTS. Transverse section. Leaf of Thistle. ‘Carduus lanceolatus,’ affected with uredospores of ‘Puccinia graminis ’’ x 70 dia. Fixed with 92% Alcohol. (Unstained.) 1. disorganised tissue of leaf. 2. Mycelium of fungus. 3. Uredospores on under side of leaf. 4. Disorganised palisade layer. Transverse section. Stem of Wheat affected with ‘teleuto- spores’ of ‘Puccinia graminis’x70dia. Fixed with 92% Alcohol. (Unstained.) 1. Disorganised tissue of stem. 2. Mycelium of fungus. 3. Inner layer of hollow stem. 4. Vascular bundle in normal condition. 5. Teleuto-spores. Transverse section. Leaf of Berbery. ‘Berberis vulgaris,’ affected with ecidiaspores of ‘Puccinia graminis’ x 36 dia. Fixed with 92% Alcohol. (Unstained.) 1. Ground tissue of leaf. 2. Palisade layer. 38. Mycelium of fungus. 4. Fully-developed ecidium. 5. Spermogone of unknown function. Vertical section through entire plant of ‘Peziza convexula’ x60 dia. Fixed with 92% Alcohol. Stained with Aniline- Picrate and Gossypimine. 1. Thallus. 2. Sterile tissue. 3. Ascospores in various stages of development. 4. Para- physes. 5. Upper convex surface of plant. II2 Fie. Fia. Fig. Fic. TA. 70. 76. 77. MICROSCOPICAL RESEARCH 1 PLATE XXI. CELL STRUCTURE AND CELL CONTENTS. Transverse section. Male conceptacle of ‘Fucus vesiculosus x60 dia. Fixed with Chromo-Acetic solution. Stained with Aniline-picrate and Gossypimine. J. Limiting tissue of thallus. 2. Lining of conceptacle. 38. Protective hairs, among which are distributed the antheridia, stained red. 4. Mouth of conceptacle. Transverse section of female conceptacle of ‘Fucus, sp.’ x60 dia. Fixed and stained as above. 1. Limiting tissue of thallus. 2. Protective hairs. 38. Oogonia in various stages of development. 4. Opening, or mouth of conceptacle. Entire thallus of Fern under surface. ‘Gymnogramma sp’ x10dia. Fixed with Chromo-Acetic solution. Stained with Borax Carmine. 1. Thallus. 2. Antheridia. 3. Archegonia. 4. Rhizoids (root hairs). Vertical section. Thallus of Fern, passing through the Archegonia x 160 dia. Fixed with Chromo-Acetic solution. Stained with Brazilin. 1. Upper surface of thallus. 2. The canal-cells of neck of Archegonia. 38. Ovum. (Section prepared by the ‘cold’ paraffin method and cut !/ etiee in.) « 114 Fie. 78. Fic. 79. Fria. 80. Fie. 81. MICROSCOPICAL RESEARCH PLATE XXII. CELL STRUCTURE AND CELL CONTENTS. Transverse section. Stem of Tradescantia. ‘ Tradescantia discolor’ x 85 dia. Fixed with Chromo-Acetic solution. Stained with Hematoxylin and Gossypimine. 1. Empty cells; 2. Cells containing starch. 38. Vascular bundles. Transverse section. Stem of Pellionia. ‘ Pellionia Daveauna’x65dia. Fixed with Chromo-Acetic solution. Stained with Hematoxylin and Gossypimine. Starch containing cells in primary ground tissue. Longitudinal median section. Apex of Root. ‘Aspidium filix-mas’x180dia. Fixed with Chromo-Acetic solution. Stained with Brazilin. (Cut 1/509. by the ‘cold’ paraffin method.) 1. Apical cell. 2. Zone of primary meristem tissue. 3. Protective root-cap tissue. Longitudinal section. Aerial Root of ‘Monstera deliciosa’ x180dia. Fixed with Chromo-Acetic solution. Stained with Brazilin. (Cut!/ 500) in. by the ‘cold’ paraffin method.) 1. Bundles of raphides. 2. Nucleus of crystal - sac 3. Primary nucleated ground tissue. 4. Nuscleus in division. j + an 1 : p q © \ GO | i lane eg ey 0 ee KEE cis ae 116 Fic. Fic. Fie. Fig. 83. 84. 85. MICROSCOPICAL RESEARCH PLATE XXIII. CELL STRUCTURE AND CELL CONTENTS. . Transverse section. Endosperm of ‘Ricinus communis’ x45 dia. Fixed with 92% Alcohol. Unstained. Endosperm cells containing protein-crystals and aleurone grains. Longitudinal section. Root of ‘Scorzonera hispanica’ x60 dia. Stained with Hematoxylin. 1. Primary ground tissue. 2. Laticiferous vessels. Longitudinal section. Stem of ‘Euphorbia splendens’ x180 dia. Fixed with Absolute alcohol. Stained with Borax Carmine. 1. Ground tissue—cells containing starch. 2. Coagulated contents of laticiferous cell. 3. Elongated (dumb-bell) starch grains. Karyokinesis in longitudinal section of Onion. ‘Allium cepa,’ x 200 dia. Fixed with Chromo-Acetic solution. Pre- pared by the cold paraffin method. Cut 4/4... Stained Brazilin. 52 e ‘ 3