MIOROSCOPIO BOTANY. A MANUAL OF THE MICROSCOPE VEGETABLE HISTOLOGY. BY DE. EDUARD STRASBURGER. FROM THE GERMAN BY REV. A. B. HERVEY Ac. f\fo, BOSTON : SAMUEL E. CASSINO, 1887. COPYRIGHT, 1.S87, 1!Y SAMUEL E. CASSINO, TRANSLATOR'S PREFACE. The "Kleine Botanische Practicum" of which this is a translation is an abridgment of a larger work of the same kind made by the author, Dr. Strasbnrger. I have made further condensation of the work in those chapters where the matter and expression would admit, notal)ly in Chapter VIII and those immediately preceding, and in Chapters XXX and XXXI, but in no case have I omitted essential matters from the text. The Introduction alone is shortened by omission, because portions of it were quite irrelevant to the purposes of an American edition. For a similar rea- son, and to save space, the Author's preface is not repro- duced. The first two "Registers" or Indices, those enumerat- ing the plants and reagents used in the studies, are also omitted, as being neither essential nor very important, the references being all contained in the general subject- index. The formuhe contained in register II are found in an appendix. The work is divided into thirty-two Lessons or Chap- ters to adapt it to the weeks in a German College 3'ear. It is believed that the work is well adapted to the needs of both the solitar}^ wcn-ker and students in American Colleges. A. B. Hervey. Taunton, Ma.ss., 4 July, 1887. (iii) TABLE OF CONTENTS. Page. Introduction. 1 Lesson. I Use of the Microscope. Structure of Starch. 6 II Gluten. Fatty Oils. Making Permanent Prep- arations. Use of the Simple Microscope. 18 III Protoplasm Streaming. The Nucleus. Draw- ing with the Camera. Determining Magnifi- cation. 29 IV Chromatophores. Colored Cell-sap. 38 V Tissue, Thickening of the Wall. Reaction on Sugar. Inulin Nitrates. Tannin. Wood sub- stance or Lignin. 45 VI Epidermis. Stoniata. 60 VII Epidermis. Hairs. Wax and Mucilage. 71 VIII Closed Collateral Vascular Bundles. 82 IX Open Collateral Vascular Bundles. 95 X Structure of the Coniferous Stems. 108 XI Structure of Linden. Bicollateral Vascular Bun- dles of the Cucurbita. Sieve Tubes. 118 XII Vascular Bundles of the Axile Cylinder, and the Secondary Lateral Growth of the Roots. 129 XIII Vascular Bundles of the Ferns and L3'copods. 138 XIV Cork. Lenticels. 145 XV Structure of the Foliage and Floral Leaves. The ends of the Vascular Bundles. 151 (V) VI CONTENTS. XVI Vegetative Cone of the Stem. 161 XVII Vegetative Cone of the Root. 173 XVIII Histology of the Mosses. 181 XIX Histology of the Fungi, Lichens, and Algae. Staining the Cell Contents. 191 XX Diatoms. Protococcus. Yeast. Protophytes. 202 XXI Schizomycetes. Use of the Immersion System. 214 XXII Repro^ ' by a clear boundary line. We also M^^^ ^ find smaller ones consisting of but two or three granules, and numerous single granules made by breaking up ^^^ , ^,^^^ g,.,i„3 the grains. Lamination is not to be from the oi\t,Avena sativa. , , A, conipouinl- grain; B, seen, and the nuclei are but rarely component pans of tbe discernible. . '«"^«- X ^^°- The starch grains of the Euphorbia have a peculiar ap- pearance. Cut a piece at will from the Euphorbia lielio- scopia and dip the cut surface into a drop of water on the slide. The milk-sap which runs out will mingle with the water and we shall see in it small isolated rod-like bodies, Fig. 8. They are the starch grains ^^ ,%>x i" question. They are strongly re- |P ^3^ X X tractive; lamination is only imper- - \ • <^ "''^>' fectly hinted at in the most favorable l] v:^ \ -^C^ eases ; in many instances a longitud- tl ^k ^^ ^"^' ^^^^ ''^ ^^ ^'® ^®^" '" ^^^® inside of 11*^ the grain. The size of the grain Fig. 8. starch grains varies and many appear to be smaller from the milk-sap of Eu- in the middle. pliorbiahelioscopia. X 510. , , • i t-i t t . ^ In the tropical Aup/iorbice, the starch grains are much more interesting. Make a prepa- ration in the same manner from Euphorbia sp>lendens, a plant often found in greenhouses. The starch grains look like bones, Fig. 9. They some- 16 USE OF THE MICROSCOPE. grains from the milk- sap of JSuphorbia splendens. A is partly enveloped in a thia membrane. X S^O- times show lamination. Sometimes a colorless sac appears on the lateral surface of the grain, A, the walls of which however arise not from the substance of the starch grain, but from the adhering plasma-mass. The small globules of the milk-sap, Avhich are distributed through the water, are seen to be in constant, rapid, trem- bling motion. It is the so-called Brownian molecular movement, and is not an indi- cation of life, l)ut is perhaps caused by currents in the fluid which move the gran- starch ules. Having made this general survey of the form and structure of starch grains, "we will now study the effects of reagents upon them under the microscope. Take the potato starch preparation ; and, after focussing it, apply a drop of iodine solution to the edge of the cover-glass (iodine water, iodine alcohol, or potassium iodide of io- dine). Be very careful and not get a drop on the cover- slass or the lens. If we do we must clean it at once. Select a place not too far from the edge of the cover- glass, where the solution was applied, and then move the slide to follow the progress of the reaction. Directly the grains will change to a bright blue and then rapidly darken to a black blue. At first the lamination becomes more distinct but rapidly disappears as the grain grows opaque. A larger quantity of potassium iodide of iodine at last produces a dark In-own color in the grains. Dry starch turns brown in the fumes of iodine, but water added rap- idly changes it to blue. Touching the opposite edge of the cover-glass with blotting paper will hapten the movement of the reasrent. STRUCTURE OF STARCH. 17 The blue reaction with iodine proves that tlie rod-like grains of the Euphorbia are really starch, notwithstanding their strange form and lack of lamination. Let us study the actifii of potassiiun hydroxide on starch grains. Use the potato-starch, introduce the reagent and watch its effect as before. The reaction should be very gradual if possible. We shall tirst see the lamination most distinctly, and then it will rapidly disappear as the grain swells. During this swelling the nucleus is greatly hollowed out and the walls of the anterior part of the grain are folded into the cavity. Gradually the grain be- comes a transparent mass with quite indistinct outline. Finally, we should study the effect of heat on the grains of starch. Warm the preparation over a flame till it reaches a temperature of 70 C, taking care that the water is kept in it, wdien it will be found that the grains are swollen in exactly the same way as when treated with po- tassium hydroxide. Before putting the microscope away, clean the lenses as already directed, and rub the tube and the inside of the sheath with a cloth. Cov^er the instrument with a glass bell w'hich should be bound under the edge with felt. XOTES. • (1) Compare Xaegeli, Die Stärkekörner, in Pflanzeiiph3'siol. Unter- suchungen Heft 2: E. Strasburger, Bau u. AVachsthum der Zellhjiute, p. 107. There is also other literature. 2 LESSON ir. Gluten Meal. Fatty Oils. Making Permanent Prep- arations. Use or the Slviple Microscope. With a stout pocket knife bisect the cotyledons of a ripe pea, Pisum sativum. Witli a sharp razor take a thin section from the cut surface. In cutting sections with a razor observe : 1. The surface to be cut should be moistened with the fluid in which the section is to be ex- amined, water or glyceriue, — in this case with the latter. 2. The first section is not to be used since the tissue is more or less torn by the pocket kuife. 3. In cutting hard tissue like this, very small sections of the utmost possible tenuity should be made, care being taken not to cut deep lest the edge of the razor be nicked. 4. Lay the edge of the razor upon the prepared surface and cut fiom the middle outward towards the side of the object. 5. In cutting, draw the razor in the direction of its length as well as push it forward. Both hands may be supported and steadied against the breast and yet have a sufficiently free movement. The back of the blade should rest upon the forefinger of the hand holding the object. 6. So small and hard an object as the half of a pea, and one so diffi- cult to hold firmly in the hand should be held in a small hand-vice. 7. Be not content with a sino-le section but make a considerable number and then select the best for the investigation. The section should be examined in strong glycerine or with glycerine and one-third distilled water. Pure water will not do, as it causes the appearance of disorganization in the fundamental substances of the cells. Use a hair (18) SECTIONS OF SEEDS. 19 pencil to traiisfor the section from the knife to the slide. Press the pencil clown upon it, in taking it from the knife, which will prevent it rolling up, iis it often does when seized at the edge with the forceps. Liy the section care- fully in a drop of the liquid on the slide and withdraw the pencil, turning it laterally in the fingers a little at the same time. To turn the section over upon the slide, press the pen- cil down upcm the slide so that it will touch the edge of the section and then rotate it in a direction away from the section. This will draw the section over upon the upper side of the pencil, when it may be put down again upon the slide the other side up. Wash the pencil after using. Put the section of the pea under a moderately high magnifying power. The tissue seems to he composed of rounded cells. Fig. 10. At the place where these cells meet is a triansular in- tercellular space filled with air. It is black like the edo:e of an air bubble. 1'he walls of the cells arc pretty thick. In each of the cells are large starch grains, also small eran- ules, al, lying between. The latter are, in their turn, embedded in a very tine granular sub- stance, p. At thin e ./^'':- Fig. 10. Section of cotyledon of the pea. wj.cell membrane; i, intercellular space; am, starch; al, aleuron grains; p, funilamental . . substance; n. nucleus, the last maile visi))le places in the section the only by staining. X 240. starch »rains are fallen out and we see the corresnondino- cavities in this fine, granular mass. The small grains are gluten meal, aleuron, or proteid grains (1). They lie in 20 STAINING THE SECTIONS. the fundamental substance of the cells. Apply an iodine solution to the section, and the various elements will as- sume their characteristic colors. Lift up the cover-glass and put a drop of the iodine directly on the section. The starch grains are colored blue or violet ; the aleuron grains and the fundamental substance yellow. AA'ith po- tassium iodide of iodine the color of the latter elements becomes very intense, but t'le starch grains are over col- ored and become a dark brown. Put a section in a drop of borax-carmine solution, and in a short time the funda- mental substance, and directly also the aleuron grains, will be colored a dark red, while the starch grains remain col- orless. The reaction -is more evident, it' we replace the solution with water or dilute glycerine. This may be done by drawing out the cannine by means of blotting paper, while at the same time the other liquid is supplied at the other side of the cover-glass. A drop of Millon's reagent causes the starch grains to swell up ver^' large and soon to become unrecoonizable. But the aleuron and fun- damental substance is disorganized, and the mass is col- ored a brick red, after a while. If now we lay a section in a solution of methyl-green and acetic acid, there will appear in a short time, in each cell, between the other elements, a green-blue fleck of somewhat indefinite outline. This is the nucleus, n. The other substances have not been colored. The starch o-rains are a little swollen and show radial clefts. The aleuron grains are a little enlarged and appear porous or hollow. The methyl-gieen acetic acid is thus a specific nucleus-stain for the case in hand. The cell walls have also been stained a beautifid, bright blue color and are much more distinctly seen, as are also the intercellular spaces, than in the glj'cerine preparation. Thus, we have learned to recognize albuminous sub- SECTION OF WHEAT GRAIN. 21 stnnces, for such are the aleuron o-iains and the protoplasm (cell plasma and nucleus), by the yello\v-l)ro\vn reaction of iodine, the absorption of coh)i'ing matter and the brick- red reaction of Alillon's reagent. We shall learn by and by that protoplasm will show these reactions only when it is dead. The reagents in this case have killed it. The nucleus shows a particularly strong affinity for coloring matter. For our second example, selecting a kernel of wheat, Triticum vulgare, we firs^t cut the kernel aci'oss in halves and make one-half fast in a hand-vice. Moisten with glycerine and cnt the section quite , to the outer surface of the kernel. The section examined in the same fluid will appear as in Fig. 11. Beneath the com- pressed dead cells of the skin, p, which represents the shell of fruits and seeds, lies a layer of quad- rangular cells close- y\g. ll. section of a grnin of wheat, Triticum Iv Dacked with aleu- '"'^^"''^- ?-'. outer, <, inner seeil-coat;«^, aleuron; a»» starch-grains; ?i, nucleus. X -^0. ron grains. Next to tliis are elongated, less uniform cells which contain slanli grains of all sizes. These points are all established by means of the i)roper reagents. Selecting a good section, we will now proceed to make a permanent preparation. We will mount our tirstol)ject in the simplest possible way and one suitable to the pies- 22 PREPARING-MICROSCOPES . eilt case, viz. , in glycerine jelly. Put as much of the jell}^- like substance on the centre of the slide as we judge will make a small drop. Then warm, the slide over a flame B' — Fig. 12. Smaller preparing microscope and hand rest of Zeiss, § natural size, oi, stage; rf, doublet; st, movable rod; sr, fine adjustment screw; s, minor; P, back of hand-rest. slowly till the jelly is quite liquefied. Put the section in the fluid and, having warmed a cover-glass, lay it on over it, — not exactly horizontal, but to avoid air bubbles, — lay the edge of the cover-glass upon the slide, let it down PREPARING-MICROSCOPES. 23 gradually and gently upon the fluid, pressing it horizon- tal afterwards. If still, there are air bubbles, warm the preparation to fluidity again and gently lift up one edge of the cover-glass a little. They will usually come out; if not, they must be allowed to stay. Several small sec- tions may be mounted under one cover-glass, by carefully distributing them about in the drop. If, in putting on the cover-glass, they should get displaced and overlie each other, warm the slide again, and with a stiff hair thrust in under the cover-glass push the sections into place. Before putting on the cover-glass, in the first place the preparation should be examined carefidly under suita])le magnification, and, if any particles of dust or dirt are in the fluid, they should be carefully removed with a needle. For this purpose the simple or compound mounting-mi- croscope is necessar}^, and we will now direct our atten- tion to learning the use of that. Let us suppose that the student has the small Zeiss preparing-microscope, or any other of like construction. Over the stage, oi. Fig. 1^, is an horizontal arm carrying a doublet, d. The arm is attached to a steel rod, st, Avhich rotates and moves up and down within a sheath, the latter movement giving the coarse adjustment. The fine adjust- ment is secured by turning the screw, .sr. The instru- ment is mounted on a sul)stantial and suitable block whose raised ends, p, serve as a support to the hand inmani[)ulat- ing the preparation. The instrument carries two and sometimes three doul)lets, with a magnifying power re- spectivel}' of fifteen, thirty and sixty diameters, and may also with advantage be furnished with magnifying glasses having powers of five and ten diameters. The large preparing-microsco[)e of Zeiss, or another of like construction consists of a lens system Fig. 13, I, in which three achromatic lenses are united in one objective, 24 PRRPARING-MICROSCOPES. o5, a tube, and an achromatic concave ocular, oc. For low magnification the objective may be used alone as a magni- fying glass, by removing the tube and the ocular. The Fig. is. Larger i)iei>aiing-micioscope of Zeiss, i natural size, ot, stage ; p, hand- rest; sr, rack and pinion; I, lens system; oft, objective; oc, ocular; or, an object slide on the stage under the spring clips. ♦ lenses are also separable and Ave may use 'the upper alone, or the upper two or all three together ; the resulting magni- fication being fifteen, twenty and thirty diameters, respect- MAKING PERMANENT PREPARATIONS. 25 ively. Focus with the screw, sr. On both sides of the st.'ise, ot, are rests, p, for supporting the Hands while at work. In order to use the compound, as a preparing-micro- scope, we ninst attach the erecting prism to ocuhir No. 2, or replace this ocular by one to which a prism is attached or one may use the erecting ocuhir, only that the>e go only with stands having a draw-tube. One can indeed train himself to work with the compound microscope with- out the erecting apparatus, but it is a m-itter of much dif- ficulty as all the motions seem to be reversed in the field of the micioscope." Hand- rests are also necessary in any case. Whatever microscope we use, we place the preparation, from which we are to remove the foreign sul)stances, on the stage. After arranging the mirror and focussing the object, we take a needle by the 1 older in each hand, sup- porting the hands on the rests, and bring the needle points both at the same time into the field of vision. We shall soon learn how to make the necessarily very small motions required, and shall have removed all foreign substances from our preparation with our needle points. Having again warmed our jelly we put on the cover-glass. Glycerine jelly i)reparations require no cementing and are therefore extremely simple to niid^e, and since most vegetal)le objects and the stains used upon them are Avell preserved in this medium, it may be commended before all other methods. Each permanent preparation should be labelled with the name of the pLint, the object, the preserving medium, the principalstains used and the date. We will now go on, and first cut a section, as before directed, of the seed of the whhe lupine, Lupinus alba, or of some related species, moistening the cut surface 26 ALBUMEN CRYSTALS. with water. Examined in this liquid, the section shows, in the cells, rounded aleuion grains and vacuoles. The grains are strongly refractive, angular, and towards the in- terior become gradually reticulated and granular. They lie close together and fill the cell, being embedded in a small quantity of fundamental substance, which also lines the cell walls. The latter are thickened and dotted, a structure which we shall study further, with more favor- able objects. The grains are colored a beautiful gold yel- low with iodide glycerine. We will next make a section from a Ricinus seed. The tissue of the endo- j5 sperm readily lends itself to section-mak- ing as it contains much oil and does not need to be moistened. Examined in water, this liquid expels the oil from the funda- mental substance. The grains embedded in this fatty sub- stance. Fig. 14, A, have within them mostly one but sometimes also two or more albumen crystals, and in most, also a single round body which is an inorganic sub- stance (a globoid), a combination of phosphoric acid with lime and magnesia. By the prolonged action of water, the fundamental substance will be disorganized, large drops of oil will collect on and in the object, and on the glass, and on this in irregular masses. But if they float free in the water they are globular. If we focus upon the optical transection of such an oil globule it will appear to be a bright gray and be surrounded with a slender black band. Fig. It. From endosperm ot liicinus communis. A. cell with contents nnder water; D, single aleu- ron grains in olive oil; ^, globoids; /j, albumen crystals. X ßtO. , VEGETABLE OILS. 27 Kiiise the tube and the small dark band becomes some- what bi'oader. Lower the tube and the band disappears, the disk showing itself somewhat more brightly bordered. The drop of oil exhibits an appearance the exact ojiposite to that which we o])served in the air bubble. The air refracts the light less, the oil more powerfully than the Avater, hence their contrary behavior. Adding now a drop of absolute alcohol to the Ricinus section in water, the preparation is made more clear and the crystals of albumen in the aleuron grains are brought out sharply to view. They become so very distinct that this method of examining them is highly commended. They are crystals of the tetrahedric hemiedrys of the reg- ular system (2). Prolonged action of the alcohol dissolves the Ricinus oil drops, it differing from other fatty oils in being missible with alcohol. If now we put a section of the Ricinus seed in glacial acetic acid the albumen crystals will disappear in the alenron grains, the latter in their turn increasing consid- erably in size. The globoids also become larger and show themselves very distinctly in each grain. No dro[)s of oil are seen, Ricinus oil mixing with acetic acid, unlike that of other plants. Alcohol and glacial acetic acid, in- asmuch as they dissolve the essential but not the fatty oils, make a good test for distinguishing these substances under the microscope. Turpentine among the essential oils dissolves somewhat less readily in these reagents than do the others. Chloro- form and ether dissolve fatty and essential oils in the same manner. Add a drop of dilute alcana tincture to a section in water and the fatty masses will be colored a red brown. The same result follows with essential oils and resins. To a glycerine preparation of i?^c^■?m5 seed , add a small 28 SECTION OF BRAZIL-NUT. quantity of hoematoxylin and it will color the albumen crystals a beautiful violet. With olive oil the albumen crystals are not visible. The Avhole grain appears as a strongly refracting rounded body in one end of which the globoid seems to form a vacuole, Fig 14, B. If the sec- tion be laid in a one per cent solution of perosmic acid the all)umen crystals come out beautifully. They slowly assume a brown shade. Both essential and fatty oils are slowly blackened with perosmic acid. Albumen crystals of extraordinary beauty, which read- ily exhibit all the reactions of albumen, are found in the endosperm of the seed of Bn'lJiolletia excelsa, which may be bought anywhere under the name of Brazil-nut. The section is easily made. Add absolute alcohol to an aque- ous preparation and the crystals come out most distinctly. The alcohol does not perceptibly affect the fatty oil. The latter is unchanged by the action of glacial acetic acid, but the albumen crystals soon dissolve. In a one per cent solution of perosmic acid the crystals are very distinct. The crystals are so large that they may be recognized by their form alone, even with comparatively small mngniti- cation. Globoids, in the shape of irregular masses of rounded forms, are found lying in the tissue with the crys- tals. The fundamental substance is very rich in oily mat- ter and gradually become» almost black with the one per cent osmic acid. The granular contents of the aleuron grains soon turn dark while the crystals are slowly col- ored 3^ellow. The crystals are optically uniaxial, hexag- onal rhombahedra-hemiedrich. Notes. (1). See Pfeffer, Jahrb. f. wiss. Bot., viii, p. 429. (2). Shimper, Uiitei-s. ii. d. Proteinkrystalle d. Pfl. Iiiaug. Di.ss. Strassburg, 1878. LESSON III. Streaming Motion in Protoplasm. The Nucleus. Drawing with the Camera. Determining THE Magnification. Selecting the hairs on the stamens of the Tradescantia, as a most favoraI)le object, we will now study the appear- ances of motion in the living protoplasm. Tradef^cantia Virginica and the species nearest rehited to it are cultivated in every botanic garden and bloom from May to hite autunm. Select hairs from a just- opening or recently-opened blos- som. Tear away a tuft of the hairs from the flower with the forceps and transfer it to water. The Avhole filament may be put under the cover-glass if the anther has been removed. In that case bubbles of air usually get entan- gled among the hairs which costs much trouble to get out. It may be done, however, by brushing it with a fine pencil while it is still \j^ i \\ \ held in place. Put on the cover-glass and if \ v ■ :\/j the air has been removed with sutKcient care \. jj ,•■';/ the hairs have not been iniured. The hairs >z.~:''-s will be seen to consist of a series of swollen ^^^ ^^ q^^^ cask-shaped cells ioined end to end, and sep- from staminate T , T . . . 1 , , -1 hair of Trades- arated b}' division walls at the constricted cantta virginica. places. Each cell, Fig. 15, shows a thin com- ^ ^^^" plete layer of protoplasm lining the cell Avail and numerous streams of protoplasm, of various dimensions running through the interior. Suspended within these streams, and enclosed in a coherent layer of plasma, is the nucleus (29) 30 STREAMING MOTION IN PROTOPLASM. (somewhat below the middle in the accompanying illus- tration). A violet colored cell-sap fills the interior of the cell, covering the nucleus and penetrated by the proto- plasm streams. Protoplasm consists of a colorless viscid substance called hyaloplasm which bears numerous minute granules, the microsomata. There are also larger strongly refractive bodies which appear to be of a bluish color and which we will call leucoplasts or starch-builders. If we focus on the protoplasmic wall layer, we shall see that it does not move as a whole, but that fine netlike anastomos- ing streams run through it. The movement is especially strong in the strings of plasma" whioh penetrate the cell cavity. These streams are of different thicknesses, anas- tomose Literally quite often, and show a prevailing ten- dency to meet at the nucleus. Most of the streams end in the plasma layer which surrounds that. The streaming movement is often only in one direction ; but often also in a very slender string two streams are seen moving in oppo- site directions. We perceive the movement by the motion of the micros(nnes and leucoplasts. Prolonging the ob- servation, we notice that the strings slowly change their thickness, arrangement and configuration. New connect- ing branches are pushed out, old ones grow thin, snap asunder and are withdrawn into others. So the image constantly changes. The nucleus is almost globular, in many cases oval or somewhat flattened. AVith the highest magnification which we employ, it appears finely dotted, and in it may also be distinguished some larger grains. In some cells the nucleus seems to have divided itself into two Avhich lie close together. The nucleus is towed about by the plasma strings and so gradually changes its })lace in the cell. To demonstrate this, make a rough sketch of the cell and contents, and after some little time compare it with the then position of the nucleus and the streams. ABBE CAMERA LUCIDA. 31 To make this sketch at all valual)le it should be quite ac- curate, and hence should be drawn with the camera. Let us learn how this is done. In Fig. 15^ is given an ideal section of the Abbe cam- era lucida, which, after focussing the object, should be at- tached to the ocular and fastened by the screw sr. It will perhaps be safest to take the ocular out of the tube for this purpose, and avoid the danger of pushing the object- ive down upon the preparation by the operation. Then adjust the mirror of the camera as in the illustration in- FiG. 15^. Abbe's camera Incida, natural size, longitndinal section. The rays follow direction of the lines. O, direction to the eye; S, direction to the drawing surface; sr, clamping screw. dining it at an angle of 45°. Now, we shall see an image of surrounding ol^jects in the field of the microscope, when we look into the ocular through the camera. Place a drawing board horizontally, by the side of the microscope under the mirror ; on this lay the drawing paper and on this hold a peucil point. If this is visible in the field of the microscope along with the image of the object, the iu- strument is properly adjusted. The pencil becomes visible by a double reflection, first from the mirror and a second time from the silvered surface of a small prism in the cam- era (see illustration), while the image of the object comes direct to the eye through a small opening in this silvered 32 DRAWING WITH CAMERA LUCIDA. surface. If the drawing board is not in clear view of the observer, the pencil point will be indistinct and the board shonld be raised up, seldom lowered. The distinctness of the microscopic image on the drawing surface depends upon the relative amount of light on each. That on the draw- ino- surface is regulated by a contrivance of smoked glass attached to the camera.* Having rightly adjusted the in- strument and the dlumination, trace the outlines of the ob- ject in the tield with the pencil. The second camera recommended in the introduction, has the advantage of being always attached to the micro- scope and ready for use. It consists of two prisms in one mounting, inclined to each other at a certain degree, liays from the pencil to the eye are brought to be parallel with those from the object, by a double reflection in the prisms. The camera is brought into position when the front edge of the prism, visil)le through a hole in the mounting, is di- rectly over the eye-lens of the ocular and nearly halves it. It should also be placed close down to the ocular. The drawing board is inclined, and will be at a [)roper ano-le if the circumference of the field of view in the micro- scope makes a perfect circle when drawn on the board. If it appears as an ellipse when so drawn, the board must be fixed at a greater or less inclination till the field is a per- fect circle on the drawing paper as in the microscope. The same result may l)e reached l)y using a stage microm- eter ruled to hundredths of a millimeter. Arrange the camera so that the successive rulings will be traced on the board, one beyond the other, using a considerably high magnifying power, and then carefully measure the distance between them. If that distance increase outward the *Tlie desired balance of illumination may also be obtained by tbe use of the diaphragm on tlie microscope, and a small screen whicli shall throw a shadow upon the drawing board.- A. B. H. MEASURING WITH CAMERA LUCIDA. 33 board lies too nearly horizontal ; if it diminish it is too nearly perpendicular ; if neither, it is just right, and then it will be found to be inclined at an angle of al)out 25^. The imaije which Ave have thrown down on our draw- ing board will enable us to determine the magnifying power of our combination of lenses. We know, for ex- ample, that the lines are really .01 mm. apart. But on the drawing surface they appear by careful measurement to be 2.4 mm. apart. Hence the magnification equals two hundred and forty diameters. If one has attained sufficient exactness in drawing to be able to produce a picture of equal dimensions to that of the microscopic image with its given magnification, he has only to measure this and divide the amount by the mag- nification to get the exact size of the object. This method gives in the simplest way such exact results that we may adopt it exclusively in our investigations. In the example before us the hair cell of the Tradescantia measures 9 mm. in breadth. This divided hy 240, the magnification, gives .0375 mm. as the real breadth of the cell.* We will now return and attempt to draw the hair cell of our plant by means of the image thrown down from the camera. We shall have to regulate the illumination of the second camera, by shadowing the drawing board, and properly adjusting the mirror, for we should have an * What seems to me a far better and more exact way is, to draw all objects which we wish to measure by means of the camera, in either of the following ways: 1. Have the drawing board fixed at a given standard distance of 25 cm. from the camera and so make all drawings at tliis distance. Then divide tlie various di- mensions of the drawing as above with the known magnification of tlielens combi- nation used. To save calculations one may have scales made corresponding to the magnification of the various combinations of lenses by which to measure the drawing at once. 2. Let the drawing board be at any convenient distance from the camera, draw the object, then without changing the relative position of anything replace the ob- ject witli the stage micrometer and draw the scale of .01 mm. along the edge of tlie paper. By this the various dimensions are always easily determined.— A. B. U, 34 STREAMING MOTION OF PROTOPLASM. equal illumination on the two. Draw with a lead pencil on stiff smooth drawing paper. A very thin solution of gum applied to the finished drawing will prevent it "rub- bing" and getting defaced. We Avill make a sketch of the outline of the whole, of the streams of plasma and of the nucleus, and after about an hour compare the object and the picture and see if thej 'Still coincide with each other. We shall find, as already said, that the divisions of the streams are different and the position of the nucleus has changed. In order to demonstrate that the streaming motion in the several cells is quite independent in each, and that it is not infiuenced by the cell walls we will observe the efiect on the filament, of an application of a neutral denser fluid like a concentrated solution of sugar, or strong glycerine. Allowing a drop of the fluid to run under the cover-glass and drawing out from the other side a portion of the water Avith a piece of blotting paper, we shall soon seethe efiect upon the cells of the hair. The denser fluid absorbs some of the water in the cell, which causes a corresponding contraction of the protoplasmic sac and draws it away from the cell wall at certain points. This contraction of the protoplasmic body under the influence of a water-ab- sorbing fluid is called plasmal}sis. While the contraction is not too strong it is ol)served that the streamino" on and from the places withdrawn from the cell wall still con- tinues. Soon, however, all movement in the cell ceases. It will, however, be resumed again if the denser fluid be replaced by water, and this may be accomplished hy draw- ing out the fluid from under the cover-glass by means of blotting paper, at the same time that water is allowed to run in under from the opposite edge of the glass. T-lie protoplasmic sac will then again be distended till it reaches and rests upon the cell wall. It often happens that when the STREAMING MOTION OF PROTOPLASM. 35 protoplasm is withdrawn from the cell wall, little masses of the plasma will be torn away from the cell body and lie as rounded balls on the walls of the cell. All these, however, are taken up andal)Sorbed into the mass of the protoplasm, when it is restored to its normal place and conditions. One may easily demonstrate that. during the above- mentioned contraction of the contents, the coloring mat- ter is not dilfused throusjli the living protoplasm, and that the coloring matter of the cell-sap is correspondingly darker. With dead cells the appearance is quite differ- ent. The application of absolute alcohol to the hairs im- mediately kills the protoplasm and causes it to absorb the coloring matter. The color of the cell-sap is immediately withdrawn and it becomes very clear, while the cell plasma and the nucleus are stained a dark violet. The violet coloring matter can now penetrate the protoplasmic sac and distribute itself in the surrounding fluid. In lack of Tradescaiitia, other plant hairs may be sub- stituted, as, for example, those from the youngest sprouts of the Cucurbita species. Cut them at the base from the plant, with a razor, and transfer to a drop of water on the slide. The stouter hairs are composed of several cells at the base, but change into a pointed row of cells upward, while others bear little many-celled knobs on their points. The network of protoplasm is richly developed in the cells and contains microsomes and larger, less numerous, green-colored chlorophyll grains. The nucleus is large, suspended in the threads, has bright nucleoli, and is moved about here and there in the cell. The root hairs of the Hydrocharis morsus ranee alTord a very characteristic ol)ject. Take the young, fresh roots with stiff hairs which are visible to the naked eye. Cut off the end of the root and lay it under the largest cover- glass in a sufficient quantity of water. On account of the considerable thickness of the object, all parts of it cannot 36 ROTATION OF THE PROTOPLASM. be brought within the focus of the stronger magnifications, the lens striking the cover-glass before the deepest parts of the object are in focus. The hair cells are very long, sac-like. All root hairs are single-celled. The rich pro- toplasm is in powerful motion ; but there are no tine, net-like, many -branched streams, only a single, strong, re- current wall-stream. We distinguish this kindof stream- ino-from the other, the circulation, by naming it "rotation." This stream is a broad, slightly screw-like, recurrent band which, if projected upon a plane, would form a very elongated figure 8. We may not, perhaps, represent the movement, as if the band as a whole rotated within the cell, since we observe that the neighboring particles continually change their relative position during the move- ment. The two streams, moving in opposite directions, do not indeed immediately impinge upon each other, but are separated by a thin layer of plasma which remains at rest. The rotation of the protoplasm is well illustrated in the cells of the leaf of Vallisneria spiralis. Make a section from the under side of a stout leaf, by laying the long, slender leaf over the index finger of the left hand, hold- ino- down the ends with the thumb and third finger, and then make a superficial section, with a razor, of about half the thickness of the leaf, and lay it on the slide with the cut surface up. Find a place where no attached air bubbles interfere with the observation, and then selecting as wide and long a cell as possible, look for the streaming movement. The movement is retarded by lowering the temperature and, consequently, accelerated by slightly warming the slide. The stream circles about the whole cell without essentially deviating from a direction parallel to its longer axis. The "indifference layer" has considerable breadth. The stream carries about the nucleus and the green chlorophyll grains. The former is flattened disk-shaped. It is for PROTOPLASMIC STREAMING IN OHARA. 37 the most part hidden by the chlorophyll grains, but oc- casionally comes in sight. Frequently, it gets stuck fast in some depression or turning i)lace, and then the chlorophyll grains get dammed up against it, till a moment later all get drawn into the stream again. The direction of the movement chano;es from cell to cell without reo;a- larity. By adding glycerine or sugur solution as before, one may easily see the movement continue, in the first moment of the contraction of the protoplasmic sac. The mostpowerful plasma stream known in vegetable cells is met with in the Characeae. We must use the genus JSfilella, since the internodes of the genus CJiara have an opaque outer layer which renders them unserviceable for our purpose. We should select one of the younger internodes, and we shall soon observe that the rotating stratum of pro- toplasm has a very consideral)le thickness, and that there is an outer layer in which are embedded the chlorophyll grains. This layer does not move. It is in this case, relatively, cpiite thick, but is commonly so thin as to escape observa- tion ; for, in all the other cases, there was an unmoving protoplasm-layer, the so-called "skin layer." An obliquely lying stri[)e on the wall of the JSFitella, easily seen, con- tains no chlorophjdl grains. It corresponds to the "indif- ference layer" of the protoplasm stream. It repeats here the appearance seen in the root hairs of HydrocJiaris when the " indifference stripe" of the protoplasmic layer is likewise found extremely reduced. The cells of the internodes of Chara have many nuclei, and the proto- plasm stream bears many' elongated nuclei which, only under the most favorable conditions, are discernil)le as clear spots. The rounded masses which appear in the stream, in greater or less number, are not to be con- founded with these. They have either a smooth surface or are covered with minute spines. Nothing is clearly known of their sisrniticance. LESSON IV. Chromatophores. Colored Cell-sap. "\Ye have alread}^ several times glanced at the structure and contents of the chlorophyll «[rains, and will now direct our special attention to these forms. For this purpose, we shall choose a widely distributed moss, distinguished for having very beautiful, large, lens-shaped chlorophjdl grains, and whose leaves, constituted of a single layer of cells, are, without further preparation, m'ost favorably adapted to our purposes. This moss is Funaria Jiygro- /-E. ^ metrica. Numerous chlorophyll graius of '^"W considerable size are seen in each cell ; and, in ^lt:%^ plants growing in diffused daylight, are dis- ^^"^0^ tributed only on the free cell walls, that is, (f^^ on the walls which constitute the upper and under surfaces of the leaf. They present Fig. 16. Ohio- , , -^ ^ rophyii grains their broad side to the observer. That they from tlie leaf of ii i • xii i Funaria hygro- '^^'® Smaller Avheu secu HI proüic w^e observe metrica. ji^ those occasioual instances when they are found lying on the side walls of the cells. Every stage in the process of self-division of the chlorophyll grains is easily found, often in one cell. (See Fig. 16. ) The resting grains appear almost circular. Then they Ijecome ellipti- cal, then biscuit-shaped and finally completely dissevered. The two young grains remain for a lono- time in contact. The starch contents of the chlorophyll grains are, accord- ing to their various sizes, in many leaves easily and in others with difficulty seen, but the starch comes out clearly if the chlorophyll grain is set free in the water and disor- (38) CHLOROPHYLL GRAINS. 39 ganizecl. For this purpose, cut the leaf into small pieces "with sharp shears, and the freed starch from the chloro- ph^'ll grains will increase in size in the water and may be easily detected with iodine. An uninjured chlorophyll grain treated with iodine is colored brown, this being the result of the combined blue coloring of the starch, the yellow brown of the protoplas- mic fundamental substance, and the green of the chloro- phyll. Thebetter way is to bleach a leaf by long immersion in alcohol. Then the iodine solution, gradually penetrat- ing the colorless chlorophyll grain, will color the starch within before it does the protoplasmic body. The iodine reaction is greatly assisted b}^ the use of potash which swells the starch grains and thus makes the least possible quantity of starch visible in the chlorophyll grains (1). A like result is better ol)tained in fresh grains by treating them on the slide with a solution of five parts chloralhy- drate in two parts water ( 2 ) to which a little iodine tincture is added. The chlorophyll is dissolved so that after a few minutes the leaf becomes colorless ; at the same time the chlorophyll grains and their starch contents are swollen and in the latter the blue cohn- comes out distinctly. Al- cohol-bleached leaves, so treated, behave in the same way. If treated with a very dilute aqueous solution of methyl violet or gentian violet, the cell membranes are stained, but the grains still more and become more distinct. The living chlorophyll grains of the Fnnaria leaf appear to be finely dotted under a high magnifying power and so betray a reticulated structure. The same results maybe had with the prothallium of the fern so that either object may replace the other. The pro- thallium may be found in any greenhouse where ferns are cultivated. Any species will do equally well. 40 COLORED ELEMENTS. For the study of other colored elements (3) we will take an opening blossom of Tropeolam mcijus. In the older blossoms the colored bodies are beo-inninof to disoroftmize. AVith the forceps thrust into the tissue, tear off a piece from the upper side of a sepal. Lay the strip in a drop of water on the slide, the epidermis up. Make the examination at once, before the Avater spoils the colored bodies, and select an uninjured cell. The colored bodies are yellow with a shade of orange. They are spindle-shaped three- to four-angled. (See Fig. 17.) The uninjured ones are homogeneous. ^ AA'^ater swells them, rounds them out \^;( and makes vacuoles or water-filled spaces, in their interior. These bodies are especially numerous on the inside wall of the epidermal cells of the upper side of the calyx. The brown stripe on the upper side of the sepals arises as a section would show from the red cell-sap FIG 17. From the i,p- which fills the epidermal cells. These per side of the calyx of ^ Tropeoinm mojus. vn- cclls cOutaiu also ycUow grains which der wall of an epider- ,1 i i 11 i ^ l • mal cell with color the colorcd cell-sap renders almost in- bodies l3-in; 510. on it. X visible. The nucleus of the red cells appears mostly as a clear spot. The petals show corresponding relations. The edges of the lamella, as w^ell as the cilia at their base, ma}^ be used for our observation. If attached air bubbles interfere with the examination, a slight pressure on the lamella will drive them aw^ay. But the sepals are to be preferred to the petals for examination of the color-bodies, on account of the papilla on the latter, which, by their own form and the quantity of air that they entangle between them, COLORED CELL-SAP. 41 materially interfere with the observation. The tier}" red places, at the base of the petals, arise from the rosy cell- sap and yellow grains in the epidermal cells. During the examination^ it has been observed that the upper surface of the epidermal cells of the upper side of the sepals are longitudinally striped. The stripes pay no regard to the boundaries of the several cells, and are folds of the cuticle covering the epidermis. The color-bodies are fairly well fixed with iodine wa- ter and are, at the same time, colored green and become very distinct. The nucleus is stained a yellow-brown, and its nucleoli are distinctly brought out. With methyl or gentian violet, the color-bodies are stained violet. Yellow coloring matter is almost always connected with a protoplasmic substance, but we sometimes find it dis- solved in the cell-sap, as in Verbascuvi iiigrum. Put the petal directly in the drop of water and remove the at- tached air as much as possible. In the epidermal cells, on either side of the leaf, which have a wavy outline, the yellow cell-sap is seen. The brown spots are caused by purplish-brown cell-sap. In the epidermis of the stamens, a thin lamella of wiiich may be taken off with the razor, is the yellow cell-sap ; also an irregular mass of cinnabar-red coloring mutter, and a number of colorless leucoplasts filled with starch. In the under lip of the corolla, A-niirrJiinuin majus, is a sulphur-yellow cell-sap. The red portions have, in their cells, a rosy cell-sap and, partly, also one, seldom more, carmine-red balls of coloring matter. Rlue cell-sap may be found in the epidermis cells of the corolla of Vinca major or minor. The epidermal layer maybe easily torn away w^th the forceps. The side walls of the cells have ledges projecting into the cell cavity, i 42 COLORED CELL-SAP. Fig. 18, which are swollen at their inner edge, so as to become T-shuped, and on account of the effect of unequal refraction have the appearance of folds. Ros}^ ceü-sap gives the color to the rose. The epidermis may be easily torn away. It is deeply papi Hated ir^ '^VV ^^^'^ velvety. The cuticle is marked ^^ ^( by distinct stripes. j(^^ ^r The epidermis of both sides of the ^^^Xr^^^'^'^u- " bhie sepals of Delphinum consoUda FIG. IS. Epidermal cell cousisls of Cclls with WaVV COUtour. of the linder side of the petal of vinca minor. X Oil the uppcr sidc the ccll riscs into ^*°' a papilla, so that by foTJUSsing at about half its heio'ht, we jjet a sun-like figure. The cells contain blue cell-sap, bordering somewhat on the violet. Besides this, many cells contain blue stars, which are produced by short needles crystallized from the coloring matter. The epidermis maybe torn away in small pieces. But the sepal is transparent enough to be examined through its whole thickness at the edges, after removing the air. Examples of red and blue cell-sap may almost always be met with in red and blue flowers, among the more strik- ing of which, is the intense red-colored flower of Adonis flamens. Stripping oft" a piece of the epidermis with the forceps, we see in the cells beautiful red, nearly round to elliptical grains, nearly as large as chlorophyll grains. They appear to be finely granular, and, in water, dissolve into very fine granules which exhibit the molecular mo- tion. Tne epidermal cells are elongated, the cuticle longitudinally striped, the stripes running distinctly across the boundaries of the cells. . The orange-red color of the root of the Daucus carota arises from carmine and orange-red, crystalline, colored CRYSTALLINE COLORED BODIES. 43 botlies; their commonest form is represented in Fig. 19. they are small, right-angled rhombs, often elongated to a needle shape, again prismatic and often fan-shaped. To these crystalline forms are often attached smallj lateiidly- projecting, starch grains. These crystals are, therefore, original sources of starch, like chlorophyll grains and other colored bodies ; but the cr3'stallized coloring matter deter- mines the form ; the crystals have bnt a very small quan- tity of plasma from which the starch originates. If we examine the variegated varieties of trees and shrubs, or even of the herbaceous plants which have red- brown leaves, Ave shall tind the cells of the epidermis filled with a rosy cell-sap, and the compound red-brown color is the eft'ect of the red on the surface and the green below. The red, autumn color of the leaves of the woodbine, Amjpelopsis hederacea, is caused by the rose- colored cell-sap in the cells of the tissue. Distinct yellow autumn . IT Fig. 19. Color-bodies from colors of leaves arise from the dis- the root of carrot. Part with Organization of chlorophyll grains, ^t^rch grains, x 54o. as is most beautifully shown in the leaves of GingTtO biloba,, or, lacking these, of the maple species. The brown color of some leaves comes from the correspond- ing color of the cell walls, but principally of the cell con- tents, as may be easily seen in the oak. Starch grains originate in specially individualized pro- toplasmic forms, as in chlorophyll grains, also in the color substances, where starch grains may often be detected, and, tinally, also in colorless starch generators ; the latter assists in the formation of starch in the deep layers of the plant body. We may name all these together chromato- 44 LEUCOPLASTS. phores, and, again, the chlorophyll bodies chloroplasts, the colored bodies chromoplasts, and the colorless starch generators leucnplasts. These forms are nearly related and may be transformed into one another ; they all belong to the protoplasm of the cell in which they lie embedded. On the contrary, the blue stars which are found in the cell-sap of DelpJdnum consolida do not belong here, but are a colored substance crystallized from the cell-sap. Like- wise the colored lumps which we found in the red cell-sap of Verbascum is not a chromatophore. The largest and most beautiful starch grains are pro- duced in the leucoplasts, and still the leucoplasts are not easily seen ; a relatively, favorable object and one not dif- ficult to obtain, for this purpose, is the rhizome of /ns Germanica. Make a sec- tion parallel to the surface of the rhizome ; directly, the onter-tissue layer is re- moved, w^e come to the starch layer; ex- amine in water. In nninjured cells, the leucoplasts appear as collections of plasma on the posterior end of the starch grains, Fig. 20. The latter grow here only, and, therefore, have an eccentric struct- ure. The leucoplasts become granular under the eye of the observer, and, finally, dissolve in small grains and show the molecular movement. Two starch grains are often found in one leucoplast ; such grains soon touch each other and henceforward have layers in common ; these and like causes produce in this and in other cases com- pound starch grains. NOTES. (1) Methode von Böhm, Sitzungsl)er. d. K. A. d. W. in Wien, Bd. XXII, p. 479. (2) Nach A. Meyer, das Chlorophyllkorn, p. 28. (3) A. F. W. Shimper, Bot. Ztg., 1880, Sp. 881; 1881, Sp. 185; 1883, 105 und Sp. 809 ; A. Meyer, das Chloropliyllkoru, Bot. Ztg., 1883, Sp. 489. Fig. 20. Leuco- plasts with starch- grains from root of Iris germanica. X 540. LESSON V. Tissue, Thickening of the Wall, Eeaction on Sugar, Inulin, Nitrates, Tannin, Wood Substance ok Lignin. From a piece of the white sugar-beet cut a section par- allel to the lono-er axis and in the direction of the radius at right anijles with the visible rinojs of the root. Ex- amined in water, it will be seen to consist of nearly rio-ht-anoled cells tilled with a watery colorless fluid. The cell walls are dotted with bright round or oval pits. In occasional cells the nucleus is visible. The intercellular spaces are mostly tilled with air. In places the paren- chyma cells become slenderer, are elongated lengthwise of the root and between them are tubes, mostly air filled, with peculiar thickening of the walls. These tubes are vessels. Thickened reticulated ledges cover the walls, thin places lying between. These thin places or pits are elon- gated transversely to the length of the vessel. Ring-like thickenings may be seen now and then projecting from the inside of the vessels. These are the diaphragm-like ■yemnants of originally perfect division walls, and indicate that the vessels originally consisted of a series of cells. The air may be drawn from the vessels by means of an air pump. Lacking this, put the section in recently boiled water, or better still in alcohol. This liquid will kill the cell contents but that does not matter in this case. Occasionally, we shall meet with a cell filled with small- klinorhombic calcium oxalate crystals. The test is that they do not dissolve in acetic acid but do in sulphuric (45) 46 STAINING BEET SECTIONS. acid. Make the test with two preparations. The result- ing gypsum is so small a quantity that it is dissolved in the surrounding liquid. Treat the section with an aqueous solution of methyl green or methyl green and acetic acid. The cell wall be- comes a beautifid green, and in the latter case the cell contents are fixed and quickly stained. The walls of cells and vessels are colored a bluish green. Not so the pits on the cell walls, which are the thin places on the walls of cells not otherwise much thickened. Every parenchyma cell contains a nucleus, having a distinct nucleolus, and surrounded by minute leucloplast, and a thin layer of protoplasm on the wall. The vessels have neither. To the section in water add chloriodide of zinc and Ave shall get the characteristic violet cellulose reaction. The color- ing begins on the edges of the section, but it may require hours to become complete. The vessels are colored a brownish yellow like lignitied membrane. The pits on the cell walls are uncolored and become more distinct. These pitted surfaces are always oval of various sizes ir- regularly distributed singly or in groups. The larger pit- ted places are overspread with violet bands of different breadths, making the appearance of fan-shaped irregular lattice work. Bright granules colored yellow brown by the chloriodide of zinc adhere to the pitted surface. For comparison produce the iodine and sulphuric acid cellulose reaction. Impregnate the section with potassium iodide and transfer to dilute sulphuric acid (two parts acid and one water). It will be colored a beautiful blue. The smaller pitted surfaces are still uncolored, the larger ones latticed blue. Make a section of a ripe pear. The pulp consists of reg- ular thin-walled large parenchyma cells somewhat round- ed at the corners, having colorless cell-sap, a much reduced PEAR STONES. 47 plasma sac and a nucleus. Scattered in the tissue are nests of strongly thickened cells, Fig. 21. The number of these united stone cells is different in different places and indifferent species. They form the so-called "stones" of the pear. The cells are distinguished by the consid- erable thickening of their walls and by the numerous, fine branched canals penetrating the walls. The branching is caused I)y a number of the canals uniting inwardly as the cell cavity becomes narrower, forming a common canal which opens into the cell cavity. When two thickened cells touch, the ca- nals meet. In the present condition, the.se cells have no liviiigcontents, only a watery fluid. Con- sequently they rep- resent only dead cell husks. Chlor- iodide of zinc slowly colors the parenchy- ma cells violet and the thickened cells a yellow brown. The latter are therefore lignified and on ac- ^,,. „, „ „ , , , ° , , Fig. 21. From the pnip of the pear. Much thick- COUnt of their thick- ened «ells, with brandling pore canals. .Siirrounaed IT -r- by thin-walled pai-encl)3-nia cells. V 210. ness and hgnihca- i .> /\ *». tion are called sclerenchyma cells. The structure of thick- ened cells is well brought out with chloriodide of zinc. We will use the pulp of the pear for our microscopical study of the sugar reaction. The most common is that with Fehling's solution (34.64 g. pure crystallized copper suli)hate, 200 g. tartrate of potash and soda dissolv^ed in water) . This solution may be kept on hand. Take about 48 SUGAR REACTIONS. 600 ccm. soda lye, sp. gr. 1.12, dilute to 1000 ccm. and boil. The section, which should be not less than two or three layers of cells in thickness, should be transferred from the Fehling's solution to the hot lye, and the reaction at once takes place. The microscope shows in the cells the vermilion-red precipitate of reduced cuprous oxide. There is therefore in the cells of the pear a substance which will reduce an alkaline cupric oxide solution, a sub- stance belonging- to the grape sugar group (glucose), in this case grape sugar. For comparison, make the investigation with a section of the sugar beet; this contains, as we know, cane sugar. Immersing the section, for a couple of seconds in the boiling liquid, gives no precipitate in the cells; the sec- tion becomes blue ; if the section lies for a long time in Fehling's solution, the surface begins to show the vermil- ion-red color. The cane sugar has undergone transfor- mation and now gives the cuprous oxide precipitate. Under the microscope, vermilion-red granules appear on the pe- ripheral cell layer, while, if the reaction has not continued too long, the inner cells contain a blue liquid. For microscopical purposes, the Barfoed sugar reaction with acidulated copper acetate has much to commend it. Dissolve one part neutral, crj^stallized copper acetate in fif- teen parts water; to 200 ccm. of this solution add 5 ccm. acetic acid, which contains thirty-eight per cent glacial acetic acid ; in a test-tube, holding from 5 to 8 ccm. of this solution, put a not too thin section of the pear and in another a section of the sugar beet, and boil up for a short time ; pour all out into small glass dishes and let them stand. After some hours we shall find the pear sec- tion covered with a fine precipitate of cuprous oxide and a small quantity also of the precipitate in the bottom of the dish, while the beet-root section has none of it. The efiects of the reaction should be compared after a few hours, NITRITE AND NITRATK REACTIONS. 49 since a small quantity of the precipitate is reoxidized in the air after a longer time and may then be dissolved. We, finally, use the sugar beet to observe the nitrate and nitrite reaction by means of diphenvlamin (3). This substance, uSfed by the chemist as a most delicate test for nitrates and nitrites, is very useful for histological re- search. jNIake any section of the beet which shall reach the outer surface, lay it on the slide, partly dry it and add the reagent ; this consists of 0.05 g. diphenylamin, in 10 ccm. pure sulphuric acid. Immediately, there ap- pears in the outer zone of the section, an intense blue color ; this zone contains the latest product in the r,-;^ \ developing tissue of the beet, and, consequently, is that part which con- tains the nitrate. Di- rectly, the blue color begins to spread over the rest of the section, but, at first, the reaction in the colored zone is quite sharply defined. We conclude that it is a ni- trate and not a nitrite, which we find here, because the former is much oftener found in the analysis of the juices of the plant. We partly dry the section, so that the reagent and the color will not spread so rapidly over it, and the colored zone will be more sharply defined. Take next the dahlia bulb, DaJdia variabilis. The longitudinally-halved bulb shows the central pith: a lon- gitudinal section of this shows, under the microscope, many series of nearly rectangular cells. Fig. 22, with a 4 From the pith of Dalilia variabilis. 50 IXULIN. much reduced protoplasmic sac with nucleus and cell-sap, the intercellular spaces being filled with air, and the cell walls finely striated, the striae lying at an angle of about 35° to 40°. There seem to be two opposite systems of stride on each w^all ; but, in fact, they belong to two adja- cent walls, and the extreme tenuity of these walls enables us to see the two systems at once. The cell walls are colored violet with chlorio- dide of zinc, but when the stripes do not approach each other very closely, a colorless line is seen between them. Like the pitted surfaces of the wall, these unthickened places are not colored by the chlor- iodide of zinc. Single, rel- atively large, rhombic-shaped places come out with great distinctness as pits ; these pits ahvays lie on the dividing line between two striae and at the places where the dividinglines of one system cross those of the other. FIG. 23. From the bulb of 7>«w*« p^^^ ^ scction lu absolute variabihs after lying in alcohol several months. Sphere-crystals on the walls, alcohol and a fine precipitate ^ ^*°' of inulin will be produced in the cell-sap. Eeplace the alcohol with water and warm the slide, and the precipitate will be again dissolved. In order to study the spherical crystals which the inulin forms, one should take a piece of the bulb which has been in al- cohol at least eight days ; examine the section in water and add nitric acid very slowly at the same time. The spherical crystals, Fig. 23, are always found on the cell walls; they TANNIN REACTION. 51 form more or less perfect globules which miiy be l)roken through b}'' one or more cell walls. For the most part, globules of tlitterent sizes form together a large group ; the globules exhibit more or less clearly a radial struct- ure, which becomes more distiuct after the uitric acid begins to affect them; this arises from the radially ar- ranged crystal needles, of which the spherule is built. Besides the radial structure, a concentric lamination is visible which is understood to signify an unsteadiness in the conditions of crystallization. Iodine solutions do not color these spherules. Warmed in a drop of water on the slide they soon disappear. There is nothing better than a gall apple for testing the tannin reaction. The gall apple is found on the leaves of the oak and is produced by the sting of the gall wasp, which thus lays an egg in the tissue of the leaf. Halve the green apple and make a delicate radial section. The cavity occupied by the larva is surrounded by a shell which is formed of isodiametric oval cells. These are mostly richly laden with starch grains. The tissue which incloses this consists of radially elongated, poh^gonal cells, which diminish in length toward the periphery of the apple, and finally end among the small cells, towards the outside strongly thickened, outermost cell layer of the epidermis. There is no definitely formed cell-contents in all this tis- sue which surrounds the inner shell. But lay a freshly prepared section in an aqueous solution of ferric chloride or ferric sulphate, and the whole mass Avill be colored a deep blue. This color is imparted to the surrounding liq- uid and gives us the iron-blue reaction of tannin, which may also have an iron-green reaction. Watching the re- action under the microscope, by putting a dry section under the cover-glass, and then adding a drop of the iron solu- tion, we see that at first a dark blue precipitate is thrown 52 SCLERENCHTMA TISSUE. down, which soon dissolves in the reagent and fills the cell with the blue fluid. The starch-filled cells of the inner shell give the weakest tannin reaction. For comparison Ave will lay a second section in an aqneous ten per cent solution of potassium bichromate, and we shall see-a thick floccnlent, reddish-brown, permanent precipitate form in the cells. We shall not examine the vascular bundles of the gall apple since they have nothing peculiar to do with the tannin reaction. If the stem of a st(mt Vinca major be cut ofi" near the ground and then broken across, we shall see numerous small fibres projecting from the edges of the broken parts. Seizing them with the forceps, we pull them out and put them on a slide in a drop of water. We shall find them to be long, pointed, much thickened sclerenchy ma fibres. The cell cavity is reduced to a narrow tube, and towards the ends quite obliterated. In the less thickened walls we find but one, and in the more thickened, two systems of striation, the one belonging to the inner and the other to the outer layer. In very old sclerenchyma fibres, often a third inner system is seen almost perpendicular to the longer axis of the fibre. The latter is derived from retic- ulated thickenings, the elongated dots appearing between. These innermost thickening systems are most sharply sep- arated from the outer. With chloriodide of zinc the fibres are colored a violet bordering on the brown. Especially instructive is the behavior of cuprammonia which dissolves pure cellulose. The efiect must be quickly and closely Avatched. The reagent greatly swells the walls of the fi- bres, at first making the striation more distinct, but quickly obliterating it. The outer layer is soon perfectly dissolved while the inner reticulated structure longer withstands the action of the reagent, and consequentl}' becomes at last fully isolated. At the beginning of the swelling, a still PITS IN CELL WALL. 53 finer lamination shows itself in the already visible layers. So each layer is composed of a number of extraordinarily fine lamelloe. This very fine lamination is especially well seen in the inner resisting layer. Split a seed of the Star-of-Bethlehem, Ornithogalum um- bellaium, with a pocket knife and make a very thin section, with razor, hand-vice and drop of water. The prepara- tion will shoAV nearly right angnlar cells as in Fig. 24. The walls are much thickened but they are perforated by a number of simple pits. Looking upon the sur- face of the wall, these pits resemble round pores, m. From the side they reseml)le canals, running from the cell cavity to the primary cell wall. The pits of neighboring walls meet and are separated only by the pri- mary cell wall,^;, which we call here, the closinsr membrane. The inner surface of the thickening layer is distinguished by its stronger refrac- tion, and forms the boundary mem- brane. Add sulphuric acid at the s\^erm of OrvWwr/alum vmbel- laium. m, pit from above ; j), edge of the cover-glass, an I the closing membrane ;«,nucleHs! thickened laj'er will be dissolved, ^ ^^^' while a network of very delicate walls will remain. These walls are the so-called middle-lamella, which correspond to the original walls of the cell before the beginning of the existing thickening, and they also penetrate the clos- ing membrane of the pits. By the long continued action of the acid they too would disappear. Chloriodide of zinc swells the thickened layer and so makes the middle la- mella visible. The coloring of the preparation is imper- fect in consequence of the swelling. Fig. 24. From the endo- 54 BORDERED PITS. The cells are closely packed with protoplasm and granu- lar matter, and the whole contents are colored yellow-brown with iodine solution. The nucleus in every cell may be easily demonstrated by means of acetic methyl green. It generally fails in no living cell or a cell capable of life. The thickened layer of the cells in the endosperm of the date, PJicenix dactyUfera, has a similar appearance. But the cells are elongated, their cell cavity narrower and their walls thicker. These cells are in the date germ radially arransred. Transverse and radial-lonijitiidinal sections show the cells in longitudinal section, while tangential sections, perpendicular to the radius, show the cells in transverse section. Chloriodide of zinc colors the thick- ening layer a very beautiful violet, and a prolonged swell- ing causes numerous lamelUB to appear. We will now turn to the coniferous wood to study the so- called " bordered pits." Take a piece of an old stem, a dry or alcoholic specimen, and with a sharp knife prepare to make the diiferent sections, a radial parallel to the longer axis, a tangential also longitudinal, and one transverse to this. The concentric annual rin2;s of the wood will o-ive us the necessary points for getting the desired directions. The radial-longitudinal section is cut perpendicular to these rings, the tangential as nearly as possible parallel to them, the transverse perpendicular to both the others. To make good wood sections and not spoil the razor requires the ex- ercise of the greatest caution. In case the razor is ground concave, a section can be made only on the edges of the w^ood or only so far as that the back of the razor will not strike the cutting surface. Still, razors used to cut wood should be but a little concave, else they will spring and cut unevenly. The best form is that which is ground flat on one side, but this has the fault of not being easily sharp- ened. The cutting surface should be kept moist, the sec- BORDERED PITS IX CONIFEROUS WOOD. 55 tion mtide as thin as possible. It need not be very large. If the cutting is too deep, withdraw the knife and so run no risk of nicking it. The razor should be very sharp so as not to mutilate the cell-membrane, and separate the in- ner thickened layer from the outer. An alcohol specimen cuts more easily than the dry wood, particuhirly if it be subsequently soaked in like parts of glycerine and alcohol. The first section cut by the razor should not be used, as the cell membranes of one side have been mutilated b}^ the pocket knife. Fig. -25. Pinus sylvestris. A, boi-flered pit, side view; B, same in tangential lon- gitudinal section; t, torus; C, transection a wliole traclieid; m, middle lamella; m*. a gusset in the same; i, inner cell membrane. X 510. With a low power, a radial section is seen to be built of longitudinally elongated cells pointed at the ends and at- tached to each other. Crossing over these cells run the cell rows of the medullary rays which we will not now consider. We focus a high power system on one of the broader walls of the longitudinally elongated wood cells and direct our whole attention to the bordered pits of this wall. The bordered pit appears to us in the form of two concentric circles, Fig. 25, A. The inner small circle or the inner ellipse represents only the opening of the pit into the cell cavity. The larger outer circle is the 56 BORDERED PITS. widest pui't of the pit at which it joins the primaiy wall separating the two cells. This bordered pit is in fact distinguished from the simple pit as we have seen it in the Star-of-Bethlehem and in the date, only that it widens at its base. The pits of adjacent cells meet in this as in the other cases. If the o[)ening of the pit (as in A) is an obliquely placed ellipse, we shall also find by changing the focus that the corresponding pit has its opening inclined in the opposite direction. The two opposite pit cavities are separated from each other by the primary wall which existed before the secondaiv thickening had be£>:un and was then very thin. This delicate wall is the closing membrane. This is thickened in the middle and forms the so-called "torus." By careful attention and focussing, this torus may be seen. It forms a smooth, bright, round disk of about double the diameter of the oritice of the pit (see in A) . In favorable cases and particularly in preparations from dry wood this thickening of the membrane is seen to possess a radial striation ; so that it would seem to be diflerentiated into radially running lamellte (6). We shall get a full view of the structure of the bordered pit only by means of a tangential section, since the bor- dered pits are on the radial walls (7) of the wood cells and will be cut across only by a tangential section. See Fig. 25, B. Look for the pits on the division walls of the widest wood cells, and be not led into error by the sections of the cells of the medullary rays. The image of the dissected pit will be seen only in the thinnest and most delicate part of the section. There they will appear in the form of two o]i)en pincer jaws, as in the illustration given in Fig. 25, B. Kecognizing the structure of this larger pit, other smaller ones will be easily made out. The thicker TRACHEiDES. 57 the Avail the longer the canal and the wider the pit cavity. In the most favorable eases one may see the closing mem- brane within the pit, with its thickened centre t. In the larger bordered pits it is mostly pressed over to one side of the pit cavity and seems to serve the purpose of a valve . The image becomes clearer after treating the section to chloriodide of zinc which colors the cell wall a yellow- brown. Some inner layers not yet fully lignified show^ a violet tlush. The closing membrane is not colored. This reagent demonstrates that this wood cell has neither nu- cleus nor protoplasmic sac. It consists onl}' of dead cell walls and resembles the vessels in its function of conduct- ing water as well as in the manner of its wall thickening. It is called a tracheide or more recently a hydroide. Often the coniferous wood which we are examining will be seen to have a spiral striation with an ascent of about 45°. The pit openings appear thus to be elongated in the direction of the stritB of the two opposite sides of the wall on which they are placed and so the opposite pit openings seem to cross each other. AYe will now make a very delicate transverse section of the wood. The tracheides are prevailin2;lv at rioht anales and mostly arranged in radial rows. On the radial walls of a wide cell wc find the pit cut in section Fig. 25, C, whose form is not different from that shown in the tanaen- tial section. Between the cells is a fine dividing line m, the middle lamella. AVhen more than two cells meet the middle lamella is widened into a solid or hollow gusset ??i*. The inner border of the cell w-all is more refractive and forms the boundary membrane i; that of the thick walled tracheides is especially distinct. It all becomes still clearer by the use of concentrated sulphuric acid. The thickened layer sw^ells and finally dissolves ; the boundary membrane, withstanding its action longest, comes out 58 PHLOROGLUCIN REACTION. sharply to view. Between the swelling, thickening layers are the primary walls of the cells and these at last remain a delicate network colored a yellow-brown. This acid- resisting, middle lamella is "cutiiiized." By the gradual swelling of the thickening layer in sulphuric acid we find that it is composed of numerous extremely delicate lamel- lae. Chloriodide of zinc colors the section yellow-brown but in some cases the innermost part of the thickening layer takes a violet tinge. If Ave follow the chloriodide of zinc treatment with dilute sulphuric acid (two parts acid and one of water) the whole thickening layer will be colored blue. Treat the section with chromic acid and the middle lamella will be dissolved and the cells separated. The thickening layer will swell somewhat, the lining membrane at first showing up sharply and afterwards disappearing. Phloroglucin and aniline sulphate give characteristic reactions with wood substances or lignin (8). Dissolve a small portion of the phloroglucin in alcohol and lay the section in the solution. Afterwards put it in a drop of water on the slide and add a little hydrochloric acid at the edge of the cover-glass. Directly the cell walls will be stained a beautiful violet red color. An aqueous solution of aniline sulphate colors Avood a bright yellow, but the color is heightened by adding a dilute sulphuric acid. In place of the phloroglucin, one may use an aqueous or al- coholic extract of cherry Avood, Avith almost the same re- sults (9). Treat n section from a fresh stem of fir AVood Avhich has either the pith cells or the bark cells, with con- centrated hydrochloric acid. Immediately the Avood Avill be colored yellow, Avhich afterwards gradually softens to a violet (10). This is also a phloroglucin reaction,, the phloroglucin being derived from the pith or bark cells. The medullary rays themselves in young Avood contain some phloroglucin. REACTION ON LIGNIFIED WALLS. 59 Thus the tliflerent behavior of lignifiod and unlignitied cell walls toward coloring matter is one important element in their investigation. Notes. (1) Compare Sachs, finally Jahrb. f. wiss. Bot., Bd. iii, p. 187. (2) Ban'oed de organiske Stoffers qualitative analyse Kjöbenhavn, 1878, pp. 210, 217, 223 Aum. (3) See H. Molisch : Ber. d. deut. bot. Gesell. I Jahrg. p. 150. (4) Sachs, Bot. Zeitg., ISGi, p. 77; Hausen, Arb. d. Bot. Inst, in Wurzburg, Bd. Ill, p. 108; Meyer, Bot. Ztg. 1883, Sp. 334. (5) Sauio, Jahrb. f. wiss. Bot. Bd. ix, p. 50; S'trasburger, Zell- häute, p. 38; Eussow, Bet. Centralbl. Bd. xiii, No. 1-5. There is also other literature. (6) See Eussow, Bot. Centralbl., 1883, Bd. xiii, No. 1-5. (7) Taugeutially placed, bordered pits, which are so rare in the fir, quite i-eguhuly occur in those wood cells of other Abietince, which are formed in the autumn. (8) Both introduced by Wiesner (See Stzber. d. Math. nat. Kl. d. Akad. d. Wiss. Bd. lxxvii, 1 Abth. und früher schon a. a. O.) (D) V. Höhnel, Sitzber. d. Math. n. Kl. d. Wiener, Akad. d. Wiss. Bd. Lxxvi, p. 685. (10) The same, p. 676. LESSON VI. EriDERMis. Stomata. Prepare a superficial section from the outside (under- side) of the "riding" leaf oi Iris florenlina. The section should be so thin that but traces of the underlying tissue should adhere to the epidermis ; examine in water, the outside uppermost. The epidermis consists of much elon- gated cells running parallel to the axis of the leaf. The cells end in a transverse division wall, are joined together without intercellular spaces, contain colorless cell-sap, a reduced plasma sac and a nucleus. The outside of the epidermis is covered with an extraordinarily, fine-grained wax. On a line with the epidermal cells lie the elliptical stomata indistinctly seen. The four contiguous surface cells reach over and partly cover the guard cells of the stoma, so there remains only an elongated, elliptical, mi- nute cavity, f, which leads to the stoma. Fig. 26, A; this cavity is filled with air and appears mostly black. Turn the section over now, and it will easily be seen that the stoma is formed of two semilunar shaped cells ; unlike the neighboring cells of the cuticle, they contain chloro- ph^dl grains. The nucleus is seen as a clear spot, usually for half the length of the cell. Between the two guard- cells occurs a spindle-shaped opening, s, which extends along half the length of the cells. Make a section now, crosswise of the leaf, and you will naturally make it trans- versely across the stomata. For this purpose, cut out a small piece from the leaf and make the section, while holding it between the two halves of an elder pith. A (60) STOMATA. 61 piece of the pith, 3 cm. long, may be carefully split in two, lengthwise, the piece of leaf laid between the two halves in such a way as to bring the edge to be cut just above the end of the pith ; hold the pith in the fingers or put a light rubber band around it to hold it in place, or even fasten it in the hand-vice, but hold it so that the knife will cut along the bioad surftice of the object and not Fig. 26. Epklei-niis of the underside of the leaf of Iris floreniina. ^, surface view; B, transection;/, minute depression ; s, stoma; c, cuticle; a, breathing cavity. X 210. upon the edge. For very delicate objects, the sunflower pith is better than elder pith. Cut first both the end of the pith and of the object, quite clean away, then cutting through both at the same time, make several very thin sections, keeping the cutting surface nioistened and a drop of water on the razor blade. Remove the sections from the knife to a slide by means of a hair pencil. The prep- 62 LEAF SECTIONS. ST03[ATA. aration of sufficiently delicate sections need occasion no great difficulty, but if such a difficulty arise, it ma}^ be met with a microtome ; a hand microtome of the simplest construction will be sufficient. The pith holding the ob- ject may be fitted into a cork and this into the well of the microtome, the pith rising some little above the • cork. The section nui}^ be made Avith a common or a flat-sided razor, cutting free-hand across the top on the brass or glass j)late, and lifting the object each time the required distances by means of the screvv. More elaborate micro- tomes, useful to the zoologist, are superfluous to the bota- nist.* jNlake a number of sections and put them in a watch glass. Examining a section in water, we find a stoma cut throuah the middle, as in Fig. 26, B. First, we notice that the cells of the cuticle are thicker-walled without than within. Still the inner walls are pretty thick, while the radial walls are very thin ; these are connected with the function of the cuticle which is not only to protect the leaf without, but also to be a water reservoir (2). The thin radial walls are adapted to an easy change in the vol- ume of the cells, for, acting like a bellows, they diminish their height with the loss of crater, and, again, increase it when the water is increased. The guard-cells lie deep beneath and between the cells of the cuticle. The little pit, /", leads down to the guard-cells ; these are much thickened above and below. These thickened sides jut out towards each other in the opening ; al)ove these thick- ened places are peculiar, beak-shaped projections. On the opposite side towards the inside of the cells of the cuticle, the walls of the guard-cells are relatively quite thin. On this method of wall thickening depends the ♦Taylor's freezing microtome is very serviceable for cutting soft tissue con- taining water. — A. B. H. GUARD-CELLS OF STOMATA. 63 mechanism for the movement of the guard-cells Avhich are more curved and the orifice opened, the greater their turgidity ; and, conversely, are more extended and the orifice closed or narrowed b}' a decrease of their turgidity. It is, in fact, clear that the guard-cells, by increased tur- gidity, become more convex on the side of least resist- ance and concave on the side of most resistance, like a rubber bag with a thicker wall on one side than on the other. If water or air be forced into it under high pres- sure, the side making the strongest resistance becomes concave, while that making the least becomes more con- vex. The thin place on the cleft side, where the two thickened ridges jut together, facilitates the flattening of the cells, while they curve out on this side. Therefore, lest the movement of the guard-cells be interfered with, it is joined to the epidermal wall on a suddenly, narrowed side and is fastened to it after the manner of a hinge. Under the stoma is the breathing cavity, «, of the same nature as the larger intercelhdar spaces filled with air, which is bordered with cells containing chlorophyll and is connected with the open cavities found between these cells. Testing with chloriodide of zinc shows us that the walls of the epidermal cells are colored a yellow-brown, through their whole circumference, with the exception of a thin, somewhat wrinkled membrane, and are the so- called cuticle, c; this cuticle swells outward at the stoma, forming the above mentioned beak-like projection, which stains yellow-brown ^v\Üx the chloriodide of zinc, and so appears to be cuticularized. As an extremely delicate membrane, the cuticle extends through the stoma over the guard-cells quite to the beginning of the parenchyma cells containing the chlorophyll. The guard-cells are vio- let in their whole extent. By the application of concen- trated sulphuric acid, the whole section is dissolved except 64 GUARD-CELLS OF STOMATA. the cuticle including the cuticularized projection of the stoma. An unusually favorable ol)ject for studying the stomata apparatus is found in the Tradescanlia virginica. The epidermis is formed of polygonal cells mostly somewhat extended in the direction of the leaf. With these alternate narrow stripes of slenderer and longer cells. These stripes appear green on the under side of the leaf, the others gray. The lateral walls of the epidermal cells are furnished with pores, the outer surface is faintly striated. The number of Fig. 27. Epidermis of the imderslcle of the leaf of Tradescanlia vircjinica. A, surface view; JS, transection; I, leucoplasts. X 240. stomata is considerably greater on the under than on the upper side of the leaf, so we will examine that side. The stomata are almost always surrounded by four cells. Fig. 27. The guard-cells lie at the same height as the epider- mis. The cleft which lies between is relatively large. They have chlorophyll grains between which, for the most part, the nucleus is visible ; also in the epidermal cells the nucleus is sharply distinct, surrounded with colorless leu- coplasts, I, Fig. 27, A. The cell-sap of the epidermal cells is here and there rose-colored. Make a cross section, EPIDERMIS. 65 as with the last example, and we shall have the stoma as in Fig. 27, B. The cleft side of the cells has thicker walls : the other, thinner. The walls of the next adjoining cells are thinner than the succeeding cells of the epidermis, and are the sec- ondary cells of the stoniata apparatus, forming the hinge, otherwise provided for in the Iris, as we have seen. The leucoplasts, I, surrounding the nucleus are very favorably situated for examination. It is interesting to note that though the leucoplasts are here exposed to the action of the strongest light they remain small and colorless and do not change to chlorophyll grains. Tvadescantia zebrina has a similarly constructed stomata apparatus, found on the under side alone. The cross section is very instructive even if not very easily made thin enough. The epidermal cells of both sides are of considerable size, particularly those of the upper side, which are so high that they alone make nearly half the thickness of the leaf. Many of them are divided by transverse walls. Mostly the epidermal cells have only watery cell-sap, which on the underside of the leaf is colored red. The epidermal cells of this plant constitute a water-holder of gieat capacity. The four sec- ondary'^ cells of the stomata are quite flat so that a larire breathing cavity of the height of the epidermal cells exists under the stomata. Taking a section from the under sur- face of the leaf, w^e can focus down throuo-h it and sfet a good image of it while no air -gets into it. The leuco- plasts about the nucleus are distinctly seen in the epider- mal cells. The Aloe and Agave species show an exti-aordinary de- velopment of the outer wall of the epidermal cells and a corresponding depression of the stomata deep in the epi- dermis. Take the Aloe nigricans found in greenhouses ; other 5 66 STOMATA. species of the genus may be used in lack of this. A su- perficial section shows the epidermal cells of both sides of the leaf to be regularly polygonal — mostly hexagonal. The cell cavity is a relatively small, oval space; it is us- ually filled with air and is black in the section. The sto- mata occur on both sides of the leaf at the bottom of the deep clefts which are surrounded by four cells and are rectangular and enclosed in a somewhat projecting rim. In order to see the guard-cells, the section must belaid on the slide inside up ; these cells are relatively broad and Fig. 28. Transection through the epidermis and stoma of Aloe nigricans, i, in- ner thickening layer. X 240. short and contain, among other things, strongly refractive, globular oil-drops. The epidermis being so hard, we may use cork in making the section ; make the section not the whole thickness of the leaf, but from one side only, cutting from the soft, inner part of the leaf toward the outer and harder, making the section perpendicular to the axis of the leaf. The very great thickening of the cells of the epidermis is at once seen in this section, Fig. 28. The thickening appertains exclusively to the outer wall of the cell. The cell cavity runs out to a point in the same STOMATA. 67 direction ; this thickened wall is white, strongly refrac- tive "and is overspread with a cuticular membrane, still more refractive, but not sharply distinct. The lateral boundaries of the cells are marked only by a delicate line indicated on the outside by a slight elevation ; the inside of this thick wall is covered by a relatively thinner, less refractive layer, ^, which surrounds the cone-shaped ex- tension of the cell cavity, and, thinning out wedge-like, ceases at the same time with the thickened layer on the lateral wall. The thickened part of the epidermis in this section looks like a notched curtain. Of the cavity which leads down to the stoma we note first, the projection or rim which encloses it, and also that the tooth which forms the thickened layer is here divided on one side and loses half its height. The guard-cells have a ridge-like, and, in section, beak-shaped projection, both above and on the cleft side ; above the guard-cells is a thin place in the wall which forms a membranous hinge or joint. The breathing cavity is deep and narrow. Parallel striation, more or less oblique, may often be seen on the thickened wall ; it is caused by the knife in cutting and often recurs in hard, elastic substances in the same manner. With chloriodide of zinc, the thick wall is colored yellow-brown, showing it to be cuticularized. The inner laj'er, ^, and the rest of the leaf tissue, are col- ored violet. The yellow-brown color extends through the hinge to the two projections on the guard-cells ; the remaining part of these cells is tinged violet. Sulphuric acid dissolves all that the last reao;ent has not colored yellow-brown, and this it dissolves in an hour, leaving only the delicate cuticle and the fine, middle lamella oc- curring between the epidermal cells. The cuticle extends over the guard-cells to the point where they join the inner cells containing chlorophyll. The cuticle and the epider- 68 STOMATA. mal layer are colored brown by the acid. The oil in the guard-cells gathers together in a strongly refractive ball on the entrance of the acid and after some time disap- pears. In the arrangement of the stomata within the epider- mis many modifications occur. A very remarkable one is that when the stomata are surronnded by a single ring- shaped, epidermal cell. The fern Aneimia fraxinifolia^ found in every botanic garden, will show this. The cells of the epidermis have an extremely wavy outline, Fig. 29, and gain stability b}^ this dovetailing of the edges, so common in epidermal cells. In the ferns, the epidermal cells are richly furnished with chlorophyll grains. The epidermis, therefore, belongs with the organs of assimilation as it does not in most phanerogams. The stoma is set in the surrounding epidermal cells as in a rim. A transverse section shows it to be raised somewhat above the sur- face of the epidermis ; this extreme case is connected by transitional forms with others, not treated here, much less re- markable. We should accustom our- selves to think of the stoma, as, in fact, <)nly inserted on the side walls of the surrounding epi- dermal cells; then the singularity of its insertion will cease. Nerium oleander shows a peculiar form. Stomata will not, at first, be found either on the upper or under side, but on both sides a uniformly small-celled epidermis, which, particularly on the under side, is covered with single-celled hairs, the walls so thickened as alfliost to obliterate the cell cavity. On the under side of the leaf Fig. 29. Aneimia fraximfolla. Stoma suiToumlert by epider- mal cells, n, nucleus of epidermal cells. X 210. WATER STOMA. 69 are found certain cavities filled with air, and having on their edges these before-mentioned short hairs ; the hairs interlock across the opening and so close up the cavity in front. A second superficial section, taken from the same place whence the epidermis has already been removed, will enalde us to get a look here and there into the depth of these cavities. Perhaps an air-pump will be necessary to remove the air, or else an immersion of the section in alcohol ; there will be seen projecting from the walls of this cavity small, con- ical elevations, the \^ — < °X^A===_/(^y apex of which will be formed of a stoma. The lateral walls of these small cones con- sist of epidermal cells which have a breath- ing cavity between them extending to the stoma. The same kind of hairs which we saw on the edge of this cav- ity spring from its walls between the cones. We will now glance at the water pore or water stoma. It exhibits a structure like that of the air stoma, on]y it is larger, the cleft, together with the intercellular space, at times at least, being filled with water ; the guard-cells of this water pore may be unmovable from the beginning, quickly die and then in all cases lose their movability. The best object for studying these structures is the Tro- pceolitm maj'us. The water pore is found on the u[)per side of the leaf, and, indeed, on the ends of the i3rincipal Fig. 30. Water stoma and surronmling epi- dermal cells from the edge of leaf of Tropieolum majus. X 240. 70 WATER STOMATA. nerves ; here, the edge of the leaf commonly exhibits a small depression. One may approximately see the water pore by putting a piece of the leaf, full thickness in water, under a cover-glass, and place it under the microscope ; but the peculiarities of it will really be recognized only when a surface section be made of this part of the leaf; it will then be seen as represented in Fig. 30. The con- tents of the guard-cells are reduced to a minimum. Sev- eral water pores are always found but a short distance apart. NOTKS. (1) Strasburger, Jahrb. f; wiss. Bot. v, p. 2Ö7; de Bary, Vergl. Anat., p. 82 u. ff., 70 u. fl'. ; Schwendener, Monatsber. d. Kgl. Akad. d. Wiss. in Berlin, 1881, p. 833. In the first named authors will be found the remaining literature, at the places quoted. (2) Westermaier, Jahrb. f. wiss. Bot,i Bd. Xiv^ p. 43. LESSON VII. Epidermis. Hairs. Wax and Mucilage. We are already acquainted with the root-hairs of Hy- drocharis morsus ranee, and since all root-hairs are so much alike we can omit further consideration of them. We have also seen the conical papill;« or elon- gated epidermal cells of various petals. So also the cask-like cells form- ing the filamentous hairs on the stamens of Tra- descantia, Fig. 15 ; also, finally, the hairs of the Cucurbita with its many celled base running out into a pointed filament. The manifold aspect of plant hairs is already well known to us, but our knowledge of them should be more com- plete. We meet vari- ous forms of the single- celled, many branched fig. 31. ^ and 5, hairs from under side of , , Ji 1 T '^"'^ **' Ckeiranthus cheiri; A, hair f?een from naU'S on the leaves and above.X i'O-^. transection. X 210. C, hair from stems of the CrilciferrP "'"'^r side of leaf of 3/«<l, A, we have a lance-like form with the cell cavity narrow, and disappearing towards the end, (71) 72 PLANT HAIRS. the surface thicklv beset with knobs little and larsje. The hair lies parallel to the axis of the plant, and so a good transverse section may easily be made. But as we wish to cut it through the middle, at the point of its insertion on the leaf, a number of sections should be made in order to get just the one we want. Thus we see. Fig. 31, B, that the place of insertion lies somewhat deep, and that the epidermal cell which extends outwardly to make the body of the hair is slenderer than its neighbors, swollen out and rounded at the bottom and reaches deeper into the adjacent tissue, forming the "foot" of the hair. A longitudinal section shows that the foot is no wider in the other dimen- sions than in this, and one sees clearly that the cell cavity of the foot extends without interruption into the body of the hair. B}^ a superficial section made through the foot, we shall find it to be circular. We also see that the cells containing chlorophyll are radially arranged al)out and joined to that part of the foot which is widened and ex- tended below the epidermis. Cheiranlhus alpinus, not seldom cultivated in botanic gardens, presents the same appearance onl}' that at one or both ends, the hair forks so that we see three or four proc- esses all spread out parallel to the surface of the leaf. The hairs of the stem and leaf of MattJiiola annua are repeatedly branched in one plane, Fig. 31, C. These hairs are so thick upon the under side of the leaf that their branches interlock. The walls are so much thickened that the cell cavity is almost obliterated. Knobs are scarcely developed on the surface. The ball-shaped foot of the hair is considerably swollen and the cells containing chlorophyll are beautifully grouped about it in a radial manner. A superficial section turned over will show this. The long single-celled hairs in the groove of the spur- like prolongation of the corolla of Viola tricolor have a PLANT HAIRS. 73 veiy peculiar form, Fig. 32. Make a cross section through the lower petal, close under the place where it folds up into a groove. The epidermal cells grow out into a hair for almost their entire lireadth. The hairs are covered Avith irregular knotty swellings, and the cuticle with lon- gitudinal projecting ridges. The cell-sap is colorless, but yellow color-bodies often occur in the wall-plasma. The filament of the stamens of Verhascum nigrum is covered with single-celled violet hairs. Remove the anther and immerse the filament in a drop of water on the slide. The hair is quite long, swollen, club- shaped at the end and filled with a violet-colored cell-sap. It is covered with longish knobs arransfcd some- what spirally about it. Branched, many-celled hairs are found on the under side of the edse of the corolla. From above they have a certain resemblance to those of the Matthiola, but in this case eveiy branch comes out of a common central point, and consists of an inde- pendent closed cell. It also branches not in one plane, but at all angles. Its cell walls are as thick, but there are no outside projections as in the Matthiola. On the edge of the leaf the hair is seen in a lateral view. The body of the hair is separated fi-om the epidermal cell, which bears it, by a partition wall. It consists of an almost always single-celled stalk and the attached branches. The edge of the corolla also bears glandular hairs. These consist of a two- or three-celled stalk and a flattened head, Fjg. 33. Hair from the groove in tlie petal of Viola tricolor. X 210. 74 HAIRS AND SCALES. which is occasionally covered with a highly refractive sub- stance on its top. The latter will be studied elsewhere under more favorable conditions. One needs only to think of the branched hairs of Ver- bascuni nigrum being several times set upon each other in order to have the hairs which form the felt on the leaves of Verbascum tJiapsiforme. The hairs are five stories high, each story being separated from the preceding one b}' a single-celled internode which is a continuation of the prin- cipal axis of the hair. The cells are mostly filled with air. Fig. 33. Scale from the uncler side of the leaf of Shepherdia canadensis. A, sur- face view; /?, sectional view. X 240. The best cross-section is made through the mid-rib of the leaf. The scales of Shepherdia canadensis belong to the same category as the branching hairs of Verbascum. With the magnifying glass, we shall find white, loosely built and more closelv built stars on the under side of the leaf, Fis^. 33, A. On the upper side of the leaf but a few white stars are seen ; the cells of these contain only air, spring from a common central point, but are laterally separated THORNS AND SPINES. 75 from each other. On the upper side of the leaf the rays of the star are not confined to one plane, but radiate in all directions. The cells or rays of the brown stars are connected with each other, almost to the edge, and fur- nished with living contents. The nucleus is always easily seen within. A transverse section through the leaf, where it rightly hits the brown star, shows that the stalk is many celled. Fig. 33, B, and that not the epidermis alone, but also the next succeeding laj^er of cells, passes into it. The stalk bears at the top the stellate, single lamellate, many-celled expansion. In lack of Shepherdia canadensis, Eleagniis angustifolia may, to a certain extent, be substituted. Here, only the white scales are found on the under side of the leaf, the disk consisting of cells either laterally isolated or grown together nearly to the edge. Now, make a lono^itudinal cut throuoli the stem of a rose, perhaps Rosa semperßorens of the garden, at a place whence a thorij projects ; if possible, make it so as to di- vide the thorn in halves, and then make the thinnest pos- sible section ; this is not so easy to do ; but do not neglect in cutting to moisten the surface of the object. Having made a section, one can see that the epidermis of the stem continues into the thorn. The cells are more thickened and elongated. Beneath the epidermis are nar- row, greatly thickened cells, and, beyond these, cells with wide cell cavities, the latter filling the whole middle por- tion of the spine ; all these cell walls are finely perforated. The epidermis of the stem is separated from the chloro- phyll tissue beneath b\' a layer of cells without chlorophyll, somewhat thick, elongated and joining each other with sloping sides. These cells are of like origin with those which form the inner tissue of the spine ; but the tissue elements of the spine are separated from the chlorophyll tissue of the stem by a flat-celled tissue layer; this tissue 76 STING OF NETTLE. layer arises by division from the lowermost layer of the spine tissue ; it follows the chlorophyll tissue of the stem but a little way and then turns towards the epider- mis, so as to work off the lateral edges of the base of the spine from the chlorophyll-lacking tissue of the stem. It is the one cork la3'er nearest to whose outer surfaces, b}' means of a separating layer, there ensues in older parts of the stem the separation of the spine ; so the spine splits away from the stem, quite smoothly ah)ng the inner surface of the cork layer. Spines on the petioles lack this cork layer. In examininof the bark tissue ad- joining the spine of the rose, we shall find crystals in the cells, crystals of calcium oxalate which will not dissolve with acetic acid or potash, but with hydrochloric acid without generating gas. They have the form either of a monoclinic column or of a drusen ; the latter consists of a great number of crystals accumulated upon an original crystal; these crystals surprise us l)y their size and their stelhite form. J'°.el,'^«2fSc:; I" order to obtain, u„harn,ed, tl,e also epidermis and small stinging hairs of the dioecious nettle, hair. X 90. j^^ ^. "-.. . i. i. i ^i ,• Urtica dioica, we must take them trom the 3'ounger parts of the plant. With a razor cut a hair from the rib of a young, strong leaf and put it in water on the slide ; if the hair is dead, it will be found to be filled with air, and the point is no longer intact. The unin- jured hair is represented in Fig. 34 ; the hair is single- celled, sharply pointed and the point swollen to a small GLANDULAE HAIRS. 77 head. At the base, the hair is broadened like a retort, and this bulb is embedded in a cup formed from the tissue of the leaf. The history of its development shows the hair to be derived from a single epidermal cell which lay at the same elevation as the neighboring cell, and the greatly swollen foot of the hair was lifted up upon a col- umn covered with epidermal and formed of snb-epidermal tissue. In the hair itself, protoplasmic streaming may be observed. The nucleus is commonly found suspended in the bulb by plasma threads. The cuticle is covered with oblique ridges. The walls of the hair are silicated as may be shown by incinerating it on a mica plate. One often finds hairs with the points broken off. By carelessly touching the hair, its point is driven into the skin, and, as it is very brittle, it breaks off, and the strongly acid sap flows into the Avound and causes a slight inflammation. A small, single-celled, bristle-like hair is seen near the other, Fig. 34, distinguished by its fine point and its thick walls ; these bristles may be seen on the edges of the leaf. Put a bit of the leaf under the cover-glass in a drop of water. In all leaves the bristles will be seen from which the cell cavity has almost disappeared; their surfaces are covered with small knobs. Glandular hairs, mentioned in connection with Verbas- cum nigrum, may be studied under most favorable con- ditions in Primula sinensis — primrose. jNIake a tran- section of a petiole. The body of the hair is separated from the epidermoidal foot-cells by a transverse wall outside of the epidermis and forms a cell fibre, which con- sists of mostly two, sometimes more, long and ^vide cells and one (rarely two) slenderer and shorter cells; these last cells bear the little globular head ; but upon this is a cap of strongly refractive, resinous, yellowish substance. The secretion takes place between the cuticle and the cell 78 GLANDULAR TUFTS. membrane. The cuticle is raised up, distended and finally ruptured, and the secretion is poured out over the top of the hair. An application of alcohol removes the secretion, and the raised cuticle will be seen lying in folds. The cells of the hair show a beautiful network of protoplasm with suspended nucleus in which lie large nucleoli. In the wall plasma are no chlorophyll grains. The glandular tufts on the membrane-like prolongations of the leaf sheaths of Riiraex jpati&ntia are extremely inter- esting. The matter secreted by these tufts is so consider- able that in moist weather the young leaves and the ends of the stems are covered with mucilage. To examine this membranous sheath direct, the inside should be turned upward. The tufts will look like small leaves, Fig. 35. These minute leaves originate with a short single-celled foot from a small epidermal cell. On the one cell are two, on epidermal prolongation on the i^Mmea; tlieSC mostly four, wllich CX- po itntta. X 240. tending in the direction of the Jong axis of the leaflet are repeated several times. On the further side-walls of the tuft are often seen bladder- like protrusions which occupy a part or the whole of the wall of a cell. The mucilage is formed here also I)etweeii the cuticle and the rest of the cell membrane, and lifts the cuticle up. The bladder finally opens and lets the muci- lage out. This is not colored with iodine or chloriodide DIGESTIVE GLANDS. 79 of zinc. In water it swells to a perfectly clear solution and behaves itself like a gummy body. The cells of the tuft are rich in protoplasmic contents and their nucleus is very distinct. With rose aniline violet the tuft is stained an intense violet, the mucilage mass becomes a pale red. Aqueous nigrosin solution stains the mucilage steel blue without colorino- the tuft. The structure of the glandular hairs hav- ino^ the function of tentacles and digestive organs of the Drosera rotundifoUa is also very interesting. They arise from the edges and the whole upper surface of the leaf in the form of slender filaments attenu- ated upwards and expanded into an egg- shaped termination, Fig. 36. They are constructed of delicate elongated cells, the larger hairs penetrated throughout with one or more spirally thickened tubes, the spiral vessels. The radial extension of the epidermis to form the head of the hair, the superficial arrangement of the epidermal elements and their increase till they consist of three layers, may be best seen in an op- tical section of the object, Fig. 36. The number of the spirally thickened cells are greater in the head of the hair. All cells which lie inside the envelope, formed by the division of the epidermal cells, are spi-giami oi Drosera ro rally thickened. By examination of the place of insertion, we shall see that not only the epider- mis but also the inner tissue of the leaf is continued into the hair. These digestive glands secrete a sWmy fluid which clings to the top of the little head like a drop of •dew. It is not produced from beneath the cuticle but on Fig. 36. Digestive 80 WAX-SECRETIXG TUFTS. the free outer surface. Small insects get caught and stuck fast in this slimy drop and then by the bending down of the hairs they are carried to the middle of the leaf. Now other hairs also bend over and touch the insect body with their tops. Immediately the chemical qualities of the secretion change, a free acid and a pepsin-like ferment being pro- duced which slowly digests the insect or any other albu- minous I)ody. The digested substance is then absorbed into the plant. A transection throusjh the winter bud of the horse-chest- nut, ^scidus hij)pocastanum, shows us button-shaped glan- dular tufts upon the covering scales, Fig. 37. The middle scale has these tufts on both sides, but the outside scales have these more on the inner surface, and the inside scales more on the outer. The struct- ure of the tuft is seen from the FIG. 37. Glandular tuft from an en- ßauve ; a SericS of Cclls in the veloping scale of the winter bud of ~ ^ yEscuius hippocastmmm surrounded middle divide above and from by its secretion. X 240. ^j^^^^ ^^^^ sccretiiig ccUs radi- ate. The illustration shows a longitudinal section of the tuft. The cuticle will burst and the secretion will be poured out between the scales coating them over andglu- ins: them together. This secretion is a mixture of gum and resin. The minute drops of gum swell in water, while rose aniline violet colors the resin masses a beautiful blue. The contents of the tufts become red. We have already remarked the fine-grained waxy coat- ing upon the epidermis of Iris ßorentina. We wull now investigate this point in some other plants. A suitable plant is found in Eoheveria glohosa. The w^axy coating gives the plant a hoaiy or glaucous appear- WAX INCRUSTATION. 81 ance, and may be easily rubbed ofl' from the leaf. An inspection of the upper surfiice of the epidermis shows us a net-like crust of blended grains. A wax coating, in the form of short rods massed together, may be easily observed on the epidermis of Eiwaliptus globulus. Saccharum officinarum is also a beautiful object. Here the wax coating appears in the form of long rods often curled at the ends. Prepare a superficial section from a node of the stem noticeable for its glaucous appearance. To remove the air from between the rods dip the section in Fig. 3S. Transection through a node of the stem of Saccharum offlci-narum show- ing a rod-shaped wax coating. X 510. cold alcohol for a short time. It is difficult to make a transection to which the little rods still adhere. Such an one is shown in Fig. 38. The rods stand closely pressed together, and show the before mentioned curling. Brought near a flame the rods melt as they also do in hot alcohol. Note. (1) See in de Bary's Vergl. Anat. die §§ 10, 13, 16, u. ff. aud there also for the literature. LESSON VIII. Closed Collateral Vascular Bundles. The common cornstalk, Zea Mays, furnishes an excel- lent object for studying the structure of the closed collat- eral fibrovascuhir bundles of the monocotyledons (1). An alcohol specimen should be used for studying the cell contents. Make a transection through an internode and put it on a slide in a diop of chloriodide of zinc and lay the slide on a piece of white paper. We may now with the naked eye see the bundles, in the form of oval-shaped dots, and their arrangement characteristic of the mono- cotyledons. There is no specialization of pith and rind by the distribution of the bundles. With a low power, select for examination a bundle lying not too near the surface of the stem, noting which way the latter lies so as to distinguish the inner and outer edge of the bundle. The bundle is represented in Fig. 39. The sheath, vg, consists of thickened lignitied parenchyma cells, surrounds the bundle, and is stained red-brown. The intercellular passage, I, is surrounded with narrow thin-walled cells col- ored yellow by the zinc reagent. The ring, a, belongs to a ring vessel, for the most part ruptured by stretching. The intercellular passage may be produced by either of two ways, by the rupturing of the cells or by their separa- tion from each other. The one we may call the "lysignian," the other the "schizoginian," method. The ring vessels and some other which sometimes project into this passage are the first elements formed in the fibro vascular bundles when the plant is rapidly growing in length. Upon the (82) FIBRO-VASCULAR BUNDLES. 83 outer edge of the l)imdle are one or more vessels ; one in this case, sp. This vessel has spiral walls as we shall be able to see by a longitudinal section. Further on at the right and left are two wide, open spaces, m, m', vessels with pitted walls. A ring or part of one is often seen project- FiG. 39. Section of a flbro-vascular bundle from the inner part of a corn stalk, Zea Mays. «Joint of a ring vessel; sp, spiral vessel; to and »*', pitted vessels; i;, sieve tubes; s, conducting-cells;;»-, compressed protophloem elements; I, intercel- lular passage; rgr, sheath. X ISO' irg into the interior of these vessels, m'. It is all that re- mains of a perforated division wall once existing intact between two cells. The cells lying between these large vessels are reticulated. The walls of the vessels, espec- 84 VASCULAR BUNDLES ially of these, are stained a yellow-brown. The cells be- tween the vessels are stained a darker yellow than those al)out the intercellular passage. The above described portion of the bundle is called the woody or xylem vascular part ; this does not impl}^ that these cell walls are very much thickened. The woody part or vessels are never lacking in the bundle, hence the term xylem is morphologically correct. In the example just studied, we have touched upon the woody portions, viz., the xylem, the protoxylem, the wood parenchyma and the vessels of the tibrovascular bundles. For the other or secondaiy element of the bundle, we select the term bast or phloem. Since the sieve-tubes never fail in the bast, it is morphologically more reason- able to call the bast the sieve part (2). Vessels and sieve tubes con8titute the tibrovascular bundles, and, being lat- erally united, they are called collateral bundles. By including the sheath also, we may call the whole a iibro- vascular cord (3). A solution of the chloriodide of zinc colors the bast in this bundle a distinct violet. Wide and narrow unligni- fied cells regularly alternate : the former are sieve tubes, V ; the latter conducting-cells, s. We shall see the fine, sieve-like punctures when the section is made at or near a division wall (see illustration). On the outer border of this group are a number of thick-walled cells, j^r; they were the first produced, but their activity terminated in the sieve-tubes and conducting-cells ; they are the be- ginnings of the bast, the protophloem elements, and are stained brownish. The cells of the sheath border upon these, the inner ones having Avide cell cavities, but the outer sclerenchyma cells of the sheath gradually pass over through intermediate forms into the large-celled pa- renchymatous, fundamental tissue ; the cells of the latter OF MONOCOTYLEDONS. 85 are stained yellow with an occasional violet tinge. Towards the outer surface of the stem the l)undles are more closely packed, the intercelhilar passages disappear, the elements are reduced one by one, and the sheath is greatly en- larged. Lateral unions between large and small bundles are frequent in this part of the stem, the conjunctions takinof phice on the sides at the points where the large vessels are. Enclosing the epidermis of the stem is a stout ring of tissue, like that of the sheath, and gives the same reac- tion with chloriodide of zinc. This extreme outer layer is called the hypoderma and is unbroken except by the stomata. The hypoderma and the bundle sheath protect the thin-walled tissue and give stability to the stem ; com- prehensiveW, they are the "stereids" and form the mechan- ical-tissue system, the "stereöms" (4). Mechanic.il prin- ciples require that for stiffness the stereöms be [)laced as near as possible to the periphery of the stem. The pe- ripheral bundles, including the sheaths, constitute a system of compound pillars. The sheath is the column, the bun- dle is its feeling. The hypodermal cylinder is strength- ened by the sclerenchyma tissue of the sheaths, even if, as in this case, it is not much developed ; this cylinder con- sists of a number of interblended columns placed in a circle. Coralline soda (5) rapidly stains the lignified walls in both the bundles and fundamental tissue a bright, coralline red, and the unlignified a rose color. This brings into great prominence the sheath cells, the walls of the vessels and the epidermal ring. Now, make a number of radial sections, so as to be sure to have one at least through the middle of a bundle. It should show the bast and the ring vessels ; stain with chlor- iodide of zinc; the colors will correspond to those of the 86 VASCULAR BUNDLES transection. But a coralline-stained section will be bet- ter for study, Fig. 40. Begin at the inner edge of the bundle. We first meet the nearly cubical cells of the fun- damental tissue, and then the sheath cells of the bundle, vg, deeply-stained, elongated, somewhat pointed, and dotted with slit-like pits diagonally arranged. Within Fig. 40. Longitudinal section of corn sttilk. Zea Mays, a and «', joints of a ring vessel; sp, spiral vessel; v, sieve tubes; s,conducting-cells;^?-, protophloem ; I, air passage ; vg, sheath. X 180. these elongated sclerenchyma cells is a much reduced wall layer of protoplasm and a nucleus. Next to the sheath is the intercellular passage which extends through the whole length of the bundle. It is surrounded Avith pri- mary, wood-parenchyma cells, thin-walled, shorter than those of the sheath, have more cell contents and are sep- arated by transverse walls. Isolated, small rings, a, be- OF MONOCOTYLEDONS. 87 lonofino; to a rinoc-vessel which has beeu ruptured durino; the elongation of the iuternode are seen attached to the outer side of the passage ; these and other small rings, sometimes seen, a' , constitute the remainder of the pro- toxylem elements. Next to the rings are one or more spiral vessels, sj) ; only one, and that a narrow one, in this case. Next, are somewhat, thicker- walled, relatively short, wood parenchyma cells, with pitted and mostl}'' re- ticulated walls ; beyond these are the bast cells, recog- nized by their thick, rose-colored division walls, the sieve-plates of the sieve-tubes, v, the punctures of which may be seen with a high magnifying power. The con- ducting cells, s, are placed side by side with the sieve-tubes, are slenderer, shorter, and have a nucleus which the sieve- tubes have not. The sheath cells bound the vascidar bun- dle and are so acutely pointed that we may speak of them as vascular fibres. Starch grains are not found in the cells of the vascular bundles nor in those of the fundamental tissue. All these cells except the vessels and the sieve- tubes have a nucleus. A longitudinal section through the middle of the bundle will not show the larger vessels, except in some cases by deep focussing, and then indistinctly. To see these, make a section through the side of the bundle. They are ob- liquely pitted. The pits are enlarged at the bottom but bordered only on one side, as the corresponding pits of the adjacent wood-parenchyma cells have no borders. The diaphragm, which consists of a double ring projecting into the interior of a vessel, is produced by the thickening of the outer edge of the transverse wall of the cell, and the subsequent absorption of the thin middle partition. We should now make a permanent preparation of both the transverse and longitudinal sections. Stain with saf- Iranin or iodine green. For double staining, immerse for 88 VASCULAR BUNDLES. a considerable time in iodine green and then in Grenadier's carmine (G) . For instantaneous double staining use picro- FlG. 41. Transeotion of flbro-vascular bundle from leaf of Iris florentina. Ele- ments with daik outlines ar« vessels and the shaded ones are those rich in cell contents, ss, compressed spiral vessels; sp, wide spiral vessels; sc, scaliform ves- sels ; V, sieve tubes and between them the narrow conductiug-cells ; pr, compressed protophloem elements; vg, sheath with wavy radial cell walls; k, section of a crys- tal. X-W. nigrosin, or picro-aniline blue. The iodine green and the picric acid will color the lignihed cell walls ; the carmine, STAINING SECTIONS. 89 nigrosin and aniline blue stain the unlignified walls and the cell-contents. Mount in glycerine or glycerine jelly. If in the former, clean away all the superfluous glycer- ine and cement the cover-glass down with a solution of Canada balsam in turpentine or chloroform, of the consist- ency of syrup. Use a glass rod as thick as a match for this purpose. Gold size or varnish will not answer. Gly- cerine-jelly mounts do not need to be sealed.* If a cornstalk is not available for this examination use the stem oi Avena saliva , common oat, or any other Gram- inacece. We will next make a longitudinal and transverse section of an alcohol specimen of the full-grown leaf of Iris flor- eniina. The alcohol specimen cuts better, contains no air and has the cell-contents already fixed. Let it lie for some time before cutting in a mixture of glycerine and a|- cohol. Stain the section by immersion for several hours in borax-carmine, and a short time in iodine green. The cell- contents will take the carmine. The lio^nified walls and vessels will be stained green, also commonly that part of the sheath which lies next to the bast. The proto- phloem elements will be blue. Such a section is represented in Fig. 41. The cells with red cell-contents are shaded in the ilhistration. The green walls of the vessels are dark, and the blue protophloein cells are light. The thick- ened cells of the fundamental tissue are unlignified and hence uncolored. For instantaneous staining use only iodine green, and if only the lignified walls are to be stained the exact time for the efiect must be carefully de- termined. The cell-contents in that case Avill not be stained. Our examination will proceed from the wood towards the bast elements from the inner and upper to the outer and *But they may be moi-e conveuiently handled, and are less liable to injury if they are securely cemented and nicely finished.— A. B. H. 90 VASCULAR BUNDLES. lower side of the leaf. The number of vessels is pretty large in the wood part and they increase in size towards the bast. They either touch each other, or are separated by thin walled, relatively narrow, primary wood-paren- chyma cells full of cell-contents. These cells also sur- round the vessels and separate them from the fundamental tissue. The compressed cells, ss, are protoxylem elements. In the bast the wdde cells are the sieve-tubes, and the small ones, rich in contents, are the conducting-cells. Be- yond, at pr, lie the protophloem elements, stained blue. This part of the bast is surrounded with the much thick- ened sclerenchyma of the sheath. It is wanting about the rest of the vascular bundle. The cells of the funda- mental tissue are smaller about the vascular bundle, and have no intervening air spaces. There are several inter- mediate forms between these cells and the larger ones of the fundamental tissue with air-filled intercellular spaces. At Ä; is a small cell containing a highly refractive crystal. By the use of coralline, we shall get a good and rapid staining, the lignified sclerenchyma becoming a fiery red, the as 3^et unlignified a bright rose color, the vessels a brown red and the other elements a pale 3'^ellow red. For comparison we will prepare a section from a fresh leaf. We see that the outer large fundamental cells con- tain chlorophyll ; those next the bundle do not. The ves- sels contain air which somewhat impairs their microscopic image. The radial walls of the first layer of cells next the wood part of the bimdle seem to have dark broad pits. By looking at the other section again Ave see that these walls are arched towards one side, vg, Fig. 41. By focus- sing up and down we shall see that this arched part of the wall forms a wave-like band bent back and forth. As we shall meet a similar structure elsewhere we will spend no more time on it now. A longitudinal section cut through the middle of a CRYSTALS IN CELLS. 91 bundle shows on the inner edge of the bundle a much elongated, partly compressed, spiral vessel. Fig. 41, ss; this is the protoxylun element. Beyond are closely wound spiral vessels, and, further along, narrow, scale- formed vessels. In the bast the sieve-plates show plainly only in the coralline preparation. Further towards the outside are the sclerenchyma fibres distinguished by their consideral)le length, thickness and pointed ends. V Jf Fig. 42. ^.crystal of calcium oxalate in acellof theleaf of /WsJ^oj-en^jn«. X240. B-B, figures which explain the form of the crystal; B^ and B\ and D, the optical section ; C, projection on a symmetrical plane. In the longitudinal section, the crystals are seen in profile, lying parallel to the longer axis of the leaf. Fig. 42, A-B; they are found in elongated cells of the funda- mental tissue, the crystals being nearly as long as the cells ; these cells have no chlorophyll like most of the neighboring cells. The crystals are calcium oxalate and dissolve without generating gas in muriatic acid; they have a long prismatic form, are mostly twins, D, and be- 92 CLOSED VASCULAR BUNDLES. ph~ -yiii long to the monoclinic system. The cell-contents of these cells are not stained with the coralline. The vascular bundles of the monocotyledons are mostly built on the type of these two cases. Closed, vascular bun- dles are not well adapt- ed to the lateral orowth of monocotyledons. This growth, which is limited to plants of the families of Draccenece, Ali one ce and Dioscwacece, takes place by means of a cam- bium zone lying outside of the vascular bundles. We will select Dra- coena rubra, cultivated in every market garden, and make a transection of the same. Inside the I)rown cork layer, we see with the naked eye a green, soft rind about 1 mm. thick, ao-ainst which is contrast- ed the yellowish hard tissue of the stem. On the border of this is the cambium rino-. The cir- cular centre of the stem has a lighter color. Now put the section under the microscope, wdth a low magnification, Fig. 43. The central fundamental tissue of roundish cells, m, has in it isolated round or oval vascular bundles, ß. Beyond a certain point,/", they are more numerous, compressed Fig. 43. Transection of stem of Draccena rubra. /,vascular bundle;/', primary. .r', sec- ondary bundles;/"', leaf bundles; m, imlig- nifled fundamental tissue; s, lignifled funda- mental tissue, surrounding the bundle ;<, tra- cheids;c, cambium ring; cr, rind; I, cork;pÄ, cork cambium; r, bundles of raphides. X^O. CLOSED VASCULAR BUNDLES. 93 and crowded together so as to touch, or are separated by only a single layer of fundamental tissue. In the latter, the cells are much thickened, deeply pitted, radially elon- gated, and arranged in a radial series. Beyond this we come to the boundary between the yellow tissue and the green rind, c, a zone of flattened, thin-walled cells ar- ranged in a strictly radial order. This is the cambium ring which provides for the lateral or radial growth of the stem. It belongs apparently to the fundamental tissue. In the middle of the zone is the initial layer of cells, one cell thick, whose successive cell divisions produce the new elements. These divisions, occurring tangentially, produce the radial arrangement of cells. An occasional radial division occurs also, and so starts a new radial series of cells. Vascular bundles occur in the young tissue in all stages of development. The youngest consist of a group ot thin-walled cells. The older ones are complete on their inner edge already, but on the outer are still in the proc- ess of formation. It is supposed that from the point f", the tissue is sec- ondary, produced by the activity of the cambium ring. The rind, c/% consists of roundish cells. Crystal needles in groups or in bundles are found in the cells of the inner border of the rind, principall3\ These so-called "raph- ides " are calcium oxalate. The other cells of the rind contain chlorophyll grains. The bundles, one of whose round sections are seen at /'"'', are those provided for the leaves. The stout layer of thin-Avalled, colorless, radially- ai-ranged cells, I, which pass outwardly into a brown, less regular tissue is the cork layer ; on the inside, young, colorless, and on the outside, old, irregularly elongated and browned cork-tissue. Stain a transverse section with coralline. The bundles come out sharply. The lignified, secondary, fundamental 94 CLOSED VASCULAR BUNDLES. tissue is stained another shade of red, the unlignified, a pale rose red. The raphide cells seem filled with a red liquid. So we know that the raphides are embedded in a homoo^eneous mucilaoe which absorbs coralline. This re- ao;ent stains veo;etable mucilao^e : neither cold nor hot alco- hoi will discolor this mucilage thus stained. But mucilage derived from cellulose is bleached in both hot and cold al- cohol (7). In this we have our test of starch and cellulose mucilage. Gum is not stained by coralline. A mixture of cum. and mucilao'e is or is not, accordins; to conditions.- An aqueous solution of nigrosin will not stain the mucilage of this plant, even with long treatment, but it will that of Rumex. This survey of the transection shows us the process of the radial growth of the plant. We will omit the study of details and of the longitudinal section. Notes. (1) In regard to the vascular bundles generally, see de Bary, Yergl. Anatomie 1877, especially Chapter vni, where the whole of tlie older literature maj' be found. Numerous critical investigations of the mor- phology of the vascular bundles, which have recently appeared, have not had a coherent treatment. G. Haberlandt, on the contrary, in the Encyklopiidie der Naturwissenschaften, Handbuch der Botanik. Bd. n, p. 593, has accomplished this in part by attempting a phj-siological in- terpretation of morphological facts, in his physiologico-anatomical works. (2) The designation vascular part and sieve part was introduced by de Bary, Vergl. Anat., p. 330. (3) See Haberlandt, in the history of the development of the me- chanical tissue-systems of plants. (4) Schwendener. The mechanical principles in the anatomical structure of the mouocotyledons. (5) This staining fluid was introduced by SzyszyloAvicz. See Bot. Centralbl. Bd. xii, p. 138. (6) See Tangl, Jahrb. f. wiss. Bot., Bd. xir, p. 170. (7) See Szyszylowicz at the same place. LESSON IX. Open Collateral Vascular Bundles. For our study of the open collateral bundles of the di- cotyledons we will take a runner of Ranunculus repens. Staiu the section with coralline. We tiud the bundles is- olated from each other and arranged in a simple, circle in the stem. The fundamental tissue consists of rounded cells which diminish towards the surface of the stem, contain chlorophyll and have large intercellular spaces between them. At the surface is the epidermis, but within, the stem is hollow, caused by the separating and rupturing of the cells. The vascular bundles have the same appearance as those of the monocotyledons, the same parts appearing in the same order. The woody part consists of vessels and thin- walled parenchyma cells ; the ring and spiral vessels on the innerside of the bundle next the vessels take up but little coloring matter, Fig. 44, s. The other subangular vessels are colored brown-red. In these walls are bor- dered pits, m. Between these vessels lie the soft- walled primary wood parenchyma. In the bast is again the alter- nation of large sieve-tubes and small conducting cells. But the bast is separated from the woody part by a many- layered stratum of radially-arranged cells, c. This ar- rangement betrays their cambium origin. A cambium layer separates the woody from the bast part, and distin- guishes this from the monocotyledons. The activity ot this cambium layer is indeed limited outwardly, but there is enough of it to give the bundle a place among the "open bundles," that is, those capable of a lateral growth. (95) 96 OPEN VASCULAR BUNDLES. It has formed a stratum several layers thick of thhi- walled cells and then ceased to grow. The bast is pro- tected oil the outside by a cord of sclerenchyma cells, colored a beautiful coralliue red. So also the iuner edge of the buudle is iuclosed by another but thinner-walled layer of such sheath cells. The sheath cells do not meet at the sides of the buudle. Fig. 44. Transection of the vascular bxuidle of a runner of Ranunculus repens. s, spiral vessels; m, bordered pitted vessels ;c, cambium; v, sieve-tubes; vg, sheath. Xiso. In the longitudhial section, ring, spiral and pitted ves- sels, and between them elongated wood parenchyma cells, are easily made out. Then follow thin-walled cambium cells, sieve-tubes, conducting cells, and finally sheath ele- ments with but slightly inclined, porous, transverse divi- sion walls. OPEN VASCULAR BUNDLES. 97 The vascular bundles of celandine, ühelidonium majus, are so like those of the Ranunculus repens that a cross-sec- tion can be easily understood from that. We prefer alco- hol material. The wood part shows large vessels pressed close together which in the older sterns have yellowish walls. The bast is strongly developed ; between the two lies the stratum of thin-walled, radially-arranged cells produced by the activity of the cambium. The sheath appears only in a bundle of thick-walled sclerenchyma cells on the outer border of the bast, in old stems colored yellow. Running around, just within the epidermis, and separated from it b}^ a cell-layer two cells thick, is a ring of sclerenchyma ceils, like those which protect and sup- port the bundles, making a common sheath to the whole inner tissue of the stem. In and on the bundles we meet an element not heretofore seen, the milk-tubes. They are tilled with a dark brown substance, which is the orange- red milk-sap of the plant into which the alcohol has run. They are found in the bast of the vascular bundles, also on the inner bolder of the wood part ; especially numerous on the sides of the bundles and the outer edge of the sclerenchyma tissue, and scattered singly through the fun- damental tissue between the bundles. They are all thin- walled, even those intercalated in the outer edge of the sclerench3ma tissue. It is impossible not to see them. In the longitudinal sections they are easily recognized also by their yellow-brown contents. They run parallel to the axis of the stem, are lurnished with transverse walls which are perforated with one or more pores, and yet these walls are quite lacking sometimes in places where we expect to tind them. Often some of the vessels in the bundles are found filled with coagulated milk-sap. Stain a transverse section with coralline, then ap[)ly a drop of potash \ye to the edge of the cover-glass. The 98 OPEN VASCULAR BUNDLES. vessels will appear fuchsiu-red, the sclerenchj'ina cells rose-red, while the milk-tubes and their dark brown con- tents will come out ver}" distiuctly. Put a very thin lon- gitudinal section in 45 per cent acetic acid carmine, and nuclei may be detected in the milk-tubes, but not very easily. Lateral unions of the milk-tubes have not been seen in this plant. Aristolochia siplio, or Dutchman's pipe, affords an un- commonly good object in which to study the lateral growth of the dicotyledons. Make a section of a branch 3 to 4 mm. thick. AVith a lens observe the iuner loose pith; about this a circle of isolated vascular bundles ; about this a contiuuous white ring; then green rind-tissue ; finally, a yellowish-green peripheral envelope. With a low power under the microscope we see that the pith is composed of large round cells in part filled with air. In the vascular buudles the wood part is darker and much broken by the wide cavities of the vessels. The cambium zone follows, composed of narrow, radially-arranged light cells, and here- upon the bast cells, not so light nor so regularly arranged but much larger. Each bundle is bordered about, espec- ially on its outer part, by parenchymatous tissue contain- ing some chlorophyll grains, aud eventually also reserve substances. The white ring beyond is formed of much thickened sclerenchyma cells which project inward some- what in wedge-shaped masses between the vascular buu- dles. Abutting upon this ring is the tissue which contains chlorophyll and air-filled intercellular spaces. Beyond this comes narrow-celled tissue containing chlorophyll, the cell-walls white and thickened at the corners, on account of which they are called " collenchyma " cells. Finally, the epidermis. Now, make a very thin section of a bundle from alco- hol material, that has previously lain iu a mixture of equal OPEN VASCULAR BUNDLES. 99 parts alcohol and glycerine. Stain, by immersing for some time in coralline. Fig. 45 gives a representation of a sec- tion of a bundle made from a growing, this year's branch, about the besrinnino' of June. The vascular bundle bemns on the inner edge with thin-wallcd, primary wood paren- Fig. 45. Transection through a young twig of this year's growth of Aristolochia siplio, showing a vascular bundle after the beginning of the active growth of the cambium, j:?, parenchymatous elements on the inner edge of tlie wood part; m' and m", vessels with bordered pits; ic, interfascicular cambium extending into the fas- cicular cambium, that is, into the cambium within the vascular bundle; v. sieve- tubes; c, rind-parenchyma; sk, inner part of the ring of sclerenchyma fibres. X 130- chynia,^, in which narrow vessels, which gradually become wider (protoxylem elements), are inclosed, the cells them- selves also becoming thicker-walled. The same also may be said of the vessels, while the intervening spaces are 100 OPEN VASCULAR BUNDLLS. taken up by tracheids, with thickened and border-pitted walls. The thick-walled wood parenchyma, vessels, and tracheids take an intense red, while the thin- walled are col- ored a faint rose. The two largest vessels are seen in the process of development. Between these lies thjn-walled, serially-arranged, secondary tissue, of cambium origin. On the outer border of the two large vessels is the cam- bium zone, in which an especially flat, not very sharply defined cell-layer represents the initial stratum. Follow- ing this, are like cells of the bast, the radial arrangement of which betrays their secondary cambium oiigin. In the middle of the bast are the sieve-tubes from which the ac- companying conducting cells are distinguished by the con- tents existing in a majority of them. The outer part of the bast, the protophloem, is taken up by narrower sieve- tubes, which are, therefore, not so sharply contrasted wdth the conducting cells. The bast is separated from the sole- renchyma ring, sk, by large-celled rind-parenchyma. The sclerenchyma is as intensely colored as the wood part of the vascular bundle. The protophloem elements are com- pressed by the multiplication and growth of the new cells from the cambium. Such a section shows very perfectly the formation of the interfascicular cambium. With the beginning of the activity of the cambium in the vascular bundle, the cells of the fundamental tissue adjoining the same at the sides also increase by self-divi- sion through the introduction of dividing w\alls, ic. So the elements of the fundamental tissue are formed into a cambium stripe which unites the cambium layer in the cir- cularly placed vascular bundle into a contuiuous cambium ring. As the figure shows, it is very easy to follow the formation of the interfascicular cambium in this plant, and to recognize for a long distance the original outline of the divided fundamental-tissue cells. The sheath is altooether lacking in this plant about the single vascular bundles. OPEN VASCULAR BUNDLES. 101 The sclerenchyma ring forms a common sheath about the whole inner tissue of the stem. A radial lonrntudinal sec- tion cut through the exact middle of a vascular bundle and stained with coralline shows, on the innermost side, elongated primary wood parenchyma with transverse divi- sion walls, between which are very narrow, somewhat compressed ring vessels ; then somewhat wider ring vessels, which in part pass into spiral vessels ; then closely spiralled wider vessels which pass into reticulated vessels ; finally, the enlarged border-pitted vessels. Between these vessels are much elongated, border-pitted, empty tracheids ; sin- gle fibre cells, like the tracheids in ftn-m, but having un- bordered pits and filled with starch ; thick-walled wood parenchyma, shorter, with transverse walls, likewise un- bordered pits and starch. The incomplete, large vessels are still wide, cylindrical, thin-walled cells, separated by transverse division walls, with rich protoplasmic wall-lin- ing, and a nucleus. In the fully completed vessels, noth- ing of their cell-contents is to be found, and of the division wall, in the pitted vessels, there remains only the ring-like projecting diaphragm. The flat cells of the caiiibium zone are rich in protoplasmic contents, nucleus and delicate transverse division walls. The sieve-plates are quite extra- ordinarily beautiful. When they are somewhat inclined, the}' present to the ol)server their whole rosy surface with dark, sparkling points. In those sieve-plates which are much inclined, the plate is divided by clear belts without pores, into several rose-colored, dotted and superimposed sections. The side walls of the sieve-tubes are also cov- ered with small, transversely extended, finely dotted, rose colored sieve-pits. In the periphery of the bast occurs, in the most astonishing manner, the formation of the cal- lus-plates, those bright, rose-colored, strongly refractive masses, which are rounded upon the free side, and which 102 OPEN VASCULAR BUNDLES. cover both sides of the sieve-pl;ite in like mass, or are prepoiideratingly on one side only. The sieve-pits on the lateral walls also have minute callus-plates. We also find here with the sieve-tubes, the narrower conduct! ng-cells tilled with their conteuts. The bast is separated from the sclerenchyma ring by the broader — and, as this. section shows, also relatively shorter — pareuchyma cells. The sclerenchyma fibres of the ring are very long, pointed at their ends, comb-like with their ends interlocked and pro- vided with pores. And, finally, we notice that the coUen- chyma cells bordering on the epidermis, are several times longer than broad and are joined with transverse walls. Now cut a section from an older branch, say one 10 mm. in diameter and examine it with the lens. The pith and the medullary rays are white, the wood-bodies yellowish. The thickest medullary rays, some ten or twelve in num- ber, open into the pith, and are those primary rays which in the beginning separated the vascular bundles. The old- est wood part of the bundles border on the pith. Since the wide vessels are lacking in them, they seem to be a thicker, darker ring penetrated by the primary medullary rays. To these succeed the concentric yearly rings. The width of the vessels increases in the first year's growth till it reaches a definite greatest diameter. The boundary of the yearly ring is clearly marked by the larger vessels, since those of widest cavity are produced in the begin- ning of the development in the spring. The outer part of the yearly ring contains no vessels distinguishable with the lens. As the secondary Wood-bodies increase in cir- cumference, new medullary rays are intercalated Avhich we designate as secondary rays of the second, third, fourth^ etc., orders. The intercalation of secondary rays follows with the greatest regularity. The farther we go from the centre, the more numerous are the medulhuy rays and the OPEN VASCULAR BUNDLES. 103 shorter are the newly adclecl ones. On the onter border of the wood bod}^ we see the caniljium ring as a dark circle, the medullary rays within which are indicated l^y delicate lines. Before the secondary wood parts, Ave see the clear brown-colored secondaiy bast lying, formed from succes- sive growths. The medulhiry rays extend be3'ond the cam- bium in consequence of its supplementary lateral growth caused b}^ the increase of the thickness of the stem. The bast is not capable of this supplementary lateral growth and appears thence from the outside to be nari'owed and rounded. The original continuous sclerenchj'ma ring is dispersed in single olive-green colored pieces of unlike size ; likewise, also the original continuous darker olive- green collenchyma layer. The periderm now undertakes the protection of the interior, and as a brown, distinctly laminated sheath covers the surface of the stem. The whole of that part subsequently produced by the cam- bium, which includes the secondary bast and the extended medullary rays, becomes secondary rind, which confronts the primary rind previously existing before the beginning of the lateral growth of the stem. No sharp boundary exists between the primary and secondary rinds. Now apply a higher power to the investigation of a thin cross-section of this stem. The pith tissue is unchanged from its young state, only that it has numerous crystal masses of calcium oxalate. The primary wood parts project into the pith tissue, formingthe so-called "medulhuy crown" or "medullary sheath." The hand-lens will not show this, as the inner parts arec(miposed of thin-walled compressed cells. First on entering the solid part we find the wood-bod- ies clearly marked off from the large pitted vessels. The vascular bundles increase in breadth correspondingly to the narrowing of the medullary rays. The vessels formed in the spring, up to the third or fourth annual ring, show an 104 OPEN VASCULAR BUNDLES. increase in volume. From the spring toward the fall, the width of the vessels rapidly decreases in each annual ring. Shortly before the close of the year's growth, only very narrow vessels are produced. The great mass of the wood consists of tracheids, narrow, thickened, empty, border- pitted cells. They contain air or water. If starch grains are ever seen in them, the knife has carried them there from neighl)oring cells. Distributed about the circumfer- ence of the vessels mainly, but also among the tracheids, are thinner-walled cells with protoplasmic contents ; also, commonly, starch and flat pits. These are wood-paren- chyma and fibre cells. The vessels are provided Avith bordered pits, only when they touch each other or the tracheids. When a vascular-pit or a tracheid-pit meets the pit of a wood-parenchyma or fibre cell, it is one-sided, that is, bordered only on the side of the vessel or tra- cheid, or, so to say, narrowed in its opening only on this side. The closing membrane of such one-sided pits is without central thickening (torus) and, unlike such thickened mem- brane, is colored blue with chloriodide of zinc (1). The cells of the medullary rays are radiall}^ extended, relatively thin-walled, and have numerous pores. On the outer border of the wood substance, we easily recognize the cambium formed from flat, thin-walled, radially-ar- ranged cells, and beyond that the thin-walled bjist. Be- sides sieve-tubes and conducting-cells, we find in this also starch-bearing bast parenchyma. The secondary bast produced by the cambium has con- sequently gained the latter additional elements. With an extremely delicate section one can follow in the bast the alternation of uncompressed with fully compressed cell layers. Similarly, flatly compressed elements have already been seen in the one-year-old branches on the OPEN VASCULAR BUNDLES. 105 periphery of the primary bast, the appearance repeating itself consequently in the bast growth of subsequent years. These bands of flatly compressed cells are after- wards broken into parts which always, and after a time more distinctly, take the form of a bow. By the inter- calation of new medullary rays the bast is constantly being divided so that every outer bast part spans two in- ner. Outside of the sieve parts in the rind are the sep- arated pieces of the ring of sclerenchyma fibres. The pieces are separated by parenchymatous tissue. The ring has been radially broken in consequence of the progres- sively lateral growth of the cambium, and the adjoining tissue of the rind has pushed itself in. The collenchyma ring also is distributed in parts. Still, there is no essential breaking of it up; rather, in single places, a tangential ex- tension of the cells takes place which then in parting came in and so the parenchymatous tissue masses get their ori- gin. The surface of the stem is covered with the periderm which presents the beautiful alternation of broad zones of wide thin-walled, and narrow zones of small, thick-walled cork cells. As in the pith and medullary rays so in the rind are found scattered crystal masses of calcium oxalate. The radial longitudinal section shows, in the first place, the wide and narrow vessels, border-pitted, with annular diaphragms ; the border-pitted tracheids ; the fibre cells, shallow-pitted and with cell contents ; the wood-paren- chyma cells, likewise with cell contents, with flat pits, shorter, less thickened than the tracheids and joined together in continuous threads. If the medullary rays have been hit, radial lines of their thin-walled cells will be seen run- ning across the section. On the outer border of the wood, we recognize the cambium cells, rich in contents, thin- walled with transverse walls between ; then the still active l)ast, and here, upon the flat cells of the older bast, the 106 OPEN VASCULAR BUNDLES. compressed alternating with the uncompressed parts. The laminated periderm shows up very beautifully in the mar- gin of the section. The longitudinal section of this layer is exactly like the transverse section, the cells being of the same breadth or height. By the cutting of the wood the exact course of the medullary rays is apparent to the unaided eye. This comes from the considerable length of the internode within which both the vascular bundles and the medullary rays retain their direction unchanged. The tangential section shows us under the microscope, the med- ullary rays in the form of broader or narrower stripes more or less parallel to each other separated by corresponding stripes of wood. As it is not a little difficult always to distinguish the different tissues in the complicated image shown by sec- tions of the wood, we will try another method, viz., that of maceration. For this purpose take a wide test-tube and over some fragments of potassium chlorate pour enough nitric acid to cover the pieces fully. Put into this a not too thin secti(m of the wood and heat to boiling over a flame. Let it stand for some minutes and pour the whole into a laro;er dish of water. With the glass rod remove the floating section into another dish of water and thence into a drop upon the slide. This experiment should not be made in the same room with the microscope else the gas may damage the instrument. The section should now be disintegrated with needles so as to have its elements separated. If the reagent has properly worked, the mid- dle lamella will be dissolved and the cells will easily come apart. Now we shall find all of the elements, heretofore studied in connection, entirely isolated. They are mostly well preserved, only that they have been robbed of their wood substance and will be stained violet for the most part with chloriodide of zinc. First of all we shall see the pit- OPEN VASCULAR BUNDLES. ' 107 ted vessels mostly sepurated into pieces corresponding to the annular diaphragms. The tracheids are especially numerous with attenuated rounded ends and bordered pits. These pits present them- selves now in the smaller walls as narrow oblique slits. But by proper focussing it is always possible to demon- strate that they widen outwardly. When some of the tra- cheids are found still joined together, the pits appear in the form of a cross, the corresponding pits on the two ad- jacent cells being inclined in opposite directions. Besides vessels and tracheids are thin-walled wood-parenchyma cells with large flat pits. They are also recognizable by their compacted and knotty cell contents. We find also isolated forms which are like those of the fibre-cells, occa- sionally with but one cell cavity but commonly divided into several shorter parts one above the other by transverse or oblique walls. Those with a single cavity are what we have heretofore known as fibre-cells, but which may be better known as "substitute fibre-cells" since they re- place the wood-parenchyma cells. The compound forms which together replace the wood- parenchyma are apparentl}^ produced by the division of a single mother-cell. The transverse division walls must have been formed at an early period when the mother-cell w^as still thin-walled, for they show the same thickness and the same pits as the side walls and must therefore have been thickened at the same time with these. NOTK. (1) See Rnssow, Bot. Ceiitralbl. Bd. xiii, p. 140. LESSON X. Structure of the Coniferous Stems. Wn shall now undertake the thorough study of the structure of the stems of the fir tree, Pinus sylvestris. We shall find the lateral growth entirely different from that of the Aristolochia. In the pine the secondary growth of the wood consists entirely of the formation of one ele- ment, the tracheids. Vessels are fonnd in the pine, only in the medullary sheath, in the primary wood of the vas- cular bundles. A transection shows that the inner edges of the dark-colored wood which projects into the pith con- sist of narrow elements with somewhat brown walls. A thin longitudinal section shows them to be spiral vessels. Some such vessels, which likewise have bordered pits and spiral bands, constitute a transitional form to the trache- ids with bordered pits o\\\j. We will use alcohol material in making a section of the cambium, the fresh being liable to tear, and the dry diffi- cult to cut. La}^ the wood about twenty-four hours in a mixture of equal parts alcohol and glycerine before cut- ting. Alcohol material has the advantage of having the cell-contents fixed also. The wood should be cut in June or July when the cambium is in full activity and put into alcohol. Make the section from the periphery of a good sized stem, as the tracheids in the later annual rings are larger. We will examine the section in glycerine. But in case we use reagents with it we shall previously wash it Avitli wa- (108) STRUCTURE OF CONIFEROUS STEMS. 109 ter. We begin by making a section of the stem from the periphery inward across several of the annual rings, the cambium and the rind. We see the tracheids arranged in a radial series and occasionally a row is doubled. These elements are quad- rangular ; sometimes five-and*six-angled. In the fall the Avails become thicker and the tracheid narrower. Imme- diately adjoining these are the wider and thinner-walled ceHs of the following spring, thus distinctly marking even to the naked eye the limit of the year's growth. Parallel to the radial rows of tracheids are the medullary rays, nar- row and of one layer of cells, seldom of more, distin- guished by their cell-contents of starch. On the radial walls of the tracheids stand the bordered pits whose structure we already know. Between the tracheids and the medullary-ray cells are very wide, half- bordered, or one-sided pits, so wide thit they cover almost the whole breadth of the wall of the tracheids. They must be called one-sided because the border is developed only in the tracheid. The closing membrane is bent forward in the tracheid and has no torus. Treated with chloriodide of zinc the closing membrane colors blue (1), while it re- mains uncolored in the two-sided bordered pits. The cells of the medullary rays at those points where they are touched b}^ the tangential walls of the tracheids are provided with a thickened ledge. (See the medullary ray m, Fig. 47, and the tracheids bearing on it.) If the section shall strike a zone in which the cells of the medullary rays are empty, we shall find them united to the adjoining tracheids with two-sided bordered pits. In the immediate neighborhood of the cambium we see the incomplete tracheids of the young wood. The walls of the cells increase rapidly in thickness toward the cam- bium zone. In sections from much older stems we see the radial walls within the cambium zone again become no STRUCTURE OF CONIFEROUS STEMS. Fig. 46.- Part of a transection of an old stem of Pinus sylvestris. The section crosses the cambium (i, initial layer) and ends on the one side in the young wood and on the other in the young bast. 1, 2, 3, developmental stages of bordered pits ; vi. medullary ray ; c,sieve plate ; /.•, flat cells with brown contents afterwards bearing crystals. X 510- thicker (2). See Fig. 4(i. Tliat which we must cull the cambium consists of an initial layer theoretically jone- cell thick, ^, which by continuous tangential division furnishes the tissue mother-cells on both the wood and bast sides, and out of these, by the division of the mother-cells, the wood and bast have their origin. No distinct boundary can be drawn between the initial layer and the tissue mother- cells of bast and wood on each side. The youngest partition walls are sharply joined to the radial side walls, i. Somewhat older parti- tion walls are, on the contrary, a little thick- ened at the points of juncture. Upon the wood side the develop- ment of the bordered pit may be followed (1, 2, 3). The series of tracheids is continued through the cambium into a row of bast STRUCTURE OF CONIFEROUS STEMS. Ill cells which maintain the radial arrangement quite as fully. The cell walls thicken rapidly on the liast side, but are of a duller white and less sparkling than the wood cells. On the radial walls of the wide bast cells are sieve-plates, corresponding to the places occupied by bordered pits in the wood. In very thin sections they may be recognized by fine pores which penetrate these spots. Mainly, bands of single flattened cells alternate with the thicker layers of sieve-tubes. These bands represent the bast paren- chyma. The majority of these cells are indicated by their strongly refractive brown contents, k. In cells far- ther removed from the cambium, one or two crystals may be seen in the brown substance. Since in this tree but one bast parenchyma band is formed in the whole year, these may be used for determining the age of the bast part. Between the cells with crystals lie those tilled with starch. So between the sieve-tubes are distributed starch cells and crystal cells, singly or in numbers. Med- ullary rays continue from the wood through the cambium into the bast, and in the latter a part of its cells contain starch. Only a comparatively narrow zone of the bast will keep the original arrangement taken by the elements. Beyond that zone the radial series is bent, the cell walls begin to be browned, the cell cavity more or less com- pressed together so that the radial walls appear bent and wavy. Only those cells of the bast and* the medullary rays which contain starch are rounded out* and full. Fi- nall^^ the sieve-tubes and crystal- bearing ^cells are quite compressed and tangentially extended, and like a lami- nated membrane separate the large starch-bearing cells. The outer rind now seems to consist entirely of the lat- ter cells. Farther towards the outside of the rind one comes to small leaves of cork and from these deeply browned dead tissue is tangentially separated. The resin-ducts have not been mentioned. Every sec- 112 STRUCTURE OF CONIFEROUS STEMS. tioii of the wood ^hows them. But in the alcohol prepara- tion they have lost their resin contents, and consequently show their structure all the better. In a transection it appears as an intercellular passage, Fig. 47, ^, surrounded by a layer of large thin-walled cells, e. The walls of these cells are browned, have a nucleus, and a wall layer of proto- plasm. Bordering on these is a second layer, flatter, and poorer in cell contents, then a more or less perfect, and •^ indeed here and there a double Ji-j-.'L-Wr^f /< ,, tjß, layer of large starch-bearins: ji? n H: jL~^^^:=;^--^^:^i cells, a. The latter will be ^v ; ^^ ':_i4 -^^fe^^lK — surrounded by tracheids, and _J^|?^^^'j?-'jF|j^'jv..-|^ will eventually rest against a Ji^'f^ g /«; C ^^o\ '^ medullary ray. Conjunction ~^Z;*^-T =•' -^ /■ • . J fe with one such is generally de- _^y;;, ^ . ' ' l_ sirable for each resin- duct at V-'4;(y,CC::l /«^^^ some one place. The resin- I^JW^^-^II if duct is produced, as their life- ^ "'''^^n^'^i ''■* history shows, by the drawing Fig. 47. Resin-duct from the wood apart of Certain coutlguous €)f Piiiuf sylvestris, i, the (\\\ct üUeiX with resin; e, the cells surrounding CellS. theduct;«, starch-bearing cells;«, ^y^ ^y-^ ^^^^^^ j^j.^j.^ ^ ^q^_ trachiids, m, medullary lay cells. X240. tion of a fre.sh stem, and find that the passage is filled with resin. It is very refractive and takes the form of irregularly-shaped drops. Alcohol causes them to disappear. Alcanna tincture colors them as it does oil drops. Upon the section on the slide in a drop of water, lay a thin shaving of the bark of a dry al- canna root. Put on the cover-glass, and add 50% alco- hol, and let it stand for an hour. Then remove the alcanna, and it will be found that the resin elements are stained a beautiful, dark red, while the other parts of the section remain colorless.* *It is much more convenient to use alcanna extract, which may be had of most aiJothecavies, certainly of all dealers in microscopical goods. — A. B. H . STRUCTURE OF CONIFEROUS STEJMS. 113 Chloriodide of zinc colors the trache'id walls of alcohol- material sections yellow-brown; the innermost thickening layer which touches the boundary membrane is, in part, colored violet. Protoplasmic contents and nuclei are eas- ily seen in the imperfectly developed tracheids near the cambium. Thus it is very easy to demonstrate that the fully developed tracheids have lost all their living con- tents. The cambium, with the 3'oungest adjoining cells, is stained a light violet, the older bast walls a dark violet. The contents of the crystal-bearing cells remain brown, the cells of the periderm red-brown. The thin-walled cells which surround the inner surface of the resin-duct are col- ored a dull violet. By staining a section made through the cambium with coralline, we easily observe the gradual extinction of the lignin in the cell walls in the neighborhood of the cambium, the coralline coloring the lignified differently from the un- lignified membrane. Lay the section for some time in coralline soda, and then examine in glycerine. The lig- nified membranes are colored an intense red, but losing that gradually towards the cambium, the color changes from a red to a pale yellow. The bast has the cell walls a pale, reddish-yellow, the sieve-plates a pronounced rose, especially where they are covered with the callus masses, and the starch grains being stained a rose color bring this tint into prominence in the. outer bast. Now, make a radial section again from the alcohol material, and the tracheids pointed, interlocked, border- pitted, are seen as before. The superficial view of the bordered pit is well known. The bordered pits are small and infrequent in the tracheids formed in the autumn. The medullary rays run across the tracheids. The rays sometimes occur sixteen cells high but are usually much less. They consist (4) of radially-extended, serially~ar- 8 114 STRUCTUKE OF CONIFEROUS STEMS. ranged cells, the middle ones having starch, and on their broad sides, next the tracheids, showing one-sided bor- dered pits. The upper and under three rows of cells are empty, with small bordered pits. In this respect they agree somewhat in structure and behavior with tracheids, and might be so named, but we prefer to limit this term to the elements in the wood part of the vascular bundles. The cambium shows, in the longitudinal view, narrow, elongated cells with end surfaces more or less inclined, and touching each other, out of which the Avood and bast proceed, and low broader cells which continue into the '0 <^^ »^ ■hi medullary rays on both sides. r ' *""^^^ . f^^^^ In order to examine the sieve-plates ^ii^'^^^^-'.n ■ ^^i (5), we make a radial section again Irom the alcohol material, and lay it in an aqueous solution of aniline blue (6). eIIj "^^ bij^rtj After a few miiuites, transfer it to glyc- pl/^|l' 11 if ei'iiie on the slide. The glycerine takes Fi*^'^. I'l Pi the coloring matter from all of the tissue I ' "] ; 5- ^ ||? except the sieves, and makes it impos- k^ ^ JjJ v^ sible to overlook them. The color is a beautiful, durable blue, and the prepara- FiG. 48. rinus syl- .- i j t -\tit vestris. Parts of two ^'ou may be made permanent. We can sieve-tubes with sieve distinguish the sievc-plates in the near vicinity of the cambium, and follow out the same into the region where the sieve-tubes are crushed, and the sieve-plates lose thereby their radial position. Still, before that, the sieve-plates have lost their stainable quality. The sieve-tubes have the form of cambium cells and have the sieve-plates on their radial walls, as the tra- cheids have the bordered pits. The sieve-plates are, for the most part, smaller than the bordered pits ; they appear as round or oval spots, which are divided info an indeünite number of finely-dotted fields with angular outHne, Fig. 48. STRUCTURE OF CONIFEROUS STEMS. 115 At some distance from the cambium, the sieve-plates are covered with the callus-plate, a brilliant blue substance. Farther away these disappear, the sieve-plate is naked and colorless. The sieve-tubes are out of function here. It is not difficult to see that the active sieve-tubes contain pro- toplasm ; still, the nucleus is wanting, most surprisingly, and has disappeared even from the youngest sieve-tube. The crystal-bearing cells of the bast are recognized by their brown contents, are relatively short, meet principally with transverse walls, and probably are produced by the transverse division of the cambium cells. They have im- merous prismatic crystals lying near and over each other. Beyond these are the starch-bearing cells. They are shorter than the crystal cells, lie in threads over each other, and are intercalated singly or in a long series between the crystal-bearing cells. They afterwards swell very consid- erably. The medullary rays may be easily followed from the wood through the bast. They retain their essential structure, only losing the characteristic pitting. The starch-bearing series are always inclosed by a row of empty cells above and below. The resin-duct in the longitudinal section appears as a long, continuous tube, inclosed by shorter cells with trans- verse division walls arching more or less into the duct. Sometimes a resin passage is found in a medullary ray. Naturally, it follows a radial course and passes the cambium from the wood to the bast. Now make a tangential section from the alcohol mate- rial. It should be made both in the bast and in the wood. The wood section shows the tracheids and the severed medullary ray. The latter have a spindle-shaped outline Avhile the cells towards the ends become smaller. The narrowest medullary rays have three cells, the majority 116 STRUCTURE OF CONIFEROUS STEMS. eight ; but some bave as many as twenty. The narowest are one layer of cells in thickness, the others may bave sev- eral layers in the middle, and in that case may have a resin passage in the centre, which will of course be cut across. Make a bast section by cutting away from the outside a number of sections of the old bast till at last we come to the young wood. Examining this section with a low power we inquire first what the still active «ieve-tubes contain. We look for the callus-plates, and we easily see \ them, without staining or high magni- fication, refractive pads lying on the cell walls. Treat the sieve-plate with chloriodide of zinc to which a like quantity of potassic iodide of iodine di- luted with half water is added. The image of the sieve-plates is in this view much the same as in cross-sections, still the number of them is much greater and il! ' -"i'V j<7(i ^" *^'^^ ^^ more likely to hit upon one i"l IK I ' i,'J favorably situated. AVe shall find what we are looking for soonest in the edges of the section. The sieve-plate, Fig. 49, A, presents itself in profile within the ^, before the foiniation t i n /• ^i • i i mi of ti,e sieve i.iate; B, I'-^^li'il ^alls of the sievc-tubc. The after the same; c, sieve walls themsclves are souicwhat swollen tube whicli has passed ii-i ii-ti beyoudihcactivestage. and colored violct by the chloriodide X^^^- of zinc. The sieve-plate is stained a red-broAvn if it belongs to a still active sieve-tube. This staining comes from the presence of plasma strings which penetrate both sides of the sieve region. The sieve-plates look as though they were traced over with red-brown cray- ons (see the figui-e). The callus-plate, JJ, in case the zinc solution is not strong enough to dissolve it, is stained Fig. 49. Pinus sylves- tris. Walls of sieve- , longi- tudinal section; A, a sieve-plate from above; B and C the adjoining parts of two sieve-tubes from the side; D, the connecting part of the strings of mucilage from two sieve-tubes after treatment with sulphuric acid; s, conducting cells; u, muci- lage string; ;;r, protoplasmic utricle; c, callus-plate; c*, small lateral callus plate of a lateral sieve spot. X 510. wide sieve-tubes, narrow conducting-cells and bast-paren- chyma. The transversely-placed sieve-plate may be eas- ily seen from above, Fig. 51, A. The conducting-cells, A, s, show very plainly with their contents tinged a dark blue. On the outer side of the wood the thin-walled, radi- ally-arranged, cambium cells follow directly upon the two large vessels and the wood-parenchyma lying between. VASCULAR BUNDLES OF CUCURBITA. 125 Then comes the outer bast which is constructed like the inner. The sieve-phites are easily found in both bast regions, and, according to our magnification appear to be perforated with small or large pores. In the older sieve- tubes, the pores are narrower and divested of strongly- refractive substances. So in A, Fig. 51, the sieve-plates are often covered with masses of violet-blue matter. In the narrower sieve-tubes on the outer and inner edges of the vascular bundles appear the callus-plates as homo- geneous, azure-blue masses. Focussing deep enough, we strike the meshwork of the sieve-plate. Using a low power on a transection, we see that the vascular bundle stands arranged in two rings. The vascular bundles of the outer ring stand before the edge ; those of the inner ring alternate with those of the outer. A rinof of scle- renchyma fibres, whose elements are of much darker color than the large-celled, fundamental tissue, protects the inner part. Upon these follow, towards the outside, rind- parenchyma containing chlorophyll, and then typically developed, here and there interrupted, uncolored, bril- liant-white collenchyma. At the points of interruption, the rind parenchyma reaches through to the epidermis, which latter bears the stomata at these places. The stem is hollow on the in- side. Cross-sections of the stem, where it is not more than 5 or 6 mm. thick, show the large vessels and the cells lying between in the state of formation. It often happens that of the two larger vessels, only one is being formed, the other (m the contrary being obliterated ; then the one attains an almost colossal diameter. In many cases also, both vessels are obliterated. Finally, there are in- dividual instances in which both vessels occur and both are as large as is usual where there is only one. 126 VASCULAR BUNDLES OF CUCURBITA. Radial sections, rightly taken, show us that the nar- rowest vessels are the ring and spiral vessels : the widest, the pitted with ring-like, transversely-placed diaphragms. The two largest vessels have walls with irregular, net-like thickenings with numerous pits between the meshes of the net. Sometimes these vessels will be found with entire partition walls, in which case a nucleus and a thin layer of protoplasm on the walls will be found. Many partition walls will be found swollen in the middle in the form of a biconvex lens. Longitudinal sections of the next older parts of the stem will show us in place of these partition walls small rings affixed to the side walls, the nucleus and protoplasmic layer having disappeared. Between the nar- row vessels is thin-walled, primary, wood-parenchyma tissue. The cells between the large vessels belong to the thick-walled, primary, wood-parenchyma tissue ; they are thickly pitted, even on the partition walls. The walls of these cells which join the vessels perpendicularly are wavy ; this causes them to modify the pits of the vessels. In these wood-parenchyma cells are nuclei and a protoplasmic sac. At both sides of the vascular bundles we may conven- iently study the wide sieve-tubes (2), Fig. 51, B. Stain the section with aniline blue and examine in glycerine. The latter fluid will withdraw the color somewhat from the cell walls after a little while, but not from the cell con- tents. Most of the sieve-plates are transversely placed, few inclined. Most of them also are covered with a cal- lus substance, and are correspondingly thickened. See Fig. 51, C. Use a comparatively low power. The sieve-plates are colored a pure blue. In the tubes which show the sieve-plates is a sac-like axillary string, u. It is a muci- lasrinous cord widened at the end so as to cover the whole VASCULAR BUNDLES OF CUCURBITA. 127 of the sieve-plate, and colored an indigo blue. The end setting on the sieve-plate is more thickly filled with con- tents. See in B. The collection of cell contents is to be remarked in one or both ends of the sieve-tube; and, if in but one, at the upper end. Besides this, a layer of pro- toplasm may be found on the walls of the sieve-tubes,^)'. No nucleus exists. In somewhat younger sieve-tubes the mucilaginous cord may be seen by low magnification press- ing through the pores of the sieve-plate into the adjoin- ing sieve-tube. In each plate the strands are all moving in one direction, but in successive plates they may be going in opposite directions. The phenomenon is not seen in older sieve- tubes, the callus substance having increased on the sieve- plate and narrowed the sieve portion, and through these narrowed pores the slimy contents of the cell continually pass (see B) from one to the other. On the outer and inner edges of the vascular bundle the callus-plates cover the sieve-plates, Fig. 51, C. They are colored azure blue, and show the sieve-plate in their midst more or less clearly. The callus-plates consist of two halves which belono- respectively to two neighboring sieve-tubes and are con- nected through the pores in the sieve-plates. A delicate striation is often observable which extends throuo-h the connecting pores. See the Figure. When two sieve- tubes are laterally joined, small sieve-spots appear on the common wall and afterwards a one-sided, c*, or two-sided callus-plate occurs. Condncting-cells, s, of the same length as the sieve-tubes, follow their course. They have rich contents and a nucleus. Sieve-tubes, in the process of development, show indigo-blue colored drops of muci- lage in their protoplasmic wall-layer. For comparison, it 128 VASCULAR BUNDLES OF CUCURBITA. is necessary to make a longitudinal section of fresh ma- terial. The sio^e-plates appear as plainly as in the alco- hol material. The slimy collections on the sieve-plates are easily seen ; but the mucilage nowhere shows itself drawn back from the side walls of the sieve-tubes in the form of a string. This appearance is due to the influence of the alcohol. Notes. (1) See Scliweudener, Das mech. Princip., p. 8. (2) Fortius compare principally de Bary, Vergl. Anatom., p. 179; K. Willielm, Beiträge znr Kenntniss des Siebröliren-Apparates dicoty- ler Pflanzen ; E. \. Janczewski, Etudes compar^es siu* les tubes cri- breux, Mem. de la soc. nat. des sc. nat. de Cherbourg, T. xxin: Russow, Stzber., der Dorp, naturf. Gesellsch., Jahrg. 1881, u. 1882. LESSON XII. Vascular Bundles of the Axile Cylinder, and the Secondary Lateral Growth of the Roots. For the study of the axile, vascular-bundle cylinder of the roots, we w'ill take a root of the common onion, Allium cejM. One may have plenty of material at any Fig. 5'2. Transection from the base of a large ;idventive root of AUmm cepa. c, rind; e, endoderm; p, pericambium; fjf+Uj I'^'S vessel; sp, spiral vessel; sc and sc, scaliform vessels ; v, bast. X 240. time by putting an onion in a glass of w^ater and letting the roots sprout. Fig. 52 is a section of such a root near the base. The epidermis and the very stout rind tissue are left out of the drawing. Still one may see some of the cells 9 (129) 130 VASCULAR BUNDLES OF ONION ROOT. bordering the endoderm at c. The endoderm, e, shows in a characteristic manner, dark shadows on its radial walls. These shadows are caused by the wavy bending of the middle part of the walls. Such an endoderm al- ways consists of one layer. We have already met it in the envelope of the vascular bundles of the leaf of Iris, which shows it to be not confined to the roots. In the middle of the vascular-bundle cylinder there are, in this case, two wide, scaliform vessels, sc. In other cases more or less than two may be seen. If the root is not old enough, the central and perhaps adjoining vessels will be thin-walled, not fully developed. Adjoining the central vessel or vessels are almost always six narrower scaliform vessels, sc*, and upon the latter follows a group of quite narrow spiral andring vessels, sj), S2)-\-ci. The size of the vessels diminishes from the centre outward and the outer- most are the ring and spiral vessels. Herewith the root diflers from the stem. The wood occupies about 180° of the circumference of the cylinder, being arranged in the form of a six-rayed star. The bast, v, alternates with the wood. This is the general law with respect to the axile, cylindrical, vascular bundle of the roots. A layer of parenchymatous fundamental tissue laterally separates the wood from the bast. The latter is recognized b}^ the M'hite sparkling walls of its cells. It consists of sieve- tubes and conductino'-cells not distinguishable with cer- tainty in a cross-section. The vessels and the bast are separated from the endoderm by a single layer of peri- cambium cells. Concentrated sulphuric acid will dissolve the whole section with the exception of the epidermis and the cell-layer bordering upon it, and the vessels and the endoderm. The latter will be colored a beautiful yellow. The endoderm, which will indeed in part bend about dur- ing the action of the acid, shows the middle band in its ra- VASCULAR BUNDLE OF FLAG EOOT. 131 dial walls beautifully uiKlulatecl. A similar appearance is observable on the outer layer of rind cells bordering ou the epidermis. The cells in question are bound fast to- gether under each other and form a sort of outer endoderm which is sometimes known as the epidermoidal layer(2). A longitudinal section shows us the vessels already men- tioned ; and staining with coralline, the sieve-tubes and sieve-plates colored rose-red are easily made visible. The conducting-cells, shorter and tilled with contents, are easily distingnished. The undulation of the mid- dle band of the radial Avails of the endoderm cells, looked at from the surface, appear like a scab form thick- ening. The pericam- bium cells have the same form as the en- doderm cells, yet of greater length. The inner endoderm takes the color (coralline) with some avidity, while the outer endoderm is contrasted from the surround- ing tissue by its coloilessness. We will take for further study a section of the root of Aco7'us calamus, sweet Hag, shown in Fig. 53. The vas- cular rays do not meet in the centre of the cylinder, but are arranged in eijjht seö-nients of a circle, while the mid- BOO ' die is tilled with medullary tissue. The larger vessels lie nearest the centre, the smaller toward the periphery. The bast, V, alternates with the vascular rays. The two are laterally separated by a single or double layer of paren- FiG. 53. Transection through the root of Aco- rus calnmus. m, pilh; s, wood; v. bast;p, peri- cauibiuai ; e, endoderm ; c, rind. X 'j^- 132 VASCULAR BUNDLE OF FLAG ROOT. cbymatous fundamental tissue, and from the endoderm, e, ])y a single layer of pericamhium. The endoderm con- sists of fiat thin-walled cells, and it and the pericambium and all the remaining fundamental tissues of the vascular- bundle cylinder are filled with starch, which makes the starchless bast appear verj'' distinct in the image. The cells of the inner rind are separated into single-celled layers by numerous air passages. In the periphery the rind cells are compacted together into a solid layer, several cells thick. The outer hypodermal rind layer consists of radially-elongated cells and forms in this, as in other roots, an outer endoderm which persists, while the epidermis dies and disintegrates. Add potash lye, and dissolve the starch, and the dark shadows in the radial walls of the endoderm are distinctly seen. Treat with sulphuric acid, and we see that the whole cell-wall of the outer endoderm is cuticularized, but only the shadow-forming band of the inner endoderm. The cells of the outer endoderm con- tain resin. There is a mechanical significance to these en- doderms. They protect both the surface and the axile vascular-bundle cylinder. By the suberization of their cell-walls they attain great solidity and little extensibility. But the interpassage of fluids between the vascular-bundle cylinder and the rind is not thereby interfered with, since the cells of the inner endoderm are suberized only or prin- cipally on their radial walls (3). A cross-section of a root of Iris florentina shows us an axile vascular-bundle cylinder, quite exactly like that of the Acorus, except that the endoderm is differently built. See Fig. 54. The cells are unilaterally thickened, U-shaped and the thickening beautifully laminated, e. Exactly in front of the vascular ray is a single unthickened cell, f. It is known as a transit-cell (4), and being permeable maintains the connection with the surrounding rind, c. VASCULAR BUNDLE OF IRIS ROOT. 133 The thickened laj'er swells and dissolves in concentrated sulphuric acid, the cuticularized middle lamella only re- maining and forming a delicate envelope about the endo- derm and transit cells. The middle lamelhi between the vessels and in the pith is not dissolved, but forms a delicate, yellowish-brown network. A tangential section, which just grazes the endoderm, teaches us that the longitudinal stripe, which lies in front of the wood parts, consists of long, thickened, alternating Avith short, unthickened, transit cells, full of cell contents. Some- times, two short transit cells follow each other. The roots of dicotyledons are less favorable for study than those of the monocoty- ledons, but, having become acquainted with the latter, we shall have no difficulty with the former. Make a cross-section from the base of an adventive root of a run- ner of Ranimculus rej)enS. fig. 54. Part of a transection thronüli m\ -1 11 II the root of Iris ßoreniina. e, endoilerm ; The axile vascular-bundle ;,, pericambium; /transit cell; .-.bast; cylinder is not so sharply «, vessels in the wood; c, rind, x 210. diflerentiated from the rind tissue as in the monocotyledons, but by attentive examination we shall find on the border of the two the dark shadows which mark the endoderm. The axile cylinder is divided into four or five vascular rays, according to the size of the root. The laro'er vessels lie here towards the inside and the smaller towards the outside. In monocotyledons, the innermost vessels are distinguished by their large size. This is rarely seen in the dicotyledons and not at all in the lianunculus. The vascular rays ex- tend to the middle of the cylinder, and abut more or less 134 VASCULAR BUNDLES OF JCJXIPER ROOT. fiill_y against each other. The innermost vessels are latest in developing, and remain in the condition of thin-walled elongated cells. The bast alternates in the ordinär}^ way with the wood. The roots of the vascnlar cryptogams are simpler, and 3^et are constrncted on the same type as those of the phan- erogams. Take next a rootlet of Taxus baccafa, abont 1 mm. thick, and make a cross-section. The rind consists of about ten thicknesses of parenchyma cells. The outer cell layer of the rind is not especially ditferentiated, there being no dis- tinct epidermis. The inside of the section is filled with the axile vascular-bundle cylinder, which is surrounded by an endoderm. The latter consists of flat, thin-walled, sub- erized cells, whose walls are browned, and wdiose diameter is considerabl}-^ less than that of the rind cells, the radial walls being characteristically shaded. A single-celled, thickened layer is developed about the endoderm. The cells are of the same width as those of the rest of the rind, but the radial walls are furnished with a thick, bright, yellow ring. These ring-like thickenings correspond to that in the neighboring cells, which give them in section the form of a biconvex-lens. The axile vascular-bundle cylin- der shows a double-arched wood body, extending across it, at the opposite ends of which is a narrow, spiral ves- sel. Inward, and joining these, is a strip of tracheids with bordered pits, characteristic of the conifers. They are easily recognized by their clear yellow, strongly-thick- ened walls. These tracheids almost always meet in the middle of the cylinder, forming a plate. On each side of the tracheids, lies a strip of narrow, thin-walled, funda- mental-tissue cells, bearing starch. Upon these, borders a somewhat small-celled tissue of thin-walled bast. Fi- nally, beyond this, a large-celled starch-bearing layer, about AXILE VASCULAR BUNDLE OF JUNIPER. 135 four cells thick. The latter are joined together, making a complete circle, somewhat reduced before the spiral ves- sels. They represent the pericambium. If now we examine a cross-section about 1.3 mm. in diameter, we shall find the two sides of the plate of tra- chekls dividing and becoming transformed into cambium, which produces tracheids on the inside and bast on the outside, and on both sides mednllary rays. Now examine Fig. 55. Transection of root of Taxus baccata after the beginning of the lateral growth, c, rind ; m, tliickening layer; e, enrtodenn; jo, pericambiuni ; s, spiral ves- sels; t, primary trachcid plate;/, stripe of fundamental tissue; t", secondary tracheids with medullary rays; v", secondary bast; v', compressed priniai-y bast; k, cells in secondary bast with crystals in the walls; r, resiubearing cells in peri- cambium. X -l-' the further activity of the cambium in a section of a root 2 mm. wide, as shown in Fig. 55. It shows the already well-known relations : the rind, c, which has lost its hairs from the onter layer of cells ; the outer strengthening layer, m; the endoderm, e; and the axile cylinder. The 136 AXILE VASCULAR CYLINDER OF JUNIPER. outer cell-layer of the pericambium bus in the mean n bile begun to divide and bas been transformed into a few layers of peritlerm. Ou botb sides of tbe tracbeid plate we see tbe inner inactive layer of fuudaniental tissue,/, the so- called connective tissue : beyond, tbe newly-formed radi- ally-arranged tracbeids, /'", witb numerous intercalated medullary vays. Tbese relations are more easily seen if one adds a little potash lye to tbe preparation. Tbe vessels, 6;, on the edges of tbe middle plate come out distinct and black. The tracbeid plate, /', as well as tbe tracbeids formed by the cambium, y^ is cobn-cd a beautiful yellow. Tbe connective tissue remains white. The secondary wood layers have a plano-convex outline Avbicb runs to a point at tbe edges but not here in front of tbe vessels. On the outer side of the wood body we find the cambium and out- side of this the secondary bast, v", which after treatment Avitlitbe potash appears white but in which single cells are black, k, having crystals of oxalate of lime in tbeir walls. The primary bast forms a layer of compressed cells on the outside of the secondary. Tbe potasb brings out tbe peri- cambium more distinctly than before, also the resin-bear- ing cells witb tbeir j^ellow-brown contents. The cork layer produced from the pericam])ium is colored a yellow-green, tbe thickening ring of the strengthening layer a brigbt yellow. Tbe endoderm is compressed by tbe cork layer. Making a section now of a root 2 mm. thick wbicb bas thrown off its rind and shows a dark brown surface, we tind tbe section has a fully closed wood part, and makes an image indistinguishable from that of a branch of like size, were it not that here the place of the pith is occupied by tbe primary tracbeid plate. The vessels at the edges of this plate are somewhat dif- ficult to make out. Tbe plate is inclosed hy the starch- AXILE VASCULAR CYLINDER JUNIPER. 137 bcarina' connective tissue which here to a certain extent displaces the medulhiry crown and into which the oldest medullar}' rays open. The two wood bodies have united in front of the vascular groups and the medullary rays at that place are scarcely noticeable by special width. The surface receives the inclosing cork layer produced from the outermost pericambium layer. The secondary rind consists of secondary bast and the elongated medullary rays. That which represents the primary rind here will consist of the enlarged and numerically increased pericam- bium cells closely packed with starch. Longitudinal sections show that the middle tracheid plate consists of the same elements as the secondary wood. We find the spiral vessels on the edges of the plate, and observe that the cells of the endoderm are quite short while those of the strengthening layer are far larger and are even longer than the adjoining cells of the rind. Coralline stains the tracheids a beautiful coral-red and brings out the sieve-plates in the primary and secondary bast. The rings of the strengthening layer also absorb the coralline. Notes. (1) De Bary, Vergl. Auat., p. 365, where the older literature will be fouud; Olivier, Ann. d. Sc. nat. Bot., vi Ser., xi Bd., p. 5, Ö'. (2) See V, Höhuel, Stzber. d. k. Ak. d. Wiss. iu Wien, math, uatur- wiss. CI. Bd. Lxxvi, i Abth. 1877, p. 642; Olivier, I.e. (3) Schweudeuer, Abh. d. kgl. Ak. d. Wiss. iu Berlin, 1882. The pro- tective sheath and its strengthening. (4) See last work quoted, p. 13. LESSON XIII. Vascular Bundles of the Ferns and Lycopods. In the leaves and stems of the ferns the vascular bnn- dles are concentrically built, whereby the wood is almost or quite fully surrounded by the bast. Make a section of Plens aquilina, in which it is possi- ble to gat a good knowledge of the vascular bundle, even when the numerous, sclerenchyma strings in the funda- mental tissue do not permit us to make a good section. Make the section from the rhizome directly behind the growing point or through the petiole of a young leaf. The vascular bundle will be sufBcientl}' developed, while the fundamental tissue will not be much hardened. The bundle will be the same in the rhizome and the petiole, and a cross-section of it from the base of the latter is shown in Fig. 56. Choose a small bundle. We first no- tice the large, border-pitted, scaliform vessels, sc; still, the smaller vessels are thickened also and only the few on the two ends of the wood which adjoin the protoxylem elements have spiral thickenings, sp. The vessels are surrounded, when they do not touch each other, by starch- bearing, wood-parenchyma cells, Ip. Wood-parenchyma and vessels form the wood part which is almost perfectly enclosed by the bast. The latter borders on the wood parenchyma with sieve-tubes, v, which are succeeded out- wardly by narrow conducting-cells, s, wiiich are filled with protoplasm — not starch, as iodine will show. But single, starch-bearing cells are sparsely distributed through this tissue. (138) VASCULAR BUNDLES OF FERNS. 139 The periphery of the bast takes on a layer of still nar- rower, thick-walled protophloeni elements. The bast is surrounded by a simple layer of cells, j-jjj, filled with starch, which in its i)Osition, but not in its origin, resem- bles pericambinm and may be called periphloem. Around this preliminary sheath is the endoderm, e, thin-walled, free from starch and suberized, and showinir the dark Fig. 5(i. Transection of a vascular bundle from the petiole of Pteris aquUina. sc, .^calilbi-m vessels; sp. sjjiral vessels. In sc* a piece of tlie scaliform perfoi'ated wall is seen; //>, wood paroncliyma; v, sieve tubes; s, conducting-cells;pr, proto- phluL'm;^^;, periphloem; e, endoderm. X 2l0. shadows on the radial w^alls. The periphloem and en- doderm cells correspond to one another and suggest a common origin in the same mother-cell. The wood at its two edges, together with the covering of w^ood-p;iren- chyma, borders directly on the periphloem or on the proto- 140 VASCULAR BUNDLES IN FERNS. phloem. At these two points, the bast is either wholly or nearly interrupted, but in other ferns this break may not occur. The walls of the endoderm cells are often broken by the cutting and then the vascular bundle will be separated from the fundamental tissue. The cells of this tissue, bordering on the endoderm, are sometimes much thickened and then are colored a yellow-brown. The cross-section through the rhizome shows a browned and cuticularized parenchymatous tissue under the deep brown epidermis which, further towards the inside, is colorless and full of starch. This starch-bearing, funda- mental tissue is penetrated with the vascular bundles and the red-brown sclerenchyma fibres ; the latter form plates which run between the vascular bundles more or less par- allel to them. The outer bundles are in immediate con- tact with the endoderm on the outside, supported by such sclerenchyma fibres, which here represent the mechanical tissue. In the inside of the bast the relations are simi- lar, there being a hypodermal ring of red-brown scleren- ch^'ma fibres which rest on the epidermis. A longitudinal section shows us all the wide, scali- form vessels again. The ends are much inclined, ladder- like, border-pitted and in part perforated (1). On the side walls separating the two vessels, it is easy to see that the pits are bordered on both sides, and the closing mem- brane has a thickened torus; but on the walls bordering on the wood-parenchyma cells the pits are bordered only on one side, and the closing membrane has no torus. The section may also hit one or the other of the spiral ves- sels, and the plates of the sieve-tubes may also be discov- ered, but only by the most careful examination ; the latter may be found much more easily b}^ the help of coralline staining, which also will shoAV the sieve-plates much in- clined and parted into numerous fields by thickened bands ; besides these, the lateral walls of the sieve-tubes bear sieve- VASCULAR BUNDLES OF FERNS. 141 spots. Together with the sieve-tubes, we find the slender, condncting-cells with finely, gi-anul.ir contents and nucleus, and in contact with the vessels the starch-bearing, rela- tively-short, wood-parenchyma cells. Resembling the lat- ter, are the starch-bearing cells of the periphloem. Small pores are seen in the walls of the long-pointed, scleren- chyma fibres of the fundamental tissue. It will be interesting to make a transection of the petiole of Pohjpodium vulgare. The vascular bundles are very thickly sheathed about, but the sheath corresponds not to the endoderm but to a strengthening layer. This layer is but a single cell thick and these cells are thickened only (m the inside walls and are there colored a dark brown. The essential endoderm lies immediately within the strength- ening layer and is scarcely recognizable on account of its cells being flattened down by the pressure of this layer. Next within comes the starch-bearing, single stratum of periphloem cells, then the bast tissue consisting of cells, of almost the same width. The condncting-cells are dis- tinguished by their contents and, as is apparent, are mingled with the sieve-tubes. The closely grouped vessels are surrounded without by a single laj'er of starch-bearing wood-parenchyma cells which at the two small edges ot the wood-part may extend even to the periphloem. Prepare now a cross-section of the petiole of Scolopen- drium vulgare, where Ave shall find the two vascular bun- dles reduced to one. Two wood portions lie apparently in one vascular bundle ; rather, in a compound bundle, either near each other, or, as is frequently to be seen, united at one point so as to form an X-like figure. The stouter legs of the figure are turned towards the upper side of the petiole. The small vessels are found at the ends of the legs. From the ends of the upper legs small vascular bundles are often seen branchins: out. The cells of the bast are all of uniform size, but the condncting-cells mingled 142 VASCULAR BUNDLES OF FERNS. ■with the sieve-tubes are easily recognizable l)y their con- tents. On the surface of the tignre, the periphh^em a})pears several layers thick and with somewhat thickened walls. The outer circumference of the compound bundle is deeply fluted at three points, viz., above and at the two sides ; and here follows, on the endoderm, a plate of sclerenchyma fibres, red brown, and thickened almost to the extinction of their cell cavities. Higher up in the leaf, the wood part gradually assumes the form of a T. The three strenijthenino^ scle- «-/ renchyma strings ^ are always present even though re- duced. In the Lycoj)o~ dium species the axile vascular-bun- dle cylinder ap- pears in a relatively more highly com- plicated form. Still the relations of the Fig. 57. Transection of stem of Lyc, epii lei mis; ve, outer s- heath ; H, inner , vi^ Uf . 1- sheath; pp, periplilocm; sc, scallCoim vessels ; .sp, DC UlmCUll 10 nUUve ring and spiial vessels ; v, sieve elements. X 2ö. out after ll a V i n o" seen the compound vascular-bundle in the petiole of the /Scolopendrium. We have in fact to deal with an axile vascular-bundle cylinder, in the Lycopodium, which i« formed by the blending of several vascular bundles built like that of the last example. We will take the Lycopo- dium cömjplanata, but another species will do as well. Color the section with an aqueous solution of safranin. Fig. 57 represents what we shall see. First we have the ei)ider- mis, ep; then the rind cells which at first have wide cav- ities but which toward the inside diminish in width, and VASCULAR BUNDLES OF LTCOPODIUM. 143 increase in thickness of Wiill till they form an almost solid sclerenchyma sheath which Ave will call the outer sheath, ve. Between these strongly thickened rind ele- ments are air-tilled, intercellular spaces. The outer cells of the rind are colored by the safraniu a cherry-red, the inner thickened cells a rose-red. The thickened rind cells suddenly cease and there follow two or three layers of tangentially- elongated-polygonal cells colored a cherry- red. These cells form the endoderm, but are distributed in several laj'ers, have no undulating bands and are not otherwise characteristically thiclvened. But on the other hand they are, like the Cells of the endoderm, cuticularized and withstand the action of sulphuric acid very well. AVe will designate this envelope ot cells, vi, the inner sheath. Next follow several layers of likewise wide cells, which often contain starch, and have white glittering walls ap- pearing as if swollen. By long staining they are colored an orange-red. These cells take the place of the peri- cambium and may therefore as in the ferns be called peri- phloem,j92J. Now we cometo the beautifully stained cherry- red xylem layer. It consists of wide scaliform vessels, sc, separated by no intervening cells, and at the thin edges of protoxylem elements, that is, narrow, ring and spiral vessels, sp. In this species the wood layers run across the cylinder and more or less parallel to each other. They are somewhat concave on one side and convex on the other, and one can see by reference to the stem in its pro- cumbent position that those stripes which were parallel to the surface of the ground had their concave sides turned upward. The small vascular liundles ot the leaves, when they enter the central cylinder, are joined, as in the ferns, to the group of spiral vessels of a wood laj^er. The wood stripes often anastomose as in the lower ones of the illus- tration. In the elongated stem of the Lycojjodium selago 144 VASCULAR BUNDLES OF LYCOPODIUM. all the wood stripes are iiiiitcd and form a star. The.se wood elements are surrounded by a single layer of thin- walled narrow cells which we will designate the wood pa- renchyma, as in the ferns. On the ends, they extend with their protoxy lern and wood-parenchyma to the protophloem tissue. Between the wood bands is the bast, the larger cells being the sieve-tubes, v. The cells are white, with strongly refractive walls, narrow, the middle row only being somewhat Avider. In a good staining, the walls of the sieve-tubes are colored a rose-red, while the rest of the bast is colorless. On the edges of these bands of sieve- tubes are the nnrrow protophloem elements. The sieve- tubes reach the periphloem, by these protophloem cells, the essentially wider cells of the former being clearly distin- guished from the wood and bast. In cutting the section, the inner bast and wood elements of the vascular-bundle cylinder are easily separated from the rest of the tissue at the inner edge of the protophloem. The longitudinal section shows us first the epidermis and next the rind cells running diagonally towards it. Next the sclcrenehyma fibres of the outer sheath ; and then the elongated parenchyma of the inner sheath ; the periphloem with white thick walls and diagonally-placed partition walls ; the scaliform vessels and the narrow, for the most part much-extended ring and spiral vessels, and finally the bast. The latter consists of very long cells which join each other at the ends with more or less diag- onally-placed walls. By the help of coralline or aniline blue, we may recognize the small inclined sieve-plates. Only the wide cells in the bast are sieve- tubes ; the more numerous, narrow cells with sparkling granular contents are conducting-cells. Notes. (1) See De Bary, Comparative Anatomy, p. 170. LESSON XIV. Cork. Lenticels. We have already had an opportunity in various objects to learn something of the nature and structure of cork. Nevertheless, we will now direct our particular attention to this object and learn to know the lenticels on the one side, and, on the other, the cork cell walls and their reac- tions. A cross-section through a branch of Satnbiicus nigra, about 3.^mm. thick, shows us the vascular bundles distrib- uted in the medullary crown, about the wide, large-celled pith, already united by interfascicular cambium. The cambium has already begun its activity, and is now form- ing in the usual way, secondary bast without, and second- ary wood within, both in the vascular bundles and in the interfascicular spaces. The primary bast is supported on the outside by sclerenchyma fibres. The rind is from ten to fifteen cells thick. The projecting edges of the stem have a thick, hypodermal, collenchyma layer, which in the furrows is reduced to a layer three or four cells thick. The collenchyma layer is perforated at the stomata by the green, parenchyma cells of the rind penetrating through to the epidermis. In a stem, 4 mm. in diameter, the cork for- mation has already begun by the tangential division of the collenchyma cells immediately bordering on the epidermis. The inner of the two sister cells again divides, and the middle cell thus formed, subsequently acts as the cork cam- bium cell. This is easily made out after the periderm has 10 {li5) 146 CORK CELLS. become several cells thick, Fig. 58, ph. The outermost, in each series of cells, is the outer part, and the innermost the inner part, of the original collenchyma cell, d; the flat cell next to the inner part, ^/i, is the cork-cambium or phel- logenic cell. In a favorable section, one may see a curious incident in the formation of a continuous cork layer which begins under the stomata. The primary rind cells which surround the breathiug cavity begin to divide, and the division reaches over into the adjoining collenchyma cells. Soon, under the stoma, a meniscous-shaped layer of dividing cells is formed, Fig. 59, ^?, which produce towards the surface colorless oblong cells, Z, and towards the inside cork- rind cells, jpd (phello- derma). The upper are designated "filling" cells. They take a brown color, but are not suberized, and FIG. 58. Transection through the upper ^y their numerical increase smi'ace of a young stem of Samhucus ni- , . , . ^m epidermis, ph, phellogeu; cl and ci, SO ^:reSS Upon the CpidcrmiS the upper and under parts of the original ^g ^^ mpture it. ThuS are collenchyma cells. X 240, ^ the rind pores or lenticels produced. Examined with the naked eye, the lenticels appear to be furrows surrounded by two lip-like pads. The brown color of the filling cells is quite apparent. On the younger places of the stem, the lenticels appear as oblong swollen spots. Still younger stages are indicated by a somewhat brighter color. The section should be made through these places in order to show the earliest stages of development. Directly after the rupturing of LENTICELS. 147 the epidermis, the division of the colleiichyma cells begins, which leads to the formation of the periderm. The filling cells of the lenticels separate from each other ; but, as they disorganize from without, the cambium builds them up from beneath. Tiie spaces between the filling cells are filled with air, and thus the atmospheric air is admitted to the inner tissue of the stem. It thus takes the place of the stomata in those older parts of the stem in which cork has begun. For the winter, somewhat closer formation and more resistent fillinsf cells are formed. pd y Fig. 59. Transection of a lenticel of Savibucus nigra, e, epidermis; ^j/t, phel- logen; I, filling cells ;;}?, cambium of lenticel ; ;;rf, phelloderm. X 90. An essential enveloping layer for the winter time formed of narrow cells, which touch each other, is not found in Sambucus, as in many other plants. But the intermediate laj'^ers which are intercalated between the filling cells from time to time serve the same purpose. The cells of these closing and interstitial layers are suberized, but have in- tercellular spaces between them, so that the closure is not quite perfect (2). In old stems of Sambucus, the peri- derm is ruptured longitudinally. These clefts go through 148 CORK CELLS. the lenticels without injuring them. The latter are pre- served still in quite old stenas, while the outer periderm layers are interleaved between them. We Avill next take the Cyiisus laburnum for the study of the structure of cork cells as they are remarkably thickened in this species. A cross-section through the rind of an old stem shows the periderm constructed of but one kind of cork cells regularly arranged in a radial series. The youngest cork cells are colorless, the older yellow, and the oldest a yellow-brown. The outside cells are tangential ly elongated even to the closing up of the cell cavity. All these cork cells are much thickened, espec- ially on the outside. Without the help of reagents, the delicate middle lamella is easily distinguished, also a stout clearly unlaminated, secondary, thickening layer, and on the inside of the latter a tertiary thickening layer. So each wall, which separates any two cells, consists of at least five layers : the middle lamella which here represents the primary wall and is lignified ; the two secondary thick- ening layers which are alone suberized ; and the two ter- tiary thickening layers, which often betray their cellulose character and may be designated the cellulose layer, but in this case are a little lignified. Chloriodide of zinc colors the cork cells yellow to yellow-brown, the younger darker than the older, the tertiary layers the darkest. Char- acteristic reactions on cork substance or suberine are those of potash, the maceration mixture and chromic acid (3). Treat the section with potash lye and notice that the cork cells become yellow. Careful warming on the slide under the cover-glass increases the intensity of the color. With the maceration mixture (chloric acid, potash and nitric acid) we get the eerie acid reaction. The cold mixture brings out all the parts of the cork cell in a yellow- brown color. Heat the preparation with an added quantity CORK CELL REACTIONS. 149 of the reagent, when necessary, and the whole section will be dissolved except the suberized membrane. The color- less globular masses which remain are the so-called eerie acid, soluble in alcohol, but much more easily in ether. Strong chromic acid dissolves the whole section except the suberized layer of the cork cells, and after a while this becomes so transparent as to be difficult to find. Still it does not disappear. Although the middle lamella has been dissolved, the secondary thickening layers still hang together. The common flask cork (from Quercus suber) consists of almost cubical, thin- walled, relatively large cells, which are commonly somewhat thicker and flatter near the limit of the year's production, and are succeeded by the cubical cells again. Potash colors the section yellow especially the thick-walled cells. The five layers of the double walls between the cells are traceable as in the last specimen. The tertiary layer does not give the cellulose reaction at first, but only after proper treatment. The suberine reac- tions are even more beautiful than in Cytlsus especially the eerie acid reaction. It often happens that from the phellogen not alone centrifugal cork cells, but also centripetal rind cells, are formed, the so-called "phelloderm." The phelloderm seldom reaches the thickness that it has in the Rihes species. Prepare a cross-section through an old stem of lithes rubrum, and we shall find under the thin-walled, brown cork-layer, first, the phellogen and then a thick layer of flat riud cells containing chloro- phyll ; the latter ari3 arranged in radial rows which co- incide with those of the adjoining cork. In the inner parts of the phelloderm, the radial arrangement is lost in consequence of the supplementary extension. The inner- most phelloderm cells adjoin the collenchyma of the riud. 150 CORK CELLS. All those formations which arise from the phellogen are included in the term periderm. In Hibes, therefore, the periderm is formed of cork (phellem) and cork rind (phelloderm) . It is interesting to make a section through a this year's stem of Hibes rubrum^ in which the cork formation has but just begun. We shall see the first be- ginnings of the formation of the phelloderm, and perhaps, demonstrate that in this plant the phellogen is pretty deeply embedded in the rind. The extreme outside por- tions, being cut off from the sap-bearing tissue by the cork layer, soon die, become brown and are thrown ofi" as the so-called bark. Notes. (1) Literature in de Bary, Vergl. Auat. p. 560; v. Höhnel, Stzber. d. math, naturw. CI. d. k. Ak. d. W. in Wien, Bd. lxxvi, 1877. (2) Klebahu, Jen. Zeitschr. f. Naturw., Bd. xvii. (3) Introduced by v. Höhnel. Work and Vol. quoted above, p. 522. LESSON XV. Structure of the Foliage and Floral Leaves. The ENDS OF THE VaSCULAR BuNDLES. We shall now take a series of objects which will make us acquainted with the structure of leaves. We shall be- gin with the foliage leaves and take those forms which will show us the widest possible diiferences in the inner struct- ure of the leaves. The first example shall be Ruta grav- FiG. 60. Leaf epidermis and adjoining tissue of fiwio gTrtreotens. ^, epidermis of the upper side; sc, epidermis cells over a secretory receptacle; pa, palisade pa- renchyma; B, epidermis of the under side; s, spoage-parenchyma. In A, the arc filled spaces are shaded; in B they are clear. eolens whose leaves are mostly retained through the winter. The leaves are doubly pinnated, the leaflets a re- verted ovate. Held towards the light clear jDoints are seen in the leaf. They are reservoirs of essential oil ; " inner glands" in the tissue of the leaf. We make first a super- ficial section of the epidermis and observe first that the upper side, Fig. 60, A, has but few if any stomata, while (151) 152 STRUCTURE OF FOLIAGE LEAF. there are many on the under side, Fig. 60, B. Both on the upper and under side four epidermal cells lie over the inner gland, A, sc, somewhat depressed m the middle. In thicker parts of the section, where the reser- voir has not been cut by the knife, one may find a yellow strongly refractive drop of matter. By deeper focussing one may see that the tissue of the upper side of the leaf consists of cells whose optical section is round, A, p. These cells are laterally almost entirely separated from each other and the intercellular spaces filled with air. On the epidermis of the under side, cells with alike round optical section are seen but in much smaller number, B, s. These cells also are separated with air, and free wide breath- ing cavities are seen under the stomata B. Now make a transverse section perpendicular to the longer axis of the leaflet, using a piece of elder-pith as already described for makinof the section. This section will show us the leaf- tissue or the "mesophyll." First, beneath the upper epi- dermis. Fig. 61, ep', are the "palisade cells," ^?', a double layer of parallel elongated cells containing chlorophyll, perpendicular to the surface of the lejif. We have seen that these were laterally somewhat separated from each other, but we find that the cells of the two layers are joined fast together at their ends. The cells of the sec- ond palisade layer, pi/' are somewhat less numerous than those of the first, and (jften two of the outer are united to one of the latter. Next to these layers succeeds a loose tissue of cells which forms a network with open meshes which extends quite to the under epidermis. We call this tissue the "sponge-parenchyma." It has some- what fewer chlorophyll grains than the palisade tissue. The cells of the upper layer of sponge-parenchyma, sp', are connected with the inner layer of palisade cells and indeed are united to a larger luunber of palisade cells. LEAF STRUCTURE. 153 Fig. 61. Transection throiigli the leaf of Riäa graveolens. ep', epidermis of the tipper side; e/?", of the under side; pr, pr', palisade parenchyma; sj), sponge-paren- chyma; /.-, crystal-bearing cell; j'.s, vascular bundle; sc, secretion receptacle; a, breathing cavity; si, stoma ta. X 210. 154 LEAF STRUCTURE. None of the palisade cells are free on their lower ends. When they seem to be, as in some cases in the illustration, it is only that their connecting cells are not in the plane of the image. So also in the sponge tissue there are no cells with free ends, but all are united at their ends. The un- der layer of sponge-parenchyma, sp'", extends to the epi- dermis and joins it more or less perpendicularly, thus giving us a form of tissue intermediate between the pali- sade and the sponge tissue. The breathing spaces, a, nnder the stomata, st^ &:e left free. Crystal masses of calcium oxalate, k, are found in some of the cells. These cells are swollen, tun-shaped, contain no chlorophyll and seem to be suspended between the green cells. At the edges of the leaflets the epidermis cells are greatly thickened on the outside, the palisade layers are reduced to one and grad- ually change over into the elongated sponge-parenchyma layer of the under side of the leaf, sp'". The vascular bundles lie in the sponge-parenchyma ; the largest, the middle nerve of the leaflet, extends on the one side almost to the inner palisade layer and on the other to the lowest extended sponge-parenchyma layer. In the vascular bun- dle itself we may easily recognize in the darker part the vessels and in the brighter the bast. The radial arrange- ment of these elements assures lis of the activity of the cambium at some time. About the vascular bundle is a parenchyma sheath whose cells contain chlorophyll grains and to the outer of which the sponge-parenchyma cells are attached. The same relations hold in the smaller vas- cular bundles represented in the illustration. Still smaller bundles, vs, which have but few vessels and bast cells, are met with in the transverse section. They are to the last still surrounded with a sheath of elongated parenchyma cells. The secretion reservoirs, sc, touch the upper or un- der epidermis. They are circular in outline, inclosed by LEAF STRUCTURE. 155 a layer of thin-Avalled more or less disorganized cells, upon which borders a layer ©f flat cells havmg tolerably strong, white walls and granular contents. Adjacent to these cells is the mesophyll with its chlorophyll contents. The epidermal cells which overlie the reservoir are flat like those which surround it. The volatile oil may be re- moved by alcohol. A superficial section made at the base of the common petiole shows that the epidermal cells are elongated and on the upper as well as the under side are interrupted Avith stomata. The oil reservoir is also not wanting. Under the epidermis is a layer of elongated collenchvma cells and next to that the tissue containino: chlorophyll. The transection of the petiole shows the epidermis thickened on the outside ; beneath this the sim- ple layer of thickened collench^-ma cells, which is inter- rupted only by the stomata. The two or three layers of elongated, green, palisade cells are quite uniformly de- veloped in the whole circumference, but are rather looser on the under side. Within these are, finally, round, first green, then colorless cells which become larger toward the middle. In this inner cylinder, in colorless cells, run the vascular bundles, the largest in the middle nearer the under side, the others in the circumference, diminished in size on both sides and with their wood parts turned towards the middle of the petiole. The larger of these bundles are provided with strings of sclerenchyma fibres. Apparently the activity of the cambium has been more prolonged in these vascular bundles which has produced secondary wood within and secondary thin-walled bast with- out. The larger vessels appear only in the inner part of the vascular bundles and the border-pitted tracheids in the outer portions. We will now take a leaf of jTcrgus silvaiica for our in- vestigation. On account of the greater thinness of the 156 LEAF STRUCTURE. leaf it is less easy to get a sufficiently thin section. Take therefore a very small piece of the leaf between the two pieces of elder-pith. Stomata are found only on the un- der side. Attached to the epidermis, ejo, Fig. 62, of the upper side, in leaves from f- mny localities, is a layer of elongated, palisade cells, pi, which are more or less sep- arated from each other by intercellular spaces. They are grouped together in bundles below, and each bundle sets on one or more funnel-shaped, broadened, sponge-paren- chyma cells, sp'. The latter are c(mnected into a network Fig. (i2. Transection of a leaf of Fagus silvatica. ep, epidermis ; pi, palisade parenchyma; sp. sponge-parenchyma; k, crystal bearing cells; iu k', a cluster of crystals; st, stoma. X ^60. by elongated, sponge-parenchyma cells, which reaches to the epidermis of the underside, ep'\ Single cells without chlorophyll, but with crystal clusters in them, k', are em- bedded in the sponge-parenchyma. The principal nerve and the lateral nerves of the first order project from the under side of the leaf as leaf ribs. The projecting por- tion of the nerve is about as thick as the rest part of the leaf. The vascular bundles extend into the projecting ril). The latter is covered with elongated, epidermal cells LEAF STRUCTURE. 157 which are followed by elongated, collenchyma cells. To these succeed cells, each of which bears a simple crystal, and then the many-layered sheath of sclerenchyma fibres \/hich encloses the whole vascular bundle. On the upper side, over the vascular bundle, the palisade layer is in- terrupted in a narrow place and is replaced by collenchyma on which follows a slender stripe of elongated, epidermal cells, ep'". A layer of cells, containing chlorophyll, en- closes the sclerenchyma sheath and on them are the sponge- parenchyma. The ribs represent the mechanical system of the leaf which must be built in conformity to that. The filaments are uniformly apparent in the surface of the leaf, the plane of the filaments being perpendicular to that of the leaf. The upper surface of the leaf is principally stretched by pulling, and the under surface by pressure. The fila- ments in th present case are I-shaped, the vascular bun- dles forming the filling of the filaments. The stiffness of the under girding depends greatly upon their springing as far as possible below the under surface of the leaf from the projecting midrib. The nerves expand the blade of the leaf, give it the necessary stiffness and stability and prevent its being torn. Small vascular bundles, like those in the illustration, are supported only on the upper and under sides with sclerenchyma fibres. The branches of these are without a sclerenchyma layer embedded directly in the parenchyma. The smaller vascular bundles are accompanied both on the bast and wood parts by crystal-bearing cells, k. Above and beneath it the epidermal cells are somewhat extended and form shallow, depressed stripes. Long hairs of scler- enchyma fibres grow from the epidermal cells over the nerves, but fall away from the full-grown leaves. Leaves, growing in sunny situations, are thicker than 158 LEAF STRUCTURE. those growing in deep shadows (2) . The additional thick- ening comes from an elongation of the palisade paren- chyma cells and an increase of the number of the layers. The palisade tissue is thus adapted to greater intensity of light and the sponge-parenchyma to a less. In palisade cells the chlorophyll grains are seen only in profile, on the elongated side walls, protruding less or more according to the intensity of the illumination into the cell cavity. On the contrary, the chlorophyll grains of the sponge-paren- chyma are seen in profile or on the surface according to the intensity of the illumination ; that is, they take a posi- tion parallel or perpendicular to the upper surface of the leaf. The palisade cells first receive the rays of light, while the sponge-parenchyma cells receive it only after it has been weakened by absorption in passing through the palisade cells. This disadvantage is in part compen- sated for by the sponge-parenchyma cells, exposing the greatest possible amount of surface to it. But if the light become too intense, the chlorophyll grains turn their edges to it. Such leaves which are developed in the bright sunlight are composed almost entirely of palisade cells, while those, only about one-third as thick, grown in deep shadow, consist almost exclusively of sponge-paren- chyma. Still other physiological consideration will be connected with our morphological investigations whose correctness may be tested by the microscopic image. The assimilation of carbon takes place in definitely col- ored chromatophores and in the higher plants, exclusively in the chlorophyll grains. Only these plasma bodies have the capacity, in light of sufficient intensity, to disintegrate the atoms of water and carbonic dioxide and form from them combinations which are rich in carbon. This proc- ess, taking place mainly in the palisade cells, requires us LEAF STRUCTURE. 159 to designate them physiologically as the principal assimi- lation cells. The palisade cells, as we have already seen, are more or less fully, laterally, separated from each other, and below bend together into tufts. So the as- similated matter will not pass laterally from cell to cell, but rather into the widened funnel-shaped cells of the sponge-parenchyma upon which the tufts of palisade cells rest, sp'. Figs. 61, 62, the physiological function of which therefore is that of absorbeut or collecting: cells. From the same point of view the next following cells of the sponge-parenchyma, sjt" , Figs. 60, 61, may be designated conducting cells. Since the sponge-parenchyma has wide air spaces in connection with the stomata it may be desig- nated ventilation tissue ; also as transpiration tissue, since a considerable evaporation takes place from the surface of the cells into the intercellular spaces. It is, on account or" its chlorophyll contents, rightly known also as assimilation tissue. The sponge-parenchyma cells are directly attached to the parenchyma sheaths of the vascular bundles and so c >nduct the assimilated material partly to that and partly to the bast of the vascular bundle. Sheath and bundle to- gether, therefore, are conductors. The vascular bundles also conduct water from the woody part of the plant, giv- ing it out into the surrounding tissue of the leaf, part of which finds its way into that water reservoir, the epider- mis. The conducting tissue of the parenchyma sheath about the vascular bundles, nuich thickened and giviuof solidity to the "mechanical cells," likewise forms the tis- sue of the projecting leaf ribs and is known as "nerve par- enchyma." This "nerve parenchyma" is continued into the fundamental tissue of the petiole, which, as we have seen in the Huia, is built principally of conducting and mechanical elements. Assimilating cells play but a sub- ordinate part in it. 160 LEAF STRUCTURE. Let US now study the iuner structure of a floral leaf and use the opportunity also to learn of the course and ending of the vascular bundles. Petals of Verbascmn ni- grum are especially well adapted to both of these purposes. The air bubbles which adhere to the petal may be driven out by tapping lightly on the cover-glass. Use no alco- hol. We observ^e a delicate epidermis and from two to four layers of sponge-parenchyma, two at the edges and four in the thicker part of the petal. The stoutest vascu- lar bundles, as Avell as the finest branches where they are reduced to spiral vessels, are sheathed in a layer of elon- gated, thin-walled, parenchyma cells. This parenchyma sheath closes toofether in front over the ends of the vascu- lar bundles. Protoplasmic streaming may be seen in the cells. The stout-branched, sponge-parenchyma cells are joined to the elements of the sheath. Particularly beau- tiful is the view of the ends of the bundles which exhibit a radiating juncture of the sponge-parenchyma cells on the sheath. The petals of Pajpaver Mhoeas has but one layer of sponge-parenchyma between the upper and under epider- mis. The vascular bundles never end free, but rather lock together in commin2;led arches at the edo-es of the leaf. They are surrounded in their whole course by a parenchyma sheath of a single layer of cells, to which the sponge parenchyma cells are joined on both sides. Notes. (1) See Haberlandt, iu encykl. d. Naturwiss., Handb. d. Bot., Bd. ii, p. 614; J. V. Sachs, Vorlesungen über Pflanzen-Physiologie, p, 59 ff. (2) See Stahl, zuletzt Jeu. Zeitschr. f. Naturvv. Bd. xvi, 1883; Concerning the influence of sunny and shady locations on the forma- tion of the foliage leaves. (3) See Haberlandt, work and vol. quoted above, p. G40. LESSON XVI. The Vegetative Cone of the Stem. Our next task shall be to select some suitable object which shall make us acquainted with the structure of the vegetative point of the vascular plants. We choose the phanerogam Hippuris vulgaris (Ij whose vegetative cone is strongly developed and easily prepared. Take a thrifty sprout and cut oif a piece from the top about a cen- timeter long. Remove the larsfer leaves. Now take the bud between the thumb and forefinger, holding it with the top down and with a razor held perpendicularly, and with a drawing motion, cut the bud longitudinally exactly in halves. Now take one of the halves and in the same way halve it, then the half of this lying nearest the centre of the bud, and so on till a section of sufficient tenuity be obtained. This jnanipulation may not at first be success- ful, but it is not a matter of any great difficulty and a little practice ought to make it easy enough. If, however, one does not overcome the difficulty, he may hold the severed bud between two flat pieces of elder pith and cut as he did between the thumb and finger, but hitting the right point in the object will be much more a matter of chance in this case. But objects of this kind may also be fastened between the edges of two pieces of elder pith and the cut made through them and the pith at the same time, as already explained. Select a section from the exact middle of the bud, which we recognize by the slender regularly-constructed vegetative cone. This vegetative cone forms the leaves in a many-branched whorl, which may be seen at some dis- 11 (IGl) 162 VEGETATIVE CONE OF STEM. tuiice from the top as isolated knobs set uniformly about its circumference. Beneath the youngest Avhorl but one, the node of the stem begins to be indicated by a transverse, thick tissue plate, above and below which, in the rind of the stem, the air passages enter. These air passages, which extend from one node to another, increase in size with the growth of the stem. The internodes grow rap- idly and uniformly, both in length and thickness. The vessels of the stem beoin to form somewhat below the fourth youngest whorl of leaves. The addition of a little potash lye brings them out very finely. These vessels appear in the longitudinal axis of the stem to belong to the vascular bundle which grows at the extremity and ends at the top in a single ring vessel. The vessels which belong to the leaves make their appearance iirst in the tenth or twelfth whorl, and are joined to the vessels of the vascu- lar bundle of the stem. At a point not so far removed from the apex, little flat knobs begin to appear in the axils of the leaves which are the beginnings of fan-shaped scales borne on simple, short stile-cells. Only in plants taken in their blooming season do we find here the axillary buds. In order to study thoroughly the structure of the vegeta- tive cone, we should select a good median longitudinal section, treat it with concentrated potash lye and having washed it, lay it in concentrated acetic acid. After a little while examine it in the same or in potassium acetate. It may be handled to best advantage, since we wish to ex- amine both sides of it, by putting it between two cover glasses and then laying these on a slide, but with no fluid between the lower one and the slide. It can then be turned over very readily. By strong magnification we ob- serve a definite arrano-ement of the cells in the "meristem" of the vegetative cone. See Fi«:. 63. There are mantel- like layers of cells whose division walls form a band of VEGETATIVE CONE OF STEM. 163 confocal parabolas. The outer layer which runs over the foundations of the leaves and covers the. whole cone is the derraatogen, d, and forms the epidermis. Under this there are four or more undiflerentiated layers of tissue which belong to the periblem,^?-, out of which the rind of the stem is developed. Finally, we come to a central cylinder with a reduced cone at top, which mostly ends in a single cell, and out of which, as we shall see, by looking deeper into the section, is formed the vascular bundle in the axis of the stem. This tissue we call the plerome, ^l. Thus the epidermis, rind and vascular bundle of the stem in Hij^puris have their own " h i s t o g e n." There is, indeed, no single apical cell but each histo- gen ends at the top of the veofetative cone in one or more "initial" cells. It must be added that in all phaneroo-ams, the sep- „_ „„ ^ ■, -,■^ ,■ p^, t^ o ' i Fig. 63. Longitudinal section of the veg- aration of the histOgenS in etatlve cone of mppurls vulgaris, d, der- ., ... . 1 inatogen; pr, periblem ; pi, plerome: /, the vegetative cone is by no beginning of the leai. x 240. means so distinctly marked as in this case. In many gymnosperms, Abieiinece, Gy- cadea, there is no sharp demarcation between the dermat- ogen and the periblem and often also the periblem and plerome are not distinctly separated. In the angiosperms the dermatogen is always distinctly set oif, but there often exists no boundary between the periblem and plerome. It is not in general a difference of tissue which is continued into the meristem of the cone that gives the necessary sta- bility to the young tissue, but rather the mechanical ar- 164 VEGETATIVE CONE OF STEM. rangement of the cell walls. We meet, in this arrange- ment of the cells, the two sorts of cell division : the anti- clinal, that is, perpendicular to the outer surface of the plant, and the periclinal, a division of cells parallel to that surface (2). We may retain the terms dermatogen, periblem and plerome, in all cases, because the arrangement of cell-layers which we have observed in Uippuris frequently recurs in phanerogams, and these terms will serve to designate, therefore, definite regions of the vegetative cone. The epidermis really arises, in the angiosperms, only from the dermatogen. But the vascular bundles may not always find their origin in the plerome, but also in the periblem. In the origination of the leaves we see, as in Fig. 63, first a periclinal parting of the cells and then an anticlinal, in the outer layer of the periblem. The dermatogen remains a single layer even over the arched places, and has only an anticlinal cell-parting. An anticlinal and periclinal division of cells takes place in the periblem layer, in the production of buds, but only an anticlinal in the dermat- ogen. For an example of the flat vegetative cone which occurs in most phanerogams, we will select the ornamental shrub cultivated in most gardens, Evonymus japonicus (3). This plant may be had at any time of the year and its buds readily lend themselves to section-making. Prepare a transection in order to 2;et a view of the cone from above. First treat the section with potash lye, wash with water and then for a longer time with acetic acid. With a low mag- nification, we recognize the cone as a flat knob surrounded by the youngest rudiments of leaves. These are arranged in a two-limbed, alternate decussate whorl. Each new pair ©f leaves comes forth after a corresponding growth of the vegetative cone in the spaces between the two preced- VEGETATIVE CONE OF STEM. 165 ing leaves, Fig. 64, A. By sufficient magnification the arrangement of the cells at the top of the cone is easily made out, as is seen in Fig. 64, B, There is no one end- / ^ Fig. 64. End of the stem of Evonyvius japonicus. A, view from above upon the top. X 12. B, view of the apex of the vegetative cone. X 240. C, median longi- tudinal section through the apex of the stem. X 28. D, median longitudinal sec- tion of the vegetative cone. X 240. d, dermatogen; pr, periblem; pi, plerome;/, beginning of the leaf; g, beginning of a bud ; pf, leaf trace; pc, procambium ring; m, pith; c, rind. cell. A transection, made considerably below the top, shows a rapid diflerentiatiou of the tissue, into funda- mental tissue, "procambium," which will form the vascular 166 VEGETATIVE CONE OF "STEM. bundles, antl primary rind. The procambium zone appears in cross section as a rhomboid figure, with somewhat pro- jecting and rounded edges. This figure is alternatel}' elon- gated in the direction of the newly-entering procambium cords. The procambium consists of thin-walled, narrow, radially-arranged cells. The production of the elements of the vascular bundles beij'ins at the edges of the fio;ure, protophlocm elements on the outer, spiral vessels on the inner side of the procambium zone. These regions of the diiferentiation of the elements of the vascular bundles are not distinctly marked off from the rest of the procam- bium tissue. The procambium zone opens in places to re- ceive the entering vascuhir bundles of the leaves. In the axils of the young leaves one may see the beginnings of the axillary buds. The median longitudinal section is shown with slight magnification in Fig. 64, (J. The flat vegetative cone, the leaf l)egiunings increasing in size, the axillary buds, g, the differentiation of the fundamental tis- sue, w, the procambium zone,^)c, the vascular bundles com- mon to both stem and leaves, the so-called leaf-trace,^, and the primary rind, c, are recognized at a glance. Pith and rind have a large number of ciystal masses of calcium oxalate. In a fresh section examined in water, the rind and pith appear green, wdiile the procambium zone is quite clear. Treat with potash lye and acetic acid in order to follow the arrangement of the cells of the vegetative cone. First, we come to the single layer of dermatogen cells, Fig. 64, jD, d. Next these, mantel-like layers of the peri- blem, ^7', and then the plerome, ^;?, a solid central C3'linder of tissue not sharply distinguished throughout from the periblem. The vegetative cone appears very narrow be- tween the two 3'oungest embryo leaves, but one may often try many times before he exactly hits the first beginnings of the leaf and makes a section like that represented in Fig. VEGETATIVE CONE OF STEM. 167 64, D. Then the cone appears much broader and the his- togens may be better traced out. The formation of the leaf begins with the periclinic division of the cells in the two outer layers of periblem, f, the dermatogen remain- ing a single-celled layer. The formation of the axillary buds takes place in the same way, in the axils of the third youngest pair of leaves by the periclinal division of the cells of the hypodermal layer. In general, it may be dem- onstrated that the dermatogen furnishes the epidermis, the periblem the rind, and the plerome the pith of the stem. It is less certain that the procambium ring arises from the plerome. That the formation of the vascular bundles is not exclu- sively connected with the plerome follows from the fact that that part of the vascular bundle which enters the leaf is within the rind and is therefore produced by the peri- blem and that the entire inner tissue of the leaf with its vascular bundles is a product of the periblem. To illustrate the growth of a cryptogam by an apical cell, Ave will select Equisetum arvense (4). The apical cell is easily seen. Use a growing sprout, taking a fresh one or one preserved in alcohol. Cut off a piece from the top about 10 mm. long, and make a longitudinal section between the fingers as already described. Find a section with the conical tip of the stem intact. Make it transparent by the addition of a little potash lye. Should this be so strong as to make the cell wall too transparent and therefore unrecognizable, weaken the solution by the addition of a little water. In fresh sections we are to avoid the use of all dehydrating substances or we shall shrink up the vegetative cone. Sections from alcohol mate- rial may, on the contrary, be examined direct in glycerine, but not after a previous soaking out in water. A section treated with potash may advantageously be stained with 168 VEGETATIVE CONE OF STEM. a very dilute solution of safranin. The staining should be very slight and the cell walls will come out all the more distinctly. We get the best results where we treat the section for a short time with concentrated potash solution, then Avash with water and lay it for two hours in concen- trated acetic acid. Examine in water, or, better still, in dilute acetic acid, or a concentrated solution of potassium acetate. A permanent preparation may be made with the Fig. 65. Longitudinal section of the vegetative cone of a sprout of Equisettim avense. t, apical cell; S', youngest segment; S", next older segment;/», principal wall; tn, halving wall; pr, later periclinal,«, anticlinal walls;/, first, f, second,/", third leaf whorl; g, initial cell of an axillary bud. X ^W- last named fluid. Glycerine shrinks these sections. Ex- amine the section between two cover glasses as already recommended in the case of Hippuris. With a rightly-prepared section the apical cell will ap- pear in the form of a triangular inverted pyramid with a convex base, Fig. 65, t. This apical cell divides by walls which are parallel to the existing lateral wall, and which follow each other spirally and form segments arranged in VEGETATIVE CONE OF STEM. 169 three exact series. These segments are seen in profile at Fig. 65, S. They also divide in a definite way and so the plant is gradually built up. At some distance from the apical cell a wall rises from the vegetative cone which gi'ows at its edge by wedge-shaped initial cells. In its further de- velopment the edge pro- trudes at certain places to form the free top of the leaf-whorl, which grows together at the base. The farther we go from the apical cell, the larger becomes the rudiments of the leaf- whorl, and at the same time the difierentiation of the inner tissue of the stem goes on, princi- pally by the separation into thick small-celled low nodes, and thinner long-celled elongated internodes, Fig. 66. The wide-celled pith , , . Fig. 66. Median longitudinal section througfi next OeginS to aj^pear. a vegetative sprout of Eqziisetum arvense. pv. The first rin»" vessel vegetative cone of the sprout; ff, initial cell of a , ° , bud; fir', fir", original pith; vs, entering spiral vessels; n, dif- node in theprocambium ferentiatloa of the node diaphragm. X 26. cord on the outer border of the pith and from here may be traced into the beginniusr of the next hiofher leaf-whorl. Each single vascular bundle is common to both the stem 170 VEGETATIVE CONE OF STEM. and the leaf and may hence ])e called a leaf-trace. As many vascular bundles run downward in each internode as there are leaves in the whorl. The separate leaf-traces first become connected by lateral branches somewhere about the lower half of the seventh internode, thus form- inr, pericam- 178 VEGETATIVE CONE OF ROOTS. Thuia occidentalis that they bear lateral rootlets in four, and at last in three, straight rows. By making a section of the root we find that these rows of rootlets correspond first to the four- and then to three-sided vascular bundle cylinders in the roots. By making a section through the Fig. C9. Median longitudinal section of a root of Pteris critica. t, apical cell; k, initial cell of cap; Jc", outer cap; c, e, r, p, cambium, epidermis, rind and peri- cambium walls respectively. X '^lO. root at the j)oint of insertion of the rootlet we find that the rootlets stand before a wood part of the cylinder, and since these wood parts run straight along the axis of the vascu- lar cylinder the observed arrangement of the lateral root- lets is explained. We will now undertake to learn VEGETATIVE CONE OF FERN ROOTS. 179 somethins: of the veofetative cone of a root which otows by means of an apical cell (3) There is no such variety of forms in the roots as in the stems ^vhich grow b}' this means. The three-sided pyramidal apical cell occurs, how- ever, and the articulation by the formation of segments remains constant. Take the root of ^i^enscnV2ca, Fisf. 69. It would be quite as well to select another form. By tilt- ing the flower pot we shall easily obtain, uninjured, the end of the root. The roots of Pteris critical as of ferns generali}^ are bilaterally constructed, flat bast parts alter- nating with the wood ; the pericambium consists of one layer, the endoderm is flat and the inner part much thick- ened. Prepare a median longitudinal section as already directed. It is not difficult to bring the apical cell here into view. It does not take in the apex of the root, but is covered with the root-cap. The apical cell. Fig. 69, t, like that of the stem of Equisetum, has the form of a tri- angular pyramid whose convex base is turned toward the root-cap, while the apex is sunk in the body of the root. The divisions succeed each other as in the Equisetum par- allel to the lateral surfaces, but besides this there will oc- cur from time to time, mostly after every three of the described divisions, the formation of a wall in the direc- tion of the surfiice of the base. The cell produced by this has nearly the form of a segment of a globe. This cell, a-, is an initial cell for the root-cap, forms a cap-like cell layer and is the origin of the root-cap. It divides into halves by a wall perpendicular to the under surface. These halves repeat the division, thus forming four four-sided cells. Qy a constant repetition of this process of division, that is by walls perpendicular to the basal wall, an old cap, Ä;", will consist of a large number of cells. The cells of old caps are filled with starch grains. They become grad- 180 VEGETATIVE CONE OF EOOTS. ually disorganized, while the apical cell constantly pro- duces new initial cells. The outer walls of the, for the time being, outer layer of cap-cells become much thickened. The division walls formed parallel to the sides of the api- cal cell follow, as in the stem of the Equisetum, the direc- tion of a spiral. Notes. (1) Sachs, Lehrb., iv Auflag,, p. 166; v. Janczewski, Ann. d. sc. nat. Bot., V Ser., T. xx, 1873, p. 162ff. ; Treub, Musee bot. de Leide, T. II, 1876; de Bary, vergl. Anat., 1877, p. 10. (2) Strasburger, Coniferen und Gnetaceen, p. .^40 ; de Bary, vergl, Anat., p. 14. See there also the further literature. (3) Nägeli u. Leitgeb, iuBeitr. zur wiss. Bot., 4 Heft, 1868, p. 74ff. LESSON XVIII. Histology of the Mosses. Heretofore we have studied the structure of the stem and leaves in the vascular plants only. We will now turn to the small stems and leaves of the mosses, which are quite without vessels (1). We will begin with Mnium undulatum, a relatively complicated case, in which the dif- ferentiation of tissue is quite well advanced. Make first a delicate transection through the stem. In the middle of the stem is an axilhiry cylinder formed of narrow thin-walled cells. We may consider this cylinder as the simplest "conducting bundle." Its cells have no living contents, but contain water only. They are distinguished from the surrounding cells by the yellowish-brown color of their walls. Upon the conducting bundle abut the wide cells of the rind, which are much larger, with greenish-yellow walls, and living, chlorophyll-containing contents. They increase somewhat in width towards the outside but at the periphery become suddenly narrow and thick-walled, and pass over without definite demarcation into the epidermis, which consists of one or two layers of much thickened cells. In two or three places the outer layer of cells of the stem is continued into a cell-plate of a single layer, Avhich cor- responds to the downward running leaf-wing on the stem. A section made below the leaves in the stouter brown part of the stem shows the walls of the peripheral cell layer colored a dark brown. From single cells of the surface grow long, brown- walled, many-times-branched cell fibres, designated root-hairs or rhizoids, which do duty as roots. These rhizoids arc distinguished by oblique division walls, (181) 182 HISTOLOGY OF MOSSES. and are hence an exception to the general rule which would demand an exactly transverse wall. Under many such di- vision walls and indeed beneath their elevated edge spring wider spreading lateral l)ranches. Only the growing ends of the rhizoids have colorless walls. These root fibres exhibit the greatest resemblance in re- spect to branching, and the inclined division walls, to the primary growth, the so-called protonema, of the typical mass, which is first developed from a sprouting spore. Still these branches, when they do not penetrate the soil, are colorless and bear chlorophyll grains. The leaf buds which develop into moss stems are lateral branches of this protonema. The near relation of rhizoids and protonema is seen also from the circumstance that the rhizoids damp- ened and set out in the light can produce protonema which will give rise to numerous new plants. It is only nec- essary to lay a tuft of Milium bottom side up and keep it damp in order to luoduce a rich green protonema mass from the rhizoids, which resembles terrestrial VaucJieria tufts in its general appearance. If the section should be made through some point in the stem of the Mnium which had been injured we shall not find the injury repaired by being closed up with a layer of cork, for the cryptogams with the exception of Botry- chium cannot form cork, but the walls of the adjacent cells will be thickened and browned so that they will, with the exception of their greater interior diameter, resemble the other cells of the outer surface. The transection will show near the surface of the stem single small strings of thin-walled cells which agree in color and in their function as carriers of water, Avith the cells of the central cylinder. These are the conducting bundles belonging to the leaves and end blindly in the rind of the stem. In PolytricJium, however, they extend HISTOLOGY OF MOSSES. 183 further inward and connect themselves with the central conducting bundle of the stem. Put a leaf in a drop of water on the slide. We shall find it to consist of a single layer of cells with a middle nerve of several layers, the latter ending in a terminal tooth which consists of a num- ber of rhombic cells. The cells of the mid-rib are much elongated, and the outer ones contain chlorophyll grains. The cells of the leaf are polygonal and also contain chloro- phyll. The bandlike hem around the edge of the leaf is formed of elongated, much-thickened cells. At nearly regular intervals on the outer edge are sharply pointed teeth one or two cells long. We may get sections of the leaf at the same time that we make sections of the stem. But if we wish to make sections of the leaf separately, we may fasten a number of them together with glycerine gum, and without waiting for the gum to dry, make sections of the whole between pieces of elder pith. Then lay the sections in water and the gum will be dissolved. This method is recommended for very thin flat sections. We see from one section that the leaf consists of one layer of cells and that the cells of the leaf-hem are very much thickened. The nerve projects more on the back than on the front of the leaf, and in the middle of it somewhat nearer the under side lies a string of thin walled cells, the conducting bundle which we before saw in the rind of the stem. This string is protected behind by some much thickened narrow cells. The image reminds us not a little of certain much reduced monocotyledonous vascular bun- dles, which consist of a few bast elements and a thin layer of sclerenchyma cells. If the stem of a wilted plant be put in the water the plant will remain wilted, but if the leaves be put in the water the plant will rapidly become turgescent. The leaves, therefore, are the principal absorbents of water, which fact 184 HISTOLOGY OF MOSSES. renders a direct connection of the condncting bundle of the leaf Avith that of the stem quite superfluous. The turf moss offers certain striking peculiarities which we will now consider. Make a transection of the stem of /Sphagnum aculifolium. The section shows us a wide cen- tral cylinder consisting of wide somewhat collenchyma- tously thickened cells ; towards the periphery the cells become gradually narrower, and in the outermost layer are colored a yellow brown. There is no specialized conduct- ing bundle in the interior of the cA'linder, which is inclosed by an outer rind of large cells three layers thick. These cells lie next the narrow yellow-brown cells of the inner cylinder. They are distinguished by their large round or oval orifices and their delicate spiral bands. The open- ings in the walls really connect the cell cavities of adjacent cells as may be seen when the section touches one of them. One often sees the mycelium of a fungus passing through these openings from cell tocell without hindrance. These porous cells of the outer walls of Sphagnum contain only Avater or air and have no living contents. They serve the plant only as capillary apparatus by which the water is con- veyed to the place where it is to be used. The plant has no cutinized cell walls ; concentrated sulphuric acid dis- solves the whole tissue, but the middle lamella and the pores of the yellow-brown outer cells of the central cylin- der resist the action of the acid longest. The extended leaf is ovate, bordered, one layer of cells thick, and consists, as a superficial view will teach, of two kinds of elements : one, of living cells with proto- plasm nucleus and chlorophyll grains ; the other of dead cells filled with water or air, and furnished with rings or spiral bands and openings between the cell cavities. The reason why dead cells used to carry water or air so often have their cell walls strengthened with spiral bands, rings HISTOLOGY OF LIVERWORTS. 185 or reticulations is because they have lost their turgiclity and must have some such mechanical support for their walls in order not to collapse or become compressed. The green cells of the leaf-blade are all connected together and form a network with elegant, bent walls whose meshes are occupied each by an empty cell. The green cells serve for the assimilation of carbon, the empty ones, like those of the stem, for conducting water. The outer edge of the leaf is occupied by slender green cells, and at the conclu- sion of these, by a slender border of cells, a single layer thick, bearing watery contents, slightly thickened on the outside and somewhat collapsed. Only the ends of these cells seem much thicker and project a little. There is no nerve in the leaves as there is no conduct- ing bundle m the stem. The plant is therefore much more simply constructed than the Mnium in this respect, but more complicated, on the other hand, in being provided with a special capiUary apparatus. The well-known MarcJiantia polymorpha (2) presents a pretty complicated structure. The lack of a cormophytic articulation does not necessarily imply a simple anatomi- cal structure. The thaUus is hard and leathery. It branches by the forking of the growing point which lies at the bottom of the apical sinus. If a sprout has but re- cently forked, the middle of the anterior indentation will be occupied by a thallus-lobe, at the two sides of which lie the apical sinuses. In the middle of each ]ol)e on the under side, an indistinctly-outlined mid-rib projects. Stripes run out diagonally forward from these, bending toward the edge of the frond. At some distance from the end, fine rhizoitls spring from the middle of the thallus and serve to fix it to the ground. By examining the luider side of the plant, inider the simplex, we can demonstrate, by the help of a needle, the presence of scales springing 186 HISTOLOGY OF LIVERWORTS. from the surface of the thalhis. There are three distinct forms of these ventral scales : those which grow on the edge of the frond, those which grow in the middle and those which are inserted between. The second and third sorts of scales give the stripes which we have observed with the naked eye. Viewed with a lens, the back side of the frond appears to be divided into small rhomboid fields, the boundaries of which are dark green, the fields them- selves more grayish. In the middle of each is a minute opening. Examining a section made parallel to the back Fig. 70. Marchantia polymoiyha. A, stoma seenffom above; B, in transection. side of the thallus with a higher magnification and we see that the outer cells are polygonal, closely connected and contain numerous large chlorophyll grains. The round opening in the middle of each field is bordered by, at most, four slender, bent, sickle-form, chlorophyll-free cells, Fig. 70, A. When the section reaches deeper, air collects under the surface of the fields. Into these air cavities, "air chambers," chlorophyll-containing cell fibres project. The lateral walls of the air chambers are constructed of closely connected cells, one to several layers thick and contain chlorophyll. In single cells of the surface are HISTOLOGY OF LIVERWORTS. 187 seen certain bodies characterized by their strong refractive power, their irregular outline and cluster-like form. In young shoots these bodies are faintly brown, in older, brown, containiug mostly only fatty oil and form the so- called "oil bodies" of the liverworts (3). A superficial section made from the under side of the thallus shows no fields, while the cells are elongated and coutain less chlo- rophyll than those of the upper side. The rhizoids ex- hibit a double structure. They are slender and provided with conical projectious, or thicker and without these. The former take their rise from that portion of the frond covered by the middle and the intermediate scales or by the former only. They lie close to the frond, quite up to the middle nerve, are covered with the scales and serve to stiffen the thallus. The common rhizoids arise princi- pally from the middle nerve aud turn with a uniformly acute angle toward the substratum upon which they fasten the thallus. At their points they are often lobed and at their base colored purple. All ventral scales consist of one layer, the middle of living, the other two of dead cells. A cross-section of the thallus shows it to consist on the upper side first of a zone of chlorophyll-containing tissue, then within, of wide cells almost destitute of chlorophyll. On the under side, the last two layers of cells are again narrow, flat, and rich in chlorophyll, forming the so-called "ventral rind layer." Oil bodies are scattered through the whole tissue. Muciperous cells, which are distinguished by their size and refractive qualities, are but poorly devel- oped in the Marchantia, but much more richly in other related genera. Looking now at the upper portion of the transection, we first see a simple layer of flat cells which over the air chamber are set free on the walls which form the side?* of the chamber. In the middle of the free outer wall is the breathing place, which, as it now shows 188 HlSTOLOGr OF LIVERWORTS. itself, is inclosed by several, from four to eight stories of cells (4). See Fig. 70, B. The opening is narrowed above and below. The cells of the upper layer are elon- gated into a membraneous border. Get the air all out of the breathing places, if possible, since it very materially injures»the image. Branched cell fibres, two or three cells high project into the air-chamber, arising from the next lower layer of cells which are flat and mostly free from chlorophyll. On the lower side of the thallus at the mid- dle nerve, are the lateral, alternating, overlapping scales. Between the scales lie the transections of the bundles of rhizoids. A middle longitudinal section shows the inser- tion of the stronger common rhizoids which uniformly de- scend from the thallus, and the cone-bearing rhizoids. Metzgeria fwxata (5) is a very simply constructed thal- lus and in many respects very instructive. The incon- spicuous plant is widely distributed, and on the bark of deciduous trees is not diflicult to find. The thallus is ribbon-shaped, clear green dichotomously divided, and has a mid-rib distinguishable by the naked eye. Aside from this mid-rib the thallus consists of one layer of cells, which are polyhedric, and filled with long chlorophyll grains. The slender mid-rib projects much more on the under than on the upper side. It consists, as one may see, by focussing down through it, first of bioad and but little elongated cells, then of slender elongated, and finally again of broad cells. The two outer layers contain chlorophyll. At the vegetative point on the under side of the nerve are a few short club-shaped hairs, filled with a strongly refractive contents. Out of the older parts of the nerve and also out of the marginal cells of the thallus come the so-called bristle hairs which, under favorable ciroumstauces, form a lobed holding-disk and thus serve as rhizoids. They are always placed on the posterior end of the cell from APICAL SINUS OF LIVERWORTS. 189 which they are separated by a bent division wall which does not pass through the whole height of the cell, but rather cuts oft* but a corner or edge of it. The inner cells of the mid-rib are somewhat strongly thickened, with al- most colleuchyma sparkling- white walls. The dividing process at the vegetative point may be followed in Metz- geria in the easiest and most instructive manner (6). In Fig. 71. Terminal growth of Metzgeria furcata. t, apical cell; s'—s'", series of consecutive segments; m' and m", marginal cells of the first and second order; p\ flat or outer midrib cells of the first order; ii, inner cells of the midrib; c, club- shaped hairs. The picture is made with the lens focussed on the inner cells of the midrib. X 510. the Metzgeria the growing point is but very slightly re- entrant. The bottom of this apical sinus, exactly at the point where the mid-rib ends, will be occupied with the apical cell. Examine it from above so as not to be dis- turbed by the club-shaped hairs. The apical cell is two edged. Fig. 11, t, and has the form of an isosceles triangle, 190 APICAL SINUS OF LIVERWORTS. with the base towards the front, mostly somewhat convex and the sides slightly bent. It is divided by walls which run parallel to the lateral walls and gives off segments al- ternately right and left, which consequently all lie in one plane. Notes. (1) See P. G. Lorentz, Jahrb. f. wlss. Bot., Bd. vi, 1867-8, p. 3G3; Goebel, Grundriss der systematischen und speciellen Pflanzenmor- phologie, 1882, p. 184; there also the literature p. 179. Later studies also G. Fritsche, Ber.d. deutsch, bot. Gesell., i Jahrg., p. 83 und Hab- erlandt; the same, p. 263. (2) See Leitgeb, Untersuchung über die Lebermoose, \i Heft, 1881. There the rest of the literature. (3) Pfeffer, die Oelkörper der Lebermoose, Flora 1874, No. 2. (4) Voigt, Beitrag zur vergl. Anat. der Marchantien, Bot. Zeitg. 1879, sp, 729. (5) See Leitgeb, quoted above, Heft in, p. 34. There also the other literature. (6) See Kny, Jahrb. f. wiss. Bot. Bd. iv, p. 85. LESSON XIX. Histology of the Fungi, Lichens and Alg-^. Staining the Cell Contents. The vegetative organs of the Fungi, with the exception of a few of the simplest forms, consist of threadlike, elon- gated, more or less elaborately branched cells, the "hyphfs " so called. They are either with or without division walls. The most massive funo-us bodies consist of aijijre orations of this hyphse. Ttie hyphse may really be so solidly united into a mass as to form a tissue, called pseudoparenchyma which very strikingly imitates the appearance of the par- enchymatous tissue of the higher plants. Still this pseu- doparenchyma is the product of a union of cell fibres and not of the progressive division of cells in three directions. For the study of this kind of structure we will take the fruit body of a toadstool, Agaricus campestris (1) , a plant found the year round and of comparatively simple struct- ure. First make a longitudinal section of the pedicel of a full grown plant. We find a structure of longitudinally running hyphse, which we can easily unravel in that direc- tion with a needle. The hyphae are arranged more or less parallel with each other, single ones occasionally run- ning obliquely between the others. Each hypha forms a cell thread which is laterally branched, here and there by branches which spring from under the division walls or else farther down alonof the side. Sometimes the cells of neighboring hyphse are connected by a cross branch, and communicate openly with each other. At the periphery of the stem the hyphee are slenderer, more closely com- pacted together and on the surface their walls are brown, and their inner cavity more or less perfectly collapsed. (191) 192 STRUCTURE OF FUNGI. Towards the middle of the pedicel the hyphas become like- wise slenderer but their texture is more loose and their course altogether irregular. Large quantities of air fill the spaces between the hyphae. Until the disturbing in- fluence of the water on the cell contents has made itself felt, little is to be remarked of it ; sometimes on the trans- verse walls a collection of it may be seen. Afterwards large vacuoles form in the cells. Infrequently small crys- tals may be found in the cells. A transection of the pedicel gives a parenchymatous ap- pearance which is lost only in the middle of the section where the hyphse present their sides to view. This pseudoparen- chymatous tissue appears to be formed from cells of various sizes irregularly po- lygonal, with more or less numerous in- FiG. 72. Agaricus^^^'^^^^^^^^' spaccs and Openings between cavipestris. Part of a them. Fig. 72. Sometimcs the section transection of the ped- , n . icei. Two of the hy- Will cut closc to the trausvcrsc wall in ph.-e are cut near the ^j^-^j^ ^^gg ^ jj^j. ^^-^^ ^^ g^^^ j^^ ^j^g ^^^^ division wall, a dot '■ appearing in tiiem. X die of the cell. Scc Fig. 72. It is a pit which is covered on each side of the cell wall with a small collection of light-refracting substances. Such pits in the centre of the cell wall are quite common in the Basidiomycetece and the Ascomyceteoe (2). In the pro- toplasmic wall-lining of the cells are numerous very small nuclei but Avhich, not being easily seen, we will not fur- ther consider. For a knowledge of the structure of the frond of the lichens Ave will select Physcia ciliaris found on tree- trunks everywhere. The thallus is an ascendant leafy bush, on the back gray green to living green, on the front gray. Stiff hairs grow from the edges, are often forked and when they reach the substratum grow to it. Make a STRUCTURK OF LICHENS. 193 section between pieces of elder pith and examine with a sufficiently high power, and we shall see that the thallus consists, on the back side, of a compact layer of narrow, thick-walled hyph;e, the rind layer. Farther inward the hyplue wind about each other in order to make the loose tissue of the fundamental layer. Here it is easy to de- monstrate that the hyphjs are long, branched tubes jointed by division walls. On the border, between the rind and pith, are comparatively large green round cells, the goni- dia. They are the same as the algas Cystococcus humicola Nägl. The hyphffi lie about the gonidia and carry to them raw nutritive substances, of which they receive back a portion when it has been assimilated by the gonidia. There is here, therefore, a case of communal life between the fun- gus and the alga by which they are mutually serviceable to each other. On the underside of the thalkis the hypha3 are in this species again closely interlaced to form a lower rind, or the loose fundamental tissue extends to the lower surface, the latter being the most prevalent case ; but, on the edges, the rind-layer of the back of the frond passes around to the front side, in all cases. From these edges the hold-fasts or rhizines grow, consisting of parallel h}'- phse closely fastened together. The walls of these hyphte are brown. This string of til)res is often divided at the base. In other lichens, these rhizines grow from the low- er surface of the thallus. Chloriodide of zinc solution im- mediately colors the gonidia a beautiful blue, while the hyph^ take only a yellow or a yellow-brownish color, the so-called fungfi-cellulose reaction. The thallus of the plant before us is said to be hetero- o:eneous because the »onidia are distributed in a distinct layer. The more highly organized lichens have a homo- geneous thallus, the gonidia being evenly dispersed through 13 194 STRUCTURE OF FRESH- WATER ALG^. the whole frond. Among the latter are the gehithious lichens, in which the gonidiaare embedded in a gelatinous jiiass through which the hyphse of the fungus freely pene- trate. The algre which participate in the formation of the lichens are of different species, are green or blue 2:reen, but be- long exclusiyeiy to the lowest groups of these plants. The öladophora (3) furnish us a much branched green thread whose thickness diminishes with the degree of its branching. It is a fresh-water plant widely dis- tributed and every species is adapted to the investigation. The determination of species in this genus is very difficult. We will select the dark green, floating, tuft-forming CladopJiora glomer- ata, for particular study. It is branched in a bushy form, the lateral branches springing from the upper end of the cell. The branching is acropetal so that the end cell of the branch may be considered as the apical cell. But Fig. 73. Cladophora glomerata. • i /• A cell from a iiiament prepared there arisc also troui the oldcr ,with chromic acid and carmine. ' '^^^ additional brauchcs, iu a 11, nucleus; cvt, chromatoplioies; '' 2), amyium-ceiiues; a, starcii- certain scusc adveutivc branches. ^''^'"^' ■ By sufficiently powerful magnifi- cation the wall lining is seen to be formed of small phites. Fig. 73, ch, which are laterally separated by delicate col- orless lines. In each plate are several colorless granules, a, besides which in several of the plates are relatively STRUCTURE OF FRESH-WATER ALGiE. 195 larger, globular, strongly refractive forms, in which are to be distingiiishecl an inner nucleus and an outer enve- lope, formerly called amylum centres, but more recently "pyrenoids,"j9. (4) The cells are filled with cell-sap and divided into irregular polygonal chambers of various sizfes, by colorless extremely thin plasma plates which extend from the wall layer through the cell cavity. These plates sometimes contain chromatophores. By careful focussing, colorless plasma balls may be seen on the wall-layer pro- jecting into the cell-cavity. They are nuclei in which, with a specially favorable position, nucleoli may be made out. In this plant we have a multinuclear cell. If now we press upon the preparation pretty smartly, we shall see the wall laj'er forced back a little, and the chlorophyll plates separated from each other and rounded out. At the same time the small grains and amylum centres stand out distinctly in the chromatophores, which now seem to be aflected b}' the water the same as the chlorophyll grains of the higher plants. If we now add a solution of potas- sium iodide of iodine the small grains and also the outer covering of the amylum centres will be tinged violet, l)ut in the green chromatophores, and also the occasionally visible nuclei, a brown color appears. We must not fail to seek in this preparation for uninjured cells, in which starch grains and amylum centres are stained and stand out sharply in their natural position and in which also by deeper focussing we distinctly make out the nucleus. Suf- ficiently strong magnification gives us the angular forms of the albumen crystals (5), of which two will sometimes be found in an amylum centre. In a short time, in the chlo- rophyll plates, are seen irregularly formed brown grains, which come from the disintegrated chlorophyll coloring matter, and present us the hypochlorine, or clilorophyll reaction (6). The reaction may be had from the influence 196 FIXING AND STAINING ALG^. of other acid salts, — but we must adopt other processes for studying the nucleus more exactly, and getting a look at its method of parting. This will give us the best op- portunity to get acquainted with some approved methods of fixino; and staininsr, to which histological studies owe so much in recent times. Put parts of the plant in 1% solution of chromic acid, in concentrated picric acid, in 1% solution of chromic and acetic acid, 0. 7^ of the former and 0. 3% of the latter, respectively (7). Let the tirst and last stand several hours. No harm will come in twenty-four hours. The second may stand twenty-four hours. Then wash carefully in distilled water. They may be kept in water for a whole day changing frequently. The picric acid preparation requires very careful handling if it is to be stained with hainatein ammonia. The variously fixed and well washed preparation we now lay in Beale's carmine in watch-glasses (8), in Thiersch's or Grenacher's borax-carmine, and in Hoyer's neutral" carmine. The plant should be subjected to the action of Beale's carmine twenty-four hours, half that time to Hoyer's, several hours in the borax-carmine. Another part of the plant we will stain with Grenacher's or Boehmer's haematoxylin which to stain well should be as old as possible and should be used very dilute. It is best to test the staining from time to time by examining with the microscope, and when the requisite degree of intensity is reached to take it out of the solution. If, in spite of this precaution, the color be- comes too dark, it may be put in pure water, or in a solu- tion of alum, or in water containing a trace of muriatic acid, till the required shade of color is obtained. If acid is used in removing the color, the specimen should be afterwards transferred to a weak solution of ammonia for a few minutes. In order to stain the preparation with the ammoniacal hamatem method (9) we must remove the last FIXING AND STAINING ALG^. 197 trace of the picric acid by putting it in a large quantity of well boiled water, which we repeatedly change, for from twenty-four to forty-eight hours. For the preparatiou of the staining fluid we throw some ha^matoxylin crystals into a small quantity of distilled water and blow upon it a jet of ammonium gas. This is done by means of a wash bottle containing an ammonia solution, in which the two glass tubes do not reach the fluid. The crystals dissolve with a beautiful violet color. Dilute the solution with distilled water and let it stand two hours. The right shade of col- or may be determined directly, but it is well perhaps to make the color too high and weaken it bj' inmiersion in water for several hours. This method of staining requires care but it gives the most satisfactory^ results. Prei^arations hardened with anything else than picric acid are less suited to this staining. The other named carmine stains are most beautiful if they are over colored and then laid for some time in a watch glass with 50 to 70% alcohol, to which a drop of hj'drochloric acid is added. For this purpose one may keep on hand a ^% solution of hydrochloric acid in 70% alcohol. If the preparation has a more or less diffused stain, the addition of the acidulated alcohol will give it a sharp stain. The preparatiou should alwaj's be washed in alcohol after treatment with the acid-alcohol. If we wish to make permanent preparations of our stained objects we will select for the carmine preparations glycer- ine or glycerine-jelly or Hoyer's mounting fluid. If we use the glycerine or the glycerine-jelly for haematoxylin stains we must be sure it contains no trace of acid. The Hoyer mounting fluid is also well suited to hsematoxyliu stains. The preparation should not be put directly into the mounting fluid, else the cells will collapse by the too sudden withdrawing of the water, but it should flrst i)e put in very dilute glycerine which concentrates slowly by stand- 198 FIXING AND STAINING ALG^. ing in the air where the water may evaporate. Then the plant may be transferred to glycerine or glycerine-jelly or Hoj^er's mounting fluid without damage. The glycerine preparation should he cemented with Canada balsam. The other media named will need no cementing. Considerino^ now the different fixino; and stainin«' media for pre[)arations we may in general say, that chromic acid or its mixture goes best with the carmine stain ; and picric acid for fixing with the haematoxylin or the hamatein am- monia staining fluid. But it must be expressly emphasized that these results are restricted to the present ol)ject, and that other objects may be better treated by other methods. It also frequently happens that an already tested staining fluid, for some unknown reason fails, so it will not be safe to base a conclusion on a single case. Generally the fix- ino- and stainins: <>f cell contents is an art which can be learned onl}' by practice, and one's first attempts are often failures. We have chosen the Cladophora as one of the most suitable objects for the introduction of the different hardening and staining processes. He who will f()lh)w the method given here, strictly, will seldom fail ; hardening with ]% chromic acid, and staining part with borax-car- mine, and anothar with hsematoxylin. In the borax-carmine preparation. Fig. 73, the nucleus comes out very sharply. The amylum centres and the rest of the cell plasma remain as good as uncolored and the starch grains take no color. AVithin the amyhun centres, the albuminous crystals are quite distinct surrounded by a hollow globe which, as we saw, gave the starch reaction with iodine. The nuclei are distributed quite uniformly through the cells lying in the chlorophyll layer and project- ing into the cell-mass. The nucleus shows a darkly stained nucleous and, for the rest, seem to be finely granular or minutely porous. The hsematoxylin or hamatein prepara- CULTIVATING FR:^SH-WATER ALG^. 199 tions have the nucleus colored dark, also the crystals in the amjlum centres, even if but slightly. The starch grains are not colored, but the microsomes of the cell-plasma are, and almost as the crystals. The genus Spirogyra presents us with a simple filamen- tous cell. We will choose a species which has a central, ea!servation, and not those of Vaucheria terres- tris often found on moist ground, for here the oogonium and the antheridium are set on a common lateral branch. The Vaucheria sessilis, living in water, forms in culture the swarm-spores already studied, and after a few weeks pro- duces sexual organs also. The oogonium, Fig. 86, o, is obliquely egg-shaped, filled with plasma contain- ing oil and chlorophyll, and separated from the thallus thread by a division wall somewhat above its place . _ .. Fig 86. Vaucheria sessilis. piece of the ot msertion (7). The OÖ- tliallus with reproductive organs, o, oögo- gonium is i)rovided with "'""V "'"""'''"'^'""V '''• '"'™™^'°''''''''''' ° ^ 01, oil drops; n, nucleus, seen only when a lateral bill-shaped out- p™periy stained, x-^w. growth in which colorless protoplasm is collected. The latter occupies the whole upper third of the oogonium in some stages of its development. Bj' a continuous obser- vation of such an oogonium, we see the colorless contents at the end send out a papillate process which slowly rounds out into a sphere and separating from the oogonium slowly sinks to the bottom of the water. This observation teaches not that the membrane at the end of the oogonium is peu- forated, but rather that it swells into a jelly-like envelope and the drop of plasma is pressed out through the gelatin- ous substance. The remaining contents of the ougcjnium round up, its colorless end being the germinal vesicle. 244 EEPRODUCTIVE APPARATUS IN VAUCHERIA. The branch bearing the antheridium is more or less bent, its upper third is set off from the rest by a division wall and becomes the antheridium, Fig. 86, a. In its ripe state it is distinguished by its colorless contents, while the branch which bears it is rich in chlorophyll grains. The apex of the antheridium is usually turned away from the oogonium. In the colorless contents of the antheridium, short rods longitudinally arranged may be more or less clearly distinguished. At the time when the oogonium exudes a part of its plasmatic substance, the antheridium opens at the apex and discharges its mucilaginous con- tents. The greater part of it remains in the form of col- orless bubbles in the surrounding water where it slowly disorganizes. A smaller part assumes the form of very minute spermatozoids. These lively swarming spermato- zoids soon collect on the gelatinous mass at the end of the oogonium. Some penetrate to the colorless embryo-sac of the spore, and in favorable cases may be seen to com- mingle with that. After a short time the fertilized spore — oospore — will be surrounded by a delicate membrane which may be seen with special distinctness at the embryo- sac. In the space of a few hours the colorless protoplasm is distributed uniformly through the oospore. Older spores are filled with large oil drops, show a brown spot on the inside and possess a hard cell wall. If one fixes the mov- ing spermatozoids with potassium iodide of iodine it will be found to be provided with two laterally-inserted op- positely-arranged cilia of unequal length. Notes. (1) de Bary, Conjugaten, p. 3; Strasburger, Befr. und Zellth., p. 5; Kny, Wandtafln, Text, p. 11. (2) Schmitz, Stzber. der niederrli. Gesell., 4 Aug., 1879, p. 23. (8) Thiu-et, Ann. d. sc. nat. Bot. lu S^r., xiv T., p. 219, und LITERATURE OF THE LESSON. 245 Taf . 16 ; Schmitz, Siphoiiocladiaceen, p. 34, u. Chromatoplioren, p. 119, Anm. ; Strasburger, Zellb. u. Zelltli., iii Aufl., p. 72. (4) See Areschoug, Observ. phycol., ii, Acta soc. scient. Upsal, Vol. IX, 1874. (.5) Tliuret, Ann. d. sc. nat. Bot., 2 ser., Bd. xix, p. 270; Stras- burger, Zellb. u. Zellth., iii Aufl., p. 213, u. 84. (6) Schmitz, Stzber. d. uiederrh. Gesell., 4 Aug., 1879, Sep. Abdr., p. 4 ; Strasburger, work before quoted, p. 88. (7) See Priugsheim, Monatsber. d. kgl. Ak. d. "Wiss. zu Berlin aus dem Jalir 1855; de Bary, Ber. d. Freib. Naturf. Gesell., 1856; Stras- burger, same work quoted, p. 90. LESSON XXIII. Keproduction of the Fungi. If one puts a piece of moist bread under a glass bell, in a few days it will be covered with a thick mat of fun- gus filaments which belong to the Pity corny ceim, Mucor mucedo (1). It grows very luxuriantly on fresh dung kept in a close moist place. Its fruiting filaments rise above the substratum several millimeters high, turn towards the source of light, and are terminated each by a round, yellow or brown, minute bead which may be easily seen with the magnifying glass. By transferring some of the plant to a drop of water on the slide and suflicient- ly increasing the magnification, it may be demonstrated that the mycelium consists of thick, much-branched irreg- ularly-divided tubes, out of which arise these straight undivided and unbranched filaments which bear the spher- ical sporangia at the top. Those which are unripe pre- serve their form in water and have a yellow-brownish protoplasm. In the youngest stages, the fruit stem is not marked oflffrom the sporangium, but further on a division wall arched strongly outward is produced on the inside of the sporangium, so that the fruit-stem ends in the spo- rangium, in a so-called "columella," a club-shaped proc- ess. The ripe sporangia are disintegrated in water, only small fragments of the wall remaining, formed of fine needles', which consist of the oxalate of lime (2). The freed spores lie nearly at a uniform distance apart em- bedded in a colorless mucilage as may be demonstrated by moving the cover-glass. Beneath the columella is ii small collar which constitutes the remainder of the calcareous . (2^6) REPRODUCTION OF THE FUNGI. 247 incrustation. In the wall-linins; of the fruit-bearino- fila- ment, if it be not too old, one may follow the longitudi- nally running streams of protoplasm. Mucor mycelium are poly nucleated, the nuclei very small and seen only by staining. On the manure culture the funsus occasionally, yet rarely, develops zygospores which appear as black points. They are produced by the conjugation of the club-shaped ends of the hyphte or m^'celium thread. On the ripe, dark, warty zygospores one may see the two my- celium threads as clear circumscribed circular spots. The cause of the potato l)light is a Phy corny ceta, the Phyto])litliora infestans (3), whose germinating filaments penetrate through the epidermal cells into the intercellular spaces of the leaf, and ramifj'ing there destroy the tissue of the leaf, forming brown flecks on the surface which con- stantly increase in size. In order to obtain the plant in a fruiting state in a larger mass, put a blighted branch of the plant under a glass bell, the air in which is saturated with vapor of water, and let it lie there for two days. The blighted leaves soon become covered, especially be- neath, with a white mould, which is formed of the filamen- tous fruit-bearers, spore-stalks, of the finigus. This mould is particularly well developed on the edges of the brown spots. A superficial section shows us that the spore-bear- ing filaments grow out of the widely opened stomata. This fact may be observed indeed, though not so satisfactorily, by using a piece of the leaf of full thickness. These co- nidia-bearing filaments appear to .be delicate, unicellular threads filled with finelj' granular protoplasm and branched at top. Fig. 87, ^. The branching is monopodial, and the number of branches but two or three, which have ir- regular swellings along their course. The conidia-bearing filaments in dry air collapse and twist about on their axes. Sometimes we find on the end 248 THE POTATO BLIGHT. of the branches spores in the process of development, but the ripened citron-shaped spores always fall off when the preparation is put in water. To find the ripe spores in situ, the plant must be examined dry, but even then a slig^ht trace of wa- ter should be introduced under the cover-glass, for the plant rapidly shrinks up when dry. Specimens collected in the open air produce the conidia-bear- ing filaments only on the under side of the leaf. The filaments are not so long as those produced in the moist chamber and therefore not so easily seen with the naked eye. A cross-section through the leaf at the Ijorder of the fleck, made by means of elder-pith, will enable us to folloAv the course of the filament in its exit Fig. 87. Superficial section of the epider- i ^i + XT • mis or leaf of Solanum hiberomm, out of the thrOUgll the StOUia. r VG- etomata of which are growing the coniiiia- riUPUtlv llsO SCVCral bearing filaments of Phytophthora infestans. ■' XöO. iJ, ripe conidia; Cone with the con- hypllPB will COllcct and tents divided; A a swarm-spore. B-D, X y^^,,^^^^^ ^^ ^^^^^ pl.^^g and send up a number of spore-bearing filaments. By following out the course of the hyphffi in the tissue of the leaf, we shall find that it runs in the intercellular spaces. Phytophthora is distin- guished from the nearly related Peranospora species by THE POTATO BLIGHT. 249 forming but few and short processes for absorbing the juices, among the cells of the host plant so that one often looks in vain for them. The delicate mycelium threads, on the contrary, cling fast to the cells of the host. The chlorophyll grains of such cells first become brown and then they with the other elements of the cell contents dis- solve and miufjle and run toijether in a brown mass, and finally the Avhole cell collapses. The spores are citron- shaped. Fig. 87, B, somewkat pointed, with short stems and finel}'' granular contents. The membrane at the apex is very delicate and a little swollen. The spores are pro- duced as we have seen on the ends of the branches of the conidia-bearing filaments, but when they are fully grown, the end of the branch grows out beyond the spore, presses it over to one side so that it stands nearlv at right ano:les with the stem and finally at the end produces a new spore. See Fig. 87, A. By sowing the spores in a drop of water on a cover-glass, and being careful to get the spores immersed in the water, and suspending the drop by laying the cover-glass on a small moist chamber, in a shaded place, we shall have, in the course of an hour or uKn'e, the beginnings of the swarm-spore forming process. Since the swarm-spores are formed from the contents of these larger forms we call them conidia and not spores. Among the conidia are sporangia Avhich behave like com- mon spores, for we see some on the edge or surface of the drop of water which put out a germinating tube from the forward papilla. In the immersed spores the contents are divided into an indefinite number of cells, (7, which show in each a small central vacuole. The apex of the conidium soon swells and finally dissolves leaving a small orifice through which the masses of differentiated contents are pressed out one after another. They speedily become swarm-spores. By fixing the swarm-spores with iodine 250 SEXUAL REPEODUCTION OF FUNGI. solution we recognize tAvo cilia inserted laternlly on the spoi'e in the neighborhood of the vacuole, D. The swarm- spore continues to move for half an hour. It then comes to rest, surrounds itself with a celhilose membrane and soon puts out a germinating tube. This germinating tube from a swarm-spore or from a conidiura direct is what penetrates the epidermis of the stem or leaf of the potato, and so infects a perfectly sound plant. By the formation of conidia the rapid increase of the fungus is provided for. Sexual reproductive organs have not yet been discov- ered in this species, though they are well known in the nearest related Peranonpora species. Branches of my- celium swell mostly at the ends, forming a spherical mass within the tissue of the host plant; which is separated from the mycelium filament by a division wall. It is called the oogonium. On each oogonium there lies the end of a mycelium branch, which has been differentiated as an antheridium. The greater part of the protoplasm of the oogonium collects into a central spherical egg^ into which the antheridium thrusts a fertilizing tube, whereupon it surrounds itself with a thick membrane. Upon almost any moist object, which has the least trace of nourishment in it for the fungus, may be found the blue green mould, Penicillium crustaceum Fries (4). It is the most widely distributed of all the moulds and may be found in all sorts of places. As convenient a way as any to obtain specimens for examination is to moisten a piece of bread and put it under a glass ])ell, Mucor will first appear, but will be gradually displaced by Penicillium which will spread a blue-green cover over the substratum in about eight days. The color comes from the spores but o\\\y when they occur in large quantities. Examine a lit- tle of the material in water. The mycelium consists of branched multicellular hypha3, the cells separated by SPORE FORMATION IN FUXGI. 251 transverse walls. The immediately visible contents are finely granular protoplasm witli small vacuoles. Single filaments not distinguishable from other m^^celium filaments have formedfruit-bearers. On their tips is a whorl of short branches, Fig. 88, s', which either themselves bear whoils of basidia, or whorls of short lateral branches which do bear the basidia. This manner of branching gives to the fruitinoj filament the appearance of a hair pencil. Frequently also secondary pencils spring from beneath a division wall of the primary filament. See the fig- ure. By a sufiiciently high magnification we shall dis- cover that the basidia are cyl- indrical, prolonged at the end into a finely pointed process, called the sterigma, 6'^. This sterigma swells and rounds at the end forming a rapidly growing spore. Beneath this is a second swellin» which forms a second spore and so the chain of spores is pro- duced. The terminal spores are thrown off* Avhile those be- low are being produced. PenidlUum tufts fixed with abso- lute alcohol may be easily colored with hematoxylin, after which it will be seen that in the cells of the mycelium and Fig. 88. Penicillium crustaceum. Fruit bearer with brancli wliorls, s' and s"; basidia, 6; sterigma, st. and spores. Nuclei visible. From an al- cohol-hematoxj^lin preparation. X 510. 252 SPORE FORMING IN FUNGI. of the spore-bearing filaments, numerous nuclei occur (5) . They are so small as to require the highest magnification. They are elongated in the direction of the longer axis of the cell and connected by fine plasma strings. In the long cells tliere are several, in the short cells of the whorl on the aerial filament but one or two, in the basidia but one at the upper end. But the basidia are commonly so filled with contents at their apex that it is almost impossible to make them out. With the strono;est maofnification one may detect a nucleus in each of the spores. Other fruit bodies than these under consideration have been observed in PenicilUum. They are produced in cer- tain cultures, have the size of a small pin-head, and are of a yellowish color. After a long resting period, they form asci within, each of which produces eight spores. This places the PenicilUum among the Ascomycetm , and indeed as the representative of that division of the Cleis- tocarp Ascomycetoe with closed fruit bodies. From the spores produced in the asci, the pencil-like fruit-bearing filaments may be cultivated on the ol^ject-slide. Notes. (1) Brefekl, Schimmelpilze, Heft i, p. 10. There also the litera- ture. (2) Brefekl, 1. c, p. 18. (3) See de Baiy, Aim. de. sc. nat. Bot., iv s6r., p. 32, und Bei- träge zur Morphl. u. Phys. der Pilze, Heft ii, p. 35. (4) Brefekl, Schimmelpilze, Heft ii. (5) Strasburger, Zeilbikl. u. Zellth., iii Aufl., p. 221. (6) Brefekl, 1. c, p. 39. LESSON XXIV. "Eeproduction of the Fungi and Lichens. In the months of May and June one may frequently find on the underside of the leaves of the barberry, Berberis vulgaris, orange-colored warts which appear to the naked eye to be finely punctured. A magnifying glass will show that the pillow-like swellings are surmounted by minute orange-red cups. The corresponding place on the upper side of the leaf is marked by a reddish fleck bordered with yellow. The magnifying glass shows it to contain in the inner parts numerous brown points bordered with orange- red, similar points being found on the edges of the swell- ing on the underside of the leaf. The little cups are the aecidium fruit of ^cidium berberidis, the spermagonia of which are the above-mentioned dark points. Both together form the first generation of our common rust-fungus, be- longing to the JEcidioinyceteoß or Uridineoe, Puccinia graminis, which completes its second generation on our corn and other Graminece, producing there the rust dis- ease (1). Prepare a delicate section of the leaf through the swellinfi: and examine it with first lower and then higher powers. We assume that the material is fresh, though good alcohol material will answer. Treating with potash lye will satisfactorily clarify the fresh section. The cell- layers of the healthy part of the barberry leaf are as fol- lows — the upper epidermis, a single layer of elongated palisade parenchyma, a layer of loose sponge-parenchyma about five cells high, the lower epidermis. The tissue of the affected place is about twice the thickness of the leaf. Upon the palisade cells which are higher but otherwise (253) 254 . SPORE FORMING IN FUNGI. little changed, is joined a close tissue more or less elon- gated in a direction perpendicular to the surface of the leaf, and is distinguished from the adjoining sponge paren- chyma by its lack of intercellular spaces. The epidermis of neither surface has been chano-ed. The cell contents of all these cells have undergone disorganization, and con- sist in part of colorless drops of oil, in part of greenish- yellow and reddish drops arising from the chlorophyll grains and cell-plasma, and of granular masses. The whole tissue of the affected part shows its intercellular spaces penetrated by delicate hj'phte, occasionally branched, ar- ticulated by divisicm walls and containing drops of oil. They extend to the epidermis on both sides. With chlor- iodide of zinc and also with iodine and sulphuric acid, the blue color is not induced in them, as fungus-celUilose rarely shows that reaction. Our section of the little cups shows them to be more than half embedded in the tissue of the swelling. We may easily see that the mycelium forms a thick layer under the little cu[)s out of which arise num))er- less club-shaped hyphpe, perpendicular to the layer and parallel to each other, solidly packed together and forming the so-called hymenium. These hyphae, the basidia, are transformed at their ends into a straight series of spores, which though in the basidia colorless, and by mutual press- ure polygonal, gradually round out and become orange- red. The spores separate from each other higher up and are discharged from the opened fruit vessel. An examina- tion of the youngest spores on the basidia teaches us beyond doubt, that they are successively separated from the point of the growing basidia by means of a transverse wall. The single layer which constitutes the wall of the perid- ium, or fruit-cup, consists of cells which look like spores but which remain polygonal and adhere together laterally. Their fine, delicate, porous walls are much thickened on the SPORE FORMING IN FUNGI. 255 outside. The growing peridiiim presses through the sur- rounding tissue of the leaf, tears open the epidermis, and so comes forth. The pear-shaped spermagonia, mainly em- bedded in the upper side of the leaf are like the teeidia- spores, surrounded l)}' a thick plexus of hyphjie, from which spring closely compressed parallel tln-eads which run toAvard the middle of the organ. These filaments are very slender, and those found on the upper part form a delicate bundle which protrudes from the organ. These threads are called the sterigma, are transformed at their tips without into small globular cells, the spermatia, which are discharged from the organ as a shiny mass. The sterigma themselves bear orange-red oil drops, which lend their color to the w^hole body of the organ, particularly to the outside. The sper- matia do not germinate. Their significance is unknown. One might be inclined to consider them the product of the male organ and to suppose that a generative act introduced the formation of the fBcidium fruit. As already mentioned, this fungus lives in a second generation on the Graminem. It belongs to the "heterecious" parasites, which in opposi- tion to the "autoecious" complete their circuit of life on diflerent hosts. The proof of this is obtained by sowing the 8ecidia spores on the germinating plants of cereals (2). The uredo growth of Puccinia gramlms meets us only too often in nature from the middle of June till fall, on rye, wheat, barley, oats, and particularly on couch-grass, Trilicum repens. It attacks principally the stem and leaf sheath of the infected plant. One recognizes it ea- sily as the slender, rusty-brown colored stripes, parallel to the nerves of the leaf, several centimeters long. The epidermis of the host will be seen torn and lifted up by the underlying layer of spores. First appears the rust-colored layer of uredo spores, with which are gradually associated the brown teleutospores. Gradually the uredo spores are 256 GROWTH OF WHEAT-RUST. changed, at last fully, till the layer becomes dark, almost black, and towards the end of summer only teleutospores are to be found. If fresh material is not at hand, alcohol material, even dry plants, will serve for examination. Make a transection of the stem of an infected plant. We may easily demonstrate that the hyph^e permeate only a definite tissue of the part. It is the loose chlorophyll- conta?ning tissue stripe, which alternates with sclerenchy- matous thickened stripe in the stem, and which is covered with an epidermis that is provided with stomata. Here the cells are thickly interwoven with the jointed hypbaä and their contents disorganized. At those points Avhere the section cuts a layer of spores one sees the mycelium with many short and delicate branches spring up towards the surface, which are headed off at their swollen ends into a unicellular spore, the uredospore. The epidermis is cracked open and its edges laterally raised up. The spores are in difierent stages of development. The ri- pened ones are a longish oval and with a sufficiently strong magnification two layers may be seen in the envelope. The outer dark brown is beset with numerous small warts ; the inner and less dark shows several, mostly four, pits, regularly divided at the equator. The contents of the spore are granular and the inner portion a lively orange-red. A transection through a stalk of oats, having the dark brown teleutospores, shows us that the cause of the hyphae is the same as previously seen. The teleutospores are borne on a someAvhat thicker-walled style than the uredo spores. They are bicelhilar oval with the two large ends turned together. The envelope is dark brown. The plants investigated in the course of the season will show both kinds of spores. The teleutospores survive the winter and are capable of growth the next spring. Each of the two cells puts out STRUCTUKE OF FUNGI. 257 a delicate tube, the so-called promycelium which divides transversely into several cells and from these issue vari- ously shaped processes which divide at their ends into kid- ney-shaped sporidia. These will infect the barberry leaves ; if they fall upon one sufficiently young, the germinating tube penetrates the outer wall of the epidermal cells directly into the inside of the leaf of the host. We therefore see that the infecting of the leaf does not altogether depend upon the germinating tube entering a stoma. In order to become acquainted with tlie structure of the hymenium of the HijmenomyceteGe, (3), we will select one of the numerous species of toadstools {Amanita), mushrooms {PsaUio(a), or agarics (Ruasula). We will take a Russula because it possesses one of the already mentioned cystides. Upon the underside of the cap are the radial lamella which bear the hymenia. Cut a [nece out of the cap parallel to the course of the lamella and make the thinnest possible transection of the whole per- pendicular to the latter. The whole section will resemble a comb, the sections of the lamella forming the teeth. With a low magnification, we shall see that the hyphfB go down from the cap-disk into the middle of the lamella, thence by repeated lateral ])ranching extend to the sides of the latter. A portion of these l)ranches swell into club- shaped forms and end blindly, but a greater part of them re- main slender and form outside of the club-shaped branches a compact layer of tissue, of roundish articulations, which is known as the sub-hymeneal layer, and is more or less sharply difi'erentiated from the inner tissue mass of the lamella, the so-called "trama," or woof. The club-shaped branches of the trama serve to give the needed stiflness to the lamella. The basidia and the paraphyses spring from the sub-hymeneal tissue, Fig. 89. They are nearly parallel with each other and set perpendicular to the sides 17 258 REPRODUCTION OF FUNGI, of the lamella, forming the hymenium. The basidia, 6, are club-shaped. At their flattened ends are formed four slender branches, c, the sterigma, which swell out at their ends into an ellipsoidal cell, the basidia spore, sp. These spores, after attaining their full size, remain smooth in most cases, but in many Russula species, have short spines on their surface. See Fig. 89. They are separated from the sterigma by a division wall and finally fall ofi". The spore carries with it a small portion of the sterigma. The paraphyses,^, are smaller sterile basidia. So far the toad- FiG. 89. Itussula rubra, a portion from the hymenium. sh, sub-hymeneal layer; h, basidia; s, sterigma; sp, spores; p, paraphyse; c, a cystid. X 540. stools and mushrooms agree wdth the description of the agarics. But in the agarics occur a few cystides, c, between the basidia and the paraphyses, which are as stout as the basidia ; their pointed ends protrude beyond the general surface of the hymenium, and with their slender base pen- etrating the sub-hymeneal layer, they represent as direct branches, the median elements of the trama. AH these elements named above are separated at their base from the hyphas by division walls, contain finely granular plasma, and often single drops of oil. HYMENIUM OF THE MOREL. 259 In order to become acquainted with the highly devel- oped form of the hymenium of the Ascomycetem we will select for examination, Morchella esculenta. Dried speci- mens may be soaked out and used, but fresh plants are naturally to be preferred. This well-known morel has an irregularly egg-shaped, stalked fruit-body, which conceals a simple cavity within, and whose upper expanded part is laid in deep folds. The sunken portions are lined with hymeneal tissue, which has not been developed in the projecting ribs between. Make a section per- pendicular to the surface of some one of the depressions. The hy- menium consists of spore sacs laid almost parallel with each other, (asci) sap filaments (paraphyses), Fig. 90. The spore-tubes, «, are nearly cylindrical and contain in their upper part eight ellipsoidal single-celled spores, closely pressed together. The ascus also contains a highly refractive epiplasm. The paraphyses are brownish filaments, articulated with division walls and slightly smaller at the top. The upper cell is the longest. The filaments are not as long as those of the asci. Both elements are the ends of the hyphte of the closely-interwoven superficially-extended, sub-hymeneal tissue. This rests on the loosely-built hy- phoe tissue of the fruit body. Treating the section with potassic iodide of iodine colors the epiplasm of the asci reddish-brown. This is a characteristic reaction .for epi- plasm and has recently been designated the glycogen reac- tion (4). A characteristic peculiarity of this reaction Fig. 90. A par: of the hy- menium of Morchella esculenta. a, asci; p, paraphyse; sh, sub- liymeneal tissue. X 2J0. 260 STRUCTURE OF LICHEN FUNGUS. shoAvs itself by the application of heat. To a section in water, stained with the iodine reagent, add a little more water but not enough to remove the color. Then gradu- ally and carefully warm it Avithout bringing it to the boil- ing point, laying it over white paper occasionally to see if tlie color becomes paler. When this takes place, rapidly cool the preparation, and if it is a large one it will be seen by the naked eye to take on its dark color again (5). By means of potassic iodide of iodine, one may trace the be- ginnings of the asci from some depth in the tissue of the sub-hymeneal layer. The paraphyses, the sub-hymeneal layer, and the tissue of the inside of the fruit-body are col- ored at the same time a yellow, or yellow-brown color. The fungus in the thallus of the lichen belongs, with rare exceptions, to the jLscoviycetece. The Pliyscia ciliaris is rich in fi-uit. The apothecium is saucer-shaped with an inclosing border formed from the thallus. This diminishes under the apothecium into a pedicel a transection of Avhich shows a radial structure, Avith a uniform thickness of rind layer following Avhich is a layer of gonidia around the whole circumference. The inside of the pedicel is occu- pied by a loose texture of hyphse. We make next a median longitudinal section through the apothecium. This shows the structure of the border of the apothecium constructed out of the tissue of the thal- lus. The gonidia layer extends to the edge of this bor- der, from which at intervals cilia-like processes put out. The style Avidens to inclose the hymenium, Avhich rests on its central fundamental tissue. The hymenium is broAvnish. It consists of a great number of long, ex- tremely slender, jointed filaments, the paraphyses, between Avhich, far less numerous, stand the club-shaped spore- sacs, the asci. The latter are always of different age, the ripe ones having eight broAvn-Avalled spores. The REPRODUCTION OF LICHENS. 261 £- spores are ellipsoidal, bicelliilar and at the boundary of the two cells a little contracted. Both elements spring from a felted, uniformly colored, horizontally extended layer, the sub-hj^meneal layer. This rests upon the central tissue of the style from which it is distinguished by its brown color and its lack of air-filled spaces. While we have seen that the hyphaj of the thallus are not colored blue with chloriodide of zinc, the hymeneal tissue is colored a dark-blue by the application of a little potassic iodide of iodine. The walls of the hymeneal elements are formed out of a particular mod- ification of cellulose, which is known as starch cellulose. Examining the thallus of this lichen with a magnifvinof glass we shall find little wart- like elevations standins: (r-^^^v*^J-,,>^i>i-. v^'S here and there singly or ßii''^^P^'^'<^'^^^^m in groups. If we make delicate transections in considerable n u m b e r through the thallus we shall, with some of them, hit one of these elevations in such a way as to show a section like that represented in Fig. 91. This is the spermagonium, an egg-shaped form, sunk in the thallus and having an open pore or mouth, sj). It occupies nearly the whole depth of the thallus, is sur- rounded on the sides by the gonidia layer, and has within a mass of very delicate, nearly radially-arranged fihmients with short joints, the sterigma (see the figure). The longer axis of the organ is occupied with a cylindrical cavity which contains short rod-like spermatia which have Fig. 91. Transection of thallus of Phijscia cUiaris througli the middle of a spermago- nium, sp; c, rind layer; m, pith; ^, gonidia layer of the thallus. X 90. 262 LITERATURE OF THE LESSON. been separated from the ends of the sterigma. These es- cape through the opening at the top of the spermago- nimn. In the CoUemacece it has been demonstrated that the function of the spermatia is that of the male genera- tive product (6). In other lichens their function is still unknown. Notes. (1) See de Biiry, Monatsber. d. k. Akad. d. Wiss. in Berlin für das Jahr 1865, p. 15; Kiiy, Bot. "Wandtafeln, p. 68; Frank, die Krankheit d. Pflanz., p. 454. (2) de Bary, same work, 1866, p. 206. (3) See de Bary, Morph, u. Pliys. der Pilze, p. 112; Goebel, Grund- züge, p. 143. In both the rest of the literature. (4) Leo Errera, L'6piplasme des Ascomycetes, 1882. There also the literature relating to epiplasma. (5) 1. c, p. 45. (6) E. Stahl, Beiträge zur Entwicklungsgeschichte der Flechten, Heft I, 1S77. LESSON XXV. Reproduction of the Mosses. The Marcliantia polymoijjha, already known to us, is most rapidly propagated in an asexual or vegetative way by means of asexual buds orgemmse. They are common in the Hepaticem generally and appear in most exquisite form in this species. They are produced in the Marchan- tia in cup-shaped receptacles on the back side of the thallus. The cup has a beautifully toothed border and the vivid green gemnife are found at the bottom. A longitudinal section through the cup, parallel with the long axis of the thallus, first narrows and then pretty suddenly widens out- ward to the edge. The tissue forming the air-chambers continues up the outside of the cup to the upper half of its outer extension. The base of the cup is occupied with club-shaped papilltB whose membrane is transformed into mucilage. BetAveen these club-shaped hairs are occasional bicellular hairs whose upper cells are divided first by trans- verse walls and subsequently by longitudinal wallstill they at last attain a considerable lateral extension, and finally become several cell-layers thick in the middle and quite biscuit-shaped inform (1). The single-celled styles are easily parted leaving the gemnife loose in the cup, from which they are soon discharged by means of the swelling mucilage which is produced in the bottom of the cup l)y the club-shaped hairs. The little notches on the side of the gemm« form the vegetative points whence are pro- duced short papilke. The cells of the gemmae are rich in chlorophyll ; still on both surfaces of the organ are found large chlorophyll-free cells, which keep near the middle (263) 264 EEPRODUCTIOX OF MARCHANTIA. but are otherwise irregularly distributed. In some of the border cells are oil-bodies. When the gemmfe are sown and germinate, these chlorophyll-free cells develop in a day or two on the under side into root-hairs, and on the upper side into the tissue of that side (2). The sexual reproductive oigans of Marchantia are placed on special receptacles. We "will examine those of M.poly- morpha (3). The male and female receptacles are easily distinguished, the former presenting disk-like and the latter umbrella-like forms. The two organs are produced -f I, V^ Fig. 92. Mardiantia polymorpha. A, optical trail .«ection of a nearly ripe anthe- ridiiim ; ]), parajiliyse; B, spermatozoids fixed with a Ifc Solution of perosmic acid. A X yO; B X tiOO. on different plants. The receptacles together with their styles represent the transformed branching of the plants. By making a delicate section through the pistillate recep- tacles, we see that its structure conforms to that of the thallus, its upper surface answering to that of the back side of the frond and the under side of the receptacle to the under or ventral side of the frond, being provided like that with rhizoids and scales. The antheridia, Fig. 92, A, are sunk in special cavities in the open side of the male organ. The section shows that each cavity contains but ANTHERIDIUM OF MARCHANTIA. 265 one antheridium too-ether with a few short sinofle-celled paraphyses, p. The cavity closes over the antheridium with the exception of a narrow canal which is left open. The antheridium is an oval body Avith a short pedicel and has an outer membrane of a single layer of cells contain- ing chlorophyll. The special mother-cells of the sper- matozoids are produced by successive right-angular cell divisions, and form a series of transverse and longitud- inal rows in the nearly ripened antheridium (see the figure) . Just before the ripening of the mother-cells of the spermutozoids the}'^ are rounded out and separate and finally burst the enclosing membrane of the antheridium at its apex, and the small round cells escape. If we put a drop of water on the top of one of these gi'owing receptacles, we shall see it spread rapidly over the whole surface and soon become milky-white. A high magnifica- tion shows it to be filled with numberless spermatozoid cells. They remain for a short time at rest after which the cell membrane begins to swell up and finally bursts and the spermatozoids escape into the water. The sper- matozoids are relatively very small, have a filiform body and two long cilia, and attached to their posterior end a minute bladder which they finally lose during their swarming. In order to see them distinctly, we may add to the preparation a drop of a one per cent solution of perosmic acid which will fix them most beautifully and allow us to study them very conveniently. See Fig. 92, B. The same result is obtained by the use of a trace of polassic iodide of iodine. The female receptacle forms, as does the male, a radially arranged inflorescence generally consisting of nine rays be- tv/een which are eight rows of archegonia on the under side. This distinguishes it from the male organ. Still this difierence depends upon the earlier deflection of the 266 ARCHEGONIUM OF MARCHANTIA. vegetative point of the receptacle towards the underside. We shall see by the use of the magnifying ghiss that the row of archegonia lying between the rays is inclosed by a common, one-layered, rib-like membrane, bordered at the edge. By making a delicate, longitudinal section be- tween thumb and finger of a relatively young receptacle, o> 0^ / \n Mb I If Fig. 93. Marchantia polymorpha. A, young, B, open arcliegonium, after the for- mation of the beginning of tlie germ; k' , necli canal cells; k" , ventral canal cells; o, ovum; pr, perianthium. X 540. we shall easily find in some of them the female organ, the archegonium. The oldest lie near the border, the suc- cessively younger nearer the pedicel. The first, the ripened ones, show their necks beyond at the edge of the disk bent upwards ; the others run straight downwards. In an archegonium, which is nearly ripe, Fig. 93, ^, a short pedicel, a ventral and a neck part, may be distin- ARCHEGONIUM OF MARCHANTIA. 267 guished. The wall of the pedicel and the ventral part is composed of one layer of cells. The central cell of the ventral part is filled with an egg, o, and a ventral canal cell, k' h", which shortl}' ])efore ripening is separated from the egg. The nncleus of the egg is easily seen. The neck has a central canal running through it which is formed by four canal-cells whose division walls have been absorbed. The contents of these four cells have comminofled and formed a continuous strin«;. Between the archeo-onia are small leaf-like scales originating in the receptacle. In many preparations one will find the membrane which cov- ers and protects the whole archegonium layer. It consists of a single stratum of cells, is fringed at the border, and its cell often contains oily bodies. It is relatively easy to see the opening of the archegonium directly under the mi- croscope. Make a longitudinal section of the female flower which is raised but a little from the pedicel and lay it dry under a cover glass upon the microscope. When a ripe arche- gonium is found, add a drop of water at the edge of the cover-glass, keeping the preparation under observation, whereupon the archegonium will almost immediately open. The cause of this opening is in the swelling of the con- tents of the canal in the neck. The canal cells of the neck dissolve at the apex and their contents escape followed by that of the ventral canal cell. The homogeneous part of these contents forms a rapidly swelling mucilage which is distributed in the surroundin«? water. The OTanular con- es o tents lie in the water and gradually disorganize. Di- rectly after the emptying of the ventral canal cell, the central cell of the ventral part is rounded up. See Fig. 93, JB. On its anterior border, is often but not always found a clear spot, a germinal fleck or embryo sac. The intro- duction of the spermatozoids into the canal of the neck may 268 FERTILIZATION OF ARCHEGONIUM. be easily ohserved in this plant. For this purpose instead of pure water, we must add to the preparation a drop of water Avhich has been for a time in contact Avith a ripe male receptacle. The spermatozoids collect about and in the mucilage which has exuded from the archegonium, and one sees them enter the neck where they become invisible. A substance is secreted from the archegonium which af- fects the spermatozoid as a chemical irritant and deter- mines the direction of its movement ; so, when it reaches the exuded mucilage, it is gradually moved along in the direction of the opening of the neck of the archegonium. It is interesting to notice that the neck of an unfertilized archegonium does not close up, and that the archegonium in such a condition gradually perishes. But if water con- taining spermatozoa be added to the preparation and the egg he fertilized, the neck is shut up in a few hours by the gradual narrowing of it from above downward. If the preparation be laid by for twenty-four hours, the existence of a cellulose membrane about the fertilized egg may be easily recognized, which gradually thickens in the next following days. The fertilized archegonium, which one finds in a section, shows a brown shrunken neck, while the egg is already midergoing segmentation, Fig. 93, C. About the base of the archegonium there begins to develop from the foot of the same, a cup-shaped envelope, the perianth, pr, which incloses the whole growing^ archegonium. In a longitudi- nal section of the receptacle, which has already expanded its marginal rays, one may see fixed the living-green, fully- developed archegonia, with their l)road bases, and their apexes adorned with what remains of the neck part. From the fertilized egg, or ovule, is produced the sporogonlum which one sees, finally, in preparations made from the older receptacles. These sporogonia form a yellow-green ANTHERIDIA OF MOSSES. 269 oviil capsule with a short pedicel. The walls of the cap- sule consist of one layer of cells. By tearing the mem- brane apart with needles and examining with a higher power, we observe a characteristic thickening ring in the otherwise thin-walled cells. The 3'^ellow-brown spores are finely dotted. Between them are long slender cells, pointed at the ends and characterized by two brown, screw- shaped bands in their walls. They are the so-called "ela- ters." The interior of the capsules are filled exclusively with spores and elaters. The open capsule has the open- ing set round with several recurved teeth. The elaters are strongly hygroscopic, bend back and forth by a change in the humidity of the atmosphere and so help to scatter the spores. All Mardiantia do not have their reproduc- tive organs upon such elaborately constructed receptacles, and in other Hepaticeca these specializations generally are wanting. On the contrary, it often happens that the pedi- cel of the sporogonium is considerably elongated, and the capsule with the spores correspondingly elevated, which also promotes the scattering of the spores. For a study ot the antheridia of the true mosses, Musci, we will choose Milium Jiornum, a widely distributed plant, which forms remarkably fine and numerous male flowers in the month of May, and at the same time offers female blooms, or archegonia, for investigation. The former are much more numerous than the latter, the latter having sometimes to be sought for a long time. The male blooms are dark green, disk-shaped, inclosed in a rosette of foli- age leaves, the so-called envelope or perigoneal leaves. Towards the middle, the leaves of the bloom rapidly di- minish in size. In the axils of the outer, but also still more in that of the inner leaves, are found numerous an- theridia and paraphyses which extend over the whole apex of the stem. This ma}" be easily seen in a longitudinal 270 AXTHERIDIA OF MOSSES. section of the bloom, the section being made between the fingers, and the apex of the stem being tnrned downward in making it. This section shows that the stem is widened at the top and a little hollowed out also where the repro- ductive organs are inserted. The central conducting bun- die, peculiar to the Mnium species, is also correspondingly widened and ends in a chlorophyll-containing tissue which is spread out under the bottom of the blossom. The anthe- ridia and the paraphyses are easily made out and their structure ascertained. The antheridia are club-shaped bod- ies, somewhat contracted at the ends and borne on short pedicels. The cells of their one-layered walls contain nu- merous chlorophyll grains. The contents consist of small colorless cells whose division walls in the young state of the antheridium are clearly placed at right angles. If older an- theritlia are cut by the section, the exuding contents are seen to consist of rounded, ;idhcting cells, the spermatozoid cells in which the filamentous bodies of the spermatozoids may often be recognized. The chlorophyll grains at the apex of the ripe antheridia are l)rownish. Empty antheridia are opened at their apex. The paraphyses are simple cell fila- ments which gradually expand upward but again contract so that the uppermost cell is always sharp. The walls of the cells are often brown at the base of the paraphyses and some- times higher up also. They bear chlorophyll. Cross- sections made through the under part of the bloom show in an instructive way the distribution of the antheridia, their relations to the enveloping leaves and to the para- physes, also many transections of the antheridia them- selves. Still more satisfactory than the male blooms of the Mni- um are the red-colored ones of the PolytricJium species wdiich may be likewise found in the month of May. Se- lect Polytrichuin juniperinum. The perigoneal leaves dif- ARCHEGONIUM OF MOSSES. 271 fcr from the foliage leaves not only in color l)iit also in the fact that the single-layered sheath-part extends quite to the apex of the leaf. The formation of the green lamellae, characteristic of the Polyirichum, is limited to the nerve on the upper side of the leaf. On the red-brown perigoneal leaves which occupy the inside of the bloom and become rapidly smaller, are the green lamellee produced, and only on the outermost leaves at the point which is bent sharply outward. So the leaf appears finally to be reduced al- most to its sheath part. The antheridia and the paraphy- ses stand in the axils of the perigoneal leaves. The middle of the bloom is occupied with a vegetative bud which is a continuation of the central string of the stem. In Poly- irichum norynale, the male flower grows up through this. The antheridia have the same structure as in Mnium. The paraphyses form a long cell-filament at their under part, widen spatulate at top into a single cell layer. If one presses a bloom of the Polytrichum between the fingers, the contents of the antheridia will exude as a milky slime clearly visible on the red-brown leaves. The female blooms of Mnium Jiornum are generally not so easily seen as the male and one must hunt for them. The plant which bears them is much shorter and has darker leaves. The upper leaves close bud-like about the pis- tillate organ, the archegonium, to protect it. The median longitudinal section shows that the stem is not essentially widened at the top but is bhmted in a peculiar way. This may be a sure indication to us that we have to do with a female bloom even when we cannot find the archegonia. The central conducting-bundle of the stem is somewhat enlaroed under the bottom of the bloom and ends, as in the male flower, in a tissue containing chlorophyll. The perie- chsetal leaves while remainino; foliaceous in form diminish in size towards the middle of the bloom. In hermaphro- 272 ARCHEGONIUM OF MOSSES. dite flowers they form what is called the perigonium. There are but few archegonia in the apex of the flower so there must be an exactly median section in order to And them. The archegonia are in the main formed on the same plan as those of the Hepaticeoe, only that the pedicel is much more strongl}^ developed and but little di- minished below, forming the principal mass on the under half of the archegonium. The ovum appears relatively small on this foundation. It must be sought for imme- diately under the beginning of the neck which is here l)ut a little more slender than the ventral part. The chlo- rophyll contents of the cells make the archegonium less transparent, hence for the most part the ovum and the ca- nal cells of the neck will be visible only after the sec- tion has been treated with potash. In the axils of the perichaetal leaves are many short paraphyses. They con- sist of a series of short cells somewhat smaller towards the top, the lower ones often being brown. We will now undertake the study of the sporogonium of this plant, Mniinn Jwrnum. It is the so-called "fruit " of the moss. It consists of the capsule and the pedicel or style. The bottom of the latter is embedded in the tissue of the mother plant. The " hood " (calypti a) which arises from the enlarged archegonium, and which covers the young capsule, will be cast off" very early and will there- fore be diflicult to find. It is composed of one and in part also of two layers of elongated cells and is split on one side quite up to its slender apex. The apex ends in a brown point which corresponds to the neck of the archegonium. At the bottom where it was broken by the growing sporogonium it appears as if cut ofi". The top of the capsule from which the calyptra has been re- moved is covered with a lid which is provided with a short beak. It may easily be removed by means of a needle SPOROGONIUM OF MOSSES. 273 and then the edge of the urn-like capsule with its teeth come into view. The teeth constitute the peristome. The upper part of the style, which is transformed into the cap- sule, is called the apophysis. In the present case the lat- ter is separated from the capsule by a slight contraction and is distinguished by its brown color. In some of the foliaceous mosses the apophysis is much stouter than the capsule, as in the jSplachnacece. To study the structure of the peristome, w^e should cut a transection of the cap- sule immediately under the edge of the urn, and transfer it to the slide with the teeth upward. Adjust the mirror and view the object with reflected light using a low mag- nification. We observe that the teeth are inserted on the inner edge of the urn, are pointed, wedge-shaped and striated across. If we breathe lightly upon the object during the observation we find that the teeth l)end to- gether inward. They are hygroscopic and bending in- ward in moist weather close up the open capsule, while in dry weather they bend back outward and again open the capsule. There are sixteen teeth upon the edge of the urn. Put such a section, which has been cut open on one side, in a drop of water, laying it flat down, and place a cover-olass over it ; examininar it with transmitted liaht, first from its outside, we see that there is a double layer of cells, about the edge of the urn, which is composed of inclined, thickened, papillaceous, chlorophyll-bearing cells with colorless walls, browned only at the base, where they are easily detached all tooether from the brown edse of the urn. These cells form the so-called "rino;" at the edoe of the capsule and mark the place where the cover sepa- rates. By turning the inside of the preparation upwards, we see that the cross-markings on the teeth are caused by projecting ledges on the inside. Inside of these teeth are the so-called "cilia." Consequently, this plant has a 18 274 SPOROGONIUM OP MOSSES. double mouth-piece, while the Bryacem possess but one. The teeth and the cilia are flat lamellae which appear to be divided off" below into compartments, and to be cross- striped above with low projecting ledges on the inner surface. Below, it is united into a continuous membrane which is arched a little between each two teeth of the outer mouth parts. Each two cilia stand between two teeth and present themselves obliquely from the edge. Their edges are beset with serrate projections, the outer along their whole height, the inner only on their upper parts. The transverse ledges or projections of the free parts of the cilia end in these teeth. By means of these teeth the ed, one fixed with iodine thosc already emptied, as their solution, c and -DX 540. browu iuiier walls, and a star- shaped hole in the top clearly show. We get a com- plete view of the structure of the antheridium only when we examine it in profile. This view may be got by bend- ing the prothallium over a needle. This view is seen in Fig. 95, A, by which it is observed that the antheridium sets in the middle of a low arched prothallium cell, 2^, from which it is separated by a division wall. The wall of the antheridium consists in almost every case of two stories of lateral cells, 1 and 2, and a cover-cell, 3. The lower has a wider cell cavity than either of the upper ones. The side view of an empty antheridium, Fig. 95, B, Fig. 95. Poll/podium vulgare. A, a ripe and B, an empty antherid- ium; p, protliallium cell; 1 and 2, ring ceils; 3, cover cell. A and yond the ANTHERIDIUM OF FERNS. 283 shows the lateral cells much swollen and veiy prominent, the inner cavity of the antheiidium consequently much diminished and the cover-cell pressed flat and broken through. If, now, we return to the superficial view of the prothallium and examine the empty antheridium from above, we shall see that the side cells have no division walls, and we perceive that they are really ring-cells. The whole wall of the antheridium therefore consists of two superimposed ring-cells a;id the cover-cell. Cells of this kind are very rare elsewhere, but constantly reappear in the antheridia of the Pohjpodiacecß. The only devia- tion from this form of antheridium among the Poli/podia- cece is in the case where the antheridium is l)orne on a pedicel and consists of but one ring-cell. If we select a prothallinm which has not been wet for some little time, we shall not have to wait long for some of the ripening an- theridia to discharge their contents. The mechanism for the discharge of the antheridium depends upon the pres- sure which the ring-cells exert on the cell contents, as well as upon the swelling substance which is secreted among the peculiar contents-cells of the antheridium. The cover-cell will finally be broken, and the contents of the antheridium will be pressed out and the ring-cells will in- crease in size. The contents consist of isolated, spheri- cal cells, the spermatozoid cells, which coming out into the surrounding water remain at rest for a little while. As may be seen by a comparatively low magnification, a little filament is coiled up in each cell, the spermatozoid, and a central collection of granules may also be recog- nized. The walls of these cells after a few hours dis- solve in the surrounding water and the spermatozoids are set free. This takes place Avith a sudden movement which uncoils the spermatozoid. One spermatozoid es- 284 SPERMATOZOIÜS OF FERNS. capes after another. We follow these and observe that they move quite rapidly through the water and at the same time rotate about their axes. After al)out twenty or thirty minutes the motion begins to slacken and soon ceases altogether. During this last stage of the motion of the spermatozoid its form is not difficult to recognize, which may be done all the more easily if we add to ihe water- drop containing them a ten per cent filtered solution of gum arable and so diminish the rapidity of their motion (1). The spermatozoid, Fig. 95, (7, is formed from a band rolled into the form of a corkscrew, the twist being narrow at the front end and growing wider backwards. The forward end bears long, fine cilia. Within the poste- rior twist lie fine granules and often an inclosing sac may be seen. By the addition of a little potassic iodide of io- dine solution the spermatozoids are very beautifully fixed. At the anterior sinus of the prothallium we shall find the female reproductive organs, the archegonia. Next to the sinus are the unripe ones ; beyond, the ripe but un- opened, and beyond them still the opened and dead ones brown on the inside. These are very easily distinguished from the male organs. They rise out of the surface of the prothallium, in the form of short cylindrical elevations which bend away from the anterior sinus. These free parts of the archegonia are only the necks, while the ventral parts are sunk in the tissue of the prothallium. The neck is com- posed of a wall with a single layer of cells in four rows and a central canal whose contents in the ripe archego- nium, in the middle, are granular and in the periphery strongly refractive. This inner canal widens club-shaped above. Below it passes into the central cell of the arche- gonium in which is the ovum. The latter is scarcely dis- tinguishable. If we do not wet the prothallium for AECHEGONIUM OF FERNS. 285 several days before the investigation, we shall probably be able to witness the opening of an archegonium. Take an archegoninm, the contents of the canal of which are very strongly refractive. The opening may occur in a moment or we may have to wait a long time for it. The opening of the neck is caused by the pressure of the re- fractive, expansive substance in the canal on the walls of the neck. The four cells at the apex of the neck sud- denly separate and the contents of the canal pour out, distinfjuishinoj themselves as a colorless mucilase in the sur- rounding water, while the granular contents slowly disor- ganize. The emptying of the archegonium takes place interruptedly, first from the neck canal and then from the ventral canal cell wdiich lies next the ovum. Under especially favorable circumstances one may see the entrance of the spermatozoids into the archegonium. We shall increase our chances of this it we use an old prothallium having archegonia and a very young one rich with antheridia. Spermatozoids distributed in the water swim quietly by the unopened archegonia ; but, if the archegonium has opened, the spermatozoids for a measur- able distance round take the direction of the open mouth of the neck, and Avill be intercepted by the mucilage mass. Within this their motion lessens ; still, however, keeping the original direction and lollow down the neck to the ovum in which they are absorbed. As has been lately discov- ered, the neck of the archegonium secretes a substance which exerts a chemical irritation on the spermatozoid which determines the direction of its movement (2). The particular irritating medium in this case is malic acid, about 0.3 % of which enters into the mass which escapes from the neck of the archegonium. The spermatozoids will work their way into capillary tubes in the same man- ner if they contain a 0.01 % to 0.1 % solution of some base united with malic acid. For the spermatozoids of 286 ARCHEQONIUM OF FERNS. the true mosses, cane sugar is the specific irritating medi- um, while in Marchantia another substance, whose nature is not yet ascertained, is produced by the archegonium. It has been experimentally demonstrated that (3) a single spermatozoid is sufficient to fertilize the ovum. Several, indeed, penetrate into the archegonium, usually, of which only one is really utilized. These processes, howev&r, are not easily followed here on account of the lack of transparency in tke tissue of the prothallium. They may be much more easily seen in the Ceratopteris. We may, however, demonstrate here that the spermato- FiG. 96. Poll/podium vulgare. ^, uniipe archegonium; A", neck canal cell; A'", ventral canal cell; o, ovum; B, ripe open archegonium. X 240. zoid-s do not take their posterior sacs with them into the archegonium, but leave them in the mucilage in front of the opening. Sometimes the spermatozoids are so numer- ous that they crowd between each other and fill up the whole neck-canal with a filamentous mass and form besides a tuft about the opening. Finally, we will examine the archegonium in section. Make a median section of the prothallium. This may be facilitated by laying several of them together after first carefully removing every adhering grain of sand. The archegonium is, as we see in Fig. 96, A and B, provided with a ventral part embedded in the prothallium, and a SPORANGIA OF SELAGINELLA. 287 curved neck-portion. The neck-canal cells, W , and the ventral-canal cells, K'\ are now distinguishable. So also the ovum, o, with its nucleus. The ventral part of the archegonium is inclosed in a layer of flat cells. In the ripe, opened archegonium, B^ at the apex of the ovum, there is often seen a colorless place, the embryo sac, where the spermatozoid is received. Other sections not median v^Mll give us a sectional view of the antheridia. The Selaginella are heterosporic Lycojjodiaceoß. They have two kinds of spores and sporangia, which we will now examine in order to complete our view of the vascu- lar cryptogams. The Selaginella are also called ligulates because their leaves are provided at the base with a small tongue. Take Selaginella Martensii Sprg., a greenhouse plant. The fertile examples are easily recognizable by the spikes or ears borne on the terminal branches. The vegetative body of the phmt is spread out in a plane, and bears four rows of leaves in pairs which obliquely cross. In each pair the upper leaf is small, the lower considerably larger. The two series of upper leaves on the back side press against the stem with their upper side. The two series of under leaves on the ventral side are spread out flat laterally, with their upper side up. The vegetative bod}^ of the plant is therefore bilateral and dorsi-ventral, that is, there is but one symmetrical plane in which the plant is laid out in a right and left half, and with a dorsal and ventral surface. The fertile terminal spikes are four-sided and provided with four rows of leaf- flets of like form turned upwards. We study the struct- ure of the spikes in this wa}'. Putting it under the sim- plex and beginning at the bottom we take ofl' one leaf after another with a needle, in the axil of each of which we find an oval, somewhat flattened sporangium. We soon see that many of the sporangia are larger than the 288 SPORANGIA OF SELAGINELLA. others and are provided with a projecting knob. If Ave open one of these sporangia with a needle, four large spores which perfectly fill the sporangium, and whose walls are sometimes arched, make their appearance. If we open one of the smaller sporangia we shall find it filled with numerous small spores. The larger are female sporangia, macrosporangia ; the large spores, female spores, macro- spores. The smaller are male elements and are called microsporangia and microspores. The smaller spores are mostly produced in fours, and have three flat surfaces which come to a point on one side ; on the other, or rounded side, the wall is beset with netlike ridges. We meet the same relations in the macrospores, correspondingly larger. The walls of the microspores soon become dark brown ■while those of the macrospores remain much clearer. If we observe the leaves from which the sporangia have been removed, we shall see the ligula as tongue-shaped mem- branes close over the place of insertion of the sporangia. A further removal of the leaves from the spike shows us that the macrosporangia are much less numerous than the microsporangia, and are principal!}' confined to its lower part. The ripe sporangium opens transversely with two lips. Herbarium specimens soaked out may be used for the study of the vegetative cone and the sporangia. Sections of either fresh or soaked material are made beautifully trans- parent, by the use of potash lye. Notes. (1) See Pfefler, Uuters. a. cl. bot. Inst, zu Tübingen, Bd. i, p. 370 (2) The same work, p. 360. (3) Strasburger, Jahrb. f. wiss. Bot., Bd. yni, p. 405. LESSON XXVII. The Reproduction of the Gymnosperms. The phanerogams are divided into two large groups, those having naked seeds and those having covered seeds, the gymnosperms and the angiosperms. These groups are distinguished by differences in the structure of the flow- er, and in the processes of fertilization and germ-building, which we will consider first of all in the gymnosperms. We shall make acquaintance first with the structure of the male flower (1) of the fir tree, Pinus sylvestris. It ripens the pollen towards the end of May. Alcohol ma- terial may, however, be successfully used, but on account of its brittleness it should be soaked out a few days be- fore using in a mixture of like parts of alcohol and glyc- erine. Material thus prepared makes far better sections than when fresh. We first observe that the male flowers are placed in large numbers on the under parts of con- temporaneous or growing shoots. They are arranged in a t\ oi'der, and correspond in position to that of the two- needle branches which adjoin the blossoms in an inter- rupted series. The blossoms like the leaf bundles occupy the axils of the secondary leaves or scales. Three decus- sate pairs of scales are found on the style of the male flower. The lower pair is laterally placed in relation to the covering scale and the mother-shoot, a position deter- mined by the existing space relations involved, a position the reverse of that occupied by the first pair of leaves of the vegetative buds of the gymnosperms, almost without exception. Next to the scales of the short style come the stamens, closely compressed and mostly arranged in ten 19 Q289) 290 MALE FLOWER OF FIR. regular series. The axis of the blossom is elongated spin- dle shape. A single stamen removed and examined with the simplex appears round, the under side occupied b}^ two pollen sacs longitudinally inserted and meeting along a median line ; at the apex runs a short seam which extends upwards. A medum, longitudinal section through the blossom shortly before the flowering, Fig. 97, A, and Fig. 97. Pinus pumilio agreeing with Pinus sylvestris. D from P. sylvestris. A, longitmVmal section of a ripe male blossom. X W- Lorigitudinal section of a single stamen. X 20. C, transection of a stamen. X 27. Z>, a ripe pollen grain. X 400. treated to potash lye, will show the course of the vascular bundles in the axis of the flower, the single vascular bun- dles with which each stamen is provided and the insertion of the pollen sac on the stamen. In less perfect sections thin places will be found where the structure of single stamens may be still better made out, B. By making a tangential section of the blossom, POLLEN OF FIR. 291 we get a transection of single stamens, C, which we will take for more exact study. We see that the two pollen sacs come togethei- in the middle and when ripe are separated by a flat wall of compressed cells which are Anally inter- calated by one or more layers of flat cells containing starch. The pollen sacs are covered with the epidermis on their free surface, on the inside of which are mostly compressed cells. In the niiddle of the stamen, in the upper and under part of the wall which separates the two pollen sacs, runs a mesophyll stripe. The upper is the larger and is penetrated by a v^ery delicate vascular bün- dle. On the lateral edges of the stamens the epidermis projects in the form of minute wings. If they are suffi- ciently large they contain a little mesophyll. On the un- der side of the pollen sacs the epidermal cells diminish in size from both sides, and at the point of least development the sac opens. These pollen sacs very closely resemble the sporangia of the Lycopodiocece. In fact, the recent investigations in comparative biology have led to the con- clusion that the pollen sac of the phanerogams and the microsporangia of the cryptogams are homologous forms. If we now examine a pollen grain produced in this sac, in as fresh a condition as possible, we shall see that it has a central body on which are fixed the two lateral sacs, D. In the ripe blossom these will appear black being filled Avith air. Very pretty markings are seen on the surface. The inside of the central pollen grain proper is filled with a finely granular protoplasm and a large nucleus. Shortly before the l)lossoming, that is just prior to the opening of the pollen sac, the pollen grain is divided hy a wall, shaped like an hour-glass, which forms a lens-shaped cell on the posterior portion ; that is, on the side turned away from the [)oint where the wings are inserted. This cell is best seen when the grain lies on its side as in our figure. A cell quite like this is diflerentiated in the mi- 292 STAMINATE ORGAN OF JUNIPER. crospoie of the heterosporic PohjiJodiaceoe before the be- ginning of the process of development which leads to the formation of the sexual product. There it is considered a vegetative cell and may be so designated here. The wings of the pollen grain are produced late as^the devel- opmental history teaches, by the elevation of the cuticle between which and the inner thickening layer of the wall a watery fluid collects. "We Avill next take the male flower of Taxus haccata. It opens in March, but by means of alcohol material one may examine it at any time. The male flower is found in the axils of the leaves of last year's branches. It begins with some decussate pairs of scales which pass over to the f arrangement. The scales grow constantly larger and soon fall into a quite indefinite arrangement on the elongated axis of the shield-shaped stamen. The whole bloom as seen with a magnifying glass resembles not a little the fertile sporangia-bearing leaves of the Equisetum. By removing a stamen and examining with the simplex, we shall And that beneath the shield are inserted from five to seven pollen sacs. These have their bas^ affixed to the under side of the shield, and their inner side to the style. Laterally, they are mainly free next each other, and wholly so outwardly and at their apex. Make a median and also a tangential longitudinal section ; the former will show us the stamen and pollen sac in longitudinal section and the latter in transection. The pollen sac wi- dens outward, the section showing a wedge-shaped form. Both sections show that the w^alls of the ripe pollen sac are reduced to the epidermis and a layer of compressed cells. The walls of the epidermal cells are provided with thickened ledges, and when the pollen sac is separated from the style the epidermal cells show a considerable re- duction in size. By removing a pollen sac wall from the stamen, with a needle, we shall see that the thickened PISTILLATE ORGAN OF JUNIPER. 293 ledges on the inner and side walls of the epidermal cells are U-shaped. This thickening occnrs also on the epi- dermal cells of the outer surface of the shield. The pol- len sac is opened by the walls parting from the style and stretching. The pollen grains are ellipsoidal in form and beset with small knobs. Shortly before flowering, the end of the grain is diflerentiated into a small cell. In alcohol material the contents of the pollen grains are shrunken and misery iceable for the investigation. The pollen grains of Taxus are not provided with the bladdery inflations of the walls observed in the Pinus, nor do they occur in all the Abietince; but, on the contrary, they recur again immediately below the Taxus in the Podocar- pus. In many genera, more than one vegetative cell will be differentiated from the contents of the pollen grain whereby projecting cell bodies will be produced within the srain. Among the Abietince onlv the o-emis Pinus has simple vegetative cells. The female flowers of the Taxus baccata (2) are found on other individuals, since the plant is dioecious, and like the male flowers in the axils of the leaves of last year's branches. Fig.' 98, A. It blossoms, as we already know, in March, but alcohol preserves the blossoms very well, and such specimens serve the purpose of our investigation perfectly and are easily managed if they are permitted to lie in glycerine and alcohol for at least twenty-four hours. The blossoms apparently terminate a small shoot, but are in reality not terminal. Not seldom we find two flowers on the same shoot. Fig. 98, at *. In rare cases one meets with a malformation in which a growing foliage shoot springs out of the side of the blossom, Fig. 98, B. AVith a magnifying glass we shall perceive that the floral shoot begins wifh a lateral pair of scales upon which follow in a spiral order other scales which gradually increase in size. The blossom itself is inclosed in three decussate pair of 204 FEMALE FLOWER OF JUNIPER. scales from which only its protruding apex is seen. This apex shows a piinctiform opening, the micropyle. We must carefully arrange the shoot in order to get a median longitudinal section. Tlie section should be made through the middle of the pair of scales which stands next but one beneath the blossom. We should select for our ex- no. 98. Tnxiis haccata. A, tj'pical form of a branch with female flowers at the period of fevtillzalion ; at * are two ovules on the same primary shoot, natural size. B, a k'af with a floral bud in its axil, the primary f-hoot being turned aside. X 2. C, a longitudinal section through the common middle of a primary and secondary shoot; V, vegetative cone of the primary shoot; a, beginning of an uxilhis; e, be- ginning of an embryo-sac; n. nucellus; », integument; n», micropyle. X 18. amination a blossom towards the end of April, somewhat old and already pollinized, as it will be easier to cut and will in many respects be more instructive. If the section is made in the direction indicated, the image Avill be like that represented in Fig. 98, (7. The blossom appears FEMALE ORGAN OF JUNIPER. 295 not to be terminal on the primary shoot, this having ter- minated its (leveh)pment by the formation of a minute, secondary shoot in the axil of its uppermost scale, which shoot ends in the flower after it has produced three decus- sate pairs of scales. Laterally, from the insertion of the secondary shoot, is seen the vegetative cone, v, of the pri- mary shoot, pressed to one side. Here and there, the next scale but one to the last on the primary shoot forms a secondary shoot, which terminates with a blossom ; and, in rare cases, as we have seen, the primary shoot forms foliage leaves. Fig. 98, B. The pairs of scales which precede the blossom are to be looked upon as its foliage envelope, the blossom itself being reduced to an ovule. We see one of them in the form which terminates the secondary shoot. We distinguish in a longitudinal sec- tion the following parts : the envelope, i, with a small opening at the top of the micropyle, m, and within this the so-called bud-nucleus, the nucellus n, in the bottom of which und^r fiivorable conditions, by treatment with pot- ash, we may recognize a large cell, the beginning of the embryo sac, e (3). As the pollen sac corresponds to a microsporangium, so the ovule corresponds to a macro- sporangium, the pollen grain to a microspore and the em- bryo sac to a macrospore. Biological investigations (4) have discovered that there are important resemblances in the beginnings of these forms ; still at the same time showing that a progressive reduction befalls that which in the phanerogams lead to the first beginnings of the macrospore. On the other hand, there are no srounds for comparinof the integument with the indusium of the vascular cryptogams. The integument is a new formation appearing on the macrosporangium of the phanerogams. On the style of the ovule of Taxus is seen a small tissue-mound, a, which remains stationary 296 REPRODUCTION IN THE FIR. for a long time, till into June, but afterwards begins to grow and in the fall forms the brlMit red arilhis which covers the ripened seed. On the already pollinated bloom which we have taken for our investigation, we may find, lying at the apex of the nucellus, a pollen grain, which has driven a short tube into the tissue of the apex. It is the large cell of the pollen grain which is grown out into this tube, while the small vegetative cell is shrunken up. The inner covering of the pollen grain, the intine, forms the pollen tube, while the outer integu- ment, the warty covering of the ripe pollen, the exine, will be stripped off. The pollen grain lies here on the pa- pillose surface of the apex, while i^i other species of Taxus and its near relations, the apex is hollowed out (5) to re- ceive the pollen grain, forming the so-called pollen cavity. If we would learn about the contrivance by which the pol- len grain is brought into the ovule, we must make our observation in nature, during the flowering-time of the plant (6). If we examine a female jilant at the time the pollen is ripe and being discharged from the pollen sac, we shall see that each of these blooujs secretes a little drop of fluid from its micropyle. The pollen grain, borne by the wind, falls into this drop of fluid and is sucked in with it. The fir, Pimis sylvestris, will give us another and an extreme example of the structure of the pistillate blos- som in the conifers. It being monoecious, both forms of flowers are found on the same plant. The ovule does not, as in the Taxus, stand alone, but a cone is produced in which numerous buds are united together, inserted on scale-like processes. The small cones occupy the tip of the present year's shoots, singly or in clusters. They stand in the axils of the bracts, like the lateral, two-nee- dled branches inserted below ; but their position above, on CONE OF THE FIR. 297 Sj-r the shoot, corresponds to that of the branch forming long shoots. The small cones are for the most part capable of fertilization by the end of May and are noticeable in their relatively smaller size by their brown-red color. They are borne upright upon a stem, the stem being covered with brown scales. Use alcohol material which has been treated with glycerine for the investigation. Cut away the separate parts from the axis of the cone with a scalpel and examine them under the simplex separating them out with a needle for the purpose. It will be seen that standing in the axils of the delicate, reversed- oval, enveloping scales with a 'S^. fringed edge, Fig. 99, &, are similarly shaped, moie thickened, ,| smooth-edged scales,/^ provided |; on the inner surface with a me- "^ -^j dian projecting carina, c. The ,^^ latterare the seminiferous scales. ^' j At the right and left of these 1 i 4.1 1 ti • • i 1 Fig. 99. Pinus sylvestris. Sem- scales, at the bottom, is inserted ,^,,^^^^^^ ,^.,,^ ^,./^i,,^ ^,^ ,^.^ an ovule Avith the micropyle of ovules .« and the keel c; behind is , , . J , , , , ^ tlie covering scale, 6. On tlie each directed downward and out- ovule the integnment is grown ward. The edge of the integu- out into two processes, m. xv. ment of the micropyle is elongated into two right and left flaps, m. Bracts and seminiferous scales grow together at the base and so remain attached to each other when sepa- rated from the axis of the cone. The cones of Abieti)ice and other conifers will be considered as single flowers or as a mere receptacle for flowers according to the signifi- cance which one attaches to the seminiferous scales. They must be considered either as flattened metamorphosed axil- lary shoots partly grown to a bract, or as a placental growth of a carpel which has heretofore been known as an .«^ -:-'i^.. i^„—m 298 SEMINIFEROUS SCALES OF FIR. enveloping scale. In the first case, we treat each as a shoot in the axil of the bract, bearing two ovules ; in the other case, we consider it as a placenta bearing two ovules, placed on the upper side of its carpel. In the first instance, the inflorescence would consist of a cone composed of many fertile, axillary shoots, and in the other the cone would be a single bloom formed of numerous carpels. The remarkable structure of the seminiferous scale is explained in reference to the act of fertilization and so can be followed out only in fresh material at the time of pollination (7). As soon as the production of pollen begins in the male blossom, one will notice an elongation of the axis of the little cones, by which the seminiferous scales and the bracts which belong to them are separated a little. The pollen may now fall upon the erect seminif- erous scale, slide down, and, guided by the carina, come at last between the two processes of the integument. These subsequently roll up and conduct the pollen grains into the micropyle and to the embryo sac. After being fully pollinized the growing seminiferous scales soon glue their edges together with resin. Neither the bracts, nor the carina develop farther, the latter having no further function. The red color of the cone passes into brown and finally to green, the cone gradually taking a hanging position. We shall next examine another variation in the devel- opmentof the pollinized ovule of the conifers (8) . We have already learned that the time of pollination for the em- bryo sac of Taxus is in the first beginning of it. From this followed a fiu'ther development of the ovule so that a considerable length of time elapsed between the polli- nation and the fructification of the ovule. In Taxus the fructification takes place in the middle of June of the same year ; in the fir, not till the next year, thirteen OVULE OF RED-FIR. 299 months after the pollination. In the pine, the two acts are separated by but about six weeks. We shall use the fir for our investigation. It would lead os too far to follow step by step the development of the embryo sac, the be- ginning of the prothallium tissue, the endosperm, and the reproductive organs in the same, the increase in size and consequent diflerentiation of the wdiole ovule. But we will take it at the point when the ovum is fully developed and ripe for fertilization. This condition is reached by the common or red-fir, Picea vulgaris, about the middle of June the pollination following in the course of a few days. Alcohol material will be found better than fresh since the ovum will be tixed. It is better to put the separate scales rather than the whole cone in the alcohol ; and the material should, as heretofore recommended, be previously treated to a mixture of alcohol and glycerine, equal parts, for at least four and twenty hours before making the sections. We should first take a general view of the whole scale. It is an inverted oval, on the iuner surfiice of which the beojinnings of the two seeds are seen ; also the outline of the wings, which afterwards as thin lamella of tissue will be separated from the inner surface of the seminiferous scale. Beneath, on the outer surface of this scale, the bract is still to be found, now, however, quite small. We may easily remove the ovule uninjured from the seminiferous scale w^ith the needle in order to make a section of it. Make the longitudinal section between the thumb and fin- ger. The hardened under portion of the ovule will not so readily lend itself to section-making. So cut away the lower half with the shears and make the section through the soft upper part containing the nucellus and the embryo sac. Stainino- media are to be used with great caution since they stain the whole protoplasm of the ovum and 500 OVULE OF RED-FIR. nc easily render it untr.insparent. First use a low power and as we are looking upon a median section cut at right angles with the base of the ovule or surface of attachment, we shall see the various elements as represented in Fig. 100. The integument, ^, forms the outer envelope of the ovule and is separated from the nucelhis about half Avay up. The nucelhis bears pollen grains, ^j, on its apex, which lie partly within and partly without the tissue. The tubes, t, from these pol- len grains Avill eventu- ally penetrate the upper part of the nucellus in order to reach the em- bryo sac, e. The latter is elliptical in outline and tilled with endo- sperm, or more correct- ly prothallium tissue. The ventral part of the Fig. 100. Median longitudinal section of the archegouia, CI (corpUS- fertilized ovule of Pw-ea rwi^'firis Lk. e, embryo ßnlj^") mav be C'lsilv rCC- sac filled with endosperm; a, ventral part of '. ' ' J archegonium; c, neck part; »i, nucleus of ovum; Oguizcd but UOt SO easily nc, nucellus or kernel of the bud; i>, pollen grain .1 I- . .f . AVfl * on and in the bud-nipple; t, pollen tube which ^'^® UeClv part, C. VV llnin penetrates the nucellus; i, integument; s, seed eacll archeo"Onium is an wings. X9. . * T .• • 1 ovum, 0, (hstinguish- able in alcohol material by its yellow-brown color, in the middle of which is a nucleus, n. Finally, the attachment of the seed wings, s. If we make a section through a fresh specimen we shall OVULE OF RED-FIR. 301 find the same relations again, only that the contents of the archegonium will often be discharged. If the section touches an archegonium without opening it, the ovum will appear as a yellowish, foamy, protoplasmic mass in which the nucleus is scarcely distinguishable or at best has the appearance of a large central vacuole. The ovum soon begins to sutfer from the effects of the surrounding water. If it is desirable to preserve the section for a considerable time it is recommended to use diluted white of an egg for an examining fluid, and to make this still more durable a little camphor may be added to it (9). In such prepara- tions, the neck part of the archegonium is not difficult to see. It consists of from two to four stories of cells. Un- der the neck part is a small cell which corresponds to the ventral canal cell of the vascular cryptogam, the ovum parting to form it shortly before ripening. The ventral part of the archegonium is surrounded by a layer of flat cells rich in contents, like the covering which we saw about the ventral part of the fern. In order to know the number and position of the archegonia we must make a series of transections through the upper part of the ovule. By this means we shall see that from three to five arche- gonia are arranged in a circle at the apex of the embryo sac. Sections which touch the apex of the embryo sac present us with an apical view of the neck part of the ar- chegonia which is a rosette of from six to eight cells. If our material has been collected at the period of fertiliza- tion we shall eventually find pollen tubes which have penetrated to the ovum, and in the under part of single ovules we shall find a four-celled rosette which may be traced out into prothallium tissue in four uninterrupted tubes. The four terminal cells of these tubes produce the germ. The seeds ripen in October. It separates then easily from the seminiferous scale. The wings continue 302 EMBRYO OF RED-FIR. Oil the inside of the seed bet^reen them and the seminif- erous scale, the seed falling off later from the Avmgs, leaving a corresponding cavity in the same. Sections in both directions will show that the cells of the seminifer- ous scales are so thickened as nearly to obliterate their cell cavity. A part of the prothallium tissue is filled WMth reserve material which has been preserved as seed albu- men or endosperm in the seeds. It forms a sac inclosing the germ. This sac is open on its micropyle end and here the root end of the seed joins the remainder of the sup- planted nucellus. The germ appears like a cylinder which grows thicker toward the end of the cotyledon, and in consequence of being filled w^ith albumen is white and untransparent like the cotyledons. Make a lono-itudinal section between the fingers and examine it in carbolic acid diluted with alcohol. This makes the image very clear, far more so than potash lye or even chloral hydrate itself, so that each row of cells may be easily followed. We see Fig. 101, c, that the cotyledons do not reach quite a third of the length of the germ and between them at the base is the vegetative cone of the stem. The little stem or cauliculus which will be designated the hj'pocotyledon or h3'pocotyle, or in two planes at right angles, Fig. 102, E. These become subsequently the pollen grains, produced, like the spores, by fourfold division within their mother- cell. The walls of the anther are lined with so-called "tapestry cells", t, which are filled with yellow-brown con- tents, and arise from the innermost of the layers lining the chamber. In the next older flower-bud the walls of the pollen mother-cell are dissolved, the young pollen grains lie free, the tapestry cells have for the most part given up their independent existence, their contents being pressed in between the young pollen grains. The layer of flat cells, y, lying immediately beneath the epidermis is much developed and forms the fibrous layer, while the next in- ner layer has become compressed and disorganized. An older bud will show that the still unused tapestry cells, especially in the periphery of the chamber, have an intense yellow-brown color, and oily sparkling appearance, and thus form the oily substance which surrounds and adheres to the pollen-grains. All species of lilies behave like the Hemerocallis, but the diflerentiation of the anther comes later. The pollen mother-cells begin to divide in Liliu7n candidum, L. cro- ceum and other species, only when the flower bud is 2 cm. high. In transections of fresh buds the large tapestry cells will be very noticeable with their yellow-brown contents. The hypodermal cells as well as all others which are after- wards to be provided with ledge-like thickenings are filled with starch grains. Funkia ovata is a good plant for study, and behaves like Hemerocallis and Lilium; so also Agcqxmthus umbellatus. 310 POLLEN OF TRADESCANTIA. Tulipa und Hyacinthus orientalis are likewise good ob- jects. In Tulipa the filament is so much attenuated under the anther that the latter will rotate. In the hyacinth the anther almost sits on the perigone. Tradescantia Virginica is less easy to make sections of, but we will examine it in reference to the pollen grains. A transection of a bud about two-thirds grown shows us the tAvo halves of the antlier separated b}'^ a rather thick connective. The walls of the compartments are al- ready reduced to two layers of cells, and the thickened ledges are already developed on the inner one. The pol- len grains are embedded in a yellow-brown substance with whose origin from the tapestry cells we are already acquainted. The division wall between the two chambers is so fully developed and is so thick that there is scarcely any depression observable between them. At the point of insertion of the chamber wall upon the division wall the fibrous layer suddenly ceases, and at this point the separation takes place later on. An exami- nation of the surface of the chamljer wall shows in this case a longitudinal diminution of the epidermal cells and a transverse lessening of the fibrous layer with a most com- plete faikn-e of the thickened ledges on the outside walls of the cells. If Ave examine with the maijnifier the stamen of a bud just ready to blossom out, we shall see the beautiful yel- low anther on the violet colored filament beset Avith violet hairs. The dry pollen grains are noAV folded on one side, Fig. 103, A. In Avater, the fold is smoothed out Fig. 103. Tradescantiavirginica. A, pollen grain dry; B, in water; C, j'oung pollen grain in water showing the vegetative cell. X -^^0. STUDY OF POLLEN. 311 and the grain becomes ellipsoidal in shape, the previously folded side being more convex than the other. The mem- brane is striated with meandering lines. The folded side shows this structure also and is distinguished only by its brighter color and its somewhat weaker cutinization. In the finely granular contents are to be distinguished two clear, homogeneous spots, B. They are the two nuclei, of which one is vermiform and the other ellipsoidal. The rest of the contents of the pollen grain is pretty uniformly fine-grained. The pollen grain very soon begins to flatten, whereby the nuclei together with the rest of the contents are much compressed. Both nuclei may be seen very beau- tifully if the pollen grain be crushed in a drop of acetate of methyl green, or acetate of iodine green. The ver- miform nucleus is deeply stained and much elongated, after its exit from the pollen grain; If the pollen grain be put in the staining fluid without being crushed, the nu- clei will be seen in their natural position, the vermiform one somewhat more deeply stained than the other. The rest of the grain will remain uncolored. If the pollen grain be crushed in a drop of water to which has been added a solution of potassium iodide of iodine, numerous small blue starch granules will be seen among the yellow- brown contents. (3) If we now return to the 3^oung flower-bud and take one about 6 mm. long and crush the anther in water, we shall find that part of the grains have but one nucleus and others tw^o, l3ing close together as in Fig. 103, G. The two nu- clei are separated by a wall which incloses one of them, together Avith a little protoplasm. This flat cell which is almost circular in form always lies on the flat side of the pollen grain where the fold in the membrane is afterwards found. In a somewhat older bud this cell is found to be separated from the wall of the pollen grain and lies free in the contents of the graiu. 312 POLLEX OF CEXOTHEEA. The pollen gniins have here become longer and corres- pondingly slender and pointed at the ends. With the ex- ception of the two ends they are filled with this nnclei. (4). In nearly ripe pollen grains the definite demarca- tions of the nuclei disappear and they lie free in the grain more or less elongated in a vermiform shape. In comparison with the gynmosperms, to which they lie very near, we should hold the small cells to be vegetative ; but really it is the generative cells Avith their highly stain- able nucleus which are concerned in the fertilization. The difference in the staining quality of vegetative and gener- ative nuclei is far more striking than in Tradescantia. We may make the above described examinations, in the case of the younger pollen grains in pure water, but in the older stages we must use meth}l green or iodine green with acetic acid. The species of Lucojum act quite like the Tradescantia. If one opens a bud of OEnothera biennis which is about ready to blossom, he will find that the anthers are already open and the pollen escaped. Afterwards viscous fibres are seen between the anthers. Putting one of these on a slide, it appears under the microscope an extremely del- icate thread partly stretched out straight and in part wavy. The pollen grains wdien dry are untransparent but their triangular form is apparent. In water and with a higher power we see that they are flattened, equilateral triangu- lar bodies with warty projecting corners. At the base of each of these warts is to be seen a ring-shaped thickening of the pollen membrane. The contents of the ripe pollen grain are tinely granular, and the two nuclei are seen with great difficulty. The pol len membrane is stained a red-brown with sulphuric acid. The acid causes a very thin yellow layer to be lifted up upon the body of the grain, forming folds from an inner, thicker, red-brown la3^er. Both membranes are united in the walls of the warts. From the lateral walls POLLEN OF ALTH^A. 313 of the warts fine teeth project toward the inside so that these walls appear to be porous. The apex of the wart is dissolved by the acid. The fine filaments which connect the pollen grains are insoluble in water, alcohol, potash lye and sulphuric acid. In 25% chromic acid, the pollen mem- brane dissolves, the strongly cutinized parts rather before the others, the latter being the caps upon the projecting warts, which remain colorless and swollen. These finally dissolve and even the viscid filaments between the pollen grains cannot withstand chromic acid. A pollen grain taken from the stigma of an old flower will show the pollen tubes already grown out, commonly from only one wart, but if from another also, then only just outside the latter. The membrane of the pollen tube passes into the lateral "walls of the wart. An intine layer as distinguished from the outer membrane does not occur (5). Instead of (Enothera one may use Epilohium or Fuchsia. AVe will now examine some peculiarly formed pollen grains. Those of the Malvacece are of extraordinarily large size. The pollen of Althoea rosea in Avater are globular, untransparent and beset with colorless spines. They become beautifully transparent in carbolic acid, and in chloralhydrate, less so in oil of cloves, still less in lemon oil. The best preparation is with carbolic acid and so we will use that. A superficial vieAv shows us that the colorless pollen membrane is beset with large pointed spines at nearly uniform intervals. Between those are others ; short, blunt and of varying thickness. Eegularly distributed circular openings appear in the membrane. The surfiice of the membrane is finely dotted. The con- tents of the grain are imiformly finely granular, and the nucleus is made out with much difiiculty. An optical transection of the grain show^s us clearly the form of the 314 POLLEN OF THE PUMPKIN. large and small spines and the canals which perforate the membrane. An extraordinarily delicate but really exist- ing intine may be traced only as the outline of the con- tents. It is papillately arched a little in the canals of the exine. In concentrated sulphuric acid the exine soon stains a red-brown and shows its structure then very dis- tinctly. Most of the pollen grains of the Malvaceoi act like those of AllJicea. In Malva crispa, a frequently cultivated spe- cies, the pollen grains are like those of AliJicea, except that the spines are all alike. Between the spines are dis- tributed the openings in the membrane, the rest of which appears finely dotted. The large pollen grains of the Cucurbita species have long received special distinction on account of the cover which closes the opening in the exine. In water, yellow oil drops exude from the surface of the exine, the grain soon l)ecomes empty of its contents and tlie sti'ucture of the membrane may then be distinctly seen. The exine is beset at regular intervals with large, and between these with many small spines. The openings are round. The cover is lifted up on one side or all around, l)y the papil- lately arched intine. It has the structure of the adjoining exine and bears one or more spines. Very good prepara- tions are got by using lemon oil, less serviceable ones in oil of cloves, but those in chloral hydrate and those in car1)olic acid are preferable. In each case that medium most suitable for clarifying it is to be sought for. By op- tical transection in lemon oil or chloral hydrate prepara- tions we are able to demonstrate the position of the cover within the exine, and find it widened inward somewhat towards the base. Under the cover the swellino;s of the intine may be seen. The oil drops on the exine are col- DEVELOPMENT OF THE POLLEN TUBE. 315 ored blue with sulphuric acitl. The exine becomes grad- ually browu. The cover is pushed off by the swelling contents. In 25% chromic acid the whole pollen mem- brane is soon dissolved, but the intine withstands it longest and is, at the moment where the exine disappears, clearly discernible as a greatl}^ swollen homogeneous membrane. The pollen grain has previously become empty, which fjx- cilitates the examination of the intine. In sulphuric acid, on the contrary, the intine is immediately dissolved, while the exine remains and the exuding contents of the grain gradually assume a rosy tint as in other cases. Of compound pollen grains, which occur both in mono- and dicotyledons, we will look at those of CaUuna vulga- ris first. The grains are united into fours and mostly tetrahedrically grouped. The pollen membrane shows but small protuberances and mostly but three openings to each grain. The species of Erica, Azalia and Uliododendron are essentially the same as those of Calluna. In the Aca- cia &t^qc\^q, especially in Mimosa (6), the pollen grains form groups of four, eight, twelve, and sixteen, or even more. In a sugar solution of from 3 to 30% which contains 1.5% gelatin, most pollen grains will put out three tubes, in which the streaming of the protoplasm may be beauti- fully *eeu. The formation of pollen tubes takes place rapidly and surely in a 5% solution of sugar with 1.5% gelatin with pollen grains from the Peonia, Slaphylea and also when they are taken from a freshly opened flower of Tradescantia. The most favorable objects are furnished by the species of Latliyrus in 15% sugar solution with 1.5% gelatin. The solution must be freshly prepared and the experiment is best made in a hanging drop in a moist chamber. See page 230. 316 LITERATURE OF THE LESSON. Notes. (1) For staraeus aud pollen, see v. Mohl, Ueber deu Bau und die Formen der Pollenkörner, 1834; Fritsclie, Ueber den Pollen, Mem. de sav. Strang, 1836 ; Naegeli, Zur Entwickelungsg. d. Poll, ble den Phan., 1842; Schacht, Jahrb. f. wiss. Bot. Bd. ii, p.l09; Warming in Hanstein's bot. Abh. Bd. ii, Heft ii; Strasburger, Befr. u. Zellth., p. 15 und Bau der Zellhäute, p. 86; Elfving, Jeu. Zeitschr. f. Naturw. Bd, XIII, p. 1; Goebel, Grundz. d. Syst., etc., p. 398; Luerssen, üruudz. d. Bot., III Auf., p. 359; Med. Pharm. Bot., Bd. ii, p. 198; Prantl, Lehrb. d. Bot. iv Aufl., p. 192. In the above quoted works is the rest of the literature. (2) Sachs, bot. Ztg., 1862, p. 242. (3) "Warming in Hanstein's Bot. Abh. Bd. ii, Heft ii; Goebel, Grundzüge, p. 409. (4) See also Elfviug, Jauaische Zeitschr. f. Naturwiss. Bd. xiii, p. 12. (5) Strasburger, Bau d. Zellh. p. 95. There also the history of its development. (6) Rosanoff, Jahrb. f. wiss. Bot. Bd. iv, p. 441 ; Engler, the same periodical Bd. x, p. 277. There also the literature. LESSON XXIX. The Gyneceum of the Angiosperms. We will now take a general survey of the structure of the ovary (1) using the Delphinium ajacis or garden larkspur for our purpose. Take an old flower from which the petals and stamens may be easily removed, and notice the three pistils which remain standing in the middle. We shall see first the green swollen part, the ovary, the slender rose-colored portion into which the ovary narrows itself, the style. This tinally ends in the stigma which is not in this case especially developed but simply terminates the style. Make a transection through the three ovaries, and examine with a low power adding a little potash lye. The transection shows us for each ovary a single cavity, Fig. 104. Apparently it is a single iruit-leat or carpel which torms each ajads. Transection of ovary. The carpellary-leaf is folded to- °''-'''-\- "; """'^'^ ^''■^"= "'• •^ _ '■ •' ^ vascular bundle ; 7;, pla- gether inwardly and its edges o;rown centa; s, embryo seed. fast to each other forming the "ventral seam," so called, which we find in the middle of the ovary on the side which faces the centre of the flower. An ovary formed of one carpellary-leaf is monocarpous, but when several such ovaries are united in one, as in this case, the flower is said to be polycarpous. The ovaries are in this example free to their base where they are inserted on the receptacle and are called superior. The whole female generative apparatus, whether it consists of one or more pistils, is designated the gy necium. (317) # 318 STRUCTURE OF THE OVARY. Our transection clearly shows the furrow on the ventral side, and by the use of a higher magnificat ion w^e can eas- ily trace the epidermis at the outside at this place through the whole thickness of the wall and see that it is continued into the epidermis of the cavity of the ovary. Even sto- mata are found in this inner epidermis. The ovary walls are penetrated by a number of vascular bundles most of which show on the backside, but some near the edges of the carpellary leaf on the ventral side. The edges of the car- pellar}' leaves are somewhat swollen, and, in the cavity of the fruit receptacle, is formed into a phicenta,^^. From this the ovules, s, originate in two series, corresponding to the number of the placentae. Vie shall give particular atten- tion to the ovule later on, and to this end lay aside our preparation. In the blossom of Butomus umbellalus are six ovaries. But these ovaries are free only in their upper halves, while they are grown together laterally below and cannot be separated without injury. The pistil is ver}^ short and the upper edge of it is the stigma. We must prepare transections both of the free and the united parts of the ovary. In those of the upper part we may easily distin- guish the carpellary leaves as in the Delpldnium, but in the sections from below the}' cannot be isolated intact laterally from each other. lu the Butomus we have an interme- diate form between the polycarpous and monocarpous flow- ers, and this will serve us as an example of a compound ovary formed out of more than one carpellary leaf. A marked peculiarity' of the Butomus is seen in the fact that the ovules do not spring alone from the edges of the carpel- lary leaves, but rather from their midd le and from their whole inner surface. The whole wall of the ovary is beset with them and acts as a parietal placenta. At the point of in- sertion of each ovule a fine vascular bundle may be seen, STRUCTURE OF THE OVARY. 319 which provides for the ovule. These are branches of larger bundles lying deeper in the tissue. The carpellary leaves of the Liliacece are superior. Take for our investigation a tulip, hyacinth, a lily or a Hemei'ocallis. In the tulip the three stigmas rest on the ovary without a style. In the hyacinth the style is short, the stigma small, dark, divided into three parts. In the lily the style is long, the stigma three-parted. In Hemero- callis the style is ver\' long with a three-parted, still very small stigma. A transection will show us a compound ovary formed of three closed carpellary leaves grown to- gether. No boundary between the parts either at the side or in the middle is to be recos^nized here. A continuous epidermis covers the Avhole organ. Three carpellary leaves form a compound ovary with three cells. Each of the three carpels which form this ovary has two series of ovules, lying along its two edges. The placenta there- fore lies in the inner angle of the ovarj^ cell. The pla- centa is therefore marginal as in Delphinium, and since it springs from the angle of the ovary which turns toward the middle it is called "central." A transection of the pistil of Hemerocallis shows us a three-cornered style, in which toward the three edges are distributed three vascu- lar bundles. A longitudinal section of the pistil which cuts the stigma will show that the latter is developed into long papillte on its upper surface. This is the most com- mon appearance of the stigma. But in the Hemerocallis we find the cuticle of the papillt© raised up by the pressure of muciluge formed beneath. The cuticle is spirally striped and conformalily to this the elevations follow a spiral line. The cuticle Avill finally be separated from the inner layer and eventually removed from the papillte. The other Liliacece might likewise show us a holloAV style, but in most flowers it is solid, filled with cells with swollen 320 STRUCTURE OF THE OVARY. side walls, or with those entering from the lateral tissue, between which the pollen tube can easily grow down- wards. The Primularia species have a superior ovary. They are dimorphic, that is they show short and long styled ovaries and stamens inserted above and below on the co- rolla. A median longitudinal section through the ovary shows us that the axis of the flower continues into the cavity of the ovary and expands into the shape of a toad- stool. In the middle this expansion rises into the style of the pistil in a papillate form and the whole upper surface is beset with ovules. We have in this case a free central placenta. The walls of the ovary nowhere join this pla- centa, as is seen by a transection in which these walls ap- pear as a free ring about the central placenta. The point of juncture or suture is not visible in this ring: so, in or- der to determine the number of carpellary leaves which go to form the ovary, we must refer to the number of the other parts of the flower and to the circumstance that in many Primidaceoe the seed capsule opens at the top with five teeth, and thus conclude that there are five. In Primula itself the number of teeth with which the capsule opens is indefinite. Instead of the Primula we may take species of the Lysimachia or Anagallis for an investiga- tion, as they have all their ovules on a free central pla- centa. We shall now take an inferior ovary — that of the Epi- jpactis palustris or some other orchid. The brown ovary lies beneath the other floral parts. We Avill select for our section a young fruit over which the floral leaves have already begun to be brown. The transection is very instructive. It shows us a simple ovary which bears on the walls at equidistant points three double pairs of placentae. The placenta divides repeatedly on the edge OVARY OF THE ORCHID. 321 and bears a large number of ovules. Upon the outside of the ovary arc six projecting ril)s, three of them corre- sponding to the place of insertion of the placentai within and the other three, especially large ones, alternate with these places. Each rib is furnished with a Vascular bun- dle or with a complexity of them, and besides this a small one at the place of separation of two placentie. If we were to bestow no thought upon it, this section would seem to agree perfectly with one made from a superior ovary, the ovary would appear to be formed from three carpellary leaves and the pairs of placentae to be produced on the united edges of two such adjacent leaves, and the three ribs which alternate with the places of insertion of the placenti^ä would be held to be the midribs of the leaves. But as this is an inferior ovary the case is far less simple. TVe may suppose either that the inferior ovary is formed from an excavated floral axis terminated above with car- pellary leaves and that from the latter the placent» con- tinue downward into the excavation, or we may suppose that the carpellary leaves are grown to the hollow floral axis ; consequently the outer part of the wall of the in- ferior ovary belongs to the stem, and the inner part to the carpellary leaves. The latter supposition is decidedly to be preferred, but it has no other than a phylogenetic value, that is to say, we represent to ourselves that in the course of time the inferior ovary is thus produced. But in reality in the object itself there is no moment in its developmental history when it anatomically answers to this supposition. AVe must be content, therefore, to demonstrate that the structure of this inferior ovary is not essentially different from that of a simple, polymerous, superior ovar3\ By examining a ripe fruit capsule of Epipactus^ we shall find that, as in most orchids, the wall splits into six longitud- inal openings, the segments between remaining united at 21 322 OVULE OF ACONITUM. the top and bottom of the ovary. Three of them are broad and fertile and three narrow and sterile, the Litter corresponding to the median ribs which we saw in the transection of the ovary, forming the intermediate sections. The three fertile segments bear the placentae. We shall next nndertake to investigate the structure of the ovule and the process of its fertilization in the angi- osperms. In order to get a view of the separate parts of the ovule, make a transection of the ovary of Aconitum Napellus or of some other spe- cies of this genus. Take a flower, just turning blue, strip otF the floral parts and make a section of the three ovaries at once, care being taken that the section is really at right angles with the axis of the ovary. Make a considerable number of sections, so as to be sure to have one which cuts the ovule at the right place. Look them over and select the best section, and in case it is not thin enough a little potash lye will help make it transparent. The im- age will be almost identical with that of the previously examined Delphinium. Still the structure of the envelope of the ovule is a little diflerent, and this ditterence gives it the preference. The ovary is monomerous. The ovules spring from a placenta formed on the infolded edge of the carpellary leaves. They are inserted with a small style or funiculus, y, whose free part is quite short, but the rest of it is grown fest to the body of the ovule and forms the so-called raphe, r. In the body of the ovule we distin- FlG. 105. Aconitum Napelhis. Xiongitiidinal section of ovule;/, funiculus; r, raphe; ti, vascular bundle of funiculus; ie and ii, outer and inner integument; n, nu- cellus; ch, chalaza; e, embryo sac; a, antipodal cells; o, egg; nc, nu- cleus of the embryo sac ; m, micro- pyle; or, wall of the ovary. X 53. STRUCTURE OF OVULE. 323 gnish, first of uU, the inner cone-shaped muss of tissue, the ovule nucleus, or nucellus, n. This corresponds to the macrospore of the vascular cryptogams. It is sur- rounded by two integuments : an inner, ^^, and an outer, ie. The inner one is developed all around down to the base. The outer one fails on the side of the funiculus, with which it is laterally connected. Between the upper edges of the inner integument is a free opening or canal, the micropyle, m, down to the nucellus. In the funiculus a vascular bundle coming from the placenta may be traced, sometimes, but not always, quite down to the base of the nucellus. At the base of the nucellus is a mass of clear tissue called the base of the ovule, or chalaza, ch. In the axis of the nucellus is the lar<>:e cavitv-forming cell of the embryo sac, e. At the base of this are some spherical cells, which in the Aconitum and in Ranunculacem generally are strongly developed ; they are the so-called antipodal cells, a. In specially favorable cases they may be seen to oc- cur in threes. At the apex of the embryo sac a small cell may be made out — but only in an exactly median sec- tion — which is the ovum cell, o. The whole ovule is anatropic or recurrent because the body of the ovule is not an elongation of the funiculus, but is folded back upon it and partly surrounded by it and grown fast to it, the mi- cropyle being turned towards the base of the funiculus. This form of ovule largely prevails in angiosperms. If we now compare this preparation with that o^ Deljplilnium we shall find that the structure is almost identical with it, the only difference being that in the DeljyJdnium the two inteo-uments of the ovule are united into one. In order to make a good section of the ovule in the or- dinary way between the thumb and finger we must re- move it from the ovary. If it is rightly placed between the thumb and finger we may obtain a median view this 324 MAKING SECTIONS OF THE OVULE. way sooner than by any other. But we may with advan- tage embed the ovule in glycerine jelly or in collodion be- fore cutting. The glycerine jelly must be relatively stiff, that is, contain considerable gelatine. Only alcohol ma- terial can be embedded in collodion. Pour the collodion solution in a little box made of Avriting paper and lay the ovule in it. It must stand in the air till it stiffens so that it will not run, then put it in 60 to 90 % alcohol. Here it will, in the course of a few hours, become of the consist- ency of gristle and be transparent ; cut through the object and the collodion together, and transfer the section with- out removing the collodion to glycerine or glycerine jelly. If one has got his collodion in the form of tablets he must dissolve it in a solution of equal parts ether and absolute alcohol. In order to make the ovule visible in the em- bedding medium it may be first stained in an aqueous so- lution of ha^niatoxylin. But the water must be afterwards removed from the object by the use of absolute alcohol be- fore it can be embedded in the collodion. For the study of the interior of the embryo sac we will take the Monotropa Hypo^itys, or false beech drops (2), the pale yellow plant common in pine forests. It is such a very favorable object for our purpose that we should spare no pains to obtain it. It blossoms in June and July and must be examined fresh since alcohol turns it dark brown and makes it opaque. It may be kept for a long time in a glass of water. Species of Pyrola answer the same purpose, only that the ovules are smaller. A tran- section of the superior ovary shows it to be fom'-celled. The placentae are much swollen and bear on their surface small, very numerous, closel}' compacted ovules. The two halves of the placentae in each compartment are widely separated by radial lines. In the upper part of the ovary these lines reach the middle and touch each other. We OVULE OF FALSE BEECH DROP. 325 now see four stout pairs of placentoe fixed to the middle of the division Avails which heloug to each two neighboring compartments, the pairs being easil}' separated with the needle. Remove the ovules from the open placenta with a needle and put them in pure water or a 3% solution of sugar in which the ovules will keep a long time. If we get our material from an old flower in which the stamens Fig. 106. Monotropa FTi/popitys. A, a whole ovule; /, the funiciilns; i, the iu- tegiimeut; B and C, the whole embryo sac; s, synergUla;; o, egg; n, nucleus of em- bryo sac ; D and /i, the upper part of the embryo sac. In E, is the first division for the Ibrmatiou of tlie endosperm. A X 210- JB to E. X. 600. have already discharged their pollen, we shall find the ovules ripe, but in part already fertilized, and in part not. Between the ovules we shall find many pieces of pollen tubes. The ovule ripe for fertilization is seen as in Fig. 106, A.. It is transparent and may be seen in optical sec- tions. It is anatropic and has but one integument, i. The whole interior of the ovule is filled with .the embryo 326 FERTILIZATION OF THE OVULE. sac, the niicellus being suppressed by the growth of the em- bryo sac. We will assume that the three cells of the apex of the embryo sac are clearly seen. These three cells form the egg apparatus. They are not of equal value. The two upper ones are helpers or synergidae, Fig. 106, B; the lower one is the true egg, o. The synergidte, as may be easily seen, have large vacuoles in their lower part and are filled above with protoplasm and at that point have their nuclei. The egg, on the contrary, lies between the principal mass of the cell plasma and cell nucleus and above the cell cavity. We may not always see both syn- ergidi«, as one may cover up the other, Fig. 106, C. At the base of the embiyo, sac one may see the three antipo- dal cells. In the interior of the embryo sac, one may find in most cases a cell nucleus, Fig. 106, A. Still in other cases there are two nuclei, Fig. 106, B, or one cell nucleus with two nucleus bodies. Fig. 106, C, and we conclude that in the end the cell nucleus is made from the union of two nuclei. Ovules, whose fertilization has begun, show the fact in the changes which have taken place in the synergidte. One or both of them appear strangely refractive. A pollen tube may also be seen penetrating the embryo sac, or at least, within the micropyle, or a piece of it projecting from the micropyle, torn away in the prep- aration. But, if the pollen tube has penetrated to the synergidae, the plasma from it will be thrown in between these cells upon the ovum itself. By careful examination, it Avill be seen that an ovum which lies near these changed S3aiergidje has two nuclei : one large, the original nucleus of the ovum, and a much smaller one which is the sperm nucleus from the pollen tube. Fig. 106, D. The latter soon increases in size. It one finds the ovule at the moment of copulation between the egg nucleus and the sperm nucleus he will see but one germinal nucleus with two nuclei of un- DEVELOPMENT OF FERTILIZED OVULE. 327 like size, Fig. 106, E, of which the smaller is the spermatic nucleus. At last the germinal nucleus will have but oue nucleolus. While the fertilizatiou of the ovum is going ou the strongly refractive sul)stance in the synergicÜB cells is lessened, apparently being used up in nourishing the ovum. At the same time with these changes in the egg appara- tus, the endosperm has begun to form in the cavity of the embryo sac, by the development of division walls in the sac itself, in this case by direct cell-division ; but in other cases, as freqnently or more often, the embryo sac nucleus and its derivatives freely divide first, followed later by the formation of division walls between the nuclei. This process, as it commonly takes place, is accompanied by a gradual but not considerable increase in size. When, on the contrary, the embryo sac rapidly grows afterthe com- pletion of the fertilization of the ovum, it takes place first by nucleus and not l>y cell division, the cell-building com- ing later when the emliryo sac is nearly full grown. In consequence of the lertilization, the ovum has taken on a delicate cellulose membrane and soon beofins to elongate tube-like and after a time penetrates the endosperm body with its apex, where it forms an embryo of a few cells. AYe have examined the embryo seed only in pure water or in sugar solution. If we would have the nucleus espec- ially distinct, we should examine it in a 2% solution of acetic acid. We thei'eby fix the nucleus and make it very sharply distinct and also preserve it in that state of self- division in which it was at that moment. Staining media are not recommended since they stain also the integument and thereby hinder the examination of the interior of the nucleus. Instead of Mo7iotropa the orchids (3) would serve us. Fertilization takes place a long time after the discharge of the pollen in the already greatly swollen ovary. Cut 328 OVULE OF ORCHIS PALLEXS. away the ovary and remove an ovule from the placenta with a needle and transfer it to water or a 3 % solution of sugar. As represented in Fig. 107, m, we see that the structure of the ovule is much like that of 3Ionotropa, except that there are two integuments, and an air cavity in the vicinity of the chalaza. The air cavity hinders the observation since it is tilled with air which tinally works up be- tween the integument. It ma}'' be taken out with the air pump or perhaps by a light pressure upon the cover-glass. In the orchids the nucellus is quite sup- pressed by the embryo sac. The egg apparatus, os, is built like that of Monotropa, only that the ovum is less deeply inserted. The antipodal cells are not to be seen, but in their place a strongly refractive substance in which are nuclei very difficult to make out. It is easier to trace the pol- len tube to the synergidae than letters are the same as in the earlier in the MoilOtvOpa ; the chaUgCS which take place in the syner- gidaj are quite the same. We also find again the two nuclei in the fertilized ovum. Endosperms are not usu- ally formed. In lack of jSlonotroixi and orchids the transparent ovules of the Gesneriacece (4) are to be commended and before all others the large-flowered Gloxinia liyhrida. The ovule with an integument is so far transparent that the Qgg apparatus is distinctly visible. It shows the two syn- FiG. 107. Orchis paUens. Ovule ripe for feriilizatioii. os, egg appar- atus; ii, le, inner and outer intefrii- nients; I, air cavity. The rest of the FERTILIZATION OF TORENIA. 329 ergldte and ovum, in this case fork-shaped. Sometimes two ova appear. The embryo sac is swollen above, but is suddenly narrowed below. The antipodal cells are not made out with certainty. But one of the most favorable plants for the study of fertilization is the Torenia asiatica (5). It is cultivated in almost all gardens and bears flowers the year around. It is distinguished by having the embryo sac protrude through the micropyle of the ovule, and so the egg ap- paratus comes into view, covered only by the wall of the embryo sac. The ovary is two-celled, the placenta cen- tral, with many ovules. Remove some of the ovules and examine in 3 % sugar solution. The ovules are anatropic or more rightly somewhat campylotropic for the embryo sac, and its integuments are somewhat bent in their upper part, Fig. 108, A. The funiculus, /, is somewhat large and the single integument stout. The embryo sac, e, shows its upper end protruded through the micropyle, smaller and pointed and lying against the funiculus. By means of potash lye, at the outset of its action, the em- bryo sac may be traced downwards into the ovule, where it may be seen to lie next the integument, very slender, somewhat spindle-shaped, e*, and at the base again nar- rowed. Our preparation in sugar water shows the three cells of the egg apparatus in the apex of the embryo sac. According to the position of the ovule, will both syner- gidaB or one be shown, as in B or C ; m the latter case one covers the other. At the apex of each synei'gida a strongly-refractive, homogeneous cap, clearly defined against the fine-grained posterior part, may be easily seen. It is called the fili- form apparatus. Chloriodide of zinc shows by the violet reaction that this cap consists of cellulose. The rest of the substance of these cells and of the ovum is colored 330 FERTILIZATION OF TORENIA. yellow-brown by this reagent. By careful examination, we find that the embryo sac membrane is opened over the synergidi^ cap, B, O. The cap, therefore, forms the closing apparatns for this opening of the sac membrane. It may be remarked in passing that they are widely dis- FiG. lOS. Torenia asiatica. A, two ovules on the placentae; e, the free apex of the embryo sac; e*, the lower widened part of the same in the interior of the ovule; /, funiculus; i, integument. X 2W. B and C, free apices of embryo sac be- fore fertilization; fl, synergidse caps, filament apparatus; o, egg; D and E, during fertilization; D, with a part of the funiculus, /; t, pollen tube. J5 to £. X 600. tributed, particnlarly in monocotyledonous plants and are often found in them protruding some distance out of the embryo sac. Their striation, which is often observed in these plants, is found to consist of fine pores filled with plasmic contents. By turning back to our preparation we find the distri- FERTILIZATION OF TORENIA. 331 bution of the contents in the syncrgiclaä and the ovnm is the same as in the Monotropa and Orchids, B, C. In the synergidaä the nucleus lies in the upper part and the vacuole in the under. This is reversed in the e, vascular bundle; «;*, end of same; st, SOUIÄ potasll lye lias dead pistil; t, style; /, funiculus of seed witli beCIl added. For the vascular bundle, /r; »rep, micro pyle; cä, clialaza end;